Conditions: PFIC1 PFIC2
Keywords: Pediatric Cholestasis
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 62
Healthy volunteers: no

Brief summary:
Double blind, randomized, placebo controlled, Phase 3 study to investigate the efficacy and safety of low doses and high doses of A4250 compared to placebo in children with progressive familial intrahepatic cholestasis (PFIC) types 1 and 2.

Detailed description:
Up to 50 sites in the following countries will take part in this study:

Australia, Belgium, Canada, France, Germany, Israel, Italy, Netherlands, Poland, Spain, Sweden, Turkey, United Kingdom, United States, and Saudi Arabia

Arms:
arm 1: Capsules for oral administration (40 ug/kg) once daily for 24 weeks arm 2: Capsules for oral administration (120 ug/kg) once daily for 24 weeks arm 3: Capsules for oral administration (to match active) once daily for 24 weeks

Interventions: intervention 1: A4250 (odevixibat) intervention 2: Placebo
Intervention descriptions: intervention 1: A4250 is a small molecule and selective inhibitor of ileal bile acid transporter (IBAT). intervention 2: Placebo identical in appearance to active drug (A4250).

Protocol document:
Prot_000.pdf: 
 
 
 
CLINICAL STUDY PROTOCOL A4250-005 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
 
IND Number:   
130591 
EudraCT Number 
2017-002338-21 
Test Product: 
A4250 
Indication: 
Progressive Familial Intrahepatic Cholestasis (PFIC) 
Sponsor: 
Albireo AB 
Development Phase: 
Phase 3 
Sponsor Signatory: 
 
Sponsor Medical Monitor: 
 
Principal Investigator: 
 
Date of the Protocol: 
24 June 2019 
Version of the Protocol: 
Final Amendment 06 
 
 
 
 
Confidential 
The information in this document contains trade secrets and commercial information that are 
privileged or confidential and may not be used, divulged, published, or otherwise disclosed 
without the written consent of Albireo AB.  These restrictions on disclosure apply equally to 
all future information supplied to you or generated by you in connection with the study. 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 2 
Final Amendment 06, 24 June 2019 
 
 
SPONSOR SIGNATURE PAGE 
PROTOCOL TITLE:  A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children with Progressive 
Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
 
PROTOCOL NUMBER:  A4250-005 
 
Albireo AB 
 
 
 
 
 
 
 
 
 
 
 
Date (day/month/year) 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 3 
Final Amendment 06, 24 June 2019 
 
INVESTIGATOR SIGNATURE PAGE 
 
PROTOCOL TITLE:  A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children with Progressive 
Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
 
PROTOCOL NUMBER:  A4250-005 
I have read this protocol and agree that it contains all necessary details for performing this 
study.  I will conduct the study as outlined herein and will complete the study within the time 
designated, in accordance with all stipulations of the protocol and in accordance with Good 
Clinical Practice (GCP), local regulatory requirements, and the Declaration of Helsinki. 
I will provide copies of the protocol and all pertinent information to all individuals 
responsible to me who assist in the conduct of this study.  I will discuss this material with 
them to ensure that they are fully informed regarding the study drug and the conduct of the 
study. 
I will use only the informed consent approved by the Independent Ethics Committee (IEC) 
and will fulfil all responsibilities for submitting pertinent information to the IEC responsible 
for this study. 
I agree that the sponsor (Albireo AB) shall have access to any source documents from which 
case report form information may have been generated. 
I further agree not to originate or use the name of Albireo AB, A4250 in any publicity, news 
release, or other public announcement, written or oral, whether to the public, press, or 
otherwise, relating to this protocol, to any amendment to the protocol, or to the performance 
of this protocol without the prior written consent of Albireo AB. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature 
 
Date (day/month/year) 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 4 
Final Amendment 06, 24 June 2019 
 
1 
ADMINISTRATIVE INFORMATION 
A Double-blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
Protocol No.: 
A4250-005 
Date of the initial Protocol: 
20 September 2017 
Date and Number of Amendment(s): 
Amendment 01, 06 December 2017 
 
Amendment 02, 10 May 2018 
 
Amendment 03, 22 November 2018 
 
Amendment 04, 01 March 2019 
 
Amendment 05, 29 April 2019 
 
Amendment 06, 24 June 2019 
Sponsor: 
Albireo AB 
 
Arvid Wallgrens backe 20 
 
413 46 Gӧteborg 
Sweden 
Clinical Research Organization: 
 
 
 
 
 
 
 
Sponsor Signatory: 
 
 
  
 
  
 
  
 
 
  
Sponsor Medical Monitor: 
 
 
 
 
Principal Investigator: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 5 
Final Amendment 06, 24 June 2019 
 
2 
STUDY SYNOPSIS 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Title of Study: 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate Efficacy and Safety of A4250 
in Children with Progressive Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
Principal Investigator: 
 
Study Centers: 
Up to 50 sites will be initiated for this study in the United States (US), Canada, European Union (EU), Middle 
East, and Australia. 
Publication(s): 
None. 
Planned Study Period: 
Q2 2018 to Q4 2019 
Development Phase: 
Phase 3 
Objectives: 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day A4250 in children with 
progressive familial intrahepatic cholestasis (PFIC) Types 1 and 2, as determined by the following: 
• 
Proportion of patients experiencing at least a 70% reduction in fasting serum bile acid (s-BA) 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L compared to placebo 
after 24 weeks of treatment 
• 
The proportion of positive pruritus assessments at the subject level over the 24-week Treatment Period 
Secondary Objectives 
• 
To evaluate the effect of A4250 on serum alanine aminotransferase (ALT) concentration 
• 
To evaluate the effect of A4250 on growth 
• 
To evaluate the effect of A4250 on sleep disturbance 
• 
To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or liver 
transplantation) 
• 
To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 
Methodology: 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the efficacy and safety of 
40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in children with PFIC (deficiencies of familial 
intrahepatic cholestasis-1 or bile salt export pump).  
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment Period and a 4-week 
Follow-up Period.  After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of A4250, or a 
matching placebo.  Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008) in which all patients will receive 
active treatment. 
There will be up to 10 clinic visits during the study and one scheduled telephone contact. 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
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Final Amendment 06, 24 June 2019 
 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Number of Patients: 
Approximately 60 patients diagnosed with PFIC-1 or PFIC-2 will be randomized.  The study will target 15% of 
enrolled patients to be PFIC-1 patients.  If enrollment of all PFIC-2 patients is completed and the 15% 
enrollment of PFIC-1 patients has not yet been achieved, then enrollment of PFIC-2 patients will continue in 
order to reach the total study target enrollment. 
Diagnosis and Main Criteria for Inclusion: 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages of ≥6 months 
and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have clinical genetic confirmation of PFIC-1 or PFIC-2 through identification of biallelic 
pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, taken as the 
average of 2 samples at least 7 days apart (Visits 1 and 2) prior to randomization  
4. Patient must have history of significant pruritus and a caregiver-reported observed scratching in the 
electronic diary (eDiary) average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  Patients who 
turn 18 years of age (or legal age per country) during the study will be required to re-consent in order 
to remain in the study 
6. Patients are expected to have a consistent caregiver for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use an eDiary 
device as required by the study 
Test Product, Dose and Mode of Administration: 
A4250, 40 µg/kg/day or 120 µg/kg/day, orally administered 
Reference Therapy, Dose and Duration of Administration: 
Matching placebo, orally administered 
Duration of Treatment: 
24 weeks 
Variables 
Efficacy:  
Primary Efficacy Endpoints 
The primary efficacy endpoints are: 
• 
EU and rest of world (RoW): Proportion of patients experiencing at least a 70% reduction in fasting 
s-BA concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L compared to 
placebo after 24 weeks of treatment. 
• 
US: Proportion of positive pruritus assessments at the subject level over the 24-week Treatment Period. 
Secondary Efficacy Endpoints 
Secondary efficacy endpoints include the following: 
• 
EU and RoW: Proportion of positive pruritus assessments at the subject level over the 24-week 
Treatment Period.  
• 
US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA concentration from 
baseline to the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 weeks of 
treatment. 
 
All Regions: 
All secondary endpoints will be compared to placebo. 
• 
Change from baseline to Week 12 and to Week 24 in fasting s-BA 
• 
Change from baseline to Week 12 and to Week 24 in serum ALT concentration 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 7 
Final Amendment 06, 24 June 2019 
 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
• 
Change in growth from baseline to Week 12 and Week 24 
• 
Proportion of responders for pruritus scores at Weeks 12 and 24 based on the Albireo PRO and ObsRO 
instruments  
• 
Change in sleep parameters measured with the Albireo PRO and ObsRO instruments from baseline 
over the 24-week Treatment Period  
• 
Proportion of individual assessments meeting the definition of a positive pruritus assessment at the 
subject level over the 24-week Treatment Period. A positive pruritus assessment includes an itch score 
≤1, or at least a one-point drop from baseline based on the Albireo PRO instrument; only patients ≥8 
years of age will complete the Albireo PRO instrument 
• 
Proportion of individual assessments meeting the definition of a positive pruritus assessment at the 
subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, respectively, or the 
proportion of positive pruritus assessments at each 4-week interval.  
• 
Proportion of individual AM assessments meeting the definition of a positive pruritus assessment at the 
subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, respectively, or the 
proportion of positive pruritus assessments at each 4-week interval.  
• 
Proportion of individual PM assessments meeting the definition of a positive pruritus assessment at the 
subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, respectively, or the 
proportion of positive pruritus assessments at each 4-week interval.  
• 
Number of patients undergoing biliary diversion surgery or liver transplantation 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• 
Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, caregiver, and 
clinician via the patient global impression of change (PGIC) items 
• 
Change from baseline in fasting s-BA at Week 4  
• 
Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline by each 
4-week interval of the Treatment Period as measured by the Albireo ObsRO instrument 
• 
Change from baseline by each 4-week interval of the Treatment Period in patient-reported and 
observer-reported night time itching and scratching severity scores, respectively 
• 
Change from baseline by each 4-week interval of the Treatment Period in patient-reported and 
observer-reported morning time itching and scratching severity scores, respectively 
• 
Change from baseline by each 4-week interval of the Treatment Period in pooled pruritus score 
including observer-reported scratching for patients <8 years of age and patient-reported itch severity 
for patients ≥8 years of age 
• 
Change from baseline by each 4-week interval of the Treatment Period in additional patient-reported 
and observer-reported sleep parameters (e.g., tiredness and number of awakenings) 
• 
Change from baseline to Week 24 in Pediatric Quality of Life Inventory (PedsQL) 
• 
Change from baseline in serum ALT concentration at Week 4  
• 
Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• 
Change from baseline in AST and gamma-glutamyl transferase at Weeks 4, 12, and 24 
• 
Change from baseline for other biochemical markers and measures of bile acid synthesis (autotaxin, 
plasma 7α-hydroxy-4-cholesten-3-one concentration [p-C4]) at Week 24 
• 
Change in pediatric end-stage liver disease/model for end-stage liver disease (PELD/MELD) score, 
AST to platelet ratio index (APRI) score, and Fib-4 score from baseline to Week 24 
• 
Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® (where available) 
• 
Liver-related mortality and liver decompensation events. 
• 
All-cause mortality 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 8 
Final Amendment 06, 24 June 2019 
 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Safety:   
Safety criteria are as follows: 
• 
Occurrence of treatment-emergent adverse events including severity and relatedness to study drug at all 
visits 
• 
The incidence of treatment-emergent serious adverse events (SAEs), based upon information from 
patient reports, including the description, incidence, and severity of an SAE 
• 
Trends evaluated for the following assessments: physical examinations, concomitant medications, vital 
signs, laboratory test results (including clinical chemistry, hematology, urinalysis, alfa-fetoprotein, 
vitamins A and E, 25-hydroxy vitamin D and INR), and abdominal ultrasound 
Statistical Methods: 
The primary objective of this study is to demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 
120 µg/kg/day A4250 in children with PFIC Types 1 and 2. 
  
The Cochran Mantel Haenzel (CMH) test stratified by PFIC class and age class will be performed to compare 
the proportion in fasting bile acid responders at the end of treatment (Week 22 and 24) in the two A4250 dose 
groups to the placebo group. To ascertain that all data are used in the CMH analysis, neighboring strata will be 
pooled if all subjects in a stratum have the same response. The proportion together with the corresponding 95% 
CI, odds ratio and corresponding 95% Clopper-Pearson exact CI and p-value for the CMH test will be presented.  
 
For the primary efficacy variable of the proportion of positive pruritus assessments at subject level over the 24-
week treatment period, an ANCOVA model will be used to analyse the comparisons between the treatment 
groups. The model will include treatment arm, AM baseline pruritus score, PM baseline pruritus score, and 
randomization stratification factors, i.e. PFIC class and age class. LS mean (SE) by treatment arm and LS mean 
difference (SE), 95% confidence intervals, and p-values (where applicable) between treatments (120 µg/kg/day 
and 40 µg/kg/day, respectively) vs. placebo will be provided. If there are concerns on model assumptions, i.e. 
normality and homogeneity of variances, a non-parametric ANCOVA based on the rank scores can be used as a 
sensitivity analysis. LS mean/SE on the outcome by treatment arm and LS mean difference/SE between active 
dose and placebo will be provided.  
 
For each primary endpoint by region (EU & RoW and US), a pooled analysis for the closed testing procedure 
will be applied to control the 1-sided overall type I error rate for two treatment comparisons vs. the placebo at 
the 0.025 level, as specified below: 
 
In the closed testing procedure, the low and high dose groups will be pooled to compare with the placebo group 
first. If the 1-sided p-value is ≤0.025, the 1-sided p-values for low dose vs. placebo and high dose vs. placebo 
will be calculated respectively. If both individual p-values are ≤0.025, a significant treatment effect will be 
declared on both dose groups. If only one of them is ≤0.025, a significant treatment effect will be declared on 
the corresponding dose group.  
 
Other secondary and exploratory outcome variables will provide supportive efficacy and safety information 
regarding the differences between the treatment groups. No adjustments will be performed for multiple 
comparisons when testing secondary and exploratory efficacy variables. 
 
Date of the Protocol:  24 June 2019 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 9 
Final Amendment 06, 24 June 2019 
 
3 
TABLE OF CONTENTS 
1 
ADMINISTRATIVE INFORMATION ........................................................................ 4 
2 
STUDY SYNOPSIS ...................................................................................................... 5 
3 
TABLE OF CONTENTS .............................................................................................. 9 
3.1 
List of Tables .................................................................................................. 13 
3.2 
List of Figures................................................................................................. 13 
3.3 
List of Appendices .......................................................................................... 13 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ................................ 14 
5 
INTRODUCTION ....................................................................................................... 16 
5.1 
Investigational Medicinal Product .................................................................. 16 
5.2 
Background..................................................................................................... 16 
5.2.1 
Progressive Familial Intrahepatic Cholestasis ........................................... 16 
5.2.2 
Summary of Clinical and Nonclinical Studies ........................................... 18 
5.3 
Rationale ......................................................................................................... 19 
5.3.1 
Risk/Benefit ............................................................................................... 20 
6 
STUDY OBJECTIVES ............................................................................................... 21 
6.1 
Primary Objectives ......................................................................................... 21 
6.2 
Secondary Objectives ..................................................................................... 21 
7 
INVESTIGATIONAL PLAN ...................................................................................... 22 
7.1 
Overall Study Design and Plan....................................................................... 22 
7.1.1 
Description ................................................................................................. 22 
7.1.2 
Schedule of Assessments ........................................................................... 24 
7.1.3 
Study Procedures and Assessments ........................................................... 28 
7.1.3.1 
Screening Period (Days -56 to 0) ............................................... 28 
7.1.3.2 
Treatment Period (Day 0 to Day 168) ........................................ 29 
7.1.3.3 
Follow-Up Period ....................................................................... 32 
7.2 
Study Population ............................................................................................ 32 
7.2.1 
Inclusion Criteria ....................................................................................... 33 
7.2.2 
Exclusion Criteria ...................................................................................... 33 
7.2.3 
Withdrawal of Patients............................................................................... 35 
7.2.4 
Study Termination by Sponsor .................................................................. 36 
8 
TREATMENT OF PATIENTS ................................................................................... 37 
8.1 
Identity of Study Drug .................................................................................... 37 
8.2 
Administration of Study Drug ........................................................................ 37 
8.3 
Study Treatment Packaging and Labeling ...................................................... 38 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
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M5.3.5.1 – Protocol A4250-005 
 
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8.3.1 
Packaging and Labeling ............................................................................. 38 
8.3.2 
Storage ....................................................................................................... 38 
8.3.3 
Blinding and Randomization of Study Drug ............................................. 38 
8.4 
Procedure for Breaking the Randomization Code .......................................... 39 
8.5 
Patient Compliance ......................................................................................... 40 
8.6 
Study Drug Accountability ............................................................................. 40 
8.7 
Concomitant Therapy and Prohibited Medications ........................................ 40 
9 
ASSESSMENT OF EFFICACY ................................................................................. 41 
9.1 
Efficacy Endpoints ......................................................................................... 41 
9.1.1 
Primary Efficacy Endpoint ........................................................................ 41 
9.1.2 
Secondary Efficacy Endpoint .................................................................... 41 
9.1.3 
Exploratory Efficacy Endpoints ................................................................. 42 
9.2 
Efficacy Assessments ..................................................................................... 43 
9.2.1 
Serum Bile Acids ....................................................................................... 43 
9.2.2 
Itching, Scratching and Sleep Score .......................................................... 44 
9.2.3 
Growth ....................................................................................................... 44 
9.2.4 
Quality of Life Questionnaire (PedsQL) ................................................... 45 
9.2.5 
Biomarkers and PK Samples ..................................................................... 45 
9.2.6 
PELD/MELD Score ................................................................................... 45 
9.2.7 
Fibroscan® .................................................................................................. 45 
9.2.8 
Markers of Fibrosis .................................................................................... 45 
9.2.9 
Global Impression of Change and Global Impression of Symptom 
Measures .................................................................................................... 45 
10 
ASSESSMENT OF SAFETY...................................................................................... 47 
10.1 
Adverse Events ............................................................................................... 47 
10.1.1 
Definitions and Investigator Assessments ................................................. 47 
10.1.1.1 Clinical Significance .................................................................. 47 
10.1.1.2 Serious Adverse Events .............................................................. 47 
10.1.1.3 Severity Assessment ................................................................... 48 
10.1.1.4 Causality Assessment ................................................................. 48 
10.1.2 
Recording of Adverse Events .................................................................... 49 
10.1.3 
Recording and Reporting of Serious Adverse Events ................................ 50 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions .............. 51 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments .................................................................................................... 52 
10.2.1 
Laboratory Assessments ............................................................................ 52 


 
 
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M5.3.5.1 – Protocol A4250-005 
 
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10.2.2 
Individual Patient Safety Monitoring ........................................................ 53 
10.2.2.1 Liver Monitoring ........................................................................ 53 
10.2.2.2 Close Observation ...................................................................... 54 
10.2.2.3 De-challenge/Re-challenge for Liver and Clinical Hepatitis 
Monitoring .................................................................................. 55 
10.2.2.4 Diarrhea ...................................................................................... 55 
10.2.2.5 Monitoring after Permanent Discontinuation of Study Drug 
Due to Safety .............................................................................. 55 
10.2.3 
Demographics/Medical and Surgical History ............................................ 55 
10.2.4 
Clinical Genetic Testing ............................................................................ 56 
10.2.5 
Physical Examination ................................................................................ 56 
10.2.6 
Vital Signs ................................................................................................. 56 
10.2.7 
Abdominal Ultrasound ............................................................................... 56 
10.2.8 
Overdose .................................................................................................... 56 
10.2.9 
Pregnancy .................................................................................................. 57 
11 
STATISTICAL EVALUATION ................................................................................. 58 
11.1 
Sample Size and Power .................................................................................. 58 
11.2 
Statistical Methods ......................................................................................... 58 
11.2.1 
Statistical Analysis Sets ............................................................................. 58 
11.2.2 
Methods of Statistical Analyses ................................................................. 59 
11.2.2.1 General Principles ...................................................................... 59 
11.2.2.2 Missing Data............................................................................... 60 
11.2.2.3 Demographic and Baseline Characteristics ................................ 60 
11.2.2.4 Subject Disposition..................................................................... 60 
11.2.2.5 Evaluation of Primary Efficacy Variables .................................. 61 
11.2.2.6 Evaluation 
of 
Secondary 
and 
Exploratory 
Efficacy 
Variables ..................................................................................... 61 
11.2.2.7 Evaluation of Safety Variables ................................................... 62 
11.2.2.8 Compliance and Exposure .......................................................... 63 
11.2.3 
Blinded Analyses to Confirm Responder Definitions ............................... 63 
11.2.4 
Data Safety Monitoring Board ................................................................... 64 
12 
DIRECT ACCESS TO SOURCE DATA/NOTES ..................................................... 65 
13 
QUALITY CONTROL AND QUALITY ASSURANCE .......................................... 66 
13.1 
Conduct of the Study ...................................................................................... 66 
13.2 
Study Monitoring............................................................................................ 66 


 
 
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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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14 
ETHICS ....................................................................................................................... 68 
14.1 
Independent Ethics Committee/Institutional Review Board .......................... 68 
14.2 
Written Informed Consents and Assents ........................................................ 68 
15 
DATA HANDLING AND RECORD KEEPING ....................................................... 70 
15.1 
Case Report Forms/Source Data Handling..................................................... 70 
15.2 
Retention of Essential Documents .................................................................. 70 
16 
FINANCING AND INSURANCE .............................................................................. 71 
17 
PUBLICATION POLICY ........................................................................................... 72 
18 
REFERENCE LIST ..................................................................................................... 73 
19 
APPENDICES ............................................................................................................. 74 
 


 
 
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3.1 
List of Tables 
Table 1 
Schedule of Assessments ................................................................................... 25 
Table 2 
Dosing and Capsule Strength ............................................................................. 38 
Table 3 
Routine Laboratory Parameters .......................................................................... 53 
 
3.2 
List of Figures 
Figure 1 
Study Design ...................................................................................................... 24 
 
3.3 
List of Appendices 
Appendix 1 Concomitant Medication Guidelines .................................................................. 74 
Appendix 2 Diary Questions including Albireo Patient-Reported 
Outcomes/Observer-Reported Outcome Instrument .......................................... 75 
Appendix 3 Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) ....................................................................... 76 
Appendix 4 Pediatric Quality of Life Inventory (PedQL) Questionnaire .............................. 77 
Appendix 5 Guideline for Fat-Soluble Vitamin Supplementation......................................... 78 
Appendix 6 Contraceptive Requirements .............................................................................. 79 
Appendix 7 Blood Volumes ................................................................................................... 80 
 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
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M5.3.5.1 – Protocol A4250-005 
 
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4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
ADME 
absorption, distribution, metabolism, and excretion 
AE(s) 
adverse event(s) 
AFP 
alfa-fetoprotein 
ALT 
alanine aminotransferase 
APRI 
AST to platelet ratio index 
ASBT 
apical sodium-dependent bile transporter (also known as IBAT) 
AST 
aspartate aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BMI 
body mass index 
BSEP 
bile salt export pump 
CPK 
creatine phosphokinase 
CRA 
clinical research associate 
CYP 
cytochrome P450 
DSMB 
Data and Safety Monitoring Board 
eCRF 
electronic case report form 
eDiary 
electronic diary 
EOT 
End of Treatment 
EU 
European Union 
EudraCT 
European Union drug regulatory agency clinical trial 
FAS 
full analysis set 
FDA 
United States Food and Drug Administration 
FIC1 
familial intrahepatic cholestasis-1 
GCP 
Good Clinical Practice 
GGT 
gamma-glutamyl transferase 
GI 
gastrointestinal 
GLMM 
generalized linear mixed modelling 
IB 
Investigator’s Brochure 
IBAT 
ileal bile acid transporter (also known as ASBT) 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
 
 Clinical Research Organization 
IEC 
Independent Ethics Committee 
INR 
international normalized ratio 
IRB 
Institutional Review Board 


 
 
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IWRS 
Interactive Web Response System 
LDH 
lactate dehydrogenase 
LFT 
liver function test 
LPLV 
last patient last visit 
MDR3 
multidrug-resistance protein 3 
MedDRA 
Medical Dictionary for Regulatory Activities 
MELD 
model for end-stage liver disease 
MMRM 
mixed model repeated measures 
ObsRO 
observer-reported outcome 
PBC 
primary biliary cholangitis 
p-C4 
plasma 7α-hydroxy-4-cholesten-3-one concentration 
PCS 
potentially clinically significant 
PedsQL 
Pediatric Quality of Life Inventory 
PELD 
pediatric end-stage liver disease 
PFIC 
progressive familial intrahepatic cholestasis 
PGIC 
patient global impression of change 
PGIS 
patient global impression of symptoms 
PK 
pharmacokinetic(s) 
PP 
per protocol 
PRO 
patient-reported outcome 
PT 
prothrombin time 
PVM 
pharmacovigilance manager 
QoL 
quality of life 
RoW 
rest of world 
RR 
respiratory rate 
SAE(s) 
serious adverse event(s) 
SAP 
statistical analysis plan 
s-BA 
serum bile acid 
SD 
standard deviation 
s-FGF19 
serum fibroblast growth factor 19 concentration 
SOC 
system organ class 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE(s) 
treatment-emergent adverse event(s) 
ULN 
upper limit of normal 
US 
United States 
WHO 
World Health Organization 


 
 
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5 
INTRODUCTION 
5.1 
Investigational Medicinal Product 
A4250 is a small molecule and a potent selective inhibitor of the ileal bile acid transporter 
(IBAT), also known as the apical sodium-dependent bile acid transporter (ASBT).  IBAT is 
an integral brush border membrane glycoprotein that co-transports sodium and bile acids and 
appears to be a critical component in the regulation of the bile acid pool size in animals and 
man.  This transporter, expressed in the distal ileum, is a key element in the enterohepatic 
circulation of bile acids since it facilitates the high-affinity, high-capacity reabsorption of bile 
acids.  Indeed, 95% of secreted bile acids are reabsorbed via IBAT [Hofmann 2009; 
Miethke 2016].  A4250 is orally administered and acts locally in the gut where it binds 
reversibly to IBAT to decrease the reuptake of bile acids.  A4250 has minimal systemic 
exposure at the predicted therapeutic dose ranges. 
No safety signals were found with A4250 in preclinical safety and toxicity studies.  In the 
first clinical study of A4250 in healthy volunteers, A4250 was given as a single oral dose up 
to 10 mg or daily oral doses up to 3 mg/day for 7 days.  A4250 was well tolerated and the 
only findings were gastrointestinal (GI) related effects, such as diarrhea, at higher doses. 
In an exploratory Phase 2 study in children with cholestatic pruritus, A4250 was given as oral 
doses of 10 to 200 µg/kg/day for 4 weeks.  A4250 reduced mean levels of serum bile acids 
(s-BA) in all cohorts.  In 11 of 13 progressive familial intrahepatic cholestasis 
(PFIC) patients, substantial reductions (ranging from 43% to 98%) were observed.  
Patient-reported diary data documented improvement of pruritus during treatment.  A4250 
was well tolerated.  No serious adverse events (SAEs) were deemed to be related to study 
drug, and most adverse events (AEs), including some increased transaminases, were 
considered unrelated to study drug and transient in nature. 
5.2 
Background 
5.2.1 
Progressive Familial Intrahepatic Cholestasis 
PFIC is a rare autosomal recessive cholestatic liver disease estimated to affect between 1 in 
every 50,000 to 100,000 children born worldwide.  PFIC represents 10% to 15% of causes of 
cholestasis in children and 10% to 15% of liver transplantation indications in children.  All 
types of PFIC exist worldwide and both sexes appear to be equally affected. 
The common underlying pathogenesis of PFIC is disruption of bile formation and bile 
transport through the liver [Jacquemin 2000].  The classification of PFIC has evolved over 
the years.  The most commonly used sub-classifications are PFIC Types 1, 2, and 3, which 
are based on the associated affected gene and described in more detail below. 
• PFIC, Type 1:  also referred to as “Byler disease” or “familial intrahepatic 
cholestasis-1 (FIC1) protein deficiency.” FIC1 protein is located on the canalicular 


 
 
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membrane of hepatocytes and facilitates movement of aminophospholipids from the outer 
to inner leaflet of the plasma membrane of the hepatocyte.  The ATP8B1 gene encodes 
FIC1 protein.  Biallelic pathologic variants in the ATP8B1 gene are associated with FIC1 
dysfunction and classified clinically as PFIC Type 1 disease. 
• PFIC, Type 2:  also referred to as “Byler syndrome” or “bile salt export pump (BSEP) 
deficiency.”  BSEP is a transporter protein that is expressed at the canalicular membrane 
of hepatocytes and is the primary exporter of bile acids.  The ABCB11 gene encodes the 
BSEP protein.  Biallelic pathologic variations in the ABCB11 gene are associated with 
BSEP dysfunction and classified clinically as PFIC Type 2 disease. 
• PFIC, Type 3:  is caused by a deficiency of the multidrug-resistance protein 3 (MDR3) 
due to mutations in the ABCB4 gene.  MDR3 is a phospholipid translocase critical for 
phospholipid secretion. 
Severe pruritus is common in children diagnosed with PFIC.  Itching (and subsequent 
scratching) is a significant morbidity for these patients and their families [Suchy 2007].  A 
more severe degree of pruritus is experienced compared to patients with other forms of liver 
disease and to other pruritic conditions such as atopic dermatitis [Murray 2011].  In patients 
with PFIC, liver biopsy reveals canalicular cholestasis and, later, the appearance of portal 
fibrosis.  Serum biochemistry indicates cholestasis with hyperbilirubinemia, elevated alanine 
aminotransferase (ALT) and aspartate aminotransferase (AST).  The concentrations of bile 
acids in serum are very high, while serum gamma-glutamyl transferase (GGT) activity 
(exception being MDR3 variants) and cholesterol are normal [Hori 2010].  Symptoms of 
portal hypertension and liver failure will develop during the course of the disease [Davit-
Spraul 2009; Alissa 2008].  Symptoms develop early; median age at onset of symptoms is 
2 months, and 78% of PFIC patients present with jaundice [Pawlikowska 2010].  The 
life-threatening and debilitating nature of PFIC is reflected by the fact that survival in those 
not resorting to surgery is 50% at 10 years of age and almost zero at 20 years of age.  
Approximately half of PFIC patients undergo liver transplantation [Davit-Spraul 2009] and 
treatment-resistant pruritus is the leading indication for the surgical procedure partial external 
biliary diversion, mostly in PFIC Type 1 and 2 patients. 
PFIC is life-threatening and debilitating.  Currently, the treatment for PFIC is palliative and 
there is no pharmaceutical treatment approved for use in PFIC.  The therapeutic choices are 
restricted to non-specific therapy of the symptoms and signs of the disease such as nutritional 
support, preventing vitamin deficiencies, and treatment of extrahepatic features.  Medical 
treatment options include off-label use of ursodeoxycholic acid, rifampin, antihistamines, and 
naltrexone.  A minority of patients respond nominally and transiently to these interventions.  
Biliary diversion is used to decrease systemic bile acids through interruption of the 
enterohepatic circulation and so avoid transplantation.  Liver transplantation is typically only 
viewed as an option when patients have failed medical treatment and/or biliary diversion and 
have liver failure or persistent uncontrolled pruritus. 


 
 
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5.2.2 
Summary of Clinical and Nonclinical Studies 
This is a summary of nonclinical and clinical studies.  More detail is provided in the 
Investigator’s Brochure ([IB] see A4250 IB).  
A4250 is a potent, selective IBAT inhibitor with no safety signals seen in the safety 
pharmacological studies.  In addition, no safety signals were seen in repeat-dose oral toxicity 
studies in rats with up to 26 weeks of daily dosing and in dogs with up to 39 weeks of daily 
dosing.  No safety signals were identified when A4250 was administered daily to juvenile 
rats from age 14 days to 63 days. 
A4250 is minimally absorbed with very low systemic exposure in both humans and other 
species.  In blood, protein binding is high (>99.7%).  Systemically absorbed A4250 is slowly 
metabolized in humans and in preclinical studies by hydroxylation only, a metabolite that 
retains IBAT activity.  A4250 did not show any significant inhibition of cytochrome 
P450 (CYP) 
enzymes 
in 
the 
rat. 
 
A4250 
inhibited 
human 
CYP3A4 
and 
CYP2D6 (IC50: 16 µmol/L for both enzymes), and CYP2C9 (IC50:1.2 µmol/L).  
To date, A4250 has been studied in 3 Albireo-sponsored clinical studies: a study in healthy 
volunteers evaluating single and multiple administration of A4250; a single-dose absorption, 
distribution, metabolism, and excretion (ADME) study; and a Phase 2 study in children with 
cholestatic pruritus.  In addition, an investigator-sponsored study has been conducted in 
patients with primary biliary cholangitis (PBC). 
In the first clinical study (A4250-001), A4250 was given to healthy volunteers as a single oral 
dose up to 10 mg or daily oral doses up to 3 mg/day for 7 days.  Following a single oral dose 
of 10 mg, the median maximum drug concentration was 0.2 nmol/L and the area under the 
curve was 0.8 nmol × h/L (n=6).  Pharmacodynamic evaluation of bile acid synthesis 
biomarkers 
(serum 
fibroblast 
growth 
factor 
19 [s-FGF19] 
and 
plasma 
7α Hydroxy 4-cholesten-3-one [p-C4]) identified the expected decrease in s-FGF19 and an 
increase in p-C4.  In addition, total plasma bile acids decreased and total fecal bile acids 
increased in A4250-treated patients compared to placebo. A4250 was generally well 
tolerated.  The only findings were GI-related effects, such as diarrhea at higher doses. 
In the ADME study (A4250-007), healthy subjects were administered a single oral dose of 
3 mg [14C]-A4250 capsule.  The main excretion route was determined to be fecal.  There 
were no quantifiable concentrations of A4250 in plasma or total radioactivity in plasma or 
whole blood.  A4250 was well tolerated.  AEs were reported and most were mild and not 
related to A4250. The 3 AEs experienced by 2 subjects reported as related to study drug were 
GI in nature, including diarrhea (2 AEs; 2 subjects) and abdominal pain (1 AE; 1 subject). 
In an exploratory Phase 2 study in children with cholestatic pruritus (A4250-003), A4250 was 
administered as oral daily doses of 10 to 200 µg/kg/day for 4 weeks.  Twenty 
patients (8 females and 12 males) aged 1 to 17 years were enrolled in this open-label 
dose-escalation study.  Four patients were re-enrolled into a second dose level.  No 


 
 
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treatment-related SAEs were reported, and most AEs, including some increased 
transaminases, were transient.  Four-week, once-daily treatment with A4250 reduced mean 
levels of s-BA in all cohorts.  Substantial s-BA reductions in 11 of 13 PFIC patients (ranging 
from 43% to 98%) were observed.  A4250 also reduced s-BA in the 11 non-PFIC patients, 
but to a lesser extent than in PFIC patients.  Patient-reported diaries documented 
improvement of the visual analogue scale itch score in 17 of 24 patients during treatment.  
The dose with the greatest improvement showed a mean decrease (± standard error of the 
mean) of 2.9 ±1.1 from baseline.  The results from this study form the basis of the current 
(A4250-005) study design. 
5.3 
Rationale 
In this investigational study, A4250 is under development for treatment of PFIC and is also 
being considered for other orphan cholestatic liver diseases. 
There is currently no medical treatment approved for PFIC.  In PFIC patients with severe 
pruritus, biliary diversion surgery is used to decrease systemic bile acids through interruption 
of the enterohepatic circulation and thereby reducing pruritus [Whitington 1988]. A portion 
of patients who have biliary diversion surgery continue to have elevated serum bile acids and 
pruritus. Liver transplantation is typically used as an option when patients have failed 
medical treatment and/or biliary diversion and have liver failure or persistent uncontrolled 
pruritus. 
By inhibiting IBAT with high selectivity and potency, A4250 has the potential to relieve 
cholestasis and improve liver function without surgical intervention in patients with PFIC.  
Since A4250 is minimally absorbed and achieves a substantial reduction of s-BA, it has the 
potential to be a safer and less invasive alternative to surgical biliary drainage.  The rationale 
for using A4250 is to decrease s-BA levels, and to reduce the major morbidity of pruritus, 
improving the health and wellbeing of patients affected with PFIC.  Ultimately, this 
noninvasive therapeutic approach has the potential to not only improve quality of life (QoL), 
but also reduce the need for biliary diversion and subsequent liver failure and liver 
transplantation.  For those patients who have undergone biliary diversion surgery and 
continue to have elevated serum bile acids and symptomatic pruritus, IBAT inhibition has the 
potential to reduce both the serum bile acid levels and the pruritus. 
The doses selected, 40 and 120 µg/kg/day, were based on the efficacy and safety data 
generated from patients with PFIC (n=10 + 3 re-entered) and supporting data from all 
patients (n=20 + 4 re-entered) in the five dose groups dosed in the A4250-003 study.  
Improvements in s-BA and pruritus were observed with doses from 30 µg/kg to 100 µg/kg, 
with a dose-related trend.  The best dose response in all patients was at the 60 µg/kg/day for 
s-BA and 100 µg/kg/day for pruritus.  The best dose response in the PFIC subgroup was at 
30 µg/kg/day for both s-BA and pruritus.  Patients with PFIC responded well to the doses 
given from 30 µg/kg to 200 µg/kg/day; however, a clear dose relationship could not be 


 
 
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established in order to select a single dose for this Phase 3 study.  As efficacy was observed 
at both low and high doses, 40 µg/kg and 120 µg/kg/day are considered to be the most 
optimal for this study.  
This study will be conducted in compliance with the protocol and with the International 
Council for Harmonisation (ICH) guidelines on Good Clinical Practice (GCP). 
Patients completing 24 weeks of double-blind treatment will be offered to enter Study 
A4250-008 and receive open-label treatment with A4250. 
5.3.1 
Risk/Benefit 
A4250 has been evaluated in three Albireo-sponsored clinical studies: a double-blind 
placebo-controlled study in healthy volunteers, a single-dose ADME study, and a Phase 2 
study in children with cholestatic pruritus.  In addition, an investigator-sponsored study has 
been conducted in patients with PBC. 
Healthy subjects have been exposed to up to 10 mg as a single dose and up to 3 mg daily as 
part of a multiple-dose evaluation.  Children with cholestatic liver disease have been treated 
with up to 0.2 mg/kg/day (200 µg/kg/day) for 4 weeks.  In total, 2 SAEs have been reported; 
both were judged by the physician to be not related to the study drug. 
Patients with cholestatic liver diseases suffer from excess bile acids in the liver resulting in 
tissue damage.  A commonly used treatment in these patients is bile diversion surgery, 
whereby approximately 50% to 100% of the enterohepatic circulation of bile acids is 
interrupted.  Inhibition of IBAT with A4250, thereby interrupting the enterohepatic 
circulation of bile acids, is therefore a potential medical alternative to surgery which could be 
of benefit to these patients if shown to be effective and safe.  Data from the Phase 2 study 
showed efficacy of A4250 in reducing s-BA concentrations and pruritus in such patients. 
Based on the mode of action of an IBAT inhibitor, loose stools or diarrhea could be expected.  
However, in the pediatric Phase 2 study with 4 weeks daily treatment, only one patient had 
mild transient diarrhea after a single dose that did not recur on multiple dosing. 


 
 
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6 
STUDY OBJECTIVES 
6.1 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day 
A4250 in children with PFIC Types 1 and 2, as determined by the following: 
• The proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment 
• The proportion of positive pruritus assessments at the subject level over the 24-week 
treatment period.   
6.2 
  Secondary Objectives 
• To evaluate the effect of A4250 on serum ALT concentration 
• To evaluate the effect of A4250 on growth 
• To evaluate the effect of A4250 on sleep disturbance 
• To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or 
liver transplantation) 
• To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 


 
 
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7 
INVESTIGATIONAL PLAN 
7.1 
Overall Study Design and Plan 
7.1.1 
Description 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the 
efficacy and safety of 40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in 
children with PFIC (deficiencies of FIC1 or BSEP). 
Study data will be periodically (approximately on a quarterly basis) reviewed by an 
unblinded Data Safety and Monitoring Board (DSMB). 
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment 
Period and a 4-week Follow-Up Period. 
After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of 
A4250, or a matching placebo. 
Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008; EudraCT Number 
2017-002325-38, subject to a separate study approval) in which all patients will receive 
active treatment.   
Visit windows applicable to each visit are presented in the schedule of assessments (Table 1). 
There will be up to 10 clinic visits during the study (Figure 1) and one scheduled telephone 
contact, as follows: 
• Visit 1: Screening Period start. Patient is considered enrolled after signing the informed 
consent form (ICF) (Days -56 to -35) 
• Visit 2: second Screening Visit (Days -28 to -7) 
• Visit 3: Randomization Visit (Day 0) 
• Telephone Contact 1: (Day 14) 
• Visit 4: Clinic Visit (Day 28) 
• Visit 5: Clinic Visit (Day 56) 
• Visit 6: Clinic Visit (Day 84) 
• Visit 7: Clinic Visit (Day 126) 
• Visit 8: Clinic Visit (Day 154) 
• Visit 9: Clinic Visit, End of Treatment ([EOT] Day 168) 
• Visit 10: Follow-Up Visit, 28 days after last dose of study drug (Day 196).  All patients 
continuing in Study A4250-008 will not have this Follow-up Visit. 


 
 
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Additional clinic visits may be required for patients who need direct site assistance, e.g., due 
to AE monitoring, to fulfil screening requirements, and/or for safety maintenance. 
Screening Period 
Informed consent must be obtained at Visit 1 prior to performing any study procedures.  After 
signing the ICF, patients will be considered enrolled and evaluated for study eligibility. 
Daily recording of pruritus using an electronic diary (eDiary) will be started to determine 
confirmation of the magnitude and the baseline pruritus symptoms before the study.  After 
consultation with the Medical Monitor, patients failing eligibility criteria may be re-screened.  
Patients not fulfilling inclusion/exclusion criteria after 3 attempts are not allowed to 
re-screen. 
Caregivers/patients will be instructed on the daily use of the eDiary.  The eDiary will include 
patient-reported outcome (PRO) and observer-reported outcome (ObsRO) items for 
evaluation of itching (PRO), scratching (ObsRO), and sleep disturbance (PRO and ObsRO) 
during the Screening Period (study Days -56 to 0), throughout the 24-week Treatment Period 
(study Days 0 to 168), and Follow-up (Day 196). 
The 14 consecutive days prior to randomization will be used for eligibility calculation of the 
diary entries.  If a patient does not meet the minimum number of observations (see 
Section 9.2.2 for scoring and missing data definitions) to determine eligibility, the patient 
may be re-screened.  Observer-reported outcomes in patients of all ages will be recorded by a 
caregiver.  If possible, the same caregiver will complete the Albireo ObsRO items throughout 
the study. 
At a second Screening Visit (Visit 2) additional laboratory samples will be taken for 
eligibility assessments. 
Treatment Period 
Eligibility for randomization will be determined at Visit 3 using the pruritus (scratching 
reports from the Albireo ObsRO) data in the 2 weeks before Visit 3, clinical genetic 
confirmation of diagnosis, and the liver biochemistry evaluations from the previous screening 
visits.  Two weeks after Visit 3, there will be a telephone contact with the caregiver/patient to 
evaluate any AEs. 
The patient will return to the clinic at Visit 4, Visit 5, Visit 6, Visit 7, Visit 8, and Visit 9 for 
assessments.  If a new caregiver will begin to complete the Albireo ObsRO during the course 
of the trial, they will be provided with training during a clinic visit before they begin to use 
the eDiary.  Additionally, caregivers will be requested to use the diary to report the time each 
dose of study drug is administered during the Treatment Period. 
The last dose of study drug will be administered the day before Visit 9.  At study Visit 9, the 
patient has the option to enroll in an open-label extension study (A4250-008) should they 


 
 
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meet the required entry criteria.  If the patient is continuing into Study A4250-008 this will be 
the last visit in this study and the first visit in Study A4250-008.  Visit 9/EOT should also be 
performed at the time patients are prematurely withdrawn from the study. 
Clinic Visit 10 will take place 28 days after Visit 9 for all patients not continuing into Study 
A4250-008. 
Figure 1 
Study Design 
 
T: telephone contact; V: study visit 
End of Study 
The end of the study is defined as follows: 
a) End of study in one country: last patient last visit (LPLV) and sites are closed 
b) End of study globally: LPLV globally and all sites closed 
7.1.2 
Schedule of Assessments 
The schedule of assessments is presented in Table 1.  For information on blood volumes, 
please see Appendix 7. 
 


 
 
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Table 1 
Schedule of Assessments 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Informed consent c 
X 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
Concomitant medicationd 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical and surgical history 
X 
 
X 
 
 
 
 
 
 
 
 
Physical examination including 
voluntary photography 
X 
 
X 
 
 
 
X 
 
 
X 
 
Skin examination 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
Vital signse 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
eDiary: itching, scratching, and 
sleep scoresf 
Daily diary entry 
Clinical chemistryg 
X 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Hematologyg 
 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Urinalysisg 
 
X 
 
 
 
 
X 
 
 
X 
 
Serum bile acidsh 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 


 
 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
INR 
 
X 
 
 
X 
 
X 
 
X 
 
X 
Autotaxini 
 
 
X 
 
X 
 
 
 
 
X 
 
p-C4i 
 
 
X 
 
X 
 
 
 
 
X 
 
AFP 
 
 
X 
 
 
 
 
 
 
X 
 
Vitamins A & Eh 
 
X 
 
 
 
 
X 
 
 
X 
 
25-hydroxy vitamin D 
 
 
X 
 
 
 
X 
 
 
X 
 
Blood sample for A4250 PKi 
 
 
 
 
X 
 
 
 
 
X 
 
Abdominal Ultrasound 
 
 
X 
 
 
 
 
 
 
X 
 
Fibroscan® (where available) 
 
 
X 
 
 
 
 
 
 
X 
 
QoL questionnaire (PedsQL) 
 
 
X 
 
 
 
 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of Change 
 
 
 
 
X 
 
X 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of 
Symptoms 
 
 
X 
 
X 
 
X 
 
 
X 
 
Pregnancy testj 
Xj 
X 
X 
 
X 
X 
X 
X 
X 
Xj 
X 


 
 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Study drug dispensedk 
 
 
X 
 
X 
X 
X 
X 
X 
 
 
Adverse eventsl 
Continuous collection 
Study drug compliance 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
AFP: alfa-fetoprotein; BMI: body mass index; eCRF: electronic case report form; eDiary: electronic diary; EOT: End of Treatment; ICF: informed consent form; 
INR: international normalized ratio; p-C4: plasma 7α Hydroxy 4-cholesten-3-one; PedsQL: Pediatric Quality of Life Inventory; PK: pharmacokinetic; QoL: quality of life; 
RR: respiratory rate; SAE: serious adverse event; s-BA: serum bile acid. 
a 
This visit is the same as Visit 1 in the extension protocol (Study A4250-008).  Must also be performed at the time a patient is prematurely withdrawn from the study. 
b 
Assessments must be performed 28 days following the final dose of study drug.  This visit is omitted if patients are entering Study A4250-008. 
c 
The ICF (and assent) must be signed before any study procedures are performed. 
d 
Past (within 1 month prior to Visit 1) and current medication will be documented in the eCRF. 
e 
Includes blood pressure, pulse, RR, temperature, height (or length depending on age), and body weight (using a certified weight scale).  BMI will be calculated. 
f 
Includes training on use of the device at Screening (Visit 1) and review of patient daily entries during 14 days prior to Clinic Visit 3 to determine eligibility.  
g 
See Table 3 for detailed parameters. 
h 
Patients will fast (water intake only is permissible) for at least 4 hours prior to the collection of samples for s-BA and vitamin A.  Exceptions can be made for infants, 
less than 12 months of age, if unable to fast for the full 4 hours.  All s-BA results during the treatment period and follow-up will be blinded. 
i 
Samples will not be collected for patients ≤10 kg.  In the event of an SAE, a PK sample should be collected as close to the onset of the event as possible. 
j 
For girls who have reached menarche.  Serum test at Visit 1; urine test at other visits.  A serum pregnancy test will also be conducted at Visit 9 for patients entering 
Study A4250-008.  If a urine pregnancy test is positive, a serum pregnancy test should be performed to confirm the pregnancy. 
k 
Study drug to be taken once daily on Day 1 through Day 167 as described in Section 8.2. 
l 
Adverse event information will be collected from the time of signing of the ICF to study discontinuation. 


 
 
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7.1.3 
Study Procedures and Assessments 
7.1.3.1 
Screening Period (Days -56 to 0) 
Day -56 to Day -35/Clinic Visit 1 
Patients will undergo a Screening Visit up to 56 days prior to the planned first day of study 
treatment.  Screening procedures and assessments are as follows: 
• Obtain written informed consent 
• Assess inclusion/exclusion criteria (Section 7.2) 
• Record demographics (age, full date of birth, gender, race, and ethnicity) 
• Document concomitant medications 
• Medical and surgical history (date of diagnosis of PFIC Types 1 or 2, prior investigational 
medications for PFIC, historical liver function test [LFT] values, any surgery performed 
including biliary diversion (if applicable), any other diagnosis, and historical liver biopsy 
data) 
• Collect, if available, past confirmatory clinical genetic laboratory report for 
PFIC-1/PFIC-2 and send to central reader for review.  If a historical report is equivocal, 
unavailable, or unobtainable, a blood sample for clinical genetic testing will be collected 
to determine eligibility 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• eDiary training and compliance requirements; caregivers and, if applicable, patients begin 
daily recording of pruritus using the eDiary (Appendix 2) 
• Clinical chemistry (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Serum pregnancy test for girls who have reached menarche.  Please see Appendix 6 for 
contraceptive requirements 
• AE monitoring 
Day -28 to Day -7/Clinic Visit 2 
A second Screening Visit will be performed.  Screening procedures and assessments are as 
follows: 
• Review concomitant medications 
• Vital signs (Section 10.2.6) 


 
 
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• Albireo PRO/ObsRO eDiary review for compliance 
• Urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• International normalized ratio (INR) 
• Vitamins A and E 
• Urine pregnancy test for girls of who have reached menarche 
• AE monitoring 
7.1.3.2 
Treatment Period (Day 0 to Day 168) 
Study Day 0/Clinic Visit 3 (Randomization) 
At the randomization visit, the following assessments will be conducted: 
• Review inclusion/exclusion criteria (Section 7.2) 
• Review concomitant medications, medical and surgical history 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance and entrance criteria requirement (see 
Inclusion Criteria 4, Section 7.2.1) 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1)  
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• 25-hydroxy vitamin D 
• Alfa-fetoprotein (AFP) 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer QoL (Pediatric Quality of Life Inventory [PedsQL]) questionnaire 
• Patient/caregiver/clinician complete the patient global impression of symptoms (PGIS) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Study drug is dispensed and patients are instructed to administer daily from Day 1 
Study Day 14 (Telephone Contact) 


 
 
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A study nurse will contact the patient/caregiver by telephone for monitoring of the following:  
• Review concomitant medications 
• Albireo PRO/ObsRO eDiary review for compliance 
• AEs 
Study Day 28 and Day 84/Clinic Visits 4 and 6  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Physical examination, including voluntary photography (at Clinic Visit 6 only; 
Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• Urinalysis (at Clinic Visit 6 only; Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Autotaxin, p-C4, and A4250 pharmacokinetic (PK) assessment (at Clinic Visit 4 only; 
samples will not be taken for patients ≤10 kg) 
• Vitamins A and E, and 25-hydroxy vitamin D (at Clinic Visit 6 only) 
• Patient/caregiver/clinician complete the patient global impression of change (PGIC) and 
PGIS 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 56 and Day 126/Clinic Visits 5 and 7 
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  


 
 
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• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 154/Clinic Visit 8  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 168/Clinic Visit 9/EOT 
The last dose of study drug is taken in the morning the day before Clinic Visit 9, and the 
following procedures and assessments will be performed (also performed at the time a patient 
prematurely withdraws):  
• Review of concomitant medications 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry, hematology, and urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Autotaxin, p-C4, and A4250 PK assessment (samples will not be taken for patients 
≤10 kg) 


 
 
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• AFP  
• Vitamins A, E, and 25-hydroxy vitamin D 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer PedsQL questionnaire 
• Patient/caregiver/clinician complete the PGIC and PGIS 
• Urine pregnancy test for girls who have reached menarche, or serum pregnancy test for 
girls continuing to study A4250-008 
• AE monitoring 
• Evaluation of study drug compliance 
7.1.3.3 
Follow-Up Period 
Study Day 196/Clinic Visit 10 
All patients not continuing into Study A4250-008 will return to the study site 28 days after 
Clinic Visit 9 or the last dose of study drug for the following assessments: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
7.2 
Study Population 
A total of approximately 60 patients with a clinical diagnosis of PFIC Type 1 or 2 that have 
been genetically confirmed will be randomized.  The study will target 15% of enrolled 
patients to be PFIC-1 patients.  If enrollment of all PFIC-2 patients is completed and the 15% 
enrollment of PFIC-1 patients has not yet been achieved, then enrollment of PFIC-2 patients 
will continue in order to reach the total study target enrollment. 
An attempt will be made to randomize a minimum of 10 patients from each age category 
(6 months to 5 years, 6 to 12 years, and 13 to ≤18 years).  


 
 
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A maximum of 5 patients that were treated in Study A4250-003 will be allowed to participate 
in this study. 
7.2.1 
Inclusion Criteria 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages 
of ≥6 months and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have clinical genetic confirmation of PFIC-1 or PFIC-2 through 
identification of biallelic pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, 
taken as the average of 2 samples at least 7 days apart (Visits 1 and 2) prior to 
randomization 
4. Patient must have history of significant pruritus and a caregiver-reported observed 
scratching in the eDiary average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to 
randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  
Patients who turn 18 years of age (or legal age per country) during the study will be 
required to re-consent in order to remain in the study 
6. Patients will be expected to have a consistent caregiver for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use 
an eDiary device as required by the study 
7.2.2 
Exclusion Criteria 
1. Patient with pathologic variations of the ABCB11 gene that predict complete absence of 
the BSEP protein 
2. Patient with past medical history or ongoing presence of other types of liver disease 
including, but not limited to, the following: 
a) Biliary atresia of any kind 
b) Benign recurrent intrahepatic cholestasis, indicated by any history of normal s-BAs 
c) Suspected or proven liver cancer or metastasis to the liver on imaging studies 
d) Histopathology on liver biopsy is suggestive of alternate non-PFIC related etiology of 
cholestasis 
3. Patient with a past medical history or ongoing presence of any other disease or condition 
known to interfere with the absorption, distribution, metabolism (specifically bile acid 
metabolism), or excretion of drugs in the intestine, including but not limited to, 
inflammatory bowel disease 


 
 
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4. Patient with past medical history or ongoing chronic (i.e., >3 months) diarrhea requiring 
intravenous fluid or nutritional intervention for treatment of the diarrhea and/or its 
sequelae 
5. Patient has a confirmed past diagnosis of infection with human immunodeficiency virus 
or other present and active, clinically significant, acute, or chronic infection, or past 
medical history of any major episode of infection requiring hospitalization or treatment 
with parenteral anti-infective treatment within 4 weeks of treatment start (study Day 1) or 
completion of oral anti-infective treatment within 2 weeks prior to start of Screening 
Period 
6. Any patient with suspected or confirmed cancers except for basal cell carcinoma, and 
non-liver cancers treated at least 5 years prior to Screening with no evidence of 
recurrence 
7. Patient with a past medical history of chronic kidney disease with an impaired renal 
function and a glomerular filtration rate <70 mL/min/1.73 m2  
8. Patient with surgical history of disruption of the enterohepatic circulation (biliary 
diversion surgery) within 6 months prior to start of Screening Period. 
9. Patient has had a liver transplant or a liver transplant is planned within 6 months of 
randomization 
10. Decompensated liver disease, coagulopathy, history or presence of clinically significant 
ascites, variceal hemorrhage, and/or encephalopathy 
11. INR >1.4 (the patient may be treated with Vitamin K intravenously, and if INR is ≤1.4 at 
resampling the patient may be randomized) 
12. Serum ALT >10 × upper limit of normal (ULN) at Screening 
13. Serum ALT >15 × ULN at any time point during the last 6 months unless an alternate 
etiology was confirmed for the elevation 
14. Total bilirubin >10 × ULN at Screening 
15. Patient suffers from uncontrolled, recalcitrant pruritic condition other than PFIC.  
Examples include, but not limited to, refractory atopic dermatitis or other primary pruritic 
skin diseases 
16. Any patient who is pregnant or lactating or who is planning to become pregnant within 
24 weeks of randomization 
17. Sexually active males and females who are not using a reliable contraceptive method with 
≤1% failure rate (such as hormonal contraception, intrauterine device, or complete 
abstinence) throughout the duration of the study and 90 days thereafter (from signed 


 
 
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informed consent through 90 days after last dose of study drug).  See Appendix 6 for 
further details 
18. Patient with a past medical history of alcohol or substance abuse will be excluded.  
Patient must agree to refrain from illicit drug and alcohol use during the study 
19. Administration of bile acid or lipid binding resins and medications that slow GI motility 
(Refer to Appendix 1 – Concomitant Medications Guidelines) 
20. Patient has had investigational exposure to a drug, biologic agent, or medical device 
within 30 days prior to Screening, or 5 half-lives of the study agent, whichever is longer 
21. Patient who has been previously treated with an IBAT inhibitor whose pruritus has not 
responded to treatment 
22. Any other conditions or abnormalities which, in the opinion of the investigator or Medical 
Monitor, may compromise the safety of the patient, or interfere with the patient 
participating in or completing the study 
7.2.3 
Withdrawal of Patients 
Patients/caregivers will be informed that they have the right to withdraw from the study at 
any time, without prejudice to their medical care, and that they are not obliged to state their 
reasons. 
Any withdrawal, and reasons for withdrawal, must be fully documented in the electronic case 
report form (eCRF) and source documents and the patient followed by the 
investigator/investigative staff.  Even after the study is completed at the Follow-up Visit, the 
investigator/investigative staff will, regardless of reason for withdrawal, record any patient 
data they receive concerning SAEs, and all drug-related non-serious AEs, and subsequently 
report these according to Section 10.1.  Withdrawn patients will not be replaced. 
Patients will be withdrawn in the following circumstances: 
• A patient’s/caregiver’s desire for withdrawal for any reason 
• Lost to follow-up (every effort must be made to contact the patient/caregiver; a certified 
letter must be sent) 
• An AE that, in the opinion of the investigator, necessitates withdrawal 
• A worsening of the symptoms related to diarrhea occurs 
• Death 
• Patient/caregiver estimation of intolerable symptoms 
• A patient’s/caregiver’s substantial noncompliance (eDiary and study drug compliance) or 
protocol violation 


 
 
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• An investigator’s opinion that continuing the patient in the study is not appropriate.  The 
investigator may withdraw a patient at any time, if it is considered to be in the patient’s 
best interest 
A patient who withdraws from treatment prematurely will have EOT (Visit 9) assessments at 
the time of withdrawal and a follow-up assessment (Visit 10) 28 days following the last dose 
of study drug, and the patient will be recorded as withdrawn. 
7.2.4 
Study Termination by Sponsor 
This study may be terminated at any time by Albireo if serious side effects should occur, if 
the investigator does not adhere to the protocol; or if, in the sponsor's judgment, there are no 
further benefits to be achieved from the study.  In this event, Albireo/designee will inform the 
study investigators, institutions, and all regulatory authorities. 
Albireo may temporarily or permanently discontinue the study at an investigative site at any 
time for safety, ethical, compliance, or other reasons.  If this is necessary, Albireo will 
endeavor to provide advance notification to the site.  If a site or the study is suspended or 
discontinued, the investigator/investigative staff will be responsible for promptly informing 
the Independent Ethics Committee (IEC).  If required by local regulations, Albireo/designee 
will be responsible for informing the IEC and the regulatory authority of study or site 
discontinuation.  In such an event, all study data and unused study drug must be returned to 
Albireo. 


 
 
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8 
TREATMENT OF PATIENTS 
8.1 
Identity of Study Drug 
A4250 and placebo will be supplied as capsules for oral administration.  White opaque 
capsules filled with pellets containing A4250 or placebo will be provided.  Two different 
capsule sizes will be available, as follows: 
• Capsule size 0 that can be opened 
• Capsule size 3 that should be swallowed intact.  These size 3 capsules may be opened 
only under exceptional circumstances, e.g., patient cannot swallow capsule intact 
The content of the A4250 and placebo capsules will be identical in appearance.  Capsule 
filling weight will also be identical for high dose, low dose, and placebo.  Bottles with 
34 capsules will be given to the patient at each visit.  A patient who requires 2 or more 
capsules per day will be given multiple bottles.  Refer to the investigational product manual. 
8.2 
Administration of Study Drug 
Patients will be dosed with A4250 at a dose of 40 µg/kg/day, 120 µg/kg/day, or placebo for 
24 weeks.  Study drug will be dispensed to the patient at Visits 3, 4, 5, 6, 7, and 8, together 
with instructions on how to store and take the drug.  Study drug administration data, 
including whether each patient took each dose or partial doses of study drug, whether there 
were any delayed or missed doses, and whether the capsule was opened or swallowed whole 
will be documented through the eDiary and transferred to the study database. 
A4250 should be taken in the morning, together with food.  On clinic visits days when 
laboratory assessments are conducted, study drug should be taken after the visit and after 
laboratory samples are taken.  Patients should not crush or chew the capsule(s).  When 
swallowing the capsule intact, the patient should administer the dose with a glass of water. 
For a patient unable to swallow capsule intact, the capsule can be opened and the contents 
sprinkled and mixed in a small amount of soft room-temperature yogurt, apple sauce, or fruit 
purée.  A list of food options can be found in the Pharmacy manual. The amount of food 
should be less than what the patient/infant is expected to eat and should be followed by water.  
1. Hold the capsule over the prepared food, gently twist open and allow the content to fall 
into food 
2. Mix the entire capsule content with 1 or 2 tablespoons (15 to 30 mL) of soft food (apple 
sauce, fruit purée, or yogurt) 
3. Administer the mixture immediately after mixing. 
The number of capsules provided to the patient will be based on body weight at Visit 3.  At 
Visit 6, if a patient’s weight has shifted to another weight category (identified in Table 2), the 
number of capsules per day may be adjusted.   


 
 
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Table 2 
Dosing and Capsule Strength 
Body Weight 
(kg) 
Capsule 
Size 
Number of 
Capsules 
per Day 
Capsule 
Strength, 
Low Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Strength, 
High Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Size, 
Placebo 
(µg) 
5.0 to <7.5 
0 
1 
200 
200 
600 
600 
0 
7.5 to <12.5 
0 
2 
200 
400 
600 
1200 
0 
12.5 to <17.5 
0 
3 
200 
600 
600 
1800 
0 
17.5 to <19.5 
0 
4 
200 
800 
600 
2400 
0 
19.5 to <25.5 
3 
2 
400 
800 
1200 
2400 
3 
25.5 to <35.5 
3 
3 
400 
1200 
1200 
3600 
3 
35.5 to <45.5 
3 
4 
400 
1600 
1200 
4800 
3 
45.5 to 55.5 
3 
5 
400 
2000 
1200 
6000 
3 
>55.5 
3 
6 
400 
2400 
1200 
7200 
3 
8.3 
Study Treatment Packaging and Labeling 
8.3.1 
Packaging and Labeling 
The capsules will be packed in high-density polyethylene containers, with child-proof 
polypropylene caps.  Study drug capsules containing A4250 in the strength specified in 
Table 2 above will be manufactured. 
Packaging and labeling will be prepared to comply with applicable regulatory requirements. 
8.3.2 
Storage 
Treatment packs containing A4250 capsules should be stored and dispensed in accordance 
with regulations in their original containers.  The storage facility at the investigative site 
should be locked and the storage temperature should be between 15°C and 25°C. 
Patients/caregivers should be informed of appropriate storage conditions (i.e., room 
temperature, between 15°C and 25°C). 
Any deviations from the recommended storage conditions should be immediately reported to 
Albireo and the study drug should not be used until authorization has been given by Albireo. 
8.3.3 
Blinding and Randomization of Study Drug 
A double-blind design is employed so that both the investigators and the patients will be 
unaware of the treatment assignment during the study.  
After written informed consent is obtained from an eligible patient, an 8-digit patient 
identification number will be assigned.  The first 2 digits will denote the country, followed by 
a 3-digit site number and a 3-digit patient sequence number.  This number will be created and 


 
 
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allocated by the Interactive Web Response System (IWRS), when the patient first enters the 
system at Screening.  The randomization codes will be computer-generated by 
 
Biostatistics and kept by a statistician independent from the project team.  The randomization 
will be done in blocks and stratified according to PFIC class (Type 1 and 2) and age class 
(6 months to 5 years, 6 to 12 years, and 13 to ≤18 years) to ensure approximate balance 
between dose schemes (1:1:1).  A separate randomization list will be prepared for the 
maximum 5 patients from Study A4250-003 regardless of stratification.  Randomization 
codes will be assigned sequentially as patients become eligible for randomization. 
Patients who are eligible for randomization will be assigned a unique 4-digit randomization 
number by IWRS.  This randomization number identifies which treatment will be allocated to 
the patient.  Patients who withdraw from the study after randomization visit are not to be 
replaced and their randomization number will not be reused.   
To ensure blinding, the study drug and the matching placebo have the same shape and size.  
Patients will receive capsule(s) of A4250 according to their dose group or capsule(s) of 
matching placebo once a day during the double-blind Treatment Period.  Labels on the study 
drug containers will not identify the treatment that a patient is randomized to.  Traceability of 
the treatment is ensured by the study drug number. 
The 5-digit study drug number will identify study drug packs and will be detailed on the 
study drug label.  Dispensing of study drug will be coordinated by IWRS.  
The system will assign a study drug number corresponding to the randomization arm.  The 
randomization number will be used in the background only to ensure no unblinding and will 
not be displayed to end-users in the IWRS. 
8.4 
Procedure for Breaking the Randomization Code 
Should a situation arise where unblinding is urgently required, i.e., only where knowledge of 
the study drug is required to adequately manage a life-threatening situation, the investigator 
at that study site may perform immediate unblinding through IWRS.  The responsibility to 
break the treatment code in emergency situations resides solely with the investigator.  The 
investigator should make every effort to discuss the rationale (status and outcome) for 
emergency unblinding with the sponsor Medical Monitor or the 
 Medical Monitor as 
soon as possible to review the individual patient details. 
The study site investigator and appropriate 
 project team members will be authorized to 
access the emergency unblinding functionality within the IWRS.  Emergency unblinding 
takes place in the IWRS via a dedicated emergency unblinding module.  The system will 
require the user to enter their emergency unblinding, which is typically provided at the same 
time login credentials are distributed.  The exact description of the treatment assigned to the 
individual patient then will be accessible to the user.  Once the randomization code is broken 
for a patient, he/she must be withdrawn from the study and all assessments and procedures at 
EOT should be performed.  As a follow-up, Visit 10, will be performed according to the time 


 
 
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schedule.  Once a randomization code has been broken, the investigator must inform the 
 and sponsor Medical Monitor in writing within 24 hours.  In case of technical issues 
accessing the system online, please see the IWRS site user manual for country-specific 
contact telephone numbers for the 
 HelpDesk 24/7 system support. 
8.5 
Patient Compliance 
The study nurse will monitor eDiary compliance by routine review of the CRF health 
website.  If both diary entries on a day are missing, the study nurse will call the 
patient/caregiver to remind them to complete all scheduled entries.  Any noncompliance will 
be documented and explained in the source documents. 
Study drug compliance will also be checked through diary responses.  Additionally, patients 
will return all unused study drug at Visits 4, 5, 6, 7, 8, and 9.  The study site staff will count 
all returned drug, assess compliance, and record details in the eCRF. 
Treatment Compliance = 100 × ((Number of study drug dispensed – number of study drug 
returned)/number of study drug that should be taken). 
Treatment compliance between 80% and 120% will be acceptable. 
8.6 
Study Drug Accountability 
Records shall be maintained of the delivery of study treatment(s) to the study site(s), of the 
inventory at the study site(s), of each use of the study treatment(s) for each patient, and of the 
return of unused study treatment(s) to Albireo. 
These records shall include dates, quantities, batch numbers, expiry dates, and the unique 
code numbers assigned to the study drug and to the study patients. 
The investigator will be responsible for ensuring that the records adequately document that 
the patients were provided the quantities specified in the protocol and that all study drug 
received from Albireo is reconciled.  
8.7 
Concomitant Therapy and Prohibited Medications 
The investigator will note all ongoing medication and any medication recently stopped 
(within 1 month prior to Visit 1) in the eCRF.  At Visits 2 through 10, all changes in 
medication (stopping or starting new medication or changes in dose) will be recorded in the 
eCRF.  All medications (prescribed or over the counter) for pruritus and sleep will be 
recorded. 
All medications taken by a patient within 3 months prior to the first intake of study drug are 
regarded as prior medication. 
All medications taken by a patient on or after the first intake of study drug, and which 
continue to be taken during the study, are regarded as concomitant medication.  
Prohibited medications are listed in Appendix 1. 


 
 
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9 
ASSESSMENT OF EFFICACY 
9.1 
Efficacy Endpoints 
9.1.1 
Primary Efficacy Endpoint 
The primary efficacy endpoints are as follows: 
• European Union (EU) and rest of the world (RoW): Proportion of patients 
experiencing at least a 70% reduction in fasting s-BA concentration from baseline to 
the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 
weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization 
and the previous visit (Clinic Visit 2).  The end value is the average of the values at 
Weeks 22 and 24 after the start of double-blind treatment. 
• United States (US): Proportion of positive pruritus assessments at the subject level 
over the 24-week Treatment Period. A positive pruritus assessment is defined as a 
scratching score of ≤1 or at least a one-point drop from baseline on the Albireo 
ObsRO instrument. At each assessment, the AM score will be compared to the AM 
baseline, and the PM score will be compared to the PM baseline. Both AM baseline 
and PM baseline pruritus assessments will be included in the analysis of this endpoint. 
AM scores from the 14 days prior to the first dose of study medications will be 
averaged as AM baseline. PM scores from the 14 days prior to the first dose will be 
averaged as PM baseline.  
9.1.2 
Secondary Efficacy Endpoint 
Secondary efficacy endpoints include the following:  
• EU and RoW: Proportion of positive pruritus assessments at the subject level over 
the 24-week Treatment Period. A positive pruritus assessment is defined as a 
scratching score of ≤1 or at least a one-point drop from baseline on the Albireo 
ObsRO instrument. At each assessment, the AM score will be compared to the AM 
baseline, and the PM score will be compared to the PM baseline. Both AM baseline 
and PM baseline pruritus assessments will be included in the analysis of this endpoint. 
AM scores from the 14 days prior to the first dose of study medications will be 
averaged as AM baseline. PM scores from the 14 days prior to the first dose will be 
averaged as PM baseline.   
• US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment. 
• All Regions: 


 
 
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All secondary endpoints are compared to placebo. 
o Change from baseline to Week 12 and to Week 24 in fasting serum bile acids 
o Change from baseline to Week 12 and to Week 24 in serum ALT 
concentration 
o Change in growth from baseline to Week 12 and to Week 24 
o Proportion of responders for pruritus scores at Weeks 12 and 24 based on the 
Albireo PRO and ObsRO instruments  
o Change in sleep parameters measured with the Albireo PRO and ObsRO 
instruments from baseline over the 24-week Treatment Period  
o Proportion of individual assessments meeting the definition of a positive 
pruritus assessment at the subject level over the 24-week Treatment Period. A 
positive pruritus assessment includes an itch score ≤1, or at least a one-point 
drop from baseline based on the Albireo PRO instrument; only patients 
≥8 years of age will complete the Albireo PRO instrument  
o Proportion of individual assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-
12, Weeks 0-18, Weeks 0-24, respectively, or the proportion of positive 
pruritus assessments at each 4-week interval.  
o Proportion of individual AM assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-
12, Weeks 0-18, Weeks 0-24, respectively, or the proportion of positive 
pruritus assessments at each 4-week interval.  
o Proportion of individual PM assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-
12, Weeks 0-18, Weeks 0-24, respectively, or the proportion of positive 
pruritus assessments at each 4-week interval.  
o Number of patients undergoing biliary diversion surgery or liver 
transplantation 
9.1.3 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, 
caregiver, and clinician via the PGIC items (Appendix 3) 
• Change from baseline in fasting s-BA at Week 4  


 
 
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• Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
by each 4 week-interval of the Treatment Period as measured by the Albireo ObsRO 
instrument 
• Change from baseline by each 4-week interval of the Treatment Period in patient-reported 
and observer-reported night time itching and scratching severity scores, respectively  
• Change from baseline by each 4-week interval of the Treatment Period in patient-reported 
and observer-reported morning time itching and scratching severity scores, respectively 
• Change from baseline by each 4-week interval of the Treatment Period in pooled pruritus 
score including observer-reported scratching for patients <8 years of age and 
patient-reported itch severity for patients ≥8 years of age 
• Change from baseline by each 4-week interval of the Treatment Period in additional 
patient-reported and observer-reported sleep parameters (e.g., tiredness and number of 
awakenings) 
• Change from baseline to Week 24 in PedsQL (Appendix 4) 
• Change from baseline in serum ALT concentration at Week 4  
• Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• Change from baseline in AST and GGT at Weeks 4, 12, and 24 
• Change from baseline for other biochemical markers and measures of bile acid synthesis 
(autotaxin, p-C4) at Week 24 
• Change in pediatric end-stage liver disease/model for end-stage liver disease 
(PELD/MELD) score, AST to platelet ratio index (APRI) score, and Fib-4 score from 
baseline to Week 24 
• Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® 
(where available) 
• Liver-related mortality and liver decompensation events 
• All-cause mortality 
9.2 
Efficacy Assessments 
9.2.1 
Serum Bile Acids 
Blood samples for analysis of fasting total s-BA will be drawn at all visits.  Patients will fast 
(water intake only is permissible) for at least 4 hours prior to the collection of samples for 
s-BA.  Exceptions can be made for infants, <12 months of age, if unable to fast for the full 
4 hours.  Any visit at which a bile acid sample result is unreportable, an additional 
unscheduled visit for a repeat sample collection may be scheduled.  All s-BA results during 


 
 
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the treatment period and follow-up will be blinded.  Samples will be processed and 
transported to a central laboratory per instructions in the laboratory manual. 
9.2.2 
Itching, Scratching and Sleep Score 
Itching, observed scratching, and sleep disturbance will be recorded twice each day via 
eDiary.  Patients and/or caregivers will be instructed to complete the eDiary every day in the 
morning after the patient wakes and in the evening just before the patient goes to sleep 
throughout the study.  Patients and caregivers will receive a formal training session during 
Visit 1. 
The eDiary includes Albireo ObsRO and PRO items.  Patients <8 years of age will not be 
asked to complete the Albireo PRO items; only the Albireo ObsRO will be completed by 
caregivers of patients in this age group.  Older patients, 8 to 18 years of age, will complete 
the Albireo PRO items and the caregiver will complete the Albireo ObsRO items.  The 
Albireo PRO items assess severity of itch, aspects of sleep disturbance (morning diary only), 
and tiredness.  For patients 8 to 12 years of age, the caregiver will read the Albireo PRO 
items along with the child and record the child’s response.  A guide will be provided to the 
caregivers that provides standardized explanations of the Albireo PRO items, in case the 
patient is confused or requires clarification about the meaning of a question.  The Albireo 
ObsRO items assess severity of observed scratching, aspects of observed sleep disturbance 
(morning diary only), and observed signs of tiredness (evening diary only).  The Albireo 
ObsRO and PRO scratching and itch severity items use 0 to 4 response scales, where each 
response is distinguished by a unique facial expression, verbal anchor, number, and color 
code (Appendix 2).  To meet eligibility criteria, a scratch score will be calculated from daily 
diary entries (ObsRO items) the 14 consecutive days prior to randomization.  
A daily AM & PM score for the Albireo ObsRO scratching item will be averaged from the 2 
ratings for each day.  A daily score will be considered missing if both of the daily 
assessments are missing.  A weekly score will be calculated by averaging the daily scratching 
scores.  A weekly score will be considered missing if ≥4 out of 7 days a week of data are 
missing.  For AM & PM baseline, a 14-day daily score of AM & PM prior to the first dose of 
study medications will be averaged as the baseline score. 
For AM baseline, a 14-day AM score prior to the first dose of study medications will be 
averaged as the AM baseline score.  The same approach will be used for PM baseline and to 
calculate a patient-reported itch severity score.  The handling of missing data is described in 
Section 11.2.2.2. 
9.2.3 
Growth 
Growth will be measured as height (velocity) and weight (Z-score) using a certified weight 
scale.  BMI will be calculated by weight (kg) / height (m)2.  Change will be defined as linear 
growth deficit (weight and BMI for age) compared to a standard growth curve (Z-score, SD 
from P50). 


 
 
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9.2.4 
Quality of Life Questionnaire (PedsQL) 
Patients and caregivers will be asked to complete a QoL questionnaire (PedsQL) at Visit 3 
and Visit 9.  Details of the questions included on the questionnaire are provided in 
Appendix 4. 
9.2.5 
Biomarkers and PK Samples 
Blood samples for autotaxin and p-C4 will be drawn at Visits 3, 4, and 9 only for children 
with body weight >10 kg.  Blood for A4250 PK assessment will only be drawn at Visits 4 
and 9 (only for children with body weight >10 kg), and as close to the onset of an SAE as 
possible, should an SAE occur.  Samples will be processed and transported to a laboratory 
per instructions in the laboratory manual. 
9.2.6 
PELD/MELD Score 
At randomization and end of treatment, PELD score will be calculated for children up to 
12 years of age.  For children 13 years or older, the MELD score will be calculated. 
PELD Score = 4.80 * ln(total bilirubin) + 18.57 * ln(INR) - 6.87 * ln(albumin) + 4.36 (if 
patient <1 year: scores for patients listed for liver transplantation before the patient's first 
birthday continue to include the value assigned for age (<1 year) until the patient reaches the 
age of 24 months) + 6.67 (if the patient has growth failure [≤2 SD]) 
Laboratory values less than 1.0 will be set to 1.0 for the calculation of the PELD score. 
MELD Score = 9.57 * ln(creatinine) + 3.78 * ln(total bilirubin) + 11.2 * ln(INR) + 6.43 
Laboratory values less than 1.0 will be set to 1.0 and serum creatinine values greater than 
4.0 mg/dL (equivalent to 353.6 µmol/L) will be set to 4.0 for calculation of the MELD score. 
9.2.7 
Fibroscan® 
Where available, Fibroscan® will be performed as per institution standard practice at Visits 3 
and 9. 
9.2.8 
Markers of Fibrosis 
APRI score and Fib-4 score will be calculated at randomization and at end of treatment. 
APRI = [(AST in U/L) / (AST ULN in U/L)] / (Platelets in 109/L) 
Fibrosis 4 Score = (Age*AST in U/L) / (Platelets in 109/L * √(ALT in U/L)). 
9.2.9 
Global Impression of Change and Global Impression of Symptom Measures 
Patients (≥8 years of age), caregivers, and clinicians will complete the PGIC and the PGIS 
measures (Appendix 3) at randomization (Visit 3; PGIS only), Visits 4 and 6, and at EOT 
(Visit 9). 
The PGIC items assess change in itch (patient version), scratching (caregiver and clinician 
versions), and sleep (all versions) since starting the study drug.  The PGIS items assess itch 


 
 
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(patient version), scratching (caregiver and clinician versions), and sleep (all versions) in the 
past week.  Caregivers and clinicians will complete the PGIC and PGIS for all patients; those 
patients ≥8 years of age will complete the patient version. 


 
 
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10 
ASSESSMENT OF SAFETY 
The timing and frequency of safety assessments are described in Section 7.1.3 and in Table 1 
above. 
The primary safety analysis for this study will include the incidence of total 
treatment-emergent adverse events (TEAE) and TEAEs categorized by causality, severity, 
and seriousness assessments made by the investigator by comparing study drug exposure to 
placebo. 
Trends in safety will also be evaluated for the following assessments: 
• Physical examinations 
• Concomitant medications 
• Vital signs 
• Laboratory test results (including clinical chemistry, hematology, urinalysis, AFP, and 
vitamins A and E, 25-hydroxy vitamin D, and INR) 
• Abdominal ultrasound 
• Discontinuations due to AEs 
10.1 
Adverse Events 
10.1.1 
Definitions and Investigator Assessments 
An AE is defined as any untoward medical occurrence in an enrolled patient regardless of 
causal relationship with study drug.  An AE can therefore be any clinically significant 
unfavorable and unintended sign, symptom, or disease that occurs once a patient is enrolled 
(ICF is signed) in the study until the patient is discharged from the study, whether or not 
related to the study drug. 
10.1.1.1 
Clinical Significance 
Clinical significance is defined as any abnormal finding that results in further clinical 
investigation(s), treatment(s), or the diagnosis of new condition.  Patient-reported events and 
protocol-mandated laboratory values, vital signs, and physical examination findings can be 
considered clinically significant (i.e., an AE) if there is a deterioration as compared to 
baseline.  Examples of clinically significant worsening from baseline could include, but is not 
limited to, events causing withdrawal from the study and events requiring medical 
intervention outside of the study causing apparent clinical manifestations, or judged relevant 
by the investigator.   
10.1.1.2 
Serious Adverse Events 
Serious criteria are applied by the investigator to each AE as specifically defined below.  
These criteria are used to determine whether an AE is serious or non-serious.  The assessment 


 
 
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is made independently of severity assessment (Section 10.1.1.3).  For example, the 
development of a severe rash that occurs after signing of informed consent may not meet 
serious criteria as defined below and therefore would be considered a severe, non-serious AE. 
Any AE that meets any 1 of the following 6 criteria is considered an SAE: 
• The outcome of the AE is death 
• The AE is immediately life-threatening.  Life-threatening means that the patient is, in the 
opinion of the investigator, at immediate risk of death from the reaction as it occurred.  
This does not include an AE that, if more severe, might have caused death 
• The AE results in persistent or significant disability/incapacity.  Disability means a 
substantial disruption of a person's ability to conduct normal life functions 
• The AE requires or prolongs hospitalization  
• The AE results in a congenital anomaly/birth defect 
• The AE is an important medical event.  Important medical events may meet serious 
criteria should the investigator assess that they may significantly jeopardize the patient, 
represent a significant hazard, or requires medical/surgical intervention to prevent one of 
the outcomes listed above.  Examples of potential SAEs based on this criteria include, but 
are not limited to, allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions even if they do not result in inpatient 
hospitalization, or the development of drug dependency and drug abuse. 
10.1.1.3 
Severity Assessment 
Severity assessments are based on the intensity of the event in relation to expectation.  The 
investigator will assess the intensity of AEs based on the following definitions: 
• Mild (awareness of sign or symptom, but easily tolerated) 
• Moderate (discomfort sufficient to cause interference with normal activities) 
• Severe (incapacitating, with inability to perform normal activities) 
Severity is a measure of intensity where seriousness is defined by the criteria outlined in 
Section 10.1.1.2.  An AE of severe intensity need not necessarily be considered serious.  For 
example, nausea that persists for several hours may be considered severe nausea, but not meet 
serious criteria, and therefore would be assessed as a severe AE but not an SAE. 
10.1.1.4 
Causality Assessment 
The investigator determines the causality of all AEs to the study drug using medical judgment 
and considering all relevant factors such as (but not limited to) the underlying study 
indication, coexisting disease, concomitant medication, relevant history, pattern of the AE, 
temporal relationship to the study drug, and de-challenge or re-challenge.  The causality 
assessment of the AE/SAE is to be made as follows. 


 
 
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Related to study drug (possibly, probably, or definitely related) 
Based on medical judgment, there is at least a reasonable possibility that the study drug 
caused the event; one or more of the following criteria apply: 
• The event follows a reasonable temporal sequence from administration of study drug 
• The event could not be reasonably attributed to the known characteristics of the patient's 
clinical state, environmental or toxic factors, or other modes of therapy administered to 
the patient 
• The event follows a known pattern of response to study drug 
• The event disappears or decreases on cessation or reduction in dose of the study drug.  (It 
should be noted that in some situations an AE will not disappear or decrease in intensity 
upon discontinuation of study drug despite other clear indications of relatedness) 
• The event reappears or worsens when the study drug is re-administered 
Unrelated to study drug (unlikely or unrelated) 
Based on medical judgment there is no reasonable possibility that the study drug caused the 
event; one or more of the following criteria apply: 
• The event does not follow a reasonable temporal sequence from administration of study 
drug 
• The event could be reasonably attributed to the known characteristics of the patient's 
clinical state, concurrent illness, environment or toxic factors, or other modes of therapy 
administered to the patient 
• The event does not follow a known pattern of response to study drug 
• The event does not disappear or decrease on cessation or reduction in dose of the study 
drug, and it does not reappear or worsen when the study drug is re-administered 
10.1.2 
Recording of Adverse Events 
It is the investigator’s responsibility to assess whether each untoward event is a clinically 
significant worsening from baseline, thereby considered an AE.  For all AEs, the severity, 
seriousness, and causality to study drug for each AE as outlined in Section 10.1.1 will be 
assessed and recorded in the eCRF. 
All serious and non-serious AEs are collected once the caregiver/patient has signed the ICF 
and until the post-treatment follow-up (Visit 10) or 28 days after the last dose of study drug. 
Any AEs or SAEs that are unresolved at the patient’s last AE assessment in the study are 
followed up by the investigator until resolution or stabilization up to the database lock and 
recorded in the eCRF.  Albireo retains the right to request additional information for any 
patient with ongoing AE(s) or SAE(s) at the end of the study. 


 
 
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TEAEs are defined as any AE that occurs after randomization (Day 0).  All AEs that occur in 
the Screening Period, i.e., after enrollment and prior to randomization, will be collected on 
the eCRF as non-treatment-emergent AEs. 
If there is a clinically significant deterioration of a laboratory value/vital sign or other routine 
study assessment that is associated with a diagnosis, the clinical diagnosis will be reported as 
an AE and the associated signs and symptoms will be considered additional information 
unless the sign or symptom is more severe than expected given the diagnosis.  For example, if 
an investigator diagnoses a study patient with hepatitis during the study period, hepatitis 
would be considered the AE and the concomitant signs and symptoms of abdominal pain, 
vomiting, and elevated ALT and AST would not be reported separately unless, in the opinion 
of the investigator, one of these signs or symptoms is more severe than expected and 
therefore a separate AE assessment is indicated. 
10.1.3 
Recording and Reporting of Serious Adverse Events 
Every SAE (regardless of severity and causality) that occurs once the caregiver/patient has 
signed the ICF and until the post-treatment follow-up (Visit 10), and 28 days after the final 
dose of study drug, should immediately and not later than within 24 hours of knowledge of 
the event, be reported by the investigator or delegate in the SAE report form.    
Report of a SAE must include at least the following information: 
• Patient identification information (study number, site number, initials, and date of birth 
[as per local country requirements for data protection]) 
• The last study drug administration date 
• The diagnosis of the event with the description (or a brief description of 
signs/symptoms/clinical course if the diagnosis is not available) and the date of onset 
• The action used to treat the event (i.e., treatment medications, temporary discontinuation) 
• The reason(s) for considering the event serious 
• The relationship of the event to the study drug or to the study procedure (e.g., the 
investigator's assessment of causality) 
• A brief narrative of the SAE 
Follow-up reports including all new information obtained of the subsequent course of the 
SAE must be prepared and the information collected in the SAE submitted to 
 
 (Pharmacovigilance and Safety Services Department) by e-mail within 24 hours of 
knowledge of the event. 
The pharmacovigilance manager (PVM) may contact the investigator to obtain further 
information on a reported SAE.  The investigator/investigative staff must respond to any 


 
 
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request for follow-up information or answers to questions regarding the SAE within the same 
timelines as for initial reports. 
The PVM reports the occurrence of the SAE and follow-up to the Albireo medical monitor 
for medical assessment of the case. 
Should an outcome of death occur within the study period or within 28 days after the last 
administration of study drug, an SAE form should be completed including the main and 
contributory causes of death. 
All SAE reports must be e-mailed to the following e-mail address within 24 hours: 
 
 
If email is unavailable, SAEs may be transmitted via fax to the following number: 
 
In a study-related medical emergency situation, when the assigned medical monitors for the 
study cannot be reached by a caller, an on call physician can be reached 24 hours per day, 
7 days per week, via the 
 call center.  The following number is a chargeable telephone 
number allowing a global reach from both landlines and mobile phones and the internet page 
indicated below contains a list of country-specific toll-free telephone numbers that is 
provided.  It should be noted that not all countries globally have access to toll-free numbers 
as indicated on the “24/7 Medical Help desk” index.  Countries without toll-free numbers 
need to dial the chargeable number as indicated below.  Furthermore, toll-free numbers might 
not be available from mobile phones. 
 
 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions 
Suspected unexpected serious adverse reaction (SUSAR) is an SAE that occurs in a patient, 
the nature or severity of which is not expected per the applicable product information (e.g., 
the IB for an unauthorized investigational product or summary of product characteristics for 
an authorized product).  
Reporting and tracking of SUSARs will be in accordance to all applicable competent 
authority regulations.  The Ethics Committee and all investigators involved in this study will 
be informed according to national and international requirements. 


 
 
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10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments 
10.2.1 
Laboratory Assessments 
Samples will be collected for clinical chemistry, hematology, and urinalysis testing at the 
time points specified in Table 1. The parameters assessed are presented in Table 3. 
Blood for AFP will be drawn at Visits 3 and 9.  Fat-soluble vitamin levels including vitamins 
A and E, and 25-hydroxy vitamin D will be drawn at Visit 2 (vitamins A & E only), 
Visit 3 (25-hydroxy vitamin D only), Visit 6, and Visit 9.  Blood for INR (surrogate for 
vitamin K) will be drawn at Visits 2, 4, 6, 8, and 10.  Patients will fast (water intake only is 
permissible) for at least 4 hours prior to the collection of samples for vitamin A.  Exceptions 
can be made for infants, <12 months of age, if unable to fast for the full 4 hours.  If a patient 
has any vitamin level(s) that are outside of the reference range, vitamin supplementation 
adjustments may be required.  Target ranges and supplementation strategy guidelines can be 
found in Appendix 5. 
A serum pregnancy test will be performed at Visit 1 and Visit 9 if the patient is entering the 
A4250-008 study. A urine pregnancy test will be collected at all other visits for girls who 
have reached menarche.  If a urine pregnancy test is positive, a serum pregnancy test should 
be performed to confirm the pregnancy.  Study drug administration will be held pending the 
confirmatory results.  If the serum pregnancy test is negative, the patient can resume dosing.  
If the serum pregnancy test is positive, the patient should be discontinued (see 
Section 10.2.9). 
All samples will be processed and transported to a laboratory per instructions in the 
laboratory manual. 
The observed values will be recorded and assessed as “normal” or “abnormal not clinically 
significant” or “abnormal clinically significant”. 
Additional safety blood samples may be needed due to follow-up of an abnormal value or 
analysis failure.  The blood samples collected for safety laboratory analysis will be destroyed 
after the analyses have been completed.  


 
 
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Table 3 
Routine Laboratory Parameters 
Clinical Chemistry 
Hematology 
Urinalysis 
• 
Albumin 
• 
ALT 
• 
Alkaline phosphatase  
• 
AST 
• 
Bilirubin – total and direct 
• 
Calcium 
• 
Chloride 
• 
Creatinine 
• 
Creatine kinase 
• 
Gamma-glutamyl transferase 
• 
Potassium 
• 
Sodium 
• 
Hematocrit 
• 
Hemoglobin 
• 
Platelet count 
• 
Red blood cell count 
• 
White blood cell count and 
differential (neutrophils, 
eosinophils, basophils, 
lymphocytes, and monocytes) 
• 
Blood 
• 
Glucose 
• 
Ketones 
• 
Leukocytes 
• 
Nitrites 
• 
pH 
• 
Protein 
 
10.2.2 
Individual Patient Safety Monitoring 
10.2.2.1 
Liver Monitoring 
Strategies to monitor markers of liver disease throughout the study are outlined below where 
the ULN will be based on central laboratory reference values for age and gender. 
If isolated transaminase elevations are observed, defined as: 
1. Normal bilirubin AND absence of clinical hepatitis symptoms AND 
• ALT or AST ≥5 × ULN (if normal at baseline) or an absolute threshold of 800 U/L, 
whichever comes first  
• OR ALT or AST ≥3 × baseline (if abnormal at baseline) or an absolute threshold of 
800 U/L, whichever comes first 
Then: 
a) Repeat liver profile (AST, ALT, bilirubin, and prothrombin time [PT] or INR) within 
2 to 3 days 
b) Evaluate creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) 
c) As needed (for example, persistent ALT/total bilirubin elevation or worsening of liver 
function), consider evaluation for alternative etiologies 
Monitor the patient using close observation found in Section 10.2.2.2 
If any ONE of the following criteria are met: 
1. Transaminases (ALT or AST ≥3 × baseline or 800 U/L, whichever comes first) AND 
bilirubin (total bilirubin > 2 × ULN) elevations 


 
 
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2. Transaminase elevations alone (ALT or AST >10 × ULN or 5 × baseline or absolute 
threshold of 800 U/L, whichever comes first) in presence of normal LDH and CPK 
3. Total bilirubin increased, unrelated to hemolysis (elevated reticulocyte count) or 
established genetic diseases, such as Gilbert’s syndrome 
a. Doubling if total bilirubin was <3 mg/dL (equivalent to 51.3 µmol/L) at baseline  
b. OR Increase by >3 mg/dL (equivalent to 51.3 µmol/L) if total bilirubin was ≥3 mg/dL 
(equivalent to 51.3 µmol/L) at baseline 
4. INR increase refractory to vitamin K administration 
a. INR >1.5 if INR was normal at baseline  
b. OR Increase by >0.4 if INR was abnormal at baseline 
5. Any increase in total bilirubin and transaminases if accompanied by EITHER a symptom 
of clinical hepatitis (e.g., vomiting, nausea, right upper quadrant pain) OR immunological 
reaction (rash or >5% eosinophilia) 
Then: 
a) Interrupt study medication 
b) Initiate drug-induced liver injury work-up for alternative etiologies 
c) Repeat liver profile (AST, ALT, total bilirubin, direct bilirubin) and PT or INR within 
48 to 72 hours 
d) Monitor the patient using close observation found in Section 10.2.2.2 
e) If a patient lives in a remote area, they may be tested locally and the results 
communicated to the investigator site promptly 
10.2.2.2 
Close Observation 
• Repeat liver enzyme and serum bilirubin tests two or three times weekly.  Frequency 
of re-testing can decrease to once a week or less if abnormalities stabilize or the trial 
drug has been discontinued and the subject is asymptomatic  
• Obtain a more detailed history of symptoms and prior or concurrent diseases 
• Obtain a history of concomitant drug use (including non-prescription medications and 
herbal and dietary supplement preparations), alcohol use, recreational drug use, and 
special diets  
• Consider ruling out alternate etiology including acute viral hepatitis types A, B, C, D, 
and 
E; 
autoimmune 
or 
alcoholic 
hepatitis; 
non-alcoholic 
steatohepatitis; 
hypoxic/ischemic hepatopathy; and biliary tract disease 
• Obtain a history of exposure to environmental chemical agents.  


 
 
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• Obtain additional tests to evaluate liver function, as appropriate (e.g., INR, direct 
bilirubin) 
• Consider gastroenterology or hepatology consultations. 
10.2.2.3 
De-challenge/Re-challenge for Liver and Clinical Hepatitis Monitoring 
1. Re-challenge is not recommended: 
a. If a patient has had possible/probable drug-induced liver injury 
b. If a decompensation event has occurred (i.e., variceal hemorrhage, ascites, hepatic 
encephalopathy, etc.). 
2. If due to underlying cholestatic liver disease variability or another alternative etiology is 
identified AND liver tests returned to baseline, re-challenge may be considered after 
consultation with the sponsor Medical Monitor. 
3. If the ALT/total bilirubin elevations are observed after re-challenge, then repeat 
re-challenge is discouraged. 
If patient is permanently discontinued, monitoring should be continued as outlined in 
Section 10.2.2.5.  
10.2.2.4 
Diarrhea 
Study drug should be discontinued if a patient develops diarrhea with at least 1 of the 
following concomitant signs or symptoms: grossly bloody stools, vomiting, dehydration 
requiring treatment with oral or intravenous rehydration and/or electrolyte imbalances, fever 
(≥38 C) and/or the diarrhea persists for 7 or more days. 
If there is gross blood in stool, INR and platelets should be measured.  
Study drug will be reintroduced (re-challenge) when the symptoms have resolved.  If the 
diarrhea re-occurs within 1 week after the re-challenge with no alternate etiology, the patient 
will be permanently discontinued and monitored as outlined in Section 10.2.2.5. 
10.2.2.5 
Monitoring after Permanent Discontinuation of Study Drug Due to Safety 
Once study drug is permanently discontinued for liver, hepatitis, diarrhea, or other severe AE 
related to study drug, the patient will be monitored weekly until the laboratory and clinical 
parameters have normalized/stabilized. 
10.2.3 
Demographics/Medical and Surgical History 
Demographic information per country regulations (age, full date of birth, gender, race, and 
ethnicity), along with medical and surgical history, will be obtained and recorded in the eCRF 
at Visit 1. 
Medical and surgical history will be entered in the eCRF at Visit 1.  This includes date of 
diagnosis of PFIC, prior investigational medications for PFIC, historical (3 months prior to 


 
 
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Screening) LFT values (AST, ALT, and total bilirubin), ongoing medication, any surgery 
performed, any other diagnosis, and historical biopsy data.   
If a liver biopsy has been performed within 1 year prior to Screening, or during the study, the 
results will be recorded in the eCRF. 
10.2.4 
Clinical Genetic Testing 
For previous confirmatory clinical genetic testing results for PFIC Type 1 or 2 performed 
prior to Screening Visit 1, the confirmatory clinical genetic testing laboratory report will be 
verified to determine eligibility. 
If the historical clinical genetic result is equivocal, unavailable, or unobtainable, clinical 
genetic analysis to confirm pathologic biallelic variations in the ATP8B1 or ABCB11 genes 
will be performed and verified to determine eligibility.  No other diagnostic genetic testing 
will be offered. 
10.2.5 
Physical Examination 
A physician or suitably trained qualified assistant will perform a complete physical 
examination, including voluntary photography of skin lesions due to scratching, at Visits 1, 3, 
6, and 9.  A skin examination will be conducted at Visits 1, 3, 4, 6, 9, and 10 (see Table 1).  
A complete physical examination will include assessment of general appearance, eyes, ears, 
nose, throat, head/neck/thyroid, lymph nodes, cardiovascular, lungs/chest, abdomen, 
genitourinary, extremities, skin, musculoskeletal, neurologic, and other.  
Skin will be examined thoroughly and excoriations/scratch marks recorded.  
10.2.6 
Vital Signs 
Evaluation of vital signs will be performed at all visits.  This includes blood pressure (systolic 
and diastolic), pulse, respiratory rate, temperature, height (or length depending on age), and 
body weight (using a certified weight scale) at clinic visits.  BMI will be calculated. 
10.2.7 
Abdominal Ultrasound 
Ultrasound of the liver and spleen will be performed at randomization (Visit 3) and at EOT 
(Visit 9).  Liver size, echogenicity, and presence of masses/nodules as well as spleen size will 
be recorded. 
10.2.8 
Overdose 
A4250 is minimally absorbed and has a very low systemic availability.  Based on toxicology 
data, for study purposes any dose exceeding a total of >3 mg/kg body weight of A4250 taken 
as a single dose or as a cumulative dose within 24 hours is defined as an overdose.   
The no-adverse-effect dose level in the most sensitive species (defined as 20 mg/kg/day) in 
the rat 1 month toxicity study gives a human equivalent dose of approximately 194 mg/day in 


 
 
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a 60 kg person.  This dose is approximately 10-fold higher than the maximum possible dose 
predicted in human studies (20 mg).  
The investigator/investigative staff should immediately and not later than within 24 hours of 
knowledge of the overdose, be reported by the investigator or delegate on the SAE report 
form.  In the event of an A4250 overdose, the patient should be monitored closely. 
10.2.9 
Pregnancy 
If a pregnancy is discovered in a female patient enrolled in the study before the end of dosing, 
the patient will be immediately discontinued from the study and will attend the same visits as 
a prematurely withdrawn patient.  If the pregnancy is discovered in a female patient enrolled 
in the study after the end of dosing, the patient will continue in the study per protocol.  If a 
pregnancy occurs in a male patient’s partner at any time during the study, the pregnancy 
should also be reported and followed. 
Pregnancy is not considered to be an AE.  However, if the patient has been dosed with the 
study drug, the pregnancy must be reported on the Paper Pregnancy Notification Form 
immediately within 24 hours after recognition to Safety Manager (please refer to Section 
10.1.3).  Date of exposure and as far as possible, details of the period of gestation at the time 
of exposure must be given. 
The pregnancy should be followed to determine outcome, including spontaneous termination, 
details of birth, and presence of any birth defects, congenital anomalies or newborn or 
maternal complication.  The infant who was exposed in-utero will be followed for up to 
2 years after delivery.  Individual cases with an abnormal outcome in association with the 
study drug should be reported on an expedited basis, i.e., reported rapidly to a competent 
authority. 


 
 
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11 
STATISTICAL EVALUATION 
All personnel involved with the analysis of the study will remain blinded until database lock 
for the double-blind Treatment Period and until major protocol violations have been 
identified. 
All statistical analyses will be performed using SAS version 9.3 or higher. 
11.1 
Sample Size and Power 
For the EU and RoW, the primary analysis will relate to the serum bile acid responder 
endpoint.  For the US, the primary analysis will relate to the pruritus endpoint. For each 
primary endpoint, a pooled analysis for the closed testing procedure will be applied to control 
the 1-sided overall type I error rate for two treatment comparisons vs. the placebo at the 0.025 
level (Kane et al, 2015, Lehmacher et al, 1991, and Marcus et al, 1976). The study will enroll 
60 to 70 patients in order to obtain at least least 20 evaluable patients in each arm. For each 
primary endpoint, simulations with 5000 iterations using 20 patients per arm were conducted 
to estimate the power after multiplicity adjustment, resulting in a standard error of <0.7% for 
each estimated power.   
Based on the Phase 2 A4250-003 study data both dose groups are predicted to have similar 
treatment effects for both serum bile acids and pruritus. For serum bile acids, simulated 
proportions were analyzed using the CMH test to generate the following 1-sided p-values: 
both A4250 arms pooled vs. placebo, low dose vs. placebo, and high dose vs. placebo. 
Assuming 60% responders in the A4250 arms and 10% responders in the placebo arm, the 
power to claim significance for a particular A4250 arm after multiplicity adjustment is 
approximately 94%. The power to claim significance for at least one arm or both arms are 
approximately 99% and 91%, respectively.  
For the proportion of positive pruritus assessments at the subject level in pruritus scores, 
simulated proportions were analyzed using ANCOVA to generate the following 1-sided p-
values: both A4250 arms pooled vs. placebo, low dose vs. placebo, and high dose vs. 
placebo. With an effect size of 1.0526, the simulated power to claim significance for a 
particular arm after multiplicity adjustment is approximately 89%. The probability to claim 
significance for at least one arm or both arms are approximately 95% and 83%, respectively.   
11.2 
Statistical Methods 
11.2.1 
Statistical Analysis Sets 
Safety Analysis Set 
The safety analysis set will consist of all patients who received at least 1 dose of study drug.  
Patients will be analyzed according to the treatment they actually received.  The safety 
analysis set will be the primary analysis set for safety analyses.  
Full Analysis Set 


 
 
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The full analysis set (FAS) will consist of all randomized patients who received at least 
1 dose of assigned treatment.  Patients will be analyzed in the treatment group they were 
randomized to even if they received incorrect study drug.  The FAS will be the primary 
analysis set for efficacy analyses. 
Per Protocol Analysis Set 
The per protocol (PP) analysis set is a subset of the FAS, and will consist of all randomized 
patients for whom no major protocol violation (no violation which may affect the study 
efficacy outcome) is documented.  The PP analysis set will provide supportive data for the 
efficacy analyses. 
Allocation of patients to the PP analysis set will be performed before unblinding of the study. 
11.2.2 
Methods of Statistical Analyses 
11.2.2.1 
General Principles 
The primary objective of this study is to demonstrate the efficacy of repeated daily doses of 
40 µg/kg/day and 120 µg/kg/day A4250 in children with PFIC Types 1 and 2.  
In the EU and RoW, for the primary efficacy variable of fasting bile acid responder endpoint 
(at least a 70% reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the 
end of treatment), the Cochran Mantel Haenszel (CMH) test stratified by PFIC class and age 
class will be performed to compare the proportion in fasting bile acid responder endpoint at 
the end of treatment (Week 22 and 24) in the two A4250 dose groups to placebo group. To 
ascertain that all data are used in the CMH analysis, neighboring strata will be pooled if all 
subjects in a stratum have the same response. Dropouts will be treated as non-responders. The 
proportion together with corresponding 95% CI, odds ratio and corresponding 95% Clopper-
Pearson exact CI and p-value for the CMH test will be presented.   
In the US, for the primary efficacy variable of the proportion of positive pruritus assessments 
at subject level over the 24-week treatment period, an analysis of covariance (ANCOVA) will 
be used. The model will include treatment arm, AM baseline pruritus score, PM baseline 
pruritus score, and randomization stratification factors, i.e. PFIC class and age class. LS 
mean(SE) by treatment arm and LS mean difference (SE), 95% confidence intervals, and p-
values (where applicable) between treatments (120 µg/kg/day and 40 µg/kg/day, respectively) 
vs. placebo will be provided. If there are concerns on model assumptions, i.e. normality and 
homogeneity of variances, a non-parametric ANCOVA based on the rank scores will be used 
as a sensitivity analysis. LS mean/SE on the outcome by treatment arm and LS mean 
difference/SE between active dose and placebo will be provided.  
The procedure for multiplicity adjustment is specified as follows:    
In the closed testing procedure, the low and high dose groups will be pooled to compare with 
the placebo group first. If the 1-sided p-value is ≤0.025, the 1-sided p-values for low dose vs. 


 
 
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placebo and high dose vs. placebo will be calculated, respectively. If both individual p-values 
are ≤0.025, a significant treatment effect will be declared on both dose groups. If only one of 
them is ≤0.025, a significant treatment effect will be declared on the corresponding dose 
group.  
Other secondary and exploratory outcome variables will provide supportive efficacy and 
safety information regarding the differences between the treatment groups. No adjustments 
will be performed for multiple comparisons when testing secondary and exploratory efficacy 
variables. 
If not otherwise specified, the baseline value of a parameter is defined as the last assessment 
of that parameter prior to the first intake of study treatment. 
11.2.2.2 
Missing Data 
In the primary analysis of the pruritus endpoint, all intermittently missing assessments will be 
classified as negative pruritus assessments. Similarly, all planned assessments after the 
intercurrent events (premature treatment discontinuation, death or initiation of rescue 
treatments such as biliary diversion surgery or liver transplantation) will be counted as 
negative pruritus assessments. However, as described above, all 336 assessments planned 
over the 24-week treatment period will be included in the denominator of the endpoint 
definition.  
In the primary analysis of fasting bile acid responder endpoint, dropouts and patients with a 
missing average at the end of the treatment will be treated as non-responders. Patients in need 
of surgical rescue, i.e. biliary diversion and listing for liver transplant will be classified as 
non-responders. Continued collection of efficacy data for patients who discontinue treatment 
will be made as far as possible. 
11.2.2.3 
Demographic and Baseline Characteristics 
Descriptive summaries of demographics and other baseline characteristics (including medical 
and surgical history) will be presented by treatment group and overall using the FAS.  
Prior and concomitant medication use during the study will be summarized by treatment 
group and overall using the safety analysis set. 
11.2.2.4 
Subject Disposition 
The following will be summarized (by treatment group and overall where applicable): 
• Patients enrolled (who signed the informed consent) 
• Patients randomized 
• Patients treated 
• Patients completing the study 
• Patients withdrawing early (including withdrawal reason) 


 
 
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Additionally, patients enrolled, included in the safety, in the FAS, and in the PP analysis set 
will be summarized by region. 
 
11.2.2.5 
Evaluation of Primary Efficacy Variables 
For EU and RoW, the primary responder endpoint based on change in fasting bile acids will 
be analyzed in terms of the proportion of patients achieving the event of interest. Patients in 
need of surgical rescue, i.e. biliary diversion and listing for liver transplant, will be classified 
as non-responders. Dropouts will be treated as non-responders.  
CMH stratified by PFIC class and age class will be performed to compare the proportion in 
fasting bile acid responders at the end of treatment (Week 22 and 24) in the two A4250 dose 
groups to the placebo group. To ascertain that all data are used in the CMH analysis, 
neighboring strata will be pooled if all subjects in a stratum have the same response. The 
proportion together with corresponding 95% CI, odds ratio and corresponding 95% Clopper-
Pearson exact CI and p-value for the CMH test will be presented 
Logistic regression will be performed as a supportive analysis to compare the proportion in 
fasting bile acid responders at the end of treatment (Week 22 and 24) in the two A4250 dose 
groups to the placebo group. The model will include treatment arm, baseline value, and 
randomization stratification factors, i.e. PFIC class and age class. Baseline value will be 
calculated as the average of serum bile acid values at randomization and the previous visit 
(Clinic Visit 2).   
In the US, for the primary efficacy variable of the proportion of positive pruritus assessments 
at the subject level over the 24-week treatment period, ANCOVA will be used to analyze the 
pruritus endpoint. The model will include treatment arm, AM baseline pruritus score, PM 
baseline pruritus score, and randomization stratification factors, i.e. PFIC class and age class. 
LS mean(SE) by treatment arm and LS mean difference (SE), 95% confidence intervals, and 
p-values (where applicable) between treatments (120 µg/kg/day and 40 µg/kg/day, 
respectively) vs. placebo will be provided. If there are concerns on model assumptions, i.e. 
normality and homogeneity of variances, a non-parametric ANCOVA based on the rank 
scores can be used. LS mean/SE on the outcome by treatment arm and LS mean 
difference/SE between active dose and placebo will be provided.  
Sensitivity analysis will be conducted to assess the impact of intermittently missing data as 
well as intercurrent events and will be specified in the SAP. Subgroup analyses will also be 
performed for each of the 3 age groups (6 months to 5 years, 6 to 12 years, and 13 to ≤18 
years) and for patients who were not included in the previous Study A4250-003. 
11.2.2.6 
Evaluation of Secondary and Exploratory Efficacy Variables 
The change in the secondary endpoints such as serum bile acids, total bilirubin, ALT, growth, 
Albireo PRO and ObsRO sleep parameters, and patient-reported itch severity will be 


 
 
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summarized by visit using descriptive statistics. Change in growth (weight for age and BMI 
for age) will also be displayed using graphical presentations.  
Proportion of responders at Weeks 12 and 24 based on the PRO/ObsRO instruments 
estimated from both distribution and anchor-based approaches will be analyzed using the 
same model as specified for the primary analysis for EU and RoW. 
Proportion of individual assessments meeting the definition of a positive pruritus assessment 
at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, 
respectively, or the proportion of positive pruritus assessments at each 4-week interval over 
the 24-week treatment period for AM or PM, respectively will be analyzed using the same 
model as specified for the primary analysis for US.   
Undergoing biliary diversion surgery and/or liver transplant, all-cause mortality, and liver 
decompensation events will mainly be analyzed by using descriptive statistics. Kaplan-Meier 
curves will be used when appropriate for time to event data.  
Comparisons of the change from baseline at Week 24 in PedsQL between the treatment 
groups will be conducted using ANCOVA. The model will include terms for baseline, PFIC 
class, age class, and treatment. 
Other exploratory variables (Global Symptom Relief, biochemical markers and measures of 
bile acid synthesis, additional Albireo PRO and ObsRO sleep parameters, PELD/MELD 
score, APRI score, and Fib-4 score) will be analyzed descriptively for categorical and 
continuous data, as applicable. For continuous data, the change from baseline will be 
analyzed in addition to the actual visit values. For categorical data, shift tables or frequency 
and percentages of patients will be presented as appropriate. Additional details on secondary 
and exploratory efficacy variables and subgroup analyses by age class (6 months to 5 years, 6 
to 12 years, and 13 to ≤18 years) will be specified in the SAP.  
11.2.2.7 
Evaluation of Safety Variables 
Safety data will be analyzed using descriptive statistics and summaries by treatment group of 
SAEs, AEs, vital signs, clinical laboratory tests (hematology, clinical chemistry and 
urinalysis), and concomitant medication.  Analyses will be performed using the safety 
analysis set.  
Summaries of AEs (coded according to the Medical Dictionary for Regulatory Activities 
[MedDRA] system organ class [SOC] and MedDRA preferred term) will include the 
following:  
• Overview of the incidence of TEAEs (TEAEs, drug-related TEAEs, TEAEs leading to 
study discontinuation, and treatment-emergent SAEs) 
• TEAEs by SOC and preferred term 
• Intensity of TEAEs by SOC and preferred term 


 
 
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• Drug-related TEAEs by SOC and preferred term 
• TEAEs leading to study discontinuation by SOC and preferred term 
• Treatment-emergent SAEs by SOC and preferred term 
Concomitant medication use during the Treatment Period will be summarized by Anatomical 
Therapeutic Chemical (ATC) class and World Health Organization (WHO) preferred name.  
Summaries of vital signs will be presented.  For each visit, the actual results and the change 
from baseline, and the number and percentage of patients with potentially clinically 
significant (PCS) values observed post-baseline will be presented. 
Summaries of clinical safety laboratory data will be presented.  For each visit, the actual 
result and the change from baseline, and the number and percentage of patients with PCS 
values observed post-baseline will be presented. 
Data listings will be provided for each patient for all safety parameters. 
11.2.2.8 
Compliance and Exposure 
Exposure will be analyzed by calculating the number of days with exposure to study drug.  
Results will be presented by treatment group using the safety analysis set.  
The percentage compliance will be described by treatment group and the number of patients 
with a compliance <80%, between 80% and 120%, and >120% will be presented based on the 
safety analysis set. 
11.2.3 
Blinded Analyses to Confirm Responder Definitions 
A blinded analysis of Albireo ObsRO and PRO eDiary data will be performed after 50% or 
more of the planned sample has completed the Week 24 Follow-Up Period.  The blinded 
analysis will be used to estimate a threshold of clinically meaningful change (i.e., responder 
definition) in Albireo ObsRO and PRO pruritus scores.  The analysis will be performed by a 
group independent from both the study team and Albireo.  Blinded data will be used for this 
analysis; data will be collapsed across treatment groups. 
The analyses will include distribution- and anchor-based analyses to examine a responder 
definition for the Albireo PRO/ObsRO pruritus scores.  Distribution-based analyses will 
include calculation of the SD and standard error of measurement for the baseline Albireo 
PRO and ObsRO scores.  Anchor-based analyses will involve examining the degree of 
change on the Albireo PRO and ObsRO scores from baseline to Week 24 for patients who 
experience change in pruritus according to PGIC and symptom items.  Details of this analysis 
will be included in a stand-alone SAP for assessing the measurement characteristics 
(reliability, validity, sensitivity to change, responder definition) of the Albireo PRO and 
ObsRO items. 


 
 
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11.2.4 
Data Safety Monitoring Board 
A DSMB consisting of sponsor-independent clinical and statistical experts will be 
established.  The DSMB will periodically (approximately on a quarterly basis) meet for the 
review of accumulating study data, including safety (AE and laboratory data) and liver 
values.  
The DSMB will have access to unblinded data.  The DSMB will make recommendations for 
the remaining part of the study (further details will be provided in the DSMB charter).  The 
DSMB may recommend continuing with the study as planned or stopping the study early for 
safety reasons.  The DSMB will submit its recommendations in writing to Albireo 
Medical/
 who are responsible for responding to the recommendations of the DSMB and 
taking appropriate action.  The investigators will only be informed by Albireo Medical/
 
if the study requires a protocol amendment or is stopped.  The DSMB may choose to make 
additional evaluations at any time if they feel this is warranted from a safety point of view.  
The DSMB will act according to its own written standard operating procedure described in a 
charter and will prepare written minutes of its meetings.  The charter of the DSMB will be 
stored in the trial master file.  The DSMB will maintain records of its meetings and these will 
become part of the study file when the study is complete.  In order not to disseminate 
unblinded data and to ensure that all staff involved in the conduct and final analysis of the 
study remain blind to the data, only the members of the DSMB and the unblinded DSMB 
statistician will have access to these data. 
 


 
 
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12 
DIRECT ACCESS TO SOURCE DATA/NOTES 
The investigator/institution shall provide direct access to source data/documents for 
study-related monitoring, audits, IEC/Institutional Review Board (IRB) review and 
regulatory inspection. 


 
 
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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
13.1 
Conduct of the Study 
Albireo/
 shall implement and maintain quality control and quality assurance procedures 
with written standard operating procedures to ensure that the study is conducted and data are 
generated, documented, and reported in compliance with the protocol, ICH GCP and 
applicable regulatory requirements. 
This study shall be conducted in accordance with the provisions of the Declaration of 
Helsinki (October 1996) and all revisions thereof, and in accordance with United States Food 
and Drug Administration (FDA) regulations (Code of Federal Regulations, Sections 312.50 
and 312.56) and with ICH GCP (CPMP 135/95). 
The investigator may not deviate from the protocol without a formal protocol amendment 
having been established and approved by an appropriate competent authority and IEC/IRB, 
except when necessary to eliminate immediate hazards to the patient or when the change(s) 
involve(s) only logistical or administrative aspects of the study.  Any deviations may result in 
the patient having to be withdrawn from the study and render that patient non-evaluable. 
13.2 
Study Monitoring 
Before study initiation, at a site initiation visit or at an investigator’s meeting, an Albireo 
representative or designee will review the protocol and eCRF with the investigators and the 
investigative staff.  During the study, the clinical monitor (clinical research associate [CRA]) 
will visit the site regularly to check the completeness of patient records, the accuracy of 
entries on the eCRFs, the adherence to the protocol and to GCP, the progress of enrollment, 
and to ensure that study drug is being stored, dispensed, and accounted for according to 
specifications. 
The investigator must ensure that eCRFs are completed within a timely period of patient 
visits, as per individual site agreements, and must allow the CRA and Albireo representative 
or designee periodic access to patient records and all study-related materials, including 
relevant hospital or clinical records, to confirm their consistency with the eCRF entries.  No 
information in these records about the identity of the patients will leave the study center.  
Albireo monitoring standards require full verification for the presence of the signed ICF, 
adherence to the inclusion/exclusion criteria, documentation of SAEs, and recording of 
primary efficacy and safety variables.  The CRA will review source data compared with the 
eCRFs and will verify source data according to the study-specific monitoring plan.  The 
design of the study, the frequency of patient visits, and the site enrollment rate will determine 
the frequency of monitoring visits.  Upon study completion, the CRA will visit the site to 
conduct a study termination visit, which will include collection of any outstanding 
documentation. 


 
 
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It is recommended that the investigator/investigative staff log into the eCRF system every 
working day or at minimum twice weekly in order to provide a prompt response to queries.  
The investigator/investigative staff should respond to queries and make any relevant changes 
to the study data within 3 working days. 


 
 
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14 
ETHICS 
14.1 
Independent Ethics Committee/Institutional Review Board 
Prior to the start of the study, the investigator is responsible for ensuring that the protocol and 
consent form have been reviewed and approved by a relevant IEC/IRB.  The IEC/IRB shall 
be appropriately constituted and perform its functions in accordance with FDA ICH GCP and 
local requirements as applicable. 
The IEC/IRB shall approve all protocol amendments (except for logistical or administrative 
changes), written informed consent documents and document updates, patient recruitment 
procedures (e.g., advertisements), written information to be provided to the patients and 
caregivers, IB, available safety information, information about payment and compensation 
available to patients, the investigator's curriculum vitae and/or other evidence of 
qualifications and any other documents requested by the IEC/IRB and regulatory authority 
(competent authority) as applicable. 
14.2 
Written Informed Consents and Assents 
The investigator (physician) or investigative staff, in accordance with local regulations, will 
explain to each patient (or legally authorized representative) the nature of the study, its 
purpose, the procedures involved, the expected duration, alternative treatment, the potential 
risks and benefits involved, and any discomfort that may occur.  Each caregiver/patient will 
be informed that participation in the study is voluntary and that he/she or their child may 
withdraw from the study at any time and that withdrawal of consent will not affect his/her 
subsequent medical treatment or relationship with the treating physician. 
Caregivers/patients will be informed that they/their children may experience side-effects or 
be at risk for symptoms, illnesses, or complications that cannot be foreseen by Albireo.  As 
with other medications, people treated with A4250 may be at risk of developing allergic 
reactions or anaphylaxis.  Caregivers/patients will be advised that study procedures include 
regular blood sampling for measurement of safety parameters and biological markers and that 
some minor risks are associated with these procedures. 
This informed consent should be given by means of a signed ICF, written in non-technical 
language in accordance with ICH GCP, the Declaration of Helsinki, and regulatory 
authorities.  The caregiver(s)/patient should read and consider the statements before signing 
and dating them and should be given a copy of each signed document.  If written consent is 
not possible, oral consent can be obtained if witnessed and followed by a signed statement 
from one or more persons not involved in the study, indicating why the patient was unable to 
sign the form.  No patient can enter the study before his/her or caregiver(s) informed consent 
has been obtained, as required by country regulations. 
The ICF is part of the protocol and must be submitted by the investigator/investigative staff 
with the protocol for IEC approval.  Albireo will supply an ICF which complies with 


 
 
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regulatory requirements and country laws and is considered appropriate for the study.  Any 
changes to the ICF suggested by the investigator must be agreed to by Albireo before 
submission to the IEC and a copy of the approved version must be provided to the clinical 
monitor after IEC approval. 


 
 
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15 
DATA HANDLING AND RECORD KEEPING 
15.1 
Case Report Forms/Source Data Handling 
The investigator shall be provided with standardized eCRFs and shall ensure that all data 
from patient visits are promptly entered into the eCRFs in accordance with the specific 
instructions given.  The investigator must sign each eCRF to verify the integrity of the data 
recorded. 
A list of the normal ranges for all laboratory tests to be undertaken forms part of the 
documentation to be collated prior to study start.  As a central laboratory has been selected to 
conduct any or all tests, it is essential that all samples be analyzed at that laboratory. 
The investigator must maintain source documents such as laboratory reports, consultation 
reports, and complete medical history and physical examination reports. 
As no ethnicity data can be collected in the source document for study sites in France, the 
eCRF will be the Source Document for “origines ethniques”,  
15.2 
Retention of Essential Documents 
Essential documents, as defined by ICH GCP, include: the signed protocol and any 
amendment(s); copies of the completed eCRFs (for site archiving, compact discs of eCRF 
data for specific patients will be provided); signed ICFs; hospital records and other source 
documents; IEC approvals and all related correspondence including approved documents; 
drug accountability records; study correspondence; and a list of patients’ names and 
addresses. 
The investigator/investigative staff must retain copies of these essential documents for the 
period specified by ICH GCP and by applicable regulatory requirements.  The 
investigator/investigative staff will inform Albireo of the location where the essential 
documents are stored and must contact Albireo for approval before disposing of any essential 
documents.  The investigator/investigative staff should take measures to prevent accidental or 
premature destruction of these documents. 


 
 
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16 
FINANCING AND INSURANCE 
Financing and insurance are addressed in a separate agreement. 
 


 
 
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17 
PUBLICATION POLICY 
Albireo will retain the ownership of all data.  When the study is complete, Albireo shall 
arrange the analysis, tabulation of data and preparation of a clinical study report. Albireo may 
also use the data for publication, presentation at scientific meetings and submission to 
regulatory authorities.  All proposed publications based on this study must be subject to the 
sponsor's approval requirements. 
 


 
 
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18 
REFERENCE LIST 
1. A4250 Investigator’s Brochure. 
2. Alissa FT, Jaffe R, Shneider BL. Update on progressive familial intrahepatic cholestasis. 
J Pediatr Gastroenterol Nutr. 2008;46(3):241-52. 
3. Davit-Spraul A, Gonzales E, Baussan C, et al. Progressive familial intrahepatic 
cholestasis. Orphanet J Rare Disease 2009;4:1-12. 
4. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and 
functions. Front Biosci (Landmark Ed). 2009 Jan 1;14:2584-98. 
5. Hori T, Justin H, Nguyen JH and Uemoto S. Progressive familial intrahepatic cholestasis. 
Hepatobiliary pancreat Dis Int. 2010; 9(6):570-8. 
6. Jacquemin E. Progressive familial intrahepatic cholestasis. Genetic basis and treatment. 
Clin Liver Dis. 2000;4(4):753-63. 
7. Miethke AG, Zhang W, Simmons J, et al. Pharmacological inhibition of ASBT changes 
bile composition and blocks progression of sclerosing cholangitis in mdr2 knockout mice. 
Hepatology. 2016; 63(2):512–23. 
8. Murray CS, Rees JL. Are subjective accounts of itch to be relied on? The lack of relation 
between visual analogue itch scores and actigraphic measures of scratch. Acta Derm 
Venereol. 2011;91(1):18-23. 
9. Pawlikowska L, Strautnieks S, Jankowska I, et al. Differences in presentation and 
progression between severe FIC1 and BSEP deficiencies. J Hepatol. 2010;53(1):170-8. 
10. Shneider BL, Magee JC, Bezerra JA, et al. Efficacy of fat-soluble vitamin 
supplementation in infants with biliary atresia. Pediatrics, 2012;130(3):e607-14.  
11. Suchy FJ, Sokol RJ, Balistreri WF, eds. Liver Disease in Children. 3rd Edition. New 
York, NY: Cambridge University Press; 2007:310–4. 
12. Venkat VL, Shneider BL, Magee JC, et al. Total Serum Bilirubin Predicts Fat-Soluble 
Vitamin Deficiency Better Than Serum Bile Acids in Infants with Biliary Atresia. J 
Pediatr Gastroenterol Nutr. 2014;59(6):702-7.  
13. Whitington PF, Whitington GL. Partial external diversion of bile for the treatment of 
intractable 
pruritus 
associated 
with 
intrahepatic 
cholestasis. 
Gastroenterology. 
1988;95(1):130-6. 
 
 


 
 
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19 
APPENDICES 
Appendix 1 
Concomitant Medication Guidelines 
 


 
 
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Appendix 2 
Diary Questions including Albireo Patient-Reported 
Outcomes/Observer-Reported Outcome Instrument 
 


 
 
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Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) 
 


 
 
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Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaire 


 
 
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Appendix 5 
Guideline for Fat-Soluble Vitamin Supplementation 


 
 
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Appendix 6 
Contraceptive Requirements 


 
 
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Appendix 7 
Blood Volumes 
 


 
 
 
CLINICAL STUDY PROTOCOL A4250-005 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
 
IND Number:   
130591 
EudraCT Number 
2017-002338-21 
Test Product: 
A4250 
Indication: 
Progressive Familial Intrahepatic Cholestasis (PFIC) 
Sponsor: 
Albireo AB 
Development Phase: 
Phase 3 
Sponsor Signatory: 
 
Sponsor Medical Monitor: 
 
Principal Investigator: 
 
Date of the Protocol: 
29 April 2019 
Version of the Protocol: 
Final Amendment 05 
 
 
 
 
Confidential 
The information in this document contains trade secrets and commercial information that are 
privileged or confidential and may not be used, divulged, published, or otherwise disclosed 
without the written consent of Albireo AB.  These restrictions on disclosure apply equally to 
all future information supplied to you or generated by you in connection with the study. 




 
 
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INVESTIGATOR SIGNATURE PAGE 
 
PROTOCOL TITLE:  A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children with Progressive 
Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
 
PROTOCOL NUMBER:  A4250-005 
I have read this protocol and agree that it contains all necessary details for performing this 
study.  I will conduct the study as outlined herein and will complete the study within the time 
designated, in accordance with all stipulations of the protocol and in accordance with Good 
Clinical Practice (GCP), local regulatory requirements, and the Declaration of Helsinki. 
I will provide copies of the protocol and all pertinent information to all individuals 
responsible to me who assist in the conduct of this study.  I will discuss this material with 
them to ensure that they are fully informed regarding the study drug and the conduct of the 
study. 
I will use only the informed consent approved by the Independent Ethics Committee (IEC) 
and will fulfil all responsibilities for submitting pertinent information to the IEC responsible 
for this study. 
I agree that the sponsor (Albireo AB) shall have access to any source documents from which 
case report form information may have been generated. 
I further agree not to originate or use the name of Albireo AB, A4250 in any publicity, news 
release, or other public announcement, written or oral, whether to the public, press, or 
otherwise, relating to this protocol, to any amendment to the protocol, or to the performance 
of this protocol without the prior written consent of Albireo AB. 
 
 
 
 
 
 
Name of Investigator 
 
 
 
 
 
 
Signature 
 
Date (day/month/year) 


 
 
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1 
ADMINISTRATIVE INFORMATION 
A Double-blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
Protocol No.: 
A4250-005 
Date of the initial Protocol: 
20 September 2017 
Date and Number of Amendment(s): 
Amendment 01, 06 December 2017 
 
Amendment 02, 10 May 2018 
 
Amendment 03, 22 November 2018 
 
Amendment 04, 01 March 2019 
 
Amendment 05, 29 April 2019 
Sponsor: 
 
 
 
 
 
 
Clinical Research Organization: 
 
 
 
 
 
 
 
 
Sponsor Signatory: 
 
 
  
 
  
 
  
 
 
 
  
Sponsor Medical Monitor: 
 
 
 
 
Principal Investigator: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
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2 
STUDY SYNOPSIS 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Title of Study: 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate Efficacy and Safety of A4250 
in Children with Progressive Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
Principal Investigator: 
 
Study Centers: 
Up to 50 sites will be initiated for this study in the United States (US), Canada, European Union (EU), Middle 
East, and Australia. 
Publication(s): 
None. 
Planned Study Period: 
Q2 2018 to Q4 2019 
Development Phase: 
Phase 3 
Objectives: 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day A4250 in children with 
progressive familial intrahepatic cholestasis (PFIC) Types 1 and 2, as determined by the following: 
• 
Proportion of patients experiencing at least a 70% reduction in fasting serum bile acid (s-BA) 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L compared to placebo 
after 24 weeks of treatment 
• 
The proportion of positive pruritus assessments at the subject level over the 24-week Treatment Period 
Secondary Objectives 
• 
To evaluate the effect of A4250 on serum alanine aminotransferase (ALT) concentration 
• 
To evaluate the effect of A4250 on growth 
• 
To evaluate the effect of A4250 on sleep disturbance 
• 
To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or liver 
transplantation) 
• 
To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 
Methodology: 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the efficacy and safety of 
40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in children with PFIC (deficiencies of familial 
intrahepatic cholestasis-1 or bile salt export pump).  
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment Period and a 4-week 
Follow-up Period.  After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of A4250, or a 
matching placebo.  Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008) in which all patients will receive 
active treatment. 
There will be up to 10 clinic visits during the study and one scheduled telephone contact. 


 
 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Number of Patients: 
Approximately 60 patients diagnosed with PFIC-1 or PFIC-2 will be randomized.  The study will target 15% of 
enrolled patients to be PFIC-1 patients.  If enrollment of all PFIC-2 patients is completed and the 15% 
enrollment of PFIC-1 patients has not yet been achieved, then enrollment of PFIC-2 patients will continue in 
order to reach the total study target enrollment. 
Diagnosis and Main Criteria for Inclusion: 
1. 
A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages of ≥6 months 
and ≤18 years at Visit 1 with a body weight above 5 kg 
2. 
Patient must have clinical genetic confirmation of PFIC-1 or PFIC-2 through identification of biallelic 
pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. 
Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, taken as the 
average of 2 samples at least 7 days apart (Visits 1 and 2) prior to randomization  
4. 
Patient must have history of significant pruritus and a caregiver-reported observed scratching in the 
electronic diary (eDiary) average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to randomization 
5. 
Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  Patients who 
turn 18 years of age (or legal age per country) during the study will be required to re-consent in order 
to remain in the study 
6. 
Patients are expected to have a consistent caregiver for the duration of the study 
7. 
Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use an eDiary 
device as required by the study 
Test Product, Dose and Mode of Administration: 
A4250, 40 µg/kg/day or 120 µg/kg/day, orally administered 
Reference Therapy, Dose and Duration of Administration: 
Matching placebo, orally administered 
Duration of Treatment: 
24 weeks 
Variables 
Efficacy:  
Primary Efficacy Endpoints 
The primary efficacy endpoints are: 
• 
EU and rest of world (RoW): Proportion of patients experiencing at least a 70% reduction in fasting 
s-BA concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L compared to 
placebo after 24 weeks of treatment. 
• 
US: Proportion of positive pruritus assessments at the subject level over the 24-week Treatment Period. 
Secondary Efficacy Endpoints 
Secondary efficacy endpoints include the following: 
• 
EU and RoW: Proportion of positive pruritus assessments at the subject level over the 24-week 
Treatment Period.  
• 
US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA concentration from 
baseline to the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 weeks of 
treatment. 
 
All Regions: 
All secondary endpoints will be compared to placebo. 
• 
Change from baseline to Week 12 and to Week 24 in fasting s-BA 
• 
Change from baseline to Week 12 and to Week 24 in serum ALT concentration 


 
 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
• 
Change in growth from baseline to Week 12 and Week 24 
• 
Proportion of responders for pruritus scores at Weeks 12 and 24 based on the Albireo PRO and ObsRO 
instruments  
• 
Change in sleep parameters measured with the Albireo PRO and ObsRO instruments from baseline 
over the 24-week Treatment Period  
• 
Proportion of individual assessments meeting the definition of a positive pruritus assessment at the 
subject level over the 24-week Treatment Period. A positive pruritus assessment includes an itch score 
≤1, or at least a one-point drop from baseline based on the Albireo PRO instrument; only patients ≥8 
years of age will complete the Albireo PRO instrument 
• 
Proportion of individual assessments meeting the definition of a positive pruritus assessment at the 
subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, respectively, or the 
proportion of positive pruritus assessments at each 4-week interval.  
• 
Proportion of individual AM assessments meeting the definition of a positive pruritus assessment at the 
subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, respectively, or the 
proportion of positive pruritus assessments at each 4-week interval.  
• 
Proportion of individual PM assessments meeting the definition of a positive pruritus assessment at the 
subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, respectively, or the 
proportion of positive pruritus assessments at each 4-week interval.  
• 
Number of patients undergoing biliary diversion surgery or liver transplantation 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• 
Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, caregiver, and 
clinician via the patient global impression of change (PGIC) items 
• 
Change from baseline in fasting s-BA at Week 4  
• 
Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline by each 4 
week interval of the Treatment Period as measured by the Albireo ObsRO instrument 
• 
Change from baseline by each 4-week interval of the Treatment Period in patient-reported and 
observer-reported night time itching and scratching severity scores, respectively 
• 
Change from baseline by each 4-week interval of the Treatment Period in patient-reported and 
observer-reported morning time itching and scratching severity scores, respectively 
• 
Change from baseline by each 4-week interval of the Treatment Period in pooled pruritus score 
including observer-reported scratching for patients <8 years of age and patient-reported itch severity 
for patients ≥8 years of age 
• 
Change from baseline by each 4-week interval of the Treatment Period in additional patient-reported 
and observer-reported sleep parameters (e.g., tiredness and number of awakenings) 
• 
Change from baseline to Week 24 in Pediatric Quality of Life Inventory (PedsQL) 
• 
Change from baseline in serum ALT concentration at Week 4  
• 
Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• 
Change from baseline in AST and gamma-glutamyl transferase at Weeks 4, 12, and 24 
• 
Change from baseline for other biochemical markers and measures of bile acid synthesis (autotaxin, 
plasma 7α-hydroxy-4-cholesten-3-one concentration [p-C4]) at Week 24 
• 
Change in pediatric end-stage liver disease/model for end-stage liver disease (PELD/MELD) score, 
AST to platelet ratio index (APRI) score, and Fib-4 score from baseline to Week 24 
• 
Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® (where available) 
• 
Liver-related mortality and liver decompensation events. 
• 
All-cause mortality 


 
 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Safety:   
Safety criteria are as follows: 
• 
Occurrence of treatment-emergent adverse events including severity and relatedness to study drug at all 
visits 
• 
The incidence of treatment-emergent serious adverse events (SAEs), based upon information from 
patient reports, including the description, incidence, and severity of an SAE 
• 
Trends evaluated for the following assessments: physical examinations, concomitant medications, vital 
signs, laboratory test results (including clinical chemistry, hematology, urinalysis, alfa-fetoprotein, 
vitamins A and E, 25-hydroxy vitamin D and INR), and abdominal ultrasound 
Statistical Methods: 
The primary objective of this study is to demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 
120 µg/kg/day A4250 in children with PFIC Types 1 and 2. 
  
The Cochran Mantel Haenzel (CMH) test stratified by PFIC class and age class will be performed to compare 
the proportion in fasting bile acid responders at the end of treatment (Week 22 and 24) in the two A4250 dose 
groups to the placebo group. To ascertain that all data are used in the CMH analysis, neighboring strata will be 
pooled if all subjects in a stratum have the same response. The proportion together with the corresponding 95% 
CI, odds ratio and corresponding 95% Clopper-Pearson exact CI and p-value for the CMH test will be presented.  
 
For the primary efficacy variable of the proportion of positive pruritus assessments at subject level over the 24-
week treatment period, an ANCOVA model will be used to analyse the comparisons between the treatment 
groups. The model will include treatment arm, AM baseline pruritus score, PM baseline pruritus score, and 
randomization stratification factors, i.e. PFIC class and age class. LS mean (SE) by treatment arm and LS mean 
difference (SE), 95% confidence intervals, and p-values (where applicable) between treatments (120 µg/kg/day 
and 40 µg/kg/day, respectively) vs. placebo will be provided. If there are concerns on model assumptions, i.e. 
normality and homogeneity of variances, a non-parametric ANCOVA based on the rank scores can be used as a 
sensitivity analysis. LS mean/SE on the outcome by treatment arm and LS mean difference/SE between active 
dose and placebo will be provided.  
 
For each primary endpoint by region (EU & RoW and US), a pooled analysis for the closed testing procedure 
will be applied to control the 1-sided overall type I error rate for two treatment comparisons vs. the placebo at 
the 0.025 level, as specified below: 
 
In the closed testing procedure, the low and high dose groups will be pooled to compare with the placebo group 
first. If the 1-sided p-value is ≤0.025, the 1-sided p-values for low dose vs. placebo and high dose vs. placebo 
will be calculated respectively. If both individual p-values are ≤0.025, a significant treatment effect will be 
declared on both dose groups. If only one of them is ≤0.025, a significant treatment effect will be declared on 
the corresponding dose group.  
 
Other secondary and exploratory outcome variables will provide supportive efficacy and safety information 
regarding the differences between the treatment groups. No adjustments will be performed for multiple 
comparisons when testing secondary and exploratory efficacy variables. 
 
Date of the Protocol:  29 April 2019 


 
 
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3 
TABLE OF CONTENTS 
1 
ADMINISTRATIVE INFORMATION ........................................................................ 4 
2 
STUDY SYNOPSIS ...................................................................................................... 5 
3 
TABLE OF CONTENTS .............................................................................................. 9 
3.1 
List of Tables .................................................................................................. 13 
3.2 
List of Figures ................................................................................................ 13 
3.3 
List of Appendices .......................................................................................... 13 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ................................ 14 
5 
INTRODUCTION ....................................................................................................... 16 
5.1 
Investigational Medicinal Product .................................................................. 16 
5.2 
Background .................................................................................................... 16 
5.2.1 
Progressive Familial Intrahepatic Cholestasis ........................................... 16 
5.2.2 
Summary of Clinical and Nonclinical Studies ........................................... 18 
5.3 
Rationale ......................................................................................................... 19 
5.3.1 
Risk/Benefit ............................................................................................... 20 
6 
STUDY OBJECTIVES ............................................................................................... 21 
6.1 
Primary Objectives ......................................................................................... 21 
6.2 
Secondary Objectives ..................................................................................... 21 
7 
INVESTIGATIONAL PLAN ...................................................................................... 22 
7.1 
Overall Study Design and Plan....................................................................... 22 
7.1.1 
Description ................................................................................................. 22 
7.1.2 
Schedule of Assessments ........................................................................... 24 
7.1.3 
Study Procedures and Assessments ........................................................... 29 
7.1.3.1 
Screening Period (Days -56 to 0) ............................................... 29 
7.1.3.2 
Treatment Period (Day 0 to Day 168) ........................................ 30 
7.1.3.3 
Follow-Up Period ....................................................................... 33 
7.2 
Study Population ............................................................................................ 33 
7.2.1 
Inclusion Criteria ....................................................................................... 34 
7.2.2 
Exclusion Criteria ...................................................................................... 34 
7.2.3 
Withdrawal of Patients............................................................................... 36 
7.2.4 
Study Termination by Sponsor .................................................................. 37 
8 
TREATMENT OF PATIENTS ................................................................................... 38 
8.1 
Identity of Study Drug .................................................................................... 38 
8.2 
Administration of Study Drug ........................................................................ 38 
8.3 
Study Treatment Packaging and Labeling ...................................................... 39 


 
 
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8.3.1 
Packaging and Labeling ............................................................................. 39 
8.3.2 
Storage ....................................................................................................... 39 
8.3.3 
Blinding and Randomization of Study Drug ............................................. 39 
8.4 
Procedure for Breaking the Randomization Code .......................................... 40 
8.5 
Patient Compliance ......................................................................................... 41 
8.6 
Study Drug Accountability ............................................................................. 41 
8.7 
Concomitant Therapy and Prohibited Medications ........................................ 41 
9 
ASSESSMENT OF EFFICACY ................................................................................. 42 
9.1 
Efficacy Endpoints ......................................................................................... 42 
9.1.1 
Primary Efficacy Endpoint ........................................................................ 42 
9.1.2 
Secondary Efficacy Endpoint .................................................................... 42 
9.1.3 
Exploratory Efficacy Endpoints ................................................................. 43 
9.2 
Efficacy Assessments ..................................................................................... 44 
9.2.1 
Serum Bile Acids ....................................................................................... 44 
9.2.2 
Itching, Scratching and Sleep Score .......................................................... 45 
9.2.3 
Growth ....................................................................................................... 45 
9.2.4 
Quality of Life Questionnaire (PedsQL).................................................... 46 
9.2.5 
Biomarkers and PK Samples ..................................................................... 46 
9.2.6 
PELD/MELD Score ................................................................................... 46 
9.2.7 
Fibroscan® .................................................................................................. 46 
9.2.8 
Markers of Fibrosis .................................................................................... 46 
9.2.9 
Global Impression of Change and Global Impression of Symptom 
Measures .................................................................................................... 46 
10 
ASSESSMENT OF SAFETY ..................................................................................... 48 
10.1 
Adverse Events ............................................................................................... 48 
10.1.1 
Definitions and Investigator Assessments ................................................. 48 
10.1.1.1 Clinical Significance .................................................................. 48 
10.1.1.2 Serious Adverse Events .............................................................. 48 
10.1.1.3 Severity Assessment ................................................................... 49 
10.1.1.4 Causality Assessment ................................................................. 49 
10.1.2 
Recording of Adverse Events .................................................................... 50 
10.1.3 
Recording and Reporting of Serious Adverse Events ................................ 51 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions .............. 52 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments .................................................................................................... 53 
10.2.1 
Laboratory Assessments ............................................................................ 53 


 
 
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10.2.2 
Individual Patient Safety Monitoring ........................................................ 54 
10.2.2.1 Liver Monitoring ........................................................................ 54 
10.2.2.2 Close Observation ...................................................................... 55 
10.2.2.3 De-challenge/Re-challenge for Liver and Clinical Hepatitis 
Monitoring .................................................................................. 56 
10.2.2.4 Diarrhea ...................................................................................... 56 
10.2.2.5 Monitoring after Permanent Discontinuation of Study Drug 
Due to Safety .............................................................................. 56 
10.2.3 
Demographics/Medical and Surgical History ............................................ 56 
10.2.4 
Clinical Genetic Testing ............................................................................ 57 
10.2.5 
Physical Examination ................................................................................ 57 
10.2.6 
Vital Signs ................................................................................................. 57 
10.2.7 
Abdominal Ultrasound ............................................................................... 57 
10.2.8 
Overdose .................................................................................................... 57 
10.2.9 
Pregnancy .................................................................................................. 58 
11 
STATISTICAL EVALUATION ................................................................................. 59 
11.1 
Sample Size and Power .................................................................................. 59 
11.2 
Statistical Methods ......................................................................................... 59 
11.2.1 
Statistical Analysis Sets ............................................................................. 59 
11.2.2 
Methods of Statistical Analyses ................................................................. 60 
11.2.2.1 General Principles ...................................................................... 60 
11.2.2.2 Missing Data .............................................................................. 61 
11.2.2.3 Demographic and Baseline Characteristics ................................ 61 
11.2.2.4 Subject Disposition..................................................................... 61 
11.2.2.5 Evaluation of Primary Efficacy Variables .................................. 62 
11.2.2.6 Evaluation 
of 
Secondary 
and 
Exploratory 
Efficacy 
Variables ..................................................................................... 62 
11.2.2.7 Evaluation of Safety Variables ................................................... 63 
11.2.2.8 Compliance and Exposure .......................................................... 64 
11.2.3 
Blinded Analyses to Confirm Responder Definitions ............................... 64 
11.2.4 
Data Safety Monitoring Board ................................................................... 65 
12 
DIRECT ACCESS TO SOURCE DATA/NOTES ..................................................... 66 
13 
QUALITY CONTROL AND QUALITY ASSURANCE .......................................... 67 
13.1 
Conduct of the Study ...................................................................................... 67 
13.2 
Study Monitoring ........................................................................................... 67 


 
 
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14 
ETHICS ....................................................................................................................... 69 
14.1 
Independent Ethics Committee/Institutional Review Board .......................... 69 
14.2 
Written Informed Consents and Assents ........................................................ 69 
15 
DATA HANDLING AND RECORD KEEPING ....................................................... 71 
15.1 
Case Report Forms/Source Data Handling..................................................... 71 
15.2 
Retention of Essential Documents .................................................................. 71 
16 
FINANCING AND INSURANCE .............................................................................. 72 
17 
PUBLICATION POLICY ........................................................................................... 73 
18 
REFERENCE LIST ..................................................................................................... 74 
19 
APPENDICES ............................................................................................................. 75 
 


 
 
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3.1 
List of Tables 
Table 1 
Schedule of Assessments ................................................................................... 25 
Table 2 
Dosing and Capsule Strength ............................................................................. 39 
Table 3 
Routine Laboratory Parameters .......................................................................... 54 
 
3.2 
List of Figures 
Figure 1 
Study Design ...................................................................................................... 24 
 
3.3 
List of Appendices 
Appendix 1 
Concomitant Medication Guidelines .................................................................. 75 
Appendix 2 
Diary Questions including Albireo Patient-Reported 
Outcomes/Observer-Reported Outcome Instrument .......................................... 76 
Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) ....................................................................... 77 
Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaire .............................. 78 
Appendix 5 
Guideline for Fat-Soluble Vitamin Supplementation......................................... 79 
Appendix 6 
Contraceptive Requirements .............................................................................. 80 
Appendix 7 
Blood Volumes ................................................................................................... 81 
 


 
 
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4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
ADME 
absorption, distribution, metabolism, and excretion 
AE(s) 
adverse event(s) 
AFP 
alfa-fetoprotein 
ALT 
alanine aminotransferase 
APRI 
AST to platelet ratio index 
ASBT 
apical sodium-dependent bile transporter (also known as IBAT) 
AST 
aspartate aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BMI 
body mass index 
BSEP 
bile salt export pump 
CPK 
creatine phosphokinase 
CRA 
clinical research associate 
CYP 
cytochrome P450 
DSMB 
Data and Safety Monitoring Board 
eCRF 
electronic case report form 
eDiary 
electronic diary 
EOT 
End of Treatment 
EU 
European Union 
EudraCT 
European Union drug regulatory agency clinical trial 
FAS 
full analysis set 
FDA 
United States Food and Drug Administration 
FIC1 
familial intrahepatic cholestasis-1 
GCP 
Good Clinical Practice 
GGT 
gamma-glutamyl transferase 
GI 
gastrointestinal 
GLMM 
generalized linear mixed modelling 
IB 
Investigator’s Brochure 
IBAT 
ileal bile acid transporter (also known as ASBT) 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
 
 
IEC 
Independent Ethics Committee 
INR 
international normalized ratio 
IRB 
Institutional Review Board 


 
 
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IWRS 
Interactive Web Response System 
LDH 
lactate dehydrogenase 
LFT 
liver function test 
LPLV 
last patient last visit 
MDR3 
multidrug-resistance protein 3 
MedDRA 
Medical Dictionary for Regulatory Activities 
MELD 
model for end-stage liver disease 
MMRM 
mixed model repeated measures 
ObsRO 
observer-reported outcome 
PBC 
primary biliary cholangitis 
p-C4 
plasma 7α-hydroxy-4-cholesten-3-one concentration 
PCS 
potentially clinically significant 
PedsQL 
Pediatric Quality of Life Inventory 
PELD 
pediatric end-stage liver disease 
PFIC 
progressive familial intrahepatic cholestasis 
PGIC 
patient global impression of change 
PGIS 
patient global impression of symptoms 
PK 
pharmacokinetic(s) 
PP 
per protocol 
PRO 
patient-reported outcome 
PT 
prothrombin time 
PVM 
pharmacovigilance manager 
QoL 
quality of life 
RoW 
rest of world 
RR 
respiratory rate 
SAE(s) 
serious adverse event(s) 
SAP 
statistical analysis plan 
s-BA 
serum bile acid 
SD 
standard deviation 
s-FGF19 
serum fibroblast growth factor 19 concentration 
SOC 
system organ class 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE(s) 
treatment-emergent adverse event(s) 
ULN 
upper limit of normal 
US 
United States 
WHO 
World Health Organization 


 
 
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5 
INTRODUCTION 
5.1 
Investigational Medicinal Product 
A4250 is a small molecule and a potent selective inhibitor of the ileal bile acid transporter 
(IBAT), also known as the apical sodium-dependent bile acid transporter (ASBT).  IBAT is 
an integral brush border membrane glycoprotein that co-transports sodium and bile acids and 
appears to be a critical component in the regulation of the bile acid pool size in animals and 
man.  This transporter, expressed in the distal ileum, is a key element in the enterohepatic 
circulation of bile acids since it facilitates the high-affinity, high-capacity reabsorption of bile 
acids.  Indeed, 95% of secreted bile acids are reabsorbed via IBAT [Hofmann 2009; 
Miethke 2016].  A4250 is orally administered and acts locally in the gut where it binds 
reversibly to IBAT to decrease the reuptake of bile acids.  A4250 has minimal systemic 
exposure at the predicted therapeutic dose ranges. 
No safety signals were found with A4250 in preclinical safety and toxicity studies.  In the 
first clinical study of A4250 in healthy volunteers, A4250 was given as a single oral dose up 
to 10 mg or daily oral doses up to 3 mg/day for 7 days.  A4250 was well tolerated and the 
only findings were gastrointestinal (GI) related effects, such as diarrhea, at higher doses. 
In an exploratory Phase 2 study in children with cholestatic pruritus, A4250 was given as oral 
doses of 10 to 200 µg/kg/day for 4 weeks.  A4250 reduced mean levels of serum bile acids 
(s-BA) in all cohorts.  In 11 of 13 progressive familial intrahepatic cholestasis 
(PFIC) patients, substantial reductions (ranging from 43% to 98%) were observed.  
Patient-reported diary data documented improvement of pruritus during treatment.  A4250 
was well tolerated.  No serious adverse events (SAEs) were deemed to be related to study 
drug, and most adverse events (AEs), including some increased transaminases, were 
considered unrelated to study drug and transient in nature. 
5.2 
Background 
5.2.1 
Progressive Familial Intrahepatic Cholestasis 
PFIC is a rare autosomal recessive cholestatic liver disease estimated to affect between 1 in 
every 50,000 to 100,000 children born worldwide.  PFIC represents 10% to 15% of causes of 
cholestasis in children and 10% to 15% of liver transplantation indications in children.  All 
types of PFIC exist worldwide and both sexes appear to be equally affected. 
The common underlying pathogenesis of PFIC is disruption of bile formation and bile 
transport through the liver [Jacquemin 2000].  The classification of PFIC has evolved over 
the years.  The most commonly used sub-classifications are PFIC Types 1, 2, and 3, which 
are based on the associated affected gene and described in more detail below. 
• PFIC, Type 1:  also referred to as “Byler disease” or “familial intrahepatic 
cholestasis-1 (FIC1) protein deficiency.” FIC1 protein is located on the canalicular 


 
 
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membrane of hepatocytes and facilitates movement of aminophospholipids from the outer 
to inner leaflet of the plasma membrane of the hepatocyte.  The ATP8B1 gene encodes 
FIC1 protein.  Biallelic pathologic variants in the ATP8B1 gene are associated with FIC1 
dysfunction and classified clinically as PFIC Type 1 disease. 
• PFIC, Type 2:  also referred to as “Byler syndrome” or “bile salt export pump (BSEP) 
deficiency.”  BSEP is a transporter protein that is expressed at the canalicular membrane 
of hepatocytes and is the primary exporter of bile acids.  The ABCB11 gene encodes the 
BSEP protein.  Biallelic pathologic variations in the ABCB11 gene are associated with 
BSEP dysfunction and classified clinically as PFIC Type 2 disease. 
• PFIC, Type 3:  is caused by a deficiency of the multidrug-resistance protein 3 (MDR3) 
due to mutations in the ABCB4 gene.  MDR3 is a phospholipid translocase critical for 
phospholipid secretion. 
Severe pruritus is common in children diagnosed with PFIC.  Itching (and subsequent 
scratching) is a significant morbidity for these patients and their families [Suchy 2007].  A 
more severe degree of pruritus is experienced compared to patients with other forms of liver 
disease and to other pruritic conditions such as atopic dermatitis [Murray 2011].  In patients 
with PFIC, liver biopsy reveals canalicular cholestasis and, later, the appearance of portal 
fibrosis.  Serum biochemistry indicates cholestasis with hyperbilirubinemia, elevated alanine 
aminotransferase (ALT) and aspartate aminotransferase (AST).  The concentrations of bile 
acids in serum are very high, while serum gamma-glutamyl transferase (GGT) activity 
(exception being MDR3 variants) and cholesterol are normal [Hori 2010].  Symptoms of 
portal hypertension and liver failure will develop during the course of the disease [Davit-
Spraul 2009; Alissa 2008].  Symptoms develop early; median age at onset of symptoms is 
2 months, and 78% of PFIC patients present with jaundice [Pawlikowska 2010].  The 
life-threatening and debilitating nature of PFIC is reflected by the fact that survival in those 
not resorting to surgery is 50% at 10 years of age and almost zero at 20 years of age.  
Approximately half of PFIC patients undergo liver transplantation [Davit-Spraul 2009] and 
treatment-resistant pruritus is the leading indication for the surgical procedure partial external 
biliary diversion, mostly in PFIC Type 1 and 2 patients. 
PFIC is life-threatening and debilitating.  Currently, the treatment for PFIC is palliative and 
there is no pharmaceutical treatment approved for use in PFIC.  The therapeutic choices are 
restricted to non-specific therapy of the symptoms and signs of the disease such as nutritional 
support, preventing vitamin deficiencies, and treatment of extrahepatic features.  Medical 
treatment options include off-label use of ursodeoxycholic acid, rifampin, antihistamines, and 
naltrexone.  A minority of patients respond nominally and transiently to these interventions.  
Biliary diversion is used to decrease systemic bile acids through interruption of the 
enterohepatic circulation and so avoid transplantation.  Liver transplantation is typically only 
viewed as an option when patients have failed medical treatment and/or biliary diversion and 
have liver failure or persistent uncontrolled pruritus. 


 
 
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5.2.2 
Summary of Clinical and Nonclinical Studies 
This is a summary of nonclinical and clinical studies.  More detail is provided in the 
Investigator’s Brochure ([IB] see A4250 IB).  
A4250 is a potent, selective IBAT inhibitor with no safety signals seen in the safety 
pharmacological studies.  In addition, no safety signals were seen in repeat-dose oral toxicity 
studies in rats with up to 26 weeks of daily dosing and in dogs with up to 39 weeks of daily 
dosing.  No safety signals were identified when A4250 was administered daily to juvenile 
rats from age 14 days to 63 days. 
A4250 is minimally absorbed with very low systemic exposure in both humans and other 
species.  In blood, protein binding is high (>99.7%).  Systemically absorbed A4250 is slowly 
metabolized in humans and in preclinical studies by hydroxylation only, a metabolite that 
retains IBAT activity.  A4250 did not show any significant inhibition of cytochrome 
P450 (CYP) 
enzymes 
in 
the 
rat. 
 
A4250 
inhibited 
human 
CYP3A4 
and 
CYP2D6 (IC50: 16 µmol/L for both enzymes), and CYP2C9 (IC50:1.2 µmol/L).  
To date, A4250 has been studied in 3 Albireo-sponsored clinical studies: a study in healthy 
volunteers evaluating single and multiple administration of A4250; a single-dose absorption, 
distribution, metabolism, and excretion (ADME) study; and a Phase 2 study in children with 
cholestatic pruritus.  In addition, an investigator-sponsored study has been conducted in 
patients with primary biliary cholangitis (PBC). 
In the first clinical study (A4250-001), A4250 was given to healthy volunteers as a single oral 
dose up to 10 mg or daily oral doses up to 3 mg/day for 7 days.  Following a single oral dose 
of 10 mg, the median maximum drug concentration was 0.2 nmol/L and the area under the 
curve was 0.8 nmol × h/L (n=6).  Pharmacodynamic evaluation of bile acid synthesis 
biomarkers 
(serum 
fibroblast 
growth 
factor 
19 [s-FGF19] 
and 
plasma 
7α Hydroxy 4-cholesten-3-one [p-C4]) identified the expected decrease in s-FGF19 and an 
increase in p-C4.  In addition, total plasma bile acids decreased and total fecal bile acids 
increased in A4250-treated patients compared to placebo. A4250 was generally well 
tolerated.  The only findings were GI-related effects, such as diarrhea at higher doses. 
In the ADME study (A4250-007), healthy subjects were administered a single oral dose of 
3 mg [14C]-A4250 capsule.  The main excretion route was determined to be fecal.  There 
were no quantifiable concentrations of A4250 in plasma or total radioactivity in plasma or 
whole blood.  A4250 was well tolerated.  AEs were reported and most were mild and not 
related to A4250. The 3 AEs experienced by 2 subjects reported as related to study drug were 
GI in nature, including diarrhea (2 AEs; 2 subjects) and abdominal pain (1 AE; 1 subject). 
In an exploratory Phase 2 study in children with cholestatic pruritus (A4250-003), A4250 was 
administered as oral daily doses of 10 to 200 µg/kg/day for 4 weeks.  Twenty 
patients (8 females and 12 males) aged 1 to 17 years were enrolled in this open-label 
dose-escalation study.  Four patients were re-enrolled into a second dose level.  No 


 
 
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treatment-related SAEs were reported, and most AEs, including some increased 
transaminases, were transient.  Four-week, once-daily treatment with A4250 reduced mean 
levels of s-BA in all cohorts.  Substantial s-BA reductions in 11 of 13 PFIC patients (ranging 
from 43% to 98%) were observed.  A4250 also reduced s-BA in the 11 non-PFIC patients, 
but to a lesser extent than in PFIC patients.  Patient-reported diaries documented 
improvement of the visual analogue scale itch score in 17 of 24 patients during treatment.  
The dose with the greatest improvement showed a mean decrease (± standard error of the 
mean) of 2.9 ±1.1 from baseline.  The results from this study form the basis of the current 
(A4250-005) study design. 
5.3 
Rationale 
In this investigational study, A4250 is under development for treatment of PFIC and is also 
being considered for other orphan cholestatic liver diseases. 
There is currently no medical treatment approved for PFIC.  In PFIC patients with severe 
pruritus, biliary diversion surgery is used to decrease systemic bile acids through interruption 
of the enterohepatic circulation and thereby reducing pruritus [Whitington 1988]. A portion 
of patients who have biliary diversion surgery continue to have elevated serum bile acids and 
pruritus. Liver transplantation is typically used as an option when patients have failed 
medical treatment and/or biliary diversion and have liver failure or persistent uncontrolled 
pruritus. 
By inhibiting IBAT with high selectivity and potency, A4250 has the potential to relieve 
cholestasis and improve liver function without surgical intervention in patients with PFIC.  
Since A4250 is minimally absorbed and achieves a substantial reduction of s-BA, it has the 
potential to be a safer and less invasive alternative to surgical biliary drainage.  The rationale 
for using A4250 is to decrease s-BA levels, and to reduce the major morbidity of pruritus, 
improving the health and wellbeing of patients affected with PFIC.  Ultimately, this 
noninvasive therapeutic approach has the potential to not only improve quality of life (QoL), 
but also reduce the need for biliary diversion and subsequent liver failure and liver 
transplantation.  For those patients who have undergone biliary diversion surgery and 
continue to have elevated serum bile acids and symptomatic pruritus, IBAT inhibition has the 
potential to reduce both the serum bile acid levels and the pruritus. 
The doses selected, 40 and 120 µg/kg/day, were based on the efficacy and safety data 
generated from patients with PFIC (n=10 + 3 re-entered) and supporting data from all 
patients (n=20 + 4 re-entered) in the five dose groups dosed in the A4250-003 study.  
Improvements in s-BA and pruritus were observed with doses from 30 µg/kg to 100 µg/kg, 
with a dose-related trend.  The best dose response in all patients was at the 60 µg/kg/day for 
s-BA and 100 µg/kg/day for pruritus.  The best dose response in the PFIC subgroup was at 
30 µg/kg/day for both s-BA and pruritus.  Patients with PFIC responded well to the doses 
given from 30 µg/kg to 200 µg/kg/day; however, a clear dose relationship could not be 


 
 
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established in order to select a single dose for this Phase 3 study.  As efficacy was observed 
at both low and high doses, 40 µg/kg and 120 µg/kg/day are considered to be the most 
optimal for this study.  
This study will be conducted in compliance with the protocol and with the International 
Council for Harmonisation (ICH) guidelines on Good Clinical Practice (GCP). 
Patients completing 24 weeks of double-blind treatment will be offered to enter Study 
A4250-008 and receive open-label treatment with A4250. 
5.3.1 
Risk/Benefit 
A4250 has been evaluated in three Albireo-sponsored clinical studies: a double-blind 
placebo-controlled study in healthy volunteers, a single-dose ADME study, and a Phase 2 
study in children with cholestatic pruritus.  In addition, an investigator-sponsored study has 
been conducted in patients with PBC. 
Healthy subjects have been exposed to up to 10 mg as a single dose and up to 3 mg daily as 
part of a multiple-dose evaluation.  Children with cholestatic liver disease have been treated 
with up to 0.2 mg/kg/day (200 µg/kg/day) for 4 weeks.  In total, 2 SAEs have been reported; 
both were judged by the physician to be not related to the study drug. 
Patients with cholestatic liver diseases suffer from excess bile acids in the liver resulting in 
tissue damage.  A commonly used treatment in these patients is bile diversion surgery, 
whereby approximately 50% to 100% of the enterohepatic circulation of bile acids is 
interrupted.  Inhibition of IBAT with A4250, thereby interrupting the enterohepatic 
circulation of bile acids, is therefore a potential medical alternative to surgery which could be 
of benefit to these patients if shown to be effective and safe.  Data from the Phase 2 study 
showed efficacy of A4250 in reducing s-BA concentrations and pruritus in such patients. 
Based on the mode of action of an IBAT inhibitor, loose stools or diarrhea could be expected.  
However, in the pediatric Phase 2 study with 4 weeks daily treatment, only one patient had 
mild transient diarrhea after a single dose that did not recur on multiple dosing. 


 
 
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6 
STUDY OBJECTIVES 
6.1 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day 
A4250 in children with PFIC Types 1 and 2, as determined by the following: 
• The proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment 
• The proportion of positive pruritus assessments at the subject level over the 24-week 
treatment period.   
6.2 
  Secondary Objectives 
• To evaluate the effect of A4250 on serum ALT concentration 
• To evaluate the effect of A4250 on growth 
• To evaluate the effect of A4250 on sleep disturbance 
• To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or 
liver transplantation) 
• To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 


 
 
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7 
INVESTIGATIONAL PLAN 
7.1 
Overall Study Design and Plan 
7.1.1 
Description 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the 
efficacy and safety of 40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in 
children with PFIC (deficiencies of FIC1 or BSEP). 
Study data will be periodically (approximately on a quarterly basis) reviewed by an 
unblinded Data Safety and Monitoring Board (DSMB). 
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment 
Period and a 4-week Follow-Up Period. 
After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of 
A4250, or a matching placebo. 
Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008; EudraCT Number 
2017-002325-38, subject to a separate study approval) in which all patients will receive 
active treatment.  A patient who prematurely withdraws due to intolerable symptoms and has 
completed at least 12 weeks of the Treatment Period and End of Treatment (EOT) Visit, will 
also be offered the opportunity to enter Study A4250-008. 
Visit windows applicable to each visit are presented in the schedule of assessments (Table 1). 
There will be up to 10 clinic visits during the study (Figure 1) and one scheduled telephone 
contact, as follows: 
• Visit 1: Screening Period start. Patient is considered enrolled after signing the informed 
consent form (ICF) (Days -56 to -35) 
• Visit 2: second Screening Visit (Days -28 to -7) 
• Visit 3: Randomization Visit (Day 0) 
• Telephone Contact 1: (Day 14) 
• Visit 4: Clinic Visit (Day 28) 
• Visit 5: Clinic Visit (Day 56) 
• Visit 6: Clinic Visit (Day 84) 
• Visit 7: Clinic Visit (Day 126) 
• Visit 8: Clinic Visit (Day 154) 
• Visit 9: Clinic Visit, EOT (Day 168) 


 
 
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• Visit 10: Follow-Up Visit, 28 days after last dose of study drug (Day 196).  All patients 
continuing in Study A4250-008 will not have this Follow-up Visit. 
Additional clinic visits may be required for patients who need direct site assistance, e.g., due 
to AE monitoring, to fulfil screening requirements, and/or for safety maintenance. 
Screening Period 
Informed consent must be obtained at Visit 1 prior to performing any study procedures.  After 
signing the ICF, patients will be considered enrolled and evaluated for study eligibility. 
Daily recording of pruritus using an electronic diary (eDiary) will be started to determine 
confirmation of the magnitude and the baseline pruritus symptoms before the study.  After 
consultation with the Medical Monitor, patients failing eligibility criteria may be re-screened.  
Patients not fulfilling inclusion/exclusion criteria after 3 attempts are not allowed to 
re-screen. 
Caregivers/patients will be instructed on the daily use of the eDiary.  The eDiary will include 
patient-reported outcome (PRO) and observer-reported outcome (ObsRO) items for 
evaluation of itching (PRO), scratching (ObsRO), and sleep disturbance (PRO and ObsRO) 
during the Screening Period (study Days -56 to 0), throughout the 24-week Treatment Period 
(study Days 0 to 168), and Follow-up (Day 196). 
The 14 consecutive days prior to randomization will be used for eligibility calculation of the 
diary entries.  If a patient does not meet the minimum number of observations (see 
Section 9.2.2 for scoring and missing data definitions) to determine eligibility, the patient 
may be re-screened.  Observer-reported outcomes in patients of all ages will be recorded by a 
caregiver.  If possible, the same caregiver will complete the Albireo ObsRO items throughout 
the study. 
At a second Screening Visit (Visit 2) additional laboratory samples will be taken for 
eligibility assessments. 
Treatment Period 
Eligibility for randomization will be determined at Visit 3 using the pruritus (scratching 
reports from the Albireo ObsRO) data in the 2 weeks before Visit 3, clinical genetic 
confirmation of diagnosis, and the liver biochemistry evaluations from the previous screening 
visits.  Two weeks after Visit 3, there will be a telephone contact with the caregiver/patient to 
evaluate any AEs. 
The patient will return to the clinic at Visit 4, Visit 5, Visit 6, Visit 7, Visit 8, and Visit 9 for 
assessments.  If a new caregiver will begin to complete the Albireo ObsRO during the course 
of the trial, they will be provided with training during a clinic visit before they begin to use 
the eDiary.  Additionally, caregivers will be requested to use the diary to report the time each 
dose of study drug is administered during the Treatment Period. 


 
 
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The last dose of study drug will be administered the day before Visit 9.  At study Visit 9, the 
patient has the option to enroll in an open-label extension study (A4250-008) should they 
meet the required entry criteria.  If the patient is continuing into Study A4250-008 this will be 
the last visit in this study and the first visit in Study A4250-008.  Visit 9/EOT should also be 
performed at the time patients are prematurely withdrawn from the study. 
Clinic Visit 10 will take place 28 days after Visit 9 for all patients not continuing into Study 
A4250-008. 
Figure 1 
Study Design 
 
T: telephone contact; V: study visit 
End of Study 
The end of the study is defined as follows: 
a) End of study in one country: last patient last visit (LPLV) and sites are closed 
b) End of study globally: LPLV globally and all sites closed 
7.1.2 
Schedule of Assessments 
The schedule of assessments is presented in Table 1.  For information on blood volumes, 
please see Appendix 7. 
 


 
 
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Table 1 
Schedule of Assessments 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Informed consent c 
X 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
Concomitant medicationd 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical and surgical history 
X 
 
X 
 
 
 
 
 
 
 
 
Physical examination including 
voluntary photography 
X 
 
X 
 
 
 
X 
 
 
X 
 
Skin examination 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
Vital signse 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
eDiary: itching, scratching, and 
sleep scoresf 
Daily diary entry 
Clinical chemistryg 
X 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Hematologyg 
 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Urinalysisg 
 
X 
 
 
 
 
X 
 
 
X 
 
Serum bile acidsh 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 


 
 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
INR 
 
X 
 
 
X 
 
X 
 
X 
 
X 
Autotaxini 
 
 
X 
 
X 
 
 
 
 
X 
 
p-C4i 
 
 
X 
 
X 
 
 
 
 
X 
 
AFP 
 
 
X 
 
 
 
 
 
 
X 
 
Vitamins A & Eh 
 
X 
 
 
 
 
X 
 
 
X 
 
25-hydroxy vitamin D 
 
 
X 
 
 
 
X 
 
 
X 
 
Blood sample for A4250 PKi 
 
 
 
 
X 
 
 
 
 
X 
 
Abdominal Ultrasound 
 
 
X 
 
 
 
 
 
 
X 
 
Fibroscan® (where available) 
 
 
X 
 
 
 
 
 
 
X 
 
QoL questionnaire (PedsQL) 
 
 
X 
 
 
 
 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of Change 
 
 
 
 
X 
 
X 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of 
Symptoms 
 
 
X 
 
X 
 
X 
 
 
X 
 
Pregnancy testj 
Xj 
X 
X 
 
X 
X 
X 
X 
X 
Xj 
X 


 
 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Study drug dispensedk 
 
 
X 
 
X 
X 
X 
X 
X 
 
 
Adverse eventsl 
Continuous collection 
Study drug compliance 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
AFP: alfa-fetoprotein; BMI: body mass index; eCRF: electronic case report form; eDiary: electronic diary; EOT: End of Treatment; ICF: informed consent form; 
INR: international normalized ratio; p-C4: plasma 7α Hydroxy 4-cholesten-3-one; PedsQL: Pediatric Quality of Life Inventory; PK: pharmacokinetic; QoL: quality of life; 
RR: respiratory rate; SAE: serious adverse event; s-BA: serum bile acid. 
 
a 
Must also be performed at the time a patient is prematurely withdrawn from the study.  This visit is the same as Visit 1 in the extension protocol (Study A4250-008). 
b 
Assessments must be performed 28 days following the final dose of study drug.  This visit is omitted if patients are entering Study A4250-008. 
c 
The ICF (and assent) must be signed before any study procedures are performed. 
d 
Past (within 1 month prior to Visit 1) and current medication will be documented in the eCRF. 
e 
Includes blood pressure, pulse, RR, temperature, height (or length depending on age), and body weight (using a certified weight scale).  BMI will be calculated. 
f 
Includes training on use of the device at Screening (Visit 1) and review of patient daily entries during 14 days prior to Clinic Visit 3 to determine eligibility.  
g 
See Table 3 for detailed parameters. 
h 
Patients will fast (water intake only is permissible) for at least 4 hours prior to the collection of samples for s-BA and vitamin A.  Exceptions can be made for infants, 
less than 12 months of age, if unable to fast for the full 4 hours.  All s-BA results during the treatment period and follow-up will be blinded. 
i 
Samples will not be collected for patients ≤10 kg.  In the event of an SAE, a PK sample should be collected as close to the onset of the event as possible. 
j 
For girls who have reached menarche.  Serum test at Visit 1; urine test at other visits.  A serum pregnancy test will also be conducted at Visit 9 for patients entering 
Study A4250-008.  If a urine pregnancy test is positive, a serum pregnancy test should be performed to confirm the pregnancy. 
k 
Study drug to be taken once daily on Day 1 through Day 167 as described in Section 8.2. 


 
 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
l 
Adverse event information will be collected from the time of signing of the ICF to study discontinuation. 


 
 
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7.1.3 
Study Procedures and Assessments 
7.1.3.1 
Screening Period (Days -56 to 0) 
Day -56 to Day -35/Clinic Visit 1 
Patients will undergo a Screening Visit up to 56 days prior to the planned first day of study 
treatment.  Screening procedures and assessments are as follows: 
• Obtain written informed consent 
• Assess inclusion/exclusion criteria (Section 7.2) 
• Record demographics (age, full date of birth, gender, race, and ethnicity) 
• Document concomitant medications 
• Medical and surgical history (date of diagnosis of PFIC Types 1 or 2, prior investigational 
medications for PFIC, historical liver function test [LFT] values, any surgery performed 
including biliary diversion (if applicable), any other diagnosis, and historical liver biopsy 
data) 
• Collect, if available, past confirmatory clinical genetic laboratory report for 
PFIC-1/PFIC-2 and send to central reader for review.  If a historical report is equivocal, 
unavailable, or unobtainable, a blood sample for clinical genetic testing will be collected 
to determine eligibility 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• eDiary training and compliance requirements; caregivers and, if applicable, patients begin 
daily recording of pruritus using the eDiary (Appendix 2) 
• Clinical chemistry (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Serum pregnancy test for girls who have reached menarche.  Please see Appendix 6 for 
contraceptive requirements 
• AE monitoring 
Day -28 to Day -7/Clinic Visit 2 
A second Screening Visit will be performed.  Screening procedures and assessments are as 
follows: 
• Review concomitant medications 
• Vital signs (Section 10.2.6) 


 
 
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• Albireo PRO/ObsRO eDiary review for compliance 
• Urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• International normalized ratio (INR) 
• Vitamins A and E 
• Urine pregnancy test for girls of who have reached menarche 
• AE monitoring 
7.1.3.2 
Treatment Period (Day 0 to Day 168) 
Study Day 0/Clinic Visit 3 (Randomization) 
At the randomization visit, the following assessments will be conducted: 
• Review inclusion/exclusion criteria (Section 7.2) 
• Review concomitant medications, medical and surgical history 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance and entrance criteria requirement (see 
Inclusion Criteria 4, Section 7.2.1) 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1)  
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• 25-hydroxy vitamin D 
• Alfa-fetoprotein (AFP) 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer QoL (Pediatric Quality of Life Inventory [PedsQL]) questionnaire 
• Patient/caregiver/clinician complete the patient global impression of symptoms (PGIS) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Study drug is dispensed and patients are instructed to administer daily from Day 1 
Study Day 14 (Telephone Contact) 


 
 
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A study nurse will contact the patient/caregiver by telephone for monitoring of the following:  
• Review concomitant medications 
• Albireo PRO/ObsRO eDiary review for compliance 
• AEs 
Study Day 28 and Day 84/Clinic Visits 4 and 6  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Physical examination, including voluntary photography (at Clinic Visit 6 only; 
Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• Urinalysis (at Clinic Visit 6 only; Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Autotaxin, p-C4, and A4250 pharmacokinetic (PK) assessment (at Clinic Visit 4 only; 
samples will not be taken for patients ≤10 kg) 
• Vitamins A and E, and 25-hydroxy vitamin D (at Clinic Visit 6 only) 
• Patient/caregiver/clinician complete the patient global impression of change (PGIC) and 
PGIS 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 56 and Day 126/Clinic Visits 5 and 7 
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  


 
 
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• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 154/Clinic Visit 8  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 168/Clinic Visit 9/EOT 
The last dose of study drug is taken in the morning the day before Clinic Visit 9, and the 
following procedures and assessments will be performed (also performed at the time a patient 
prematurely withdraws):  
• Review of concomitant medications 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry, hematology, and urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Autotaxin, p-C4, and A4250 PK assessment (samples will not be taken for patients 
≤10 kg) 


 
 
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• AFP  
• Vitamins A, E, and 25-hydroxy vitamin D 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer PedsQL questionnaire 
• Patient/caregiver/clinician complete the PGIC and PGIS 
• Urine pregnancy test for girls who have reached menarche, or serum pregnancy test for 
girls continuing to study A4250-008 
• AE monitoring 
• Evaluation of study drug compliance 
7.1.3.3 
Follow-Up Period 
Study Day 196/Clinic Visit 10 
All patients not continuing into Study A4250-008 will return to the study site 28 days after 
Clinic Visit 9 or the last dose of study drug for the following assessments: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
7.2 
Study Population 
A total of approximately 60 patients with a clinical diagnosis of PFIC Type 1 or 2 that have 
been genetically confirmed will be randomized.  The study will target 15% of enrolled 
patients to be PFIC-1 patients.  If enrollment of all PFIC-2 patients is completed and the 15% 
enrollment of PFIC-1 patients has not yet been achieved, then enrollment of PFIC-2 patients 
will continue in order to reach the total study target enrollment. 
An attempt will be made to randomize a minimum of 10 patients from each age category 
(6 months to 5 years, 6 to 12 years, and 13 to ≤18 years).  


 
 
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A maximum of 5 patients that were treated in Study A4250-003 will be allowed to participate 
in this study. 
7.2.1 
Inclusion Criteria 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages 
of ≥6 months and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have clinical genetic confirmation of PFIC-1 or PFIC-2 through 
identification of biallelic pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, 
taken as the average of 2 samples at least 7 days apart (Visits 1 and 2) prior to 
randomization 
4. Patient must have history of significant pruritus and a caregiver-reported observed 
scratching in the eDiary average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to 
randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  
Patients who turn 18 years of age (or legal age per country) during the study will be 
required to re-consent in order to remain in the study 
6. Patients will be expected to have a consistent caregiver for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use 
an eDiary device as required by the study 
7.2.2 
Exclusion Criteria 
1. Patient with pathologic variations of the ABCB11 gene that predict complete absence of 
the BSEP protein 
2. Patient with past medical history or ongoing presence of other types of liver disease 
including, but not limited to, the following: 
a) Biliary atresia of any kind 
b) Benign recurrent intrahepatic cholestasis, indicated by any history of normal s-BAs 
c) Suspected or proven liver cancer or metastasis to the liver on imaging studies 
d) Histopathology on liver biopsy is suggestive of alternate non-PFIC related etiology of 
cholestasis 
3. Patient with a past medical history or ongoing presence of any other disease or condition 
known to interfere with the absorption, distribution, metabolism (specifically bile acid 
metabolism), or excretion of drugs in the intestine, including but not limited to, 
inflammatory bowel disease 


 
 
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4. Patient with past medical history or ongoing chronic (i.e., >3 months) diarrhea requiring 
intravenous fluid or nutritional intervention for treatment of the diarrhea and/or its 
sequelae 
5. Patient has a confirmed past diagnosis of infection with human immunodeficiency virus 
or other present and active, clinically significant, acute, or chronic infection, or past 
medical history of any major episode of infection requiring hospitalization or treatment 
with parenteral anti-infective treatment within 4 weeks of treatment start (study Day 1) or 
completion of oral anti-infective treatment within 2 weeks prior to start of Screening 
Period 
6. Any patient with suspected or confirmed cancers except for basal cell carcinoma, and 
non-liver cancers treated at least 5 years prior to Screening with no evidence of 
recurrence 
7. Patient with a past medical history of chronic kidney disease with an impaired renal 
function and a glomerular filtration rate <70 mL/min/1.73 m2  
8. Patient with surgical history of disruption of the enterohepatic circulation (biliary 
diversion surgery) within 6 months prior to start of Screening Period. 
9. Patient has had a liver transplant or a liver transplant is planned within 6 months of 
randomization 
10. Decompensated liver disease, coagulopathy, history or presence of clinically significant 
ascites, variceal hemorrhage, and/or encephalopathy 
11. INR >1.4 (the patient may be treated with Vitamin K intravenously, and if INR is ≤1.4 at 
resampling the patient may be randomized) 
12. Serum ALT >10 × upper limit of normal (ULN) at Screening 
13. Serum ALT >15 × ULN at any time point during the last 6 months unless an alternate 
etiology was confirmed for the elevation 
14. Total bilirubin >10 × ULN at Screening 
15. Patient suffers from uncontrolled, recalcitrant pruritic condition other than PFIC.  
Examples include, but not limited to, refractory atopic dermatitis or other primary pruritic 
skin diseases 
16. Any patient who is pregnant or lactating or who is planning to become pregnant within 
24 weeks of randomization 
17. Sexually active males and females who are not using a reliable contraceptive method with 
≤1% failure rate (such as hormonal contraception, intrauterine device, or complete 
abstinence) throughout the duration of the study and 90 days thereafter (from signed 


 
 
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informed consent through 90 days after last dose of study drug).  See Appendix 6 for 
further details 
18. Patient with a past medical history of alcohol or substance abuse will be excluded.  
Patient must agree to refrain from illicit drug and alcohol use during the study 
19. Administration of bile acid or lipid binding resins and medications that slow GI motility 
(Refer to Appendix 1 – Concomitant Medications Guidelines) 
20. Patient has had investigational exposure to a drug, biologic agent, or medical device 
within 30 days prior to Screening, or 5 half-lives of the study agent, whichever is longer 
21. Patient who has been previously treated with an IBAT inhibitor whose pruritus has not 
responded to treatment 
22. Any other conditions or abnormalities which, in the opinion of the investigator or Medical 
Monitor, may compromise the safety of the patient, or interfere with the patient 
participating in or completing the study 
7.2.3 
Withdrawal of Patients 
Patients/caregivers will be informed that they have the right to withdraw from the study at 
any time, without prejudice to their medical care, and that they are not obliged to state their 
reasons. 
Any withdrawal, and reasons for withdrawal, must be fully documented in the electronic case 
report form (eCRF) and source documents and the patient followed by the 
investigator/investigative staff.  Even after the study is completed at the Follow-up Visit, the 
investigator/investigative staff will, regardless of reason for withdrawal, record any patient 
data they receive concerning SAEs, and all drug-related non-serious AEs, and subsequently 
report these according to Section 10.1.  Withdrawn patients will not be replaced. 
Patients will be withdrawn in the following circumstances: 
• A patient’s/caregiver’s desire for withdrawal for any reason 
• Lost to follow-up (every effort must be made to contact the patient/caregiver; a certified 
letter must be sent) 
• An AE that, in the opinion of the investigator, necessitates withdrawal 
• A worsening of the symptoms related to diarrhea occurs 
• Death 
• Patient/caregiver estimation of intolerable symptoms 
• A patient’s/caregiver’s substantial noncompliance (eDiary and study drug compliance) or 
protocol violation 


 
 
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• An investigator’s opinion that continuing the patient in the study is not appropriate.  The 
investigator may withdraw a patient at any time, if it is considered to be in the patient’s 
best interest 
A patient who prematurely withdraws due to intolerable symptoms and has completed at least 
12 weeks treatment in the double-blind study, will be offered the opportunity to enter 
open-label Study A4250-008.  A patient who withdraws from treatment prematurely and does 
not enter Study A4250-008 will have EOT (Visit 9) assessments at the time of withdrawal 
and a follow-up assessment (Visit 10) 28 days following the last dose of study drug, and the 
patient will be recorded as withdrawn. 
7.2.4 
Study Termination by Sponsor 
This study may be terminated at any time by Albireo if serious side effects should occur, if 
the investigator does not adhere to the protocol; or if, in the sponsor's judgment, there are no 
further benefits to be achieved from the study.  In this event, Albireo/designee will inform the 
study investigators, institutions, and all regulatory authorities. 
Albireo may temporarily or permanently discontinue the study at an investigative site at any 
time for safety, ethical, compliance, or other reasons.  If this is necessary, Albireo will 
endeavor to provide advance notification to the site.  If a site or the study is suspended or 
discontinued, the investigator/investigative staff will be responsible for promptly informing 
the Independent Ethics Committee (IEC).  If required by local regulations, Albireo/designee 
will be responsible for informing the IEC and the regulatory authority of study or site 
discontinuation.  In such an event, all study data and unused study drug must be returned to 
Albireo. 


 
 
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8 
TREATMENT OF PATIENTS 
8.1 
Identity of Study Drug 
A4250 and placebo will be supplied as capsules for oral administration.  White opaque 
capsules filled with pellets containing A4250 or placebo will be provided.  Two different 
capsule sizes will be available, as follows: 
• Capsule size 0 that can be opened 
• Capsule size 3 that should be swallowed intact.  These size 3 capsules may be opened 
only under exceptional circumstances, e.g., patient cannot swallow capsule intact 
The content of the A4250 and placebo capsules will be identical in appearance.  Capsule 
filling weight will also be identical for high dose, low dose, and placebo.  Bottles with 
34 capsules will be given to the patient at each visit.  A patient who requires 2 or more 
capsules per day will be given multiple bottles.  Refer to the investigational product manual. 
8.2 
Administration of Study Drug 
Patients will be dosed with A4250 at a dose of 40 µg/kg/day, 120 µg/kg/day, or placebo for 
24 weeks.  Study drug will be dispensed to the patient at Visits 3, 4, 5, 6, 7, and 8, together 
with instructions on how to store and take the drug.  Study drug administration data, 
including whether each patient took each dose or partial doses of study drug, whether there 
were any delayed or missed doses, and whether the capsule was opened or swallowed whole 
will be documented through the eDiary and transferred to the study database. 
A4250 should be taken in the morning, together with food.  On clinic visits days when 
laboratory assessments are conducted, study drug should be taken after the visit and after 
laboratory samples are taken.  Patients should not crush or chew the capsule(s).  When 
swallowing the capsule intact, the patient should administer the dose with a glass of water. 
For a patient unable to swallow capsule intact, the capsule can be opened and the contents 
sprinkled and mixed in a small amount of soft room-temperature yogurt, apple sauce, or fruit 
purée.  A list of food options can be found in the Pharmacy manual. The amount of food 
should be less than what the patient/infant is expected to eat and should be followed by water.  
1. Hold the capsule over the prepared food, gently twist open and allow the content to fall 
into food 
2. Mix the entire capsule content with 1 or 2 tablespoons (15 to 30 mL) of soft food (apple 
sauce, fruit purée, or yogurt) 
3. Administer the mixture immediately after mixing. 
The number of capsules provided to the patient will be based on body weight at Visit 3.  At 
Visit 6, if a patient’s weight has shifted to another weight category (identified in Table 2), the 
number of capsules per day may be adjusted.   


 
 
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Table 2 
Dosing and Capsule Strength 
Body Weight 
(kg) 
Capsule 
Size 
Number of 
Capsules 
per Day 
Capsule 
Strength, 
Low Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Strength, 
High Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Size, 
Placebo 
(µg) 
5.0 to <7.5 
0 
1 
200 
200 
600 
600 
0 
7.5 to <12.5 
0 
2 
200 
400 
600 
1200 
0 
12.5 to <17.5 
0 
3 
200 
600 
600 
1800 
0 
17.5 to <19.5 
0 
4 
200 
800 
600 
2400 
0 
19.5 to <25.5 
3 
2 
400 
800 
1200 
2400 
3 
25.5 to <35.5 
3 
3 
400 
1200 
1200 
3600 
3 
35.5 to <45.5 
3 
4 
400 
1600 
1200 
4800 
3 
45.5 to 55.5 
3 
5 
400 
2000 
1200 
6000 
3 
>55.5 
3 
6 
400 
2400 
1200 
7200 
3 
8.3 
Study Treatment Packaging and Labeling 
8.3.1 
Packaging and Labeling 
The capsules will be packed in high-density polyethylene containers, with child-proof 
polypropylene caps.  Study drug capsules containing A4250 in the strength specified in 
Table 2 above will be manufactured. 
Packaging and labeling will be prepared to comply with applicable regulatory requirements. 
8.3.2 
Storage 
Treatment packs containing A4250 capsules should be stored and dispensed in accordance 
with regulations in their original containers.  The storage facility at the investigative site 
should be locked and the storage temperature should be between 15°C and 25°C. 
Patients/caregivers should be informed of appropriate storage conditions (i.e., room 
temperature, between 15°C and 25°C). 
Any deviations from the recommended storage conditions should be immediately reported to 
Albireo and the study drug should not be used until authorization has been given by Albireo. 
8.3.3 
Blinding and Randomization of Study Drug 
A double-blind design is employed so that both the investigators and the patients will be 
unaware of the treatment assignment during the study.  
After written informed consent is obtained from an eligible patient, an 8-digit patient 
identification number will be assigned.  The first 2 digits will denote the country, followed by 
a 3-digit site number and a 3-digit patient sequence number.  This number will be created and 


 
 
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allocated by the Interactive Web Response System (IWRS), when the patient first enters the 
system at Screening.  The randomization codes will be computer-generated by 
 
Biostatistics and kept by a statistician independent from the project team.  The randomization 
will be done in blocks and stratified according to PFIC class (Type 1 and 2) and age class 
(6 months to 5 years, 6 to 12 years, and 13 to ≤18 years) to ensure approximate balance 
between dose schemes (1:1:1).  A separate randomization list will be prepared for the 
maximum 5 patients from Study A4250-003 regardless of stratification.  Randomization 
codes will be assigned sequentially as patients become eligible for randomization. 
Patients who are eligible for randomization will be assigned a unique 4-digit randomization 
number by IWRS.  This randomization number identifies which treatment will be allocated to 
the patient.  Patients who withdraw from the study after randomization visit are not to be 
replaced and their randomization number will not be reused.   
To ensure blinding, the study drug and the matching placebo have the same shape and size.  
Patients will receive capsule(s) of A4250 according to their dose group or capsule(s) of 
matching placebo once a day during the double-blind Treatment Period.  Labels on the study 
drug containers will not identify the treatment that a patient is randomized to.  Traceability of 
the treatment is ensured by the study drug number. 
The 5-digit study drug number will identify study drug packs and will be detailed on the 
study drug label.  Dispensing of study drug will be coordinated by IWRS.  
The system will assign a study drug number corresponding to the randomization arm.  The 
randomization number will be used in the background only to ensure no unblinding and will 
not be displayed to end-users in the IWRS. 
8.4 
Procedure for Breaking the Randomization Code 
Should a situation arise where unblinding is urgently required, i.e., only where knowledge of 
the study drug is required to adequately manage a life-threatening situation, the investigator 
at that study site may perform immediate unblinding through IWRS.  The responsibility to 
break the treatment code in emergency situations resides solely with the investigator.  The 
investigator should make every effort to discuss the rationale (status and outcome) for 
emergency unblinding with the sponsor Medical Monitor or the 
Medical Monitor as 
soon as possible to review the individual patient details. 
The study site investigator and appropriate 
 project team members will be authorized to 
access the emergency unblinding functionality within the IWRS.  Emergency unblinding 
takes place in the IWRS via a dedicated emergency unblinding module.  The system will 
require the user to enter their emergency unblinding, which is typically provided at the same 
time login credentials are distributed.  The exact description of the treatment assigned to the 
individual patient then will be accessible to the user.  Once the randomization code is broken 
for a patient, he/she must be withdrawn from the study and all assessments and procedures at 
EOT should be performed.  As a follow-up, Visit 10, will be performed according to the time 


 
 
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schedule.  Once a randomization code has been broken, the investigator must inform the 
 and sponsor Medical Monitor in writing within 24 hours.  In case of technical issues 
accessing the system online, please see the IWRS site user manual for country-specific 
contact telephone numbers for the 
 HelpDesk 24/7 system support. 
8.5 
Patient Compliance 
The study nurse will monitor eDiary compliance by routine review of the CRF health 
website.  If both diary entries on a day are missing, the study nurse will call the 
patient/caregiver to remind them to complete all scheduled entries.  Any noncompliance will 
be documented and explained in the source documents. 
Study drug compliance will also be checked through diary responses.  Additionally, patients 
will return all unused study drug at Visits 4, 5, 6, 7, 8, and 9.  The study site staff will count 
all returned drug, assess compliance, and record details in the eCRF. 
Treatment Compliance = 100 × ((Number of study drug dispensed – number of study drug 
returned)/number of study drug that should be taken). 
Treatment compliance between 80% and 120% will be acceptable. 
8.6 
Study Drug Accountability 
Records shall be maintained of the delivery of study treatment(s) to the study site(s), of the 
inventory at the study site(s), of each use of the study treatment(s) for each patient, and of the 
return of unused study treatment(s) to Albireo. 
These records shall include dates, quantities, batch numbers, expiry dates, and the unique 
code numbers assigned to the study drug and to the study patients. 
The investigator will be responsible for ensuring that the records adequately document that 
the patients were provided the quantities specified in the protocol and that all study drug 
received from Albireo is reconciled.  
8.7 
Concomitant Therapy and Prohibited Medications 
The investigator will note all ongoing medication and any medication recently stopped 
(within 1 month prior to Visit 1) in the eCRF.  At Visits 2 through 10, all changes in 
medication (stopping or starting new medication or changes in dose) will be recorded in the 
eCRF.  All medications (prescribed or over the counter) for pruritus and sleep will be 
recorded. 
All medications taken by a patient within 3 months prior to the first intake of study drug are 
regarded as prior medication. 
All medications taken by a patient on or after the first intake of study drug, and which 
continue to be taken during the study, are regarded as concomitant medication.  
Prohibited medications are listed in Appendix 1. 


 
 
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9 
ASSESSMENT OF EFFICACY 
9.1 
Efficacy Endpoints 
9.1.1 
Primary Efficacy Endpoint 
The primary efficacy endpoints are as follows: 
• European Union (EU) and rest of the world (RoW): Proportion of patients 
experiencing at least a 70% reduction in fasting s-BA concentration from baseline to 
the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 
weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization 
and the previous visit (Clinic Visit 2).  The end value is the average of the values at 
Weeks 22 and 24 after the start of double-blind treatment. 
• United States (US): Proportion of positive pruritus assessments at the subject level 
over the 24-week Treatment Period. A positive pruritus assessment is defined as a 
scratching score of ≤1 or at least a one-point drop from baseline on the Albireo 
ObsRO instrument. At each assessment, the AM score will be compared to the AM 
baseline, and the PM score will be compared to the PM baseline. Both AM baseline 
and PM baseline pruritus assessments will be included in the analysis of this endpoint. 
AM scores from the 14 days prior to the first dose of study medications will be 
averaged as AM baseline. PM scores from the 14 days prior to the first dose will be 
averaged as PM baseline.  
9.1.2 
Secondary Efficacy Endpoint 
Secondary efficacy endpoints include the following:  
• EU and RoW: Proportion of positive pruritus assessments at the subject level over 
the 24-week Treatment Period. A positive pruritus assessment is defined as a 
scratching score of ≤1 or at least a one-point drop from baseline on the Albireo 
ObsRO instrument. At each assessment, the AM score will be compared to the AM 
baseline, and the PM score will be compared to the PM baseline. Both AM baseline 
and PM baseline pruritus assessments will be included in the analysis of this endpoint. 
AM scores from the 14 days prior to the first dose of study medications will be 
averaged as AM baseline. PM scores from the 14 days prior to the first dose will be 
averaged as PM baseline.   
• US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment. 
• All Regions: 


 
 
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All secondary endpoints are compared to placebo. 
o Change from baseline to Week 12 and to Week 24 in fasting serum bile acids 
o Change from baseline to Week 12 and to Week 24 in serum ALT 
concentration 
o Change in growth from baseline to Week 12 and to Week 24 
o Proportion of responders for pruritus scores at Weeks 12 and 24 based on the 
Albireo PRO and ObsRO instruments  
o Change in sleep parameters measured with the Albireo PRO and ObsRO 
instruments from baseline over the 24-week Treatment Period  
o Proportion of individual assessments meeting the definition of a positive 
pruritus assessment at the subject level over the 24-week Treatment Period. A 
positive pruritus assessment includes an itch score ≤1, or at least a one-point 
drop from baseline based on the Albireo PRO instrument; only patients 
≥8 years of age will complete the Albireo PRO instrument  
o Proportion of individual assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-
12, Weeks 0-18, Weeks 0-24, respectively, or the proportion of positive 
pruritus assessments at each 4-week interval.  
o Proportion of individual AM assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-
12, Weeks 0-18, Weeks 0-24, respectively, or the proportion of positive 
pruritus assessments at each 4-week interval.  
o Proportion of individual PM assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-
12, Weeks 0-18, Weeks 0-24, respectively, or the proportion of positive 
pruritus assessments at each 4-week interval.  
o Number of patients undergoing biliary diversion surgery or liver 
transplantation 
9.1.3 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, 
caregiver, and clinician via the PGIC items (Appendix 3) 
• Change from baseline in fasting s-BA at Week 4  


 
 
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• Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
by each 4 week-interval of the Treatment Period as measured by the Albireo ObsRO 
instrument 
• Change from baseline by each 4-week interval of the Treatment Period in patient-reported 
and observer-reported night time itching and scratching severity scores, respectively  
• Change from baseline by each 4-week interval of the Treatment Period in patient-reported 
and observer-reported morning time itching and scratching severity scores, respectively 
• Change from baseline by each 4-week interval of the Treatment Period in pooled pruritus 
score including observer-reported scratching for patients <8 years of age and 
patient-reported itch severity for patients ≥8 years of age 
• Change from baseline by each 4-week interval of the Treatment Period in additional 
patient-reported and observer-reported sleep parameters (e.g., tiredness and number of 
awakenings) 
• Change from baseline to Week 24 in PedsQL (Appendix 4) 
• Change from baseline in serum ALT concentration at Week 4  
• Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• Change from baseline in AST and GGT at Weeks 4, 12, and 24 
• Change from baseline for other biochemical markers and measures of bile acid synthesis 
(autotaxin, p-C4) at Week 24 
• Change in pediatric end-stage liver disease/model for end-stage liver disease 
(PELD/MELD) score, AST to platelet ratio index (APRI) score, and Fib-4 score from 
baseline to Week 24 
• Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® 
(where available) 
• Liver-related mortality and liver decompensation events 
• All-cause mortality 
9.2 
Efficacy Assessments 
9.2.1 
Serum Bile Acids 
Blood samples for analysis of fasting total s-BA will be drawn at all visits.  Patients will fast 
(water intake only is permissible) for at least 4 hours prior to the collection of samples for 
s-BA.  Exceptions can be made for infants, <12 months of age, if unable to fast for the full 
4 hours.  Any visit at which a bile acid sample result is unreportable, an additional 
unscheduled visit for a repeat sample collection may be scheduled.  All s-BA results during 


 
 
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the treatment period and follow-up will be blinded.  Samples will be processed and 
transported to a central laboratory per instructions in the laboratory manual. 
9.2.2 
Itching, Scratching and Sleep Score 
Itching, observed scratching, and sleep disturbance will be recorded twice each day via 
eDiary.  Patients and/or caregivers will be instructed to complete the eDiary every day in the 
morning after the patient wakes and in the evening just before the patient goes to sleep 
throughout the study.  Patients and caregivers will receive a formal training session during 
Visit 1. 
The eDiary includes Albireo ObsRO and PRO items.  Patients <8 years of age will not be 
asked to complete the Albireo PRO items; only the Albireo ObsRO will be completed by 
caregivers of patients in this age group.  Older patients, 8 to 18 years of age, will complete 
the Albireo PRO items and the caregiver will complete the Albireo ObsRO items.  The 
Albireo PRO items assess severity of itch, aspects of sleep disturbance (morning diary only), 
and tiredness.  For patients 8 to 12 years of age, the caregiver will read the Albireo PRO 
items along with the child and record the child’s response.  A guide will be provided to the 
caregivers that provides standardized explanations of the Albireo PRO items, in case the 
patient is confused or requires clarification about the meaning of a question.  The Albireo 
ObsRO items assess severity of observed scratching, aspects of observed sleep disturbance 
(morning diary only), and observed signs of tiredness (evening diary only).  The Albireo 
ObsRO and PRO scratching and itch severity items use 0 to 4 response scales, where each 
response is distinguished by a unique facial expression, verbal anchor, number, and color 
code (Appendix 2).  To meet eligibility criteria, a scratch score will be calculated from daily 
diary entries (ObsRO items) the 14 consecutive days prior to randomization.  
A daily AM & PM score for the Albireo ObsRO scratching item will be averaged from the 2 
ratings for each day.  A daily score will be considered missing if both of the daily 
assessments are missing.  A weekly score will be calculated by averaging the daily scratching 
scores.  A weekly score will be considered missing if ≥4 out of 7 days a week of data are 
missing.  For AM & PM baseline, a 14-day daily score of AM & PM prior to the first dose of 
study medications will be averaged as the baseline score. 
For AM baseline, a 14-day AM score prior to the first dose of study medications will be 
averaged as the AM baseline score.  The same approach will be used for PM baseline and to 
calculate a patient-reported itch severity score.  The handling of missing data is described in 
Section 11.2.2.2. 
9.2.3 
Growth 
Growth will be measured as height (velocity) and weight (Z-score) using a certified weight 
scale.  BMI will be calculated by weight (kg) / height (m)2.  Change will be defined as linear 
growth deficit (weight and BMI for age) compared to a standard growth curve (Z-score, SD 
from P50). 


 
 
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9.2.4 
Quality of Life Questionnaire (PedsQL) 
Patients and caregivers will be asked to complete a QoL questionnaire (PedsQL) at Visit 3 
and Visit 9.  Details of the questions included on the questionnaire are provided in 
Appendix 4. 
9.2.5 
Biomarkers and PK Samples 
Blood samples for autotaxin and p-C4 will be drawn at Visits 3, 4, and 9 only for children 
with body weight >10 kg.  Blood for A4250 PK assessment will only be drawn at Visits 4 
and 9 (only for children with body weight >10 kg), and as close to the onset of an SAE as 
possible, should an SAE occur.  Samples will be processed and transported to a laboratory 
per instructions in the laboratory manual. 
9.2.6 
PELD/MELD Score 
At randomization and end of treatment, PELD score will be calculated for children up to 
12 years of age.  For children 13 years or older, the MELD score will be calculated. 
PELD Score = 4.80 * ln(total bilirubin) + 18.57 * ln(INR) - 6.87 * ln(albumin) + 4.36 (if 
patient <1 year: scores for patients listed for liver transplantation before the patient's first 
birthday continue to include the value assigned for age (<1 year) until the patient reaches the 
age of 24 months) + 6.67 (if the patient has growth failure [≤2 SD]) 
Laboratory values less than 1.0 will be set to 1.0 for the calculation of the PELD score. 
MELD Score = 9.57 * ln(creatinine) + 3.78 * ln(total bilirubin) + 11.2 * ln(INR) + 6.43 
Laboratory values less than 1.0 will be set to 1.0 and serum creatinine values greater than 
4.0 mg/dL (equivalent to 353.6 µmol/L) will be set to 4.0 for calculation of the MELD score. 
9.2.7 
Fibroscan® 
Where available, Fibroscan® will be performed as per institution standard practice at Visits 3 
and 9. 
9.2.8 
Markers of Fibrosis 
APRI score and Fib-4 score will be calculated at randomization and at end of treatment. 
APRI = [(AST in U/L) / (AST ULN in U/L)] / (Platelets in 109/L) 
Fibrosis 4 Score = (Age*AST in U/L) / (Platelets in 109/L * √(ALT in U/L)). 
9.2.9 
Global Impression of Change and Global Impression of Symptom Measures 
Patients (≥8 years of age), caregivers, and clinicians will complete the PGIC and the PGIS 
measures (Appendix 3) at randomization (Visit 3; PGIS only), Visits 4 and 6, and at EOT 
(Visit 9). 
The PGIC items assess change in itch (patient version), scratching (caregiver and clinician 
versions), and sleep (all versions) since starting the study drug.  The PGIS items assess itch 


 
 
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(patient version), scratching (caregiver and clinician versions), and sleep (all versions) in the 
past week.  Caregivers and clinicians will complete the PGIC and PGIS for all patients; those 
patients ≥8 years of age will complete the patient version. 


 
 
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10 
ASSESSMENT OF SAFETY 
The timing and frequency of safety assessments are described in Section 7.1.3 and in Table 1 
above. 
The primary safety analysis for this study will include the incidence of total 
treatment-emergent adverse events (TEAE) and TEAEs categorized by causality, severity, 
and seriousness assessments made by the investigator by comparing study drug exposure to 
placebo. 
Trends in safety will also be evaluated for the following assessments: 
• Physical examinations 
• Concomitant medications 
• Vital signs 
• Laboratory test results (including clinical chemistry, hematology, urinalysis, AFP, and 
vitamins A and E, 25-hydroxy vitamin D, and INR) 
• Abdominal ultrasound 
• Discontinuations due to AEs 
10.1 
Adverse Events 
10.1.1 
Definitions and Investigator Assessments 
An AE is defined as any untoward medical occurrence in an enrolled patient regardless of 
causal relationship with study drug.  An AE can therefore be any clinically significant 
unfavorable and unintended sign, symptom, or disease that occurs once a patient is enrolled 
(ICF is signed) in the study until the patient is discharged from the study, whether or not 
related to the study drug. 
10.1.1.1 
Clinical Significance 
Clinical significance is defined as any abnormal finding that results in further clinical 
investigation(s), treatment(s), or the diagnosis of new condition.  Patient-reported events and 
protocol-mandated laboratory values, vital signs, and physical examination findings can be 
considered clinically significant (i.e., an AE) if there is a deterioration as compared to 
baseline.  Examples of clinically significant worsening from baseline could include, but is not 
limited to, events causing withdrawal from the study and events requiring medical 
intervention outside of the study causing apparent clinical manifestations, or judged relevant 
by the investigator.   
10.1.1.2 
Serious Adverse Events 
Serious criteria are applied by the investigator to each AE as specifically defined below.  
These criteria are used to determine whether an AE is serious or non-serious.  The assessment 


 
 
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is made independently of severity assessment (Section 10.1.1.3).  For example, the 
development of a severe rash that occurs after signing of informed consent may not meet 
serious criteria as defined below and therefore would be considered a severe, non-serious AE. 
Any AE that meets any 1 of the following 6 criteria is considered an SAE: 
• The outcome of the AE is death 
• The AE is immediately life-threatening.  Life-threatening means that the patient is, in the 
opinion of the investigator, at immediate risk of death from the reaction as it occurred.  
This does not include an AE that, if more severe, might have caused death 
• The AE results in persistent or significant disability/incapacity.  Disability means a 
substantial disruption of a person's ability to conduct normal life functions 
• The AE requires or prolongs hospitalization  
• The AE results in a congenital anomaly/birth defect 
• The AE is an important medical event.  Important medical events may meet serious 
criteria should the investigator assess that they may significantly jeopardize the patient, 
represent a significant hazard, or requires medical/surgical intervention to prevent one of 
the outcomes listed above.  Examples of potential SAEs based on this criteria include, but 
are not limited to, allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions even if they do not result in inpatient 
hospitalization, or the development of drug dependency and drug abuse. 
10.1.1.3 
Severity Assessment 
Severity assessments are based on the intensity of the event in relation to expectation.  The 
investigator will assess the intensity of AEs based on the following definitions: 
• Mild (awareness of sign or symptom, but easily tolerated) 
• Moderate (discomfort sufficient to cause interference with normal activities) 
• Severe (incapacitating, with inability to perform normal activities) 
Severity is a measure of intensity where seriousness is defined by the criteria outlined in 
Section 10.1.1.2.  An AE of severe intensity need not necessarily be considered serious.  For 
example, nausea that persists for several hours may be considered severe nausea, but not meet 
serious criteria, and therefore would be assessed as a severe AE but not an SAE. 
10.1.1.4 
Causality Assessment 
The investigator determines the causality of all AEs to the study drug using medical judgment 
and considering all relevant factors such as (but not limited to) the underlying study 
indication, coexisting disease, concomitant medication, relevant history, pattern of the AE, 
temporal relationship to the study drug, and de-challenge or re-challenge.  The causality 
assessment of the AE/SAE is to be made as follows. 


 
 
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Related to study drug (possibly, probably, or definitely related) 
Based on medical judgment, there is at least a reasonable possibility that the study drug 
caused the event; one or more of the following criteria apply: 
• The event follows a reasonable temporal sequence from administration of study drug 
• The event could not be reasonably attributed to the known characteristics of the patient's 
clinical state, environmental or toxic factors, or other modes of therapy administered to 
the patient 
• The event follows a known pattern of response to study drug 
• The event disappears or decreases on cessation or reduction in dose of the study drug.  (It 
should be noted that in some situations an AE will not disappear or decrease in intensity 
upon discontinuation of study drug despite other clear indications of relatedness) 
• The event reappears or worsens when the study drug is re-administered 
Unrelated to study drug (unlikely or unrelated) 
Based on medical judgment there is no reasonable possibility that the study drug caused the 
event; one or more of the following criteria apply: 
• The event does not follow a reasonable temporal sequence from administration of study 
drug 
• The event could be reasonably attributed to the known characteristics of the patient's 
clinical state, concurrent illness, environment or toxic factors, or other modes of therapy 
administered to the patient 
• The event does not follow a known pattern of response to study drug 
• The event does not disappear or decrease on cessation or reduction in dose of the study 
drug, and it does not reappear or worsen when the study drug is re-administered 
10.1.2 
Recording of Adverse Events 
It is the investigator’s responsibility to assess whether each untoward event is a clinically 
significant worsening from baseline, thereby considered an AE.  For all AEs, the severity, 
seriousness, and causality to study drug for each AE as outlined in Section 10.1.1 will be 
assessed and recorded in the eCRF. 
All serious and non-serious AEs are collected once the caregiver/patient has signed the ICF 
and until the post-treatment follow-up (Visit 10) or 28 days after the last dose of study drug. 
Any AEs or SAEs that are unresolved at the patient’s last AE assessment in the study are 
followed up by the investigator until resolution or stabilization up to the database lock and 
recorded in the eCRF.  Albireo retains the right to request additional information for any 
patient with ongoing AE(s) or SAE(s) at the end of the study. 


 
 
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TEAEs are defined as any AE that occurs after randomization (Day 0).  All AEs that occur in 
the Screening Period, i.e., after enrollment and prior to randomization, will be collected on 
the eCRF as non-treatment-emergent AEs. 
If there is a clinically significant deterioration of a laboratory value/vital sign or other routine 
study assessment that is associated with a diagnosis, the clinical diagnosis will be reported as 
an AE and the associated signs and symptoms will be considered additional information 
unless the sign or symptom is more severe than expected given the diagnosis.  For example, if 
an investigator diagnoses a study patient with hepatitis during the study period, hepatitis 
would be considered the AE and the concomitant signs and symptoms of abdominal pain, 
vomiting, and elevated ALT and AST would not be reported separately unless, in the opinion 
of the investigator, one of these signs or symptoms is more severe than expected and 
therefore a separate AE assessment is indicated. 
10.1.3 
Recording and Reporting of Serious Adverse Events 
Every SAE (regardless of severity and causality) that occurs once the caregiver/patient has 
signed the ICF and until the post-treatment follow-up (Visit 10), and 28 days after the final 
dose of study drug, should immediately and not later than within 24 hours of knowledge of 
the event, be reported by the investigator or delegate in the SAE report form.    
Report of a SAE must include at least the following information: 
• Patient identification information (study number, site number, initials, and date of birth 
[as per local country requirements for data protection]) 
• The last study drug administration date 
• The diagnosis of the event with the description (or a brief description of 
signs/symptoms/clinical course if the diagnosis is not available) and the date of onset 
• The action used to treat the event (i.e., treatment medications, temporary discontinuation) 
• The reason(s) for considering the event serious 
• The relationship of the event to the study drug or to the study procedure (e.g., the 
investigator's assessment of causality) 
• A brief narrative of the SAE 
Follow-up reports including all new information obtained of the subsequent course of the 
SAE must be prepared and the information collected in the SAE submitted to 
 
 (Pharmacovigilance and Safety Services Department) by e-mail within 24 hours of 
knowledge of the event. 
The pharmacovigilance manager (PVM) may contact the investigator to obtain further 
information on a reported SAE.  The investigator/investigative staff must respond to any 


 
 
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request for follow-up information or answers to questions regarding the SAE within the same 
timelines as for initial reports. 
The PVM reports the occurrence of the SAE and follow-up to the Albireo medical monitor 
for medical assessment of the case. 
Should an outcome of death occur within the study period or within 28 days after the last 
administration of study drug, an SAE form should be completed including the main and 
contributory causes of death. 
All SAE reports must be e-mailed to the following e-mail address within 24 hours: 
 
 
If email is unavailable, SAEs may be transmitted via fax to the following number: 
 
In a study-related medical emergency situation, when the assigned medical monitors for the 
study cannot be reached by a caller, an on call physician can be reached 24 hours per day, 
7 days per week, via the 
 call center.  The following number is a chargeable telephone 
number allowing a global reach from both landlines and mobile phones and the internet page 
indicated below contains a list of country-specific toll-free telephone numbers that is 
provided.  It should be noted that not all countries globally have access to toll-free numbers 
as indicated on the “24/7 Medical Help desk” index.  Countries without toll-free numbers 
need to dial the chargeable number as indicated below.  Furthermore, toll-free numbers might 
not be available from mobile phones. 
 
 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions 
Suspected unexpected serious adverse reaction (SUSAR) is an SAE that occurs in a patient, 
the nature or severity of which is not expected per the applicable product information (e.g., 
the IB for an unauthorized investigational product or summary of product characteristics for 
an authorized product).  
Reporting and tracking of SUSARs will be in accordance to all applicable competent 
authority regulations.  The Ethics Committee and all investigators involved in this study will 
be informed according to national and international requirements. 


 
 
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10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments 
10.2.1 
Laboratory Assessments 
Samples will be collected for clinical chemistry, hematology, and urinalysis testing at the 
time points specified in Table 1. The parameters assessed are presented in Table 3. 
Blood for AFP will be drawn at Visits 3 and 9.  Fat-soluble vitamin levels including vitamins 
A and E, and 25-hydroxy vitamin D will be drawn at Visit 2 (vitamins A & E only), 
Visit 3 (25-hydroxy vitamin D only), Visit 6, and Visit 9.  Blood for INR (surrogate for 
vitamin K) will be drawn at Visits 2, 4, 6, 8, and 10.  Patients will fast (water intake only is 
permissible) for at least 4 hours prior to the collection of samples for vitamin A.  Exceptions 
can be made for infants, <12 months of age, if unable to fast for the full 4 hours.  If a patient 
has any vitamin level(s) that are outside of the reference range, vitamin supplementation 
adjustments may be required.  Target ranges and supplementation strategy guidelines can be 
found in Appendix 5. 
A serum pregnancy test will be performed at Visit 1 and Visit 9 if the patient is entering the 
A4250-008 study. A urine pregnancy test will be collected at all other visits for girls who 
have reached menarche.  If a urine pregnancy test is positive, a serum pregnancy test should 
be performed to confirm the pregnancy.  Study drug administration will be held pending the 
confirmatory results.  If the serum pregnancy test is negative, the patient can resume dosing.  
If the serum pregnancy test is positive, the patient should be discontinued (see 
Section 10.2.9). 
All samples will be processed and transported to a laboratory per instructions in the 
laboratory manual. 
The observed values will be recorded and assessed as “normal” or “abnormal not clinically 
significant” or “abnormal clinically significant”. 
Additional safety blood samples may be needed due to follow-up of an abnormal value or 
analysis failure.  The blood samples collected for safety laboratory analysis will be destroyed 
after the analyses have been completed.  


 
 
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Table 3 
Routine Laboratory Parameters 
Clinical Chemistry 
Hematology 
Urinalysis 
• 
Albumin 
• 
ALT 
• 
Alkaline phosphatase  
• 
AST 
• 
Bilirubin – total and direct 
• 
Calcium 
• 
Chloride 
• 
Creatinine 
• 
Creatine kinase 
• 
Gamma-glutamyl transferase 
• 
Potassium 
• 
Sodium 
• 
Hematocrit 
• 
Hemoglobin 
• 
Platelet count 
• 
Red blood cell count 
• 
White blood cell count and 
differential (neutrophils, 
eosinophils, basophils, 
lymphocytes, and monocytes) 
• 
Blood 
• 
Glucose 
• 
Ketones 
• 
Leukocytes 
• 
Nitrites 
• 
pH 
• 
Protein 
 
10.2.2 
Individual Patient Safety Monitoring 
10.2.2.1 
Liver Monitoring 
Strategies to monitor markers of liver disease throughout the study are outlined below where 
the ULN will be based on central laboratory reference values for age and gender. 
If isolated transaminase elevations are observed, defined as: 
1. Normal bilirubin AND absence of clinical hepatitis symptoms AND 
• ALT or AST ≥5 × ULN (if normal at baseline) or an absolute threshold of 800 U/L, 
whichever comes first  
• OR ALT or AST ≥3 × baseline (if abnormal at baseline) or an absolute threshold of 
800 U/L, whichever comes first 
Then: 
a) Repeat liver profile (AST, ALT, bilirubin, and prothrombin time [PT] or INR) within 
2 to 3 days 
b) Evaluate creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) 
c) As needed (for example, persistent ALT/total bilirubin elevation or worsening of liver 
function), consider evaluation for alternative etiologies 
Monitor the patient using close observation found in Section 10.2.2.2 
If any ONE of the following criteria are met: 
1. Transaminases (ALT or AST ≥3 × baseline or 800 U/L, whichever comes first) AND 
bilirubin (total bilirubin > 2 × ULN) elevations 


 
 
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2. Transaminase elevations alone (ALT or AST >10 × ULN or 5 × baseline or absolute 
threshold of 800 U/L, whichever comes first) in presence of normal LDH and CPK 
3. Total bilirubin increased, unrelated to hemolysis (elevated reticulocyte count) or 
established genetic diseases, such as Gilbert’s syndrome 
a. Doubling if total bilirubin was <3 mg/dL (equivalent to 51.3 µmol/L) at baseline  
b. OR Increase by >3 mg/dL (equivalent to 51.3 µmol/L) if total bilirubin was ≥3 mg/dL 
(equivalent to 51.3 µmol/L) at baseline 
4. INR increase refractory to vitamin K administration 
a. INR >1.5 if INR was normal at baseline  
b. OR Increase by >0.4 if INR was abnormal at baseline 
5. Any increase in total bilirubin and transaminases if accompanied by EITHER a symptom 
of clinical hepatitis (e.g., vomiting, nausea, right upper quadrant pain) OR immunological 
reaction (rash or >5% eosinophilia) 
Then: 
a) Interrupt study medication 
b) Initiate drug-induced liver injury work-up for alternative etiologies 
c) Repeat liver profile (AST, ALT, total bilirubin, direct bilirubin) and PT or INR within 
48 to 72 hours 
d) Monitor the patient using close observation found in Section 10.2.2.2 
e) If a patient lives in a remote area, they may be tested locally and the results 
communicated to the investigator site promptly 
10.2.2.2 
Close Observation 
• Repeat liver enzyme and serum bilirubin tests two or three times weekly.  Frequency 
of re-testing can decrease to once a week or less if abnormalities stabilize or the trial 
drug has been discontinued and the subject is asymptomatic  
• Obtain a more detailed history of symptoms and prior or concurrent diseases 
• Obtain a history of concomitant drug use (including non-prescription medications and 
herbal and dietary supplement preparations), alcohol use, recreational drug use, and 
special diets  
• Consider ruling out alternate etiology including acute viral hepatitis types A, B, C, D, 
and 
E; 
autoimmune 
or 
alcoholic 
hepatitis; 
non-alcoholic 
steatohepatitis; 
hypoxic/ischemic hepatopathy; and biliary tract disease 
• Obtain a history of exposure to environmental chemical agents.  


 
 
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• Obtain additional tests to evaluate liver function, as appropriate (e.g., INR, direct 
bilirubin) 
• Consider gastroenterology or hepatology consultations. 
10.2.2.3 
De-challenge/Re-challenge for Liver and Clinical Hepatitis Monitoring 
1. Re-challenge is not recommended: 
a. If a patient has had possible/probable drug-induced liver injury 
b. If a decompensation event has occurred (i.e., variceal hemorrhage, ascites, hepatic 
encephalopathy, etc.). 
2. If due to underlying cholestatic liver disease variability or another alternative etiology is 
identified AND liver tests returned to baseline, re-challenge may be considered after 
consultation with the sponsor Medical Monitor. 
3. If the ALT/total bilirubin elevations are observed after re-challenge, then repeat 
re-challenge is discouraged. 
If patient is permanently discontinued, monitoring should be continued as outlined in 
Section 10.2.2.5.  
10.2.2.4 
Diarrhea 
Study drug should be discontinued if a patient develops diarrhea with at least 1 of the 
following concomitant signs or symptoms: grossly bloody stools, vomiting, dehydration 
requiring treatment with oral or intravenous rehydration and/or electrolyte imbalances, fever 
(≥38 C) and/or the diarrhea persists for 7 or more days. 
If there is gross blood in stool, INR and platelets should be measured.  
Study drug will be reintroduced (re-challenge) when the symptoms have resolved.  If the 
diarrhea re-occurs within 1 week after the re-challenge with no alternate etiology, the patient 
will be permanently discontinued and monitored as outlined in Section 10.2.2.5. 
10.2.2.5 
Monitoring after Permanent Discontinuation of Study Drug Due to Safety 
Once study drug is permanently discontinued for liver, hepatitis, diarrhea, or other severe AE 
related to study drug, the patient will be monitored weekly until the laboratory and clinical 
parameters have normalized/stabilized. 
10.2.3 
Demographics/Medical and Surgical History 
Demographic information per country regulations (age, full date of birth, gender, race, and 
ethnicity), along with medical and surgical history, will be obtained and recorded in the eCRF 
at Visit 1. 
Medical and surgical history will be entered in the eCRF at Visit 1.  This includes date of 
diagnosis of PFIC, prior investigational medications for PFIC, historical (3 months prior to 


 
 
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Screening) LFT values (AST, ALT, and total bilirubin), ongoing medication, any surgery 
performed, any other diagnosis, and historical biopsy data.   
If a liver biopsy has been performed within 1 year prior to Screening, or during the study, the 
results will be recorded in the eCRF. 
10.2.4 
Clinical Genetic Testing 
For previous confirmatory clinical genetic testing results for PFIC Type 1 or 2 performed 
prior to Screening Visit 1, the confirmatory clinical genetic testing laboratory report will be 
verified to determine eligibility. 
If the historical clinical genetic result is equivocal, unavailable, or unobtainable, clinical 
genetic analysis to confirm pathologic biallelic variations in the ATP8B1 or ABCB11 genes 
will be performed and verified to determine eligibility.  No other diagnostic genetic testing 
will be offered. 
10.2.5 
Physical Examination 
A physician or suitably trained qualified assistant will perform a complete physical 
examination, including voluntary photography of skin lesions due to scratching, at Visits 1, 3, 
6, and 9.  A skin examination will be conducted at Visits 1, 3, 4, 6, 9, and 10 (see Table 1).  
A complete physical examination will include assessment of general appearance, eyes, ears, 
nose, throat, head/neck/thyroid, lymph nodes, cardiovascular, lungs/chest, abdomen, 
genitourinary, extremities, skin, musculoskeletal, neurologic, and other.  
Skin will be examined thoroughly and excoriations/scratch marks recorded.  
10.2.6 
Vital Signs 
Evaluation of vital signs will be performed at all visits.  This includes blood pressure (systolic 
and diastolic), pulse, respiratory rate, temperature, height (or length depending on age), and 
body weight (using a certified weight scale) at clinic visits.  BMI will be calculated. 
10.2.7 
Abdominal Ultrasound 
Ultrasound of the liver and spleen will be performed at randomization (Visit 3) and at EOT 
(Visit 9).  Liver size, echogenicity, and presence of masses/nodules as well as spleen size will 
be recorded. 
10.2.8 
Overdose 
A4250 is minimally absorbed and has a very low systemic availability.  Based on toxicology 
data, for study purposes any dose exceeding a total of >3 mg/kg body weight of A4250 taken 
as a single dose or as a cumulative dose within 24 hours is defined as an overdose.   
The no-adverse-effect dose level in the most sensitive species (defined as 20 mg/kg/day) in 
the rat 1 month toxicity study gives a human equivalent dose of approximately 194 mg/day in 


 
 
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a 60 kg person.  This dose is approximately 10-fold higher than the maximum possible dose 
predicted in human studies (20 mg).  
The investigator/investigative staff should immediately and not later than within 24 hours of 
knowledge of the overdose, be reported by the investigator or delegate on the SAE report 
form.  In the event of an A4250 overdose, the patient should be monitored closely. 
10.2.9 
Pregnancy 
If a pregnancy is discovered in a female patient enrolled in the study before the end of dosing, 
the patient will be immediately discontinued from the study and will attend the same visits as 
a prematurely withdrawn patient.  If the pregnancy is discovered in a female patient enrolled 
in the study after the end of dosing, the patient will continue in the study per protocol.  If a 
pregnancy occurs in a male patient’s partner at any time during the study, the pregnancy 
should also be reported and followed. 
Pregnancy is not considered to be an AE.  However, if the patient has been dosed with the 
study drug, the pregnancy must be reported on the Paper Pregnancy Notification Form 
immediately within 24 hours after recognition to Safety Manager (please refer to Section 
10.1.3).  Date of exposure and as far as possible, details of the period of gestation at the time 
of exposure must be given. 
The pregnancy should be followed to determine outcome, including spontaneous termination, 
details of birth, and presence of any birth defects, congenital anomalies or newborn or 
maternal complication.  The infant who was exposed in-utero will be followed for up to 
2 years after delivery.  Individual cases with an abnormal outcome in association with the 
study drug should be reported on an expedited basis, i.e., reported rapidly to a competent 
authority. 


 
 
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11 
STATISTICAL EVALUATION 
All personnel involved with the analysis of the study will remain blinded until database lock 
for the double-blind Treatment Period and until major protocol violations have been 
identified. 
All statistical analyses will be performed using SAS version 9.3 or higher. 
11.1 
Sample Size and Power 
For the EU and RoW, the primary analysis will relate to the serum bile acid responder 
endpoint.  For the US, the primary analysis will relate to the pruritus endpoint. For each 
primary endpoint, a pooled analysis for the closed testing procedure will be applied to control 
the 1-sided overall type I error rate for two treatment comparisons vs. the placebo at the 0.025 
level (Kane et al, 2015, Lehmacher et al, 1991, and Marcus et al, 1976). The study will enroll 
60 to 70 patients in order to obtain at least least 20 evaluable patients in each arm. For each 
primary endpoint, simulations with 5000 iterations using 20 patients per arm were conducted 
to estimate the power after multiplicity adjustment, resulting in a standard error of <0.7% for 
each estimated power.   
Based on the Phase 2 A4250-003 study data both dose groups are predicted to have similar 
treatment effects for both serum bile acids and pruritus. For serum bile acids, simulated 
proportions were analyzed using the CMH test to generate the following 1-sided p-values: 
both A4250 arms pooled vs. placebo, low dose vs. placebo, and high dose vs. placebo. 
Assuming 60% responders in the A4250 arms and 10% responders in the placebo arm, the 
power to claim significance for a particular A4250 arm after multiplicity adjustment is 
approximately 94%. The power to claim significance for at least one arm or both arms are 
approximately 99% and 91%, respectively.  
For the proportion of positive pruritus assessments at the subject level in pruritus scores, 
simulated proportions were analyzed using ANCOVA to generate the following 1-sided p-
values: both A4250 arms pooled vs. placebo, low dose vs. placebo, and high dose vs. 
placebo. With an effect size of 1.0526, the simulated power to claim significance for a 
particular arm after multiplicity adjustment is approximately 89%. The probability to claim 
significance for at least one arm or both arms are approximately 95% and 83%, respectively.   
11.2 
Statistical Methods 
11.2.1 
Statistical Analysis Sets 
Safety Analysis Set 
The safety analysis set will consist of all patients who received at least 1 dose of study drug.  
Patients will be analyzed according to the treatment they actually received.  The safety 
analysis set will be the primary analysis set for safety analyses.  
Full Analysis Set 


 
 
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The full analysis set (FAS) will consist of all randomized patients who received at least 
1 dose of assigned treatment.  Patients will be analyzed in the treatment group they were 
randomized to even if they received incorrect study drug.  The FAS will be the primary 
analysis set for efficacy analyses. 
Per Protocol Analysis Set 
The per protocol (PP) analysis set is a subset of the FAS, and will consist of all randomized 
patients for whom no major protocol violation (no violation which may affect the study 
efficacy outcome) is documented.  The PP analysis set will provide supportive data for the 
efficacy analyses. 
Allocation of patients to the PP analysis set will be performed before unblinding of the study. 
11.2.2 
Methods of Statistical Analyses 
11.2.2.1 
General Principles 
The primary objective of this study is to demonstrate the efficacy of repeated daily doses of 
40 µg/kg/day and 120 µg/kg/day A4250 in children with PFIC Types 1 and 2.  
In the EU and RoW, for the primary efficacy variable of fasting bile acid responder endpoint 
(at least a 70% reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the 
end of treatment), the Cochran Mantel Haenszel (CMH) test stratified by PFIC class and age 
class will be performed to compare the proportion in fasting bile acid responder endpoint at 
the end of treatment (Week 22 and 24) in the two A4250 dose groups to placebo group. To 
ascertain that all data are used in the CMH analysis, neighboring strata will be pooled if all 
subjects in a stratum have the same response. Dropouts will be treated as non-responders. The 
proportion together with corresponding 95% CI, odds ratio and corresponding 95% Clopper-
Pearson exact CI and p-value for the CMH test will be presented.   
In the US, for the primary efficacy variable of the proportion of positive pruritus assessments 
at subject level over the 24-week treatment period, an analysis of covariance (ANCOVA) will 
be used. The model will include treatment arm, AM baseline pruritus score, PM baseline 
pruritus score, and randomization stratification factors, i.e. PFIC class and age class. LS 
mean(SE) by treatment arm and LS mean difference (SE), 95% confidence intervals, and p-
values (where applicable) between treatments (120 µg/kg/day and 40 µg/kg/day, respectively) 
vs. placebo will be provided. If there are concerns on model assumptions, i.e. normality and 
homogeneity of variances, a non-parametric ANCOVA based on the rank scores will be used 
as a sensitivity analysis. LS mean/SE on the outcome by treatment arm and LS mean 
difference/SE between active dose and placebo will be provided.  
The procedure for multiplicity adjustment is specified as follows:    
In the closed testing procedure, the low and high dose groups will be pooled to compare with 
the placebo group first. If the 1-sided p-value is ≤0.025, the 1-sided p-values for low dose vs. 


 
 
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placebo and high dose vs. placebo will be calculated, respectively. If both individual p-values 
are ≤0.025, a significant treatment effect will be declared on both dose groups. If only one of 
them is ≤0.025, a significant treatment effect will be declared on the corresponding dose 
group.  
Other secondary and exploratory outcome variables will provide supportive efficacy and 
safety information regarding the differences between the treatment groups. No adjustments 
will be performed for multiple comparisons when testing secondary and exploratory efficacy 
variables. 
If not otherwise specified, the baseline value of a parameter is defined as the last assessment 
of that parameter prior to the first intake of study treatment. 
11.2.2.2 
Missing Data 
In the primary analysis of the pruritus endpoint, all intermittently missing assessments will be 
classified as negative pruritus assessments. Similarly, all planned assessments after the 
intercurrent events (premature treatment discontinuation, death or initiation of rescue 
treatments such as biliary diversion surgery or liver transplantation) will be counted as 
negative pruritus assessments. However, as described above, all 336 assessments planned 
over the 24-week treatment period will be included in the denominator of the endpoint 
definition.  
In the primary analysis of fasting bile acid responder endpoint, dropouts and patients with a 
missing average at the end of the treatment will be treated as non-responders. Patients in need 
of surgical rescue, i.e. biliary diversion and listing for liver transplant will be classified as 
non-responders. Continued collection of efficacy data for patients who discontinue treatment 
will be made as far as possible. 
11.2.2.3 
Demographic and Baseline Characteristics 
Descriptive summaries of demographics and other baseline characteristics (including medical 
and surgical history) will be presented by treatment group and overall using the FAS.  
Prior and concomitant medication use during the study will be summarized by treatment 
group and overall using the safety analysis set. 
11.2.2.4 
Subject Disposition 
The following will be summarized (by treatment group and overall where applicable): 
• Patients enrolled (who signed the informed consent) 
• Patients randomized 
• Patients treated 
• Patients completing the study 
• Patients withdrawing early (including withdrawal reason) 


 
 
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Additionally, patients enrolled, included in the safety, in the FAS, and in the PP analysis set 
will be summarized by region. 
 
11.2.2.5 
Evaluation of Primary Efficacy Variables 
For EU and RoW, the primary responder endpoint based on change in fasting bile acids will 
be analyzed in terms of the proportion of patients achieving the event of interest. Patients in 
need of surgical rescue, i.e. biliary diversion and listing for liver transplant, will be classified 
as non-responders. Dropouts will be treated as non-responders.  
CMH stratified by PFIC class and age class will be performed to compare the proportion in 
fasting bile acid responders at the end of treatment (Week 22 and 24) in the two A4250 dose 
groups to the placebo group. To ascertain that all data are used in the CMH analysis, 
neighboring strata will be pooled if all subjects in a stratum have the same response. The 
proportion together with corresponding 95% CI, odds ratio and corresponding 95% Clopper-
Pearson exact CI and p-value for the CMH test will be presented 
Logistic regression will be performed as a supportive analysis to compare the proportion in 
fasting bile acid responders at the end of treatment (Week 22 and 24) in the two A4250 dose 
groups to the placebo group. The model will include treatment arm, baseline value, and 
randomization stratification factors, i.e. PFIC class and age class. Baseline value will be 
calculated as the average of serum bile acid values at randomization and the previous visit 
(Clinic Visit 2).   
In the US, for the primary efficacy variable of the proportion of positive pruritus assessments 
at the subject level over the 24-week treatment period, ANCOVA will be used to analyze the 
pruritus endpoint. The model will include treatment arm, AM baseline pruritus score, PM 
baseline pruritus score, and randomization stratification factors, i.e. PFIC class and age class. 
LS mean(SE) by treatment arm and LS mean difference (SE), 95% confidence intervals, and 
p-values (where applicable) between treatments (120 µg/kg/day and 40 µg/kg/day, 
respectively) vs. placebo will be provided. If there are concerns on model assumptions, i.e. 
normality and homogeneity of variances, a non-parametric ANCOVA based on the rank 
scores can be used. LS mean/SE on the outcome by treatment arm and LS mean 
difference/SE between active dose and placebo will be provided.  
Sensitivity analysis will be conducted to assess the impact of intermittently missing data as 
well as intercurrent events and will be specified in the SAP. Subgroup analyses will also be 
performed for each of the 3 age groups (6 months to 5 years, 6 to 12 years, and 13 to ≤18 
years) and for patients who were not included in the previous Study A4250-003. 
11.2.2.6 
Evaluation of Secondary and Exploratory Efficacy Variables 
The change in the secondary endpoints such as serum bile acids, total bilirubin, ALT, growth, 
Albireo PRO and ObsRO sleep parameters, and patient-reported itch severity will be 


 
 
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summarized by visit using descriptive statistics. Change in growth (weight for age and BMI 
for age) will also be displayed using graphical presentations.  
Proportion of responders at Weeks 12 and 24 based on the PRO/ObsRO instruments 
estimated from both distribution and anchor-based approaches will be analyzed using the 
same model as specified for the primary analysis for EU and RoW. 
Proportion of individual assessments meeting the definition of a positive pruritus assessment 
at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, 
respectively, or the proportion of positive pruritus assessments at each 4-week interval over 
the 24-week treatment period for AM or PM, respectively will be analyzed using the same 
model as specified for the primary analysis for US.   
Undergoing biliary diversion surgery and/or liver transplant, all-cause mortality, and liver 
decompensation events will mainly be analyzed by using descriptive statistics. Kaplan-Meier 
curves will be used when appropriate for time to event data.  
Comparisons of the change from baseline at Week 24 in PedsQL between the treatment 
groups will be conducted using ANCOVA. The model will include terms for baseline, PFIC 
class, age class, and treatment. 
Other exploratory variables (Global Symptom Relief, biochemical markers and measures of 
bile acid synthesis, additional Albireo PRO and ObsRO sleep parameters, PELD/MELD 
score, APRI score, and Fib-4 score) will be analyzed descriptively for categorical and 
continuous data, as applicable. For continuous data, the change from baseline will be 
analyzed in addition to the actual visit values. For categorical data, shift tables or frequency 
and percentages of patients will be presented as appropriate. Additional details on secondary 
and exploratory efficacy variables and subgroup analyses by age class (6 months to 5 years, 6 
to 12 years, and 13 to ≤18 years) will be specified in the SAP.  
11.2.2.7 
Evaluation of Safety Variables 
Safety data will be analyzed using descriptive statistics and summaries by treatment group of 
SAEs, AEs, vital signs, clinical laboratory tests (hematology, clinical chemistry and 
urinalysis), and concomitant medication.  Analyses will be performed using the safety 
analysis set.  
Summaries of AEs (coded according to the Medical Dictionary for Regulatory Activities 
[MedDRA] system organ class [SOC] and MedDRA preferred term) will include the 
following:  
• Overview of the incidence of TEAEs (TEAEs, drug-related TEAEs, TEAEs leading to 
study discontinuation, and treatment-emergent SAEs) 
• TEAEs by SOC and preferred term 
• Intensity of TEAEs by SOC and preferred term 


 
 
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• Drug-related TEAEs by SOC and preferred term 
• TEAEs leading to study discontinuation by SOC and preferred term 
• Treatment-emergent SAEs by SOC and preferred term 
Concomitant medication use during the Treatment Period will be summarized by Anatomical 
Therapeutic Chemical (ATC) class and World Health Organization (WHO) preferred name.  
Summaries of vital signs will be presented.  For each visit, the actual results and the change 
from baseline, and the number and percentage of patients with potentially clinically 
significant (PCS) values observed post-baseline will be presented. 
Summaries of clinical safety laboratory data will be presented.  For each visit, the actual 
result and the change from baseline, and the number and percentage of patients with PCS 
values observed post-baseline will be presented. 
Data listings will be provided for each patient for all safety parameters. 
11.2.2.8 
Compliance and Exposure 
Exposure will be analyzed by calculating the number of days with exposure to study drug.  
Results will be presented by treatment group using the safety analysis set.  
The percentage compliance will be described by treatment group and the number of patients 
with a compliance <80%, between 80% and 120%, and >120% will be presented based on the 
safety analysis set. 
11.2.3 
Blinded Analyses to Confirm Responder Definitions 
A blinded analysis of Albireo ObsRO and PRO eDiary data will be performed after 50% or 
more of the planned sample has completed the Week 24 Follow-Up Period.  The blinded 
analysis will be used to estimate a threshold of clinically meaningful change (i.e., responder 
definition) in Albireo ObsRO and PRO pruritus scores.  The analysis will be performed by a 
group independent from both the study team and Albireo.  Blinded data will be used for this 
analysis; data will be collapsed across treatment groups. 
The analyses will include distribution- and anchor-based analyses to examine a responder 
definition for the Albireo PRO/ObsRO pruritus scores.  Distribution-based analyses will 
include calculation of the SD and standard error of measurement for the baseline Albireo 
PRO and ObsRO scores.  Anchor-based analyses will involve examining the degree of 
change on the Albireo PRO and ObsRO scores from baseline to Week 24 for patients who 
experience change in pruritus according to PGIC and symptom items.  Details of this analysis 
will be included in a stand-alone SAP for assessing the measurement characteristics 
(reliability, validity, sensitivity to change, responder definition) of the Albireo PRO and 
ObsRO items. 


 
 
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11.2.4 
Data Safety Monitoring Board 
A DSMB consisting of sponsor-independent clinical and statistical experts will be 
established.  The DSMB will periodically (approximately on a quarterly basis) meet for the 
review of accumulating study data, including safety (AE and laboratory data) and liver 
values.  
The DSMB will have access to unblinded data.  The DSMB will make recommendations for 
the remaining part of the study (further details will be provided in the DSMB charter).  The 
DSMB may recommend continuing with the study as planned or stopping the study early for 
safety reasons.  The DSMB will submit its recommendations in writing to Albireo 
Medical/
 who are responsible for responding to the recommendations of the DSMB and 
taking appropriate action.  The investigators will only be informed by Albireo Medical/
 
if the study requires a protocol amendment or is stopped.  The DSMB may choose to make 
additional evaluations at any time if they feel this is warranted from a safety point of view.  
The DSMB will act according to its own written standard operating procedure described in a 
charter and will prepare written minutes of its meetings.  The charter of the DSMB will be 
stored in the trial master file.  The DSMB will maintain records of its meetings and these will 
become part of the study file when the study is complete.  In order not to disseminate 
unblinded data and to ensure that all staff involved in the conduct and final analysis of the 
study remain blind to the data, only the members of the DSMB and the unblinded DSMB 
statistician will have access to these data. 
 


 
 
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12 
DIRECT ACCESS TO SOURCE DATA/NOTES 
The investigator/institution shall provide direct access to source data/documents for 
study-related monitoring, audits, IEC/Institutional Review Board (IRB) review and 
regulatory inspection. 


 
 
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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
13.1 
Conduct of the Study 
Albireo/
 shall implement and maintain quality control and quality assurance procedures 
with written standard operating procedures to ensure that the study is conducted and data are 
generated, documented, and reported in compliance with the protocol, ICH GCP and 
applicable regulatory requirements. 
This study shall be conducted in accordance with the provisions of the Declaration of 
Helsinki (October 1996) and all revisions thereof, and in accordance with United States Food 
and Drug Administration (FDA) regulations (Code of Federal Regulations, Sections 312.50 
and 312.56) and with ICH GCP (CPMP 135/95). 
The investigator may not deviate from the protocol without a formal protocol amendment 
having been established and approved by an appropriate competent authority and IEC/IRB, 
except when necessary to eliminate immediate hazards to the patient or when the change(s) 
involve(s) only logistical or administrative aspects of the study.  Any deviations may result in 
the patient having to be withdrawn from the study and render that patient non-evaluable. 
13.2 
Study Monitoring 
Before study initiation, at a site initiation visit or at an investigator’s meeting, an Albireo 
representative or designee will review the protocol and eCRF with the investigators and the 
investigative staff.  During the study, the clinical monitor (clinical research associate [CRA]) 
will visit the site regularly to check the completeness of patient records, the accuracy of 
entries on the eCRFs, the adherence to the protocol and to GCP, the progress of enrollment, 
and to ensure that study drug is being stored, dispensed, and accounted for according to 
specifications. 
The investigator must ensure that eCRFs are completed within a timely period of patient 
visits, as per individual site agreements, and must allow the CRA and Albireo representative 
or designee periodic access to patient records and all study-related materials, including 
relevant hospital or clinical records, to confirm their consistency with the eCRF entries.  No 
information in these records about the identity of the patients will leave the study center.  
Albireo monitoring standards require full verification for the presence of the signed ICF, 
adherence to the inclusion/exclusion criteria, documentation of SAEs, and recording of 
primary efficacy and safety variables.  The CRA will review source data compared with the 
eCRFs and will verify source data according to the study-specific monitoring plan.  The 
design of the study, the frequency of patient visits, and the site enrollment rate will determine 
the frequency of monitoring visits.  Upon study completion, the CRA will visit the site to 
conduct a study termination visit, which will include collection of any outstanding 
documentation. 


 
 
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It is recommended that the investigator/investigative staff log into the eCRF system every 
working day or at minimum twice weekly in order to provide a prompt response to queries.  
The investigator/investigative staff should respond to queries and make any relevant changes 
to the study data within 3 working days. 


 
 
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14 
ETHICS 
14.1 
Independent Ethics Committee/Institutional Review Board 
Prior to the start of the study, the investigator is responsible for ensuring that the protocol and 
consent form have been reviewed and approved by a relevant IEC/IRB.  The IEC/IRB shall 
be appropriately constituted and perform its functions in accordance with FDA ICH GCP and 
local requirements as applicable. 
The IEC/IRB shall approve all protocol amendments (except for logistical or administrative 
changes), written informed consent documents and document updates, patient recruitment 
procedures (e.g., advertisements), written information to be provided to the patients and 
caregivers, IB, available safety information, information about payment and compensation 
available to patients, the investigator's curriculum vitae and/or other evidence of 
qualifications and any other documents requested by the IEC/IRB and regulatory authority 
(competent authority) as applicable. 
14.2 
Written Informed Consents and Assents 
The investigator (physician) or investigative staff, in accordance with local regulations, will 
explain to each patient (or legally authorized representative) the nature of the study, its 
purpose, the procedures involved, the expected duration, alternative treatment, the potential 
risks and benefits involved, and any discomfort that may occur.  Each caregiver/patient will 
be informed that participation in the study is voluntary and that he/she or their child may 
withdraw from the study at any time and that withdrawal of consent will not affect his/her 
subsequent medical treatment or relationship with the treating physician. 
Caregivers/patients will be informed that they/their children may experience side-effects or 
be at risk for symptoms, illnesses, or complications that cannot be foreseen by Albireo.  As 
with other medications, people treated with A4250 may be at risk of developing allergic 
reactions or anaphylaxis.  Caregivers/patients will be advised that study procedures include 
regular blood sampling for measurement of safety parameters and biological markers and that 
some minor risks are associated with these procedures. 
This informed consent should be given by means of a signed ICF, written in non-technical 
language in accordance with ICH GCP, the Declaration of Helsinki, and regulatory 
authorities.  The caregiver(s)/patient should read and consider the statements before signing 
and dating them and should be given a copy of each signed document.  If written consent is 
not possible, oral consent can be obtained if witnessed and followed by a signed statement 
from one or more persons not involved in the study, indicating why the patient was unable to 
sign the form.  No patient can enter the study before his/her or caregiver(s) informed consent 
has been obtained, as required by country regulations. 
The ICF is part of the protocol and must be submitted by the investigator/investigative staff 
with the protocol for IEC approval.  Albireo will supply an ICF which complies with 


 
 
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regulatory requirements and country laws and is considered appropriate for the study.  Any 
changes to the ICF suggested by the investigator must be agreed to by Albireo before 
submission to the IEC and a copy of the approved version must be provided to the clinical 
monitor after IEC approval. 


 
 
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15 
DATA HANDLING AND RECORD KEEPING 
15.1 
Case Report Forms/Source Data Handling 
The investigator shall be provided with standardized eCRFs and shall ensure that all data 
from patient visits are promptly entered into the eCRFs in accordance with the specific 
instructions given.  The investigator must sign each eCRF to verify the integrity of the data 
recorded. 
A list of the normal ranges for all laboratory tests to be undertaken forms part of the 
documentation to be collated prior to study start.  As a central laboratory has been selected to 
conduct any or all tests, it is essential that all samples be analyzed at that laboratory. 
The investigator must maintain source documents such as laboratory reports, consultation 
reports, and complete medical history and physical examination reports. 
As no ethnicity data can be collected in the source document for study sites in France, the 
eCRF will be the Source Document for “origines ethniques”,  
15.2 
Retention of Essential Documents 
Essential documents, as defined by ICH GCP, include: the signed protocol and any 
amendment(s); copies of the completed eCRFs (for site archiving, compact discs of eCRF 
data for specific patients will be provided); signed ICFs; hospital records and other source 
documents; IEC approvals and all related correspondence including approved documents; 
drug accountability records; study correspondence; and a list of patients’ names and 
addresses. 
The investigator/investigative staff must retain copies of these essential documents for the 
period specified by ICH GCP and by applicable regulatory requirements.  The 
investigator/investigative staff will inform Albireo of the location where the essential 
documents are stored and must contact Albireo for approval before disposing of any essential 
documents.  The investigator/investigative staff should take measures to prevent accidental or 
premature destruction of these documents. 


 
 
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16 
FINANCING AND INSURANCE 
Financing and insurance are addressed in a separate agreement. 
 


 
 
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17 
PUBLICATION POLICY 
Albireo will retain the ownership of all data.  When the study is complete, Albireo shall 
arrange the analysis, tabulation of data and preparation of a clinical study report. Albireo may 
also use the data for publication, presentation at scientific meetings and submission to 
regulatory authorities.  All proposed publications based on this study must be subject to the 
sponsor's approval requirements. 
 


 
 
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18 
REFERENCE LIST 
1. A4250 Investigator’s Brochure. 
2. Alissa FT, Jaffe R, Shneider BL. Update on progressive familial intrahepatic cholestasis. 
J Pediatr Gastroenterol Nutr. 2008;46(3):241-52. 
3. Davit-Spraul A, Gonzales E, Baussan C, et al. Progressive familial intrahepatic 
cholestasis. Orphanet J Rare Disease 2009;4:1-12. 
4. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and 
functions. Front Biosci (Landmark Ed). 2009 Jan 1;14:2584-98. 
5. Hori T, Justin H, Nguyen JH and Uemoto S. Progressive familial intrahepatic cholestasis. 
Hepatobiliary pancreat Dis Int. 2010; 9(6):570-8. 
6. Jacquemin E. Progressive familial intrahepatic cholestasis. Genetic basis and treatment. 
Clin Liver Dis. 2000;4(4):753-63. 
7. Miethke AG, Zhang W, Simmons J, et al. Pharmacological inhibition of ASBT changes 
bile composition and blocks progression of sclerosing cholangitis in mdr2 knockout mice. 
Hepatology. 2016; 63(2):512–23. 
8. Murray CS, Rees JL. Are subjective accounts of itch to be relied on? The lack of relation 
between visual analogue itch scores and actigraphic measures of scratch. Acta Derm 
Venereol. 2011;91(1):18-23. 
9. Pawlikowska L, Strautnieks S, Jankowska I, et al. Differences in presentation and 
progression between severe FIC1 and BSEP deficiencies. J Hepatol. 2010;53(1):170-8. 
10. Shneider BL, Magee JC, Bezerra JA, et al. Efficacy of fat-soluble vitamin 
supplementation in infants with biliary atresia. Pediatrics, 2012;130(3):e607-14.  
11. Suchy FJ, Sokol RJ, Balistreri WF, eds. Liver Disease in Children. 3rd Edition. New 
York, NY: Cambridge University Press; 2007:310–4. 
12. Venkat VL, Shneider BL, Magee JC, et al. Total Serum Bilirubin Predicts Fat-Soluble 
Vitamin Deficiency Better Than Serum Bile Acids in Infants with Biliary Atresia. J 
Pediatr Gastroenterol Nutr. 2014;59(6):702-7.  
13. Whitington PF, Whitington GL. Partial external diversion of bile for the treatment of 
intractable 
pruritus 
associated 
with 
intrahepatic 
cholestasis. 
Gastroenterology. 
1988;95(1):130-6. 
 
 


 
 
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19 
APPENDICES 
Appendix 1 
Concomitant Medication Guidelines 
 


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Appendix 2 
Diary Questions including Albireo Patient-Reported 
Outcomes/Observer-Reported Outcome Instrument 
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Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) 
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Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaire 
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Appendix 5 
Guideline for Fat-Soluble Vitamin Supplementation 
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Appendix 6 
Contraceptive Requirements 
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Appendix 7 
Blood Volumes 
Page 112


 
 
 
CLINICAL STUDY PROTOCOL A4250-005 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
 
IND Number:   
130591 
EudraCT Number 
2017-002338-21 
Test Product: 
A4250 
Indication: 
Progressive Familial Intrahepatic Cholestasis (PFIC) 
Sponsor: 
Albireo AB 
Development Phase: 
Phase 3 
Sponsor Signatory: 
 
Sponsor Medical Monitor: 
 
Principal Investigator: 
 
Date of the Protocol: 
01 March 2019 
Version of the Protocol: 
Final Amendment 04 
 
 
 
 
Confidential 
The information in this document contains trade secrets and commercial information that are 
privileged or confidential and may not be used, divulged, published, or otherwise disclosed 
without the written consent of Albireo AB.  These restrictions on disclosure apply equally to 
all future information supplied to you or generated by you in connection with the study. 




 
 
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INVESTIGATOR SIGNATURE PAGE 
 
PROTOCOL TITLE:  A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children with Progressive 
Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
 
PROTOCOL NUMBER:  A4250-005 
I have read this protocol and agree that it contains all necessary details for performing this 
study.  I will conduct the study as outlined herein and will complete the study within the time 
designated, in accordance with all stipulations of the protocol and in accordance with Good 
Clinical Practice (GCP), local regulatory requirements, and the Declaration of Helsinki. 
I will provide copies of the protocol and all pertinent information to all individuals 
responsible to me who assist in the conduct of this study.  I will discuss this material with 
them to ensure that they are fully informed regarding the study drug and the conduct of the 
study. 
I will use only the informed consent approved by the Independent Ethics Committee (IEC) 
and will fulfil all responsibilities for submitting pertinent information to the IEC responsible 
for this study. 
I agree that the sponsor (Albireo AB) shall have access to any source documents from which 
case report form information may have been generated. 
I further agree not to originate or use the name of Albireo AB, A4250 in any publicity, news 
release, or other public announcement, written or oral, whether to the public, press, or 
otherwise, relating to this protocol, to any amendment to the protocol, or to the performance 
of this protocol without the prior written consent of Albireo AB. 
 
 
 
 
 
 
Name of Investigator 
 
 
 
 
 
 
Signature 
 
Date (day/month/year) 


 
 
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1 
ADMINISTRATIVE INFORMATION 
A Double-blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
Protocol No.: 
A4250-005 
Date of the initial Protocol: 
20 September 2017 
Date and Number of Amendment(s): 
Amendment 01, 06 December 2017 
 
Amendment 02, 10 May 2018 
 
Amendment 03, 22 November 2018 
 
Amendment 04, 01 March 2019 
Sponsor: 
Albireo AB 
 
Arvid Wallgrens backe 20 
 
413 46 Gӧteborg 
Sweden 
Clinical Research Organization: 
 
 
 
 
 
 
 
 
Sponsor Signatory: 
 
 
  
 
  
 
  
 
 
 
  
Sponsor Medical Monitor: 
 
 
 
 
Principal Investigator: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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2 
STUDY SYNOPSIS 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Title of Study: 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate Efficacy and Safety of A4250 
in Children with Progressive Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
Principal Investigator: 
 
Study Centers: 
Up to 50 sites will be initiated for this study in the United States (US), Canada, European Union (EU), Middle 
East, and Australia. 
Publication(s): 
None. 
Planned Study Period: 
Q2 2018 to Q4 2019 
Development Phase: 
Phase 3 
Objectives: 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day A4250 in children with 
progressive familial intrahepatic cholestasis (PFIC) Types 1 and 2, as determined by the following: 
•
Proportion of patients experiencing at least a 70% reduction in fasting serum bile acid (s-BA)
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L compared to placebo 
after 24 weeks of treatment 
•
The proportion of positive pruritus assessments at the subject level over the 24-week Treatment Period
Secondary Objectives 
•
To evaluate the effect of A4250 on serum alanine aminotransferase (ALT) concentration
•
To evaluate the effect of A4250 on growth
•
To evaluate the effect of A4250 on sleep disturbance
•
To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or liver
transplantation)
•
To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks
Methodology: 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the efficacy and safety of 
40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in children with PFIC (deficiencies of familial 
intrahepatic cholestasis-1 or bile salt export pump).  
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment Period and a 4-week 
Follow-up Period.  After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of A4250, or a 
matching placebo.  Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008) in which all patients will receive 
active treatment. 
There will be up to 10 clinic visits during the study and one scheduled telephone contact. 


 
 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Number of Patients: 
Approximately 60 patients diagnosed with PFIC-1 or PFIC-2 will be randomized.  The study will target 15% of 
enrolled patients to be PFIC-1 patients.  If enrollment of all PFIC-2 patients is completed and the 15% 
enrollment of PFIC-1 patients has not yet been achieved, then enrollment of PFIC-2 patients will continue in 
order to reach the total study target enrollment. 
Diagnosis and Main Criteria for Inclusion: 
1. 
A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages of ≥6 months 
and ≤18 years at Visit 1 with a body weight above 5 kg 
2. 
Patient must have clinical genetic confirmation of PFIC-1 or PFIC-2 through identification of biallelic 
pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. 
Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, taken as the 
average of 2 samples at least 7 days apart (Visits 1 and 2) prior to randomization  
4. 
Patient must have history of significant pruritus and a caregiver-reported observed scratching in the 
electronic diary (eDiary) average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to randomization 
5. 
Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  Patients who 
turn 18 years of age (or legal age per country) during the study will be required to re-consent in order 
to remain in the study 
6. 
Patients are expected to have a consistent caregiver for the duration of the study 
7. 
Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use an eDiary 
device as required by the study 
Test Product, Dose and Mode of Administration: 
A4250, 40 µg/kg/day or 120 µg/kg/day, orally administered 
Reference Therapy, Dose and Duration of Administration: 
Matching placebo, orally administered 
Duration of Treatment: 
24 weeks 
Variables 
Efficacy:  
Primary Efficacy Endpoints 
The primary efficacy endpoints are: 
• 
EU and rest of world (RoW): Proportion of patients experiencing at least a 70% reduction in fasting 
s-BA concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L compared to 
placebo after 24 weeks of treatment. 
• 
US: Proportion of positive pruritus assessments at the subject level over the 24-week Treatment Period. 
Secondary Efficacy Endpoints 
Secondary efficacy endpoints include the following: 
• 
EU and RoW: Proportion of positive pruritus assessments at the subject level over the 24-week 
Treatment Period.  
• 
US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA concentration from 
baseline to the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 weeks of 
treatment. 
 
All Regions: 
All secondary endpoints will be compared to placebo. 
• 
Change from baseline to Week 12 and to Week 24 in fasting s-BA 
• 
Change from baseline to Week 12 and to Week 24 in serum ALT concentration 


 
 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
• 
Change in growth from baseline to Week 12 and Week 24 
• 
Proportion of responders for pruritus scores at Weeks 12 and 24 based on the Albireo PRO and ObsRO 
instruments  
• 
Change in sleep parameters measured with the Albireo PRO and ObsRO instruments from baseline 
over the 24-week Treatment Period  
• 
Proportion of individual assessments meeting the definition of a positive pruritus assessment at the 
subject level over the 24-week Treatment Period. A positive pruritus assessment includes an itch score 
≤1, or at least a one-point drop from baseline based on the Albireo PRO instrument; only patients ≥8 
years of age will complete the Albireo PRO instrument 
• 
Proportion of individual assessments meeting the definition of a positive pruritus assessment at the 
subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, respectively, or the 
proportion of positive pruritus assessments at each 4-week interval.  
• 
Proportion of individual AM assessments meeting the definition of a positive pruritus assessment at the 
subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, respectively, or the 
proportion of positive pruritus assessments at each 4-week interval.  
• 
Proportion of individual PM assessments meeting the definition of a positive pruritus assessment at the 
subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, respectively, or the 
proportion of positive pruritus assessments at each 4-week interval.  
• 
Number of patients undergoing biliary diversion surgery or liver transplantation 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• 
Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, caregiver, and 
clinician via the patient global impression of change (PGIC) items 
• 
Change from baseline in fasting s-BA at Week 4  
• 
Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline by each 4 
week interval of the Treatment Period as measured by the Albireo ObsRO instrument 
• 
Change from baseline by each 4-week interval of the Treatment Period in patient-reported and 
observer-reported night time itching and scratching severity scores, respectively 
• 
Change from baseline by each 4-week interval of the Treatment Period in patient-reported and 
observer-reported morning time itching and scratching severity scores, respectively 
• 
Change from baseline by each 4-week interval of the Treatment Period in pooled pruritus score 
including observer-reported scratching for patients <8 years of age and patient-reported itch severity 
for patients ≥8 years of age 
• 
Change from baseline by each 4-week interval of the Treatment Period in additional patient-reported 
and observer-reported sleep parameters (e.g., tiredness and number of awakenings) 
• 
Change from baseline to Week 24 in Pediatric Quality of Life Inventory (PedsQL) 
• 
Change from baseline in serum ALT concentration at Week 4  
• 
Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• 
Change from baseline in AST and gamma-glutamyl transferase at Weeks 4, 12, and 24 
• 
Change from baseline for other biochemical markers and measures of bile acid synthesis (autotaxin, 
plasma 7α-hydroxy-4-cholesten-3-one concentration [p-C4]) at Week 24 
• 
Change in pediatric end-stage liver disease/model for end-stage liver disease (PELD/MELD) score, 
AST to platelet ratio index (APRI) score, and Fib-4 score from baseline to Week 24 
• 
Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® (where available) 
• 
Liver-related mortality and liver decompensation events. 
• 
All-cause mortality 


 
 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Safety:   
Safety criteria are as follows: 
• 
Occurrence of treatment-emergent adverse events including severity and relatedness to study drug at all 
visits 
• 
The incidence of treatment-emergent serious adverse events (SAEs), based upon information from 
patient reports, including the description, incidence, and severity of an SAE 
• 
Trends evaluated for the following assessments: physical examinations, concomitant medications, vital 
signs, laboratory test results (including clinical chemistry, hematology, urinalysis, alfa-fetoprotein, 
vitamins A and E, 25-hydroxy vitamin D and INR), and abdominal ultrasound 
Statistical Methods: 
The primary objective of this study is to demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 
120 µg/kg/day A4250 in children with PFIC Types 1 and 2. 
  
The Cochran Mantel Haenzel (CMH) test stratified by PFIC class and age class will be performed to compare 
the proportion in fasting bile acid responders at the end of treatment (Week 22 and 24) in the two A4250 dose 
groups to the placebo group. To ascertain that all data are used in the CMH analysis, neighboring strata will be 
pooled if all subjects in a stratum have the same response. The proportion together with the corresponding 95% 
CI, odds ratio and corresponding 95% Clopper-Pearson exact CI and p-value for the CMH test will be presented.  
 
For the primary efficacy variable of the proportion of positive pruritus assessments at subject level over the 24-
week treatment period, an ANCOVA model will be used to analyse the comparisons between the treatment 
groups. The model will include treatment arm, AM baseline pruritus score, PM baseline pruritus score, and 
randomization stratification factors, i.e. PFIC class and age class. LS mean (SE) by treatment arm and LS mean 
difference (SE), 95% confidence intervals, and p-values (where applicable) between treatments (120 µg/kg/day 
and 40 µg/kg/day, respectively) vs. placebo will be provided. If there are concerns on model assumptions, i.e. 
normality and homogeneity of variances, a non-parametric ANCOVA based on the rank scores can be used as a 
sensitivity analysis. LS mean/SE on the outcome by treatment arm and LS mean difference/SE between active 
dose and placebo will be provided.  
 
For each primary endpoint by region (EU & RoW and US), a pooled analysis for the closed testing procedure 
will be applied to control the 1-sided overall type I error rate for two treatment comparisons vs. the placebo at 
the 0.025 level, as specified below: 
 
In the closed testing procedure, the low and high dose groups will be pooled to compare with the placebo group 
first. If the 1-sided p-value is ≤0.025, the 1-sided p-values for low dose vs. placebo and high dose vs. placebo 
will be calculated respectively. If both individual p-values are ≤0.025, a significant treatment effect will be 
declared on both dose groups. If only one of them is ≤0.025, a significant treatment effect will be declared on 
the corresponding dose group.  
 
Other secondary and exploratory outcome variables will provide supportive efficacy and safety information 
regarding the differences between the treatment groups. No adjustments will be performed for multiple 
comparisons when testing secondary and exploratory efficacy variables. 
 
Date of the Protocol:  01 March 2019 


 
 
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3 
TABLE OF CONTENTS 
1 
ADMINISTRATIVE INFORMATION ........................................................................ 4 
2 
STUDY SYNOPSIS ...................................................................................................... 5 
3 
TABLE OF CONTENTS .............................................................................................. 9 
3.1 
List of Tables .................................................................................................. 13 
3.2 
List of Figures ................................................................................................ 13 
3.3 
List of Appendices .......................................................................................... 13 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ................................ 14 
5 
INTRODUCTION ....................................................................................................... 16 
5.1 
Investigational Medicinal Product .................................................................. 16 
5.2 
Background .................................................................................................... 16 
5.2.1 
Progressive Familial Intrahepatic Cholestasis ........................................... 16 
5.2.2 
Summary of Clinical and Nonclinical Studies ........................................... 18 
5.3 
Rationale ......................................................................................................... 19 
5.3.1 
Risk/Benefit ............................................................................................... 20 
6 
STUDY OBJECTIVES ............................................................................................... 21 
6.1 
Primary Objectives ......................................................................................... 21 
6.2 
Secondary Objectives ..................................................................................... 21 
7 
INVESTIGATIONAL PLAN ...................................................................................... 22 
7.1 
Overall Study Design and Plan....................................................................... 22 
7.1.1 
Description ................................................................................................. 22 
7.1.2 
Schedule of Assessments ........................................................................... 24 
7.1.3 
Study Procedures and Assessments ........................................................... 29 
7.1.3.1 
Screening Period (Days -56 to 0) ............................................... 29 
7.1.3.2 
Treatment Period (Day 0 to Day 168) ........................................ 30 
7.1.3.3 
Follow-Up Period ....................................................................... 33 
7.2 
Study Population ............................................................................................ 33 
7.2.1 
Inclusion Criteria ....................................................................................... 34 
7.2.2 
Exclusion Criteria ...................................................................................... 34 
7.2.3 
Withdrawal of Patients............................................................................... 36 
7.2.4 
Study Termination by Sponsor .................................................................. 37 
8 
TREATMENT OF PATIENTS ................................................................................... 38 
8.1 
Identity of Study Drug .................................................................................... 38 
8.2 
Administration of Study Drug ........................................................................ 38 
8.3 
Study Treatment Packaging and Labeling ...................................................... 39 


 
 
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8.3.1 
Packaging and Labeling ............................................................................. 39 
8.3.2 
Storage ....................................................................................................... 39 
8.3.3 
Blinding and Randomization of Study Drug ............................................. 39 
8.4 
Procedure for Breaking the Randomization Code .......................................... 40 
8.5 
Patient Compliance ......................................................................................... 41 
8.6 
Study Drug Accountability ............................................................................. 41 
8.7 
Concomitant Therapy and Prohibited Medications ........................................ 41 
9 
ASSESSMENT OF EFFICACY ................................................................................. 42 
9.1 
Efficacy Endpoints ......................................................................................... 42 
9.1.1 
Primary Efficacy Endpoint ........................................................................ 42 
9.1.2 
Secondary Efficacy Endpoint .................................................................... 42 
9.1.3 
Exploratory Efficacy Endpoints ................................................................. 43 
9.2 
Efficacy Assessments ..................................................................................... 44 
9.2.1 
Serum Bile Acids ....................................................................................... 44 
9.2.2 
Itching, Scratching and Sleep Score .......................................................... 45 
9.2.3 
Growth ....................................................................................................... 45 
9.2.4 
Quality of Life Questionnaire (PedsQL).................................................... 46 
9.2.5 
Biomarkers and PK Samples ..................................................................... 46 
9.2.6 
PELD/MELD Score ................................................................................... 46 
9.2.7 
Fibroscan® .................................................................................................. 46 
9.2.8 
Markers of Fibrosis .................................................................................... 46 
9.2.9 
Global Impression of Change and Global Impression of Symptom 
Measures .................................................................................................... 46 
10 
ASSESSMENT OF SAFETY ..................................................................................... 48 
10.1 
Adverse Events ............................................................................................... 48 
10.1.1 
Definitions and Investigator Assessments ................................................. 48 
10.1.1.1 Clinical Significance .................................................................. 48 
10.1.1.2 Serious Adverse Events .............................................................. 48 
10.1.1.3 Severity Assessment ................................................................... 49 
10.1.1.4 Causality Assessment ................................................................. 49 
10.1.2 
Recording of Adverse Events .................................................................... 50 
10.1.3 
Recording and Reporting of Serious Adverse Events ................................ 51 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions .............. 52 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments .................................................................................................... 53 
10.2.1 
Laboratory Assessments ............................................................................ 53 


 
 
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10.2.2 
Individual Patient Safety Monitoring ........................................................ 54 
10.2.2.1 Liver Monitoring ........................................................................ 54 
10.2.2.2 Close Observation ...................................................................... 55 
10.2.2.3 De-challenge/Re-challenge for Liver and Clinical Hepatitis 
Monitoring .................................................................................. 56 
10.2.2.4 Diarrhea ...................................................................................... 56 
10.2.2.5 Monitoring after Permanent Discontinuation of Study Drug 
Due to Safety .............................................................................. 56 
10.2.3 
Demographics/Medical and Surgical History ............................................ 56 
10.2.4 
Clinical Genetic Testing ............................................................................ 57 
10.2.5 
Physical Examination ................................................................................ 57 
10.2.6 
Vital Signs ................................................................................................. 57 
10.2.7 
Abdominal Ultrasound ............................................................................... 57 
10.2.8 
Overdose .................................................................................................... 57 
10.2.9 
Pregnancy .................................................................................................. 58 
11 
STATISTICAL EVALUATION ................................................................................. 59 
11.1 
Sample Size and Power .................................................................................. 59 
11.2 
Statistical Methods ......................................................................................... 59 
11.2.1 
Statistical Analysis Sets ............................................................................. 59 
11.2.2 
Methods of Statistical Analyses ................................................................. 60 
11.2.2.1 General Principles ...................................................................... 60 
11.2.2.2 Missing Data .............................................................................. 61 
11.2.2.3 Demographic and Baseline Characteristics ................................ 61 
11.2.2.4 Subject Disposition..................................................................... 61 
11.2.2.5 Evaluation of Primary Efficacy Variables .................................. 62 
11.2.2.6 Evaluation 
of 
Secondary 
and 
Exploratory 
Efficacy 
Variables ..................................................................................... 62 
11.2.2.7 Evaluation of Safety Variables ................................................... 63 
11.2.2.8 Compliance and Exposure .......................................................... 64 
11.2.3 
Blinded Analyses to Confirm Responder Definitions ............................... 64 
11.2.4 
Data Safety Monitoring Board ................................................................... 65 
12 
DIRECT ACCESS TO SOURCE DATA/NOTES ..................................................... 66 
13 
QUALITY CONTROL AND QUALITY ASSURANCE .......................................... 67 
13.1 
Conduct of the Study ...................................................................................... 67 
13.2 
Study Monitoring ........................................................................................... 67 


 
 
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14 
ETHICS ....................................................................................................................... 69 
14.1 
Independent Ethics Committee/Institutional Review Board .......................... 69 
14.2 
Written Informed Consents and Assents ........................................................ 69 
15 
DATA HANDLING AND RECORD KEEPING ....................................................... 71 
15.1 
Case Report Forms/Source Data Handling..................................................... 71 
15.2 
Retention of Essential Documents .................................................................. 71 
16 
FINANCING AND INSURANCE .............................................................................. 72 
17 
PUBLICATION POLICY ........................................................................................... 73 
18 
REFERENCE LIST ..................................................................................................... 74 
19 
APPENDICES ............................................................................................................. 75 
 




 
 
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4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
ADME 
absorption, distribution, metabolism, and excretion 
AE(s) 
adverse event(s) 
AFP 
alfa-fetoprotein 
ALT 
alanine aminotransferase 
APRI 
AST to platelet ratio index 
ASBT 
apical sodium-dependent bile transporter (also known as IBAT) 
AST 
aspartate aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BMI 
body mass index 
BSEP 
bile salt export pump 
CPK 
creatine phosphokinase 
CRA 
clinical research associate 
CYP 
cytochrome P450 
DSMB 
Data and Safety Monitoring Board 
eCRF 
electronic case report form 
eDiary 
electronic diary 
EOT 
End of Treatment 
EU 
European Union 
EudraCT 
European Union drug regulatory agency clinical trial 
FAS 
full analysis set 
FDA 
United States Food and Drug Administration 
FIC1 
familial intrahepatic cholestasis-1 
GCP 
Good Clinical Practice 
GGT 
gamma-glutamyl transferase 
GI 
gastrointestinal 
GLMM 
generalized linear mixed modelling 
IB 
Investigator’s Brochure 
IBAT 
ileal bile acid transporter (also known as ASBT) 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
 
 
IEC 
Independent Ethics Committee 
INR 
international normalized ratio 
IRB 
Institutional Review Board 


 
 
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IWRS 
Interactive Web Response System 
LDH 
lactate dehydrogenase 
LFT 
liver function test 
LPLV 
last patient last visit 
MDR3 
multidrug-resistance protein 3 
MedDRA 
Medical Dictionary for Regulatory Activities 
MELD 
model for end-stage liver disease 
MMRM 
mixed model repeated measures 
ObsRO 
observer-reported outcome 
PBC 
primary biliary cholangitis 
p-C4 
plasma 7α-hydroxy-4-cholesten-3-one concentration 
PCS 
potentially clinically significant 
PedsQL 
Pediatric Quality of Life Inventory 
PELD 
pediatric end-stage liver disease 
PFIC 
progressive familial intrahepatic cholestasis 
PGIC 
patient global impression of change 
PGIS 
patient global impression of symptoms 
PK 
pharmacokinetic(s) 
PP 
per protocol 
PRO 
patient-reported outcome 
PT 
prothrombin time 
PVM 
pharmacovigilance manager 
QoL 
quality of life 
RoW 
rest of world 
RR 
respiratory rate 
SAE(s) 
serious adverse event(s) 
SAP 
statistical analysis plan 
s-BA 
serum bile acid 
SD 
standard deviation 
s-FGF19 
serum fibroblast growth factor 19 concentration 
SOC 
system organ class 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE(s) 
treatment-emergent adverse event(s) 
ULN 
upper limit of normal 
US 
United States 
WHO 
World Health Organization 


 
 
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5 
INTRODUCTION 
5.1 
Investigational Medicinal Product 
A4250 is a small molecule and a potent selective inhibitor of the ileal bile acid transporter 
(IBAT), also known as the apical sodium-dependent bile acid transporter (ASBT).  IBAT is 
an integral brush border membrane glycoprotein that co-transports sodium and bile acids and 
appears to be a critical component in the regulation of the bile acid pool size in animals and 
man.  This transporter, expressed in the distal ileum, is a key element in the enterohepatic 
circulation of bile acids since it facilitates the high-affinity, high-capacity reabsorption of bile 
acids.  Indeed, 95% of secreted bile acids are reabsorbed via IBAT [Hofmann 2009; 
Miethke 2016].  A4250 is orally administered and acts locally in the gut where it binds 
reversibly to IBAT to decrease the reuptake of bile acids.  A4250 has minimal systemic 
exposure at the predicted therapeutic dose ranges. 
No safety signals were found with A4250 in preclinical safety and toxicity studies.  In the 
first clinical study of A4250 in healthy volunteers, A4250 was given as a single oral dose up 
to 10 mg or daily oral doses up to 3 mg/day for 7 days.  A4250 was well tolerated and the 
only findings were gastrointestinal (GI) related effects, such as diarrhea, at higher doses. 
In an exploratory Phase 2 study in children with cholestatic pruritus, A4250 was given as oral 
doses of 10 to 200 µg/kg/day for 4 weeks.  A4250 reduced mean levels of serum bile acids 
(s-BA) in all cohorts.  In 11 of 13 progressive familial intrahepatic cholestasis 
(PFIC) patients, substantial reductions (ranging from 43% to 98%) were observed.  
Patient-reported diary data documented improvement of pruritus during treatment.  A4250 
was well tolerated.  No serious adverse events (SAEs) were deemed to be related to study 
drug, and most adverse events (AEs), including some increased transaminases, were 
considered unrelated to study drug and transient in nature. 
5.2 
Background 
5.2.1 
Progressive Familial Intrahepatic Cholestasis 
PFIC is a rare autosomal recessive cholestatic liver disease estimated to affect between 1 in 
every 50,000 to 100,000 children born worldwide.  PFIC represents 10% to 15% of causes of 
cholestasis in children and 10% to 15% of liver transplantation indications in children.  All 
types of PFIC exist worldwide and both sexes appear to be equally affected. 
The common underlying pathogenesis of PFIC is disruption of bile formation and bile 
transport through the liver [Jacquemin 2000].  The classification of PFIC has evolved over 
the years.  The most commonly used sub-classifications are PFIC Types 1, 2, and 3, which 
are based on the associated affected gene and described in more detail below. 
• PFIC, Type 1:  also referred to as “Byler disease” or “familial intrahepatic 
cholestasis-1 (FIC1) protein deficiency.” FIC1 protein is located on the canalicular 


 
 
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membrane of hepatocytes and facilitates movement of aminophospholipids from the outer 
to inner leaflet of the plasma membrane of the hepatocyte.  The ATP8B1 gene encodes 
FIC1 protein.  Biallelic pathologic variants in the ATP8B1 gene are associated with FIC1 
dysfunction and classified clinically as PFIC Type 1 disease. 
• PFIC, Type 2:  also referred to as “Byler syndrome” or “bile salt export pump (BSEP) 
deficiency.”  BSEP is a transporter protein that is expressed at the canalicular membrane 
of hepatocytes and is the primary exporter of bile acids.  The ABCB11 gene encodes the 
BSEP protein.  Biallelic pathologic variations in the ABCB11 gene are associated with 
BSEP dysfunction and classified clinically as PFIC Type 2 disease. 
• PFIC, Type 3:  is caused by a deficiency of the multidrug-resistance protein 3 (MDR3) 
due to mutations in the ABCB4 gene.  MDR3 is a phospholipid translocase critical for 
phospholipid secretion. 
Severe pruritus is common in children diagnosed with PFIC.  Itching (and subsequent 
scratching) is a significant morbidity for these patients and their families [Suchy 2007].  A 
more severe degree of pruritus is experienced compared to patients with other forms of liver 
disease and to other pruritic conditions such as atopic dermatitis [Murray 2011].  In patients 
with PFIC, liver biopsy reveals canalicular cholestasis and, later, the appearance of portal 
fibrosis.  Serum biochemistry indicates cholestasis with hyperbilirubinemia, elevated alanine 
aminotransferase (ALT) and aspartate aminotransferase (AST).  The concentrations of bile 
acids in serum are very high, while serum gamma-glutamyl transferase (GGT) activity 
(exception being MDR3 variants) and cholesterol are normal [Hori 2010].  Symptoms of 
portal hypertension and liver failure will develop during the course of the disease [Davit-
Spraul 2009; Alissa 2008].  Symptoms develop early; median age at onset of symptoms is 
2 months, and 78% of PFIC patients present with jaundice [Pawlikowska 2010].  The 
life-threatening and debilitating nature of PFIC is reflected by the fact that survival in those 
not resorting to surgery is 50% at 10 years of age and almost zero at 20 years of age.  
Approximately half of PFIC patients undergo liver transplantation [Davit-Spraul 2009] and 
treatment-resistant pruritus is the leading indication for the surgical procedure partial external 
biliary diversion, mostly in PFIC Type 1 and 2 patients. 
PFIC is life-threatening and debilitating.  Currently, the treatment for PFIC is palliative and 
there is no pharmaceutical treatment approved for use in PFIC.  The therapeutic choices are 
restricted to non-specific therapy of the symptoms and signs of the disease such as nutritional 
support, preventing vitamin deficiencies, and treatment of extrahepatic features.  Medical 
treatment options include off-label use of ursodeoxycholic acid, rifampin, antihistamines, and 
naltrexone.  A minority of patients respond nominally and transiently to these interventions.  
Biliary diversion is used to decrease systemic bile acids through interruption of the 
enterohepatic circulation and so avoid transplantation.  Liver transplantation is typically only 
viewed as an option when patients have failed medical treatment and/or biliary diversion and 
have liver failure or persistent uncontrolled pruritus. 


 
 
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5.2.2 
Summary of Clinical and Nonclinical Studies 
This is a summary of nonclinical and clinical studies.  More detail is provided in the 
Investigator’s Brochure ([IB] see A4250 IB).  
A4250 is a potent, selective IBAT inhibitor with no safety signals seen in the safety 
pharmacological studies.  In addition, no safety signals were seen in repeat-dose oral toxicity 
studies in rats with up to 26 weeks of daily dosing and in dogs with up to 39 weeks of daily 
dosing.  No safety signals were identified when A4250 was administered daily to juvenile 
rats from age 14 days to 63 days. 
A4250 is minimally absorbed with very low systemic exposure in both humans and other 
species.  In blood, protein binding is high (>99.7%).  Systemically absorbed A4250 is slowly 
metabolized in humans and in preclinical studies by hydroxylation only, a metabolite that 
retains IBAT activity.  A4250 did not show any significant inhibition of cytochrome 
P450 (CYP) 
enzymes 
in 
the 
rat. 
 
A4250 
inhibited 
human 
CYP3A4 
and 
CYP2D6 (IC50: 16 µmol/L for both enzymes), and CYP2C9 (IC50:1.2 µmol/L).  
To date, A4250 has been studied in 3 Albireo-sponsored clinical studies: a study in healthy 
volunteers evaluating single and multiple administration of A4250; a single-dose absorption, 
distribution, metabolism, and excretion (ADME) study; and a Phase 2 study in children with 
cholestatic pruritus.  In addition, an investigator-sponsored study has been conducted in 
patients with primary biliary cholangitis (PBC). 
In the first clinical study (A4250-001), A4250 was given to healthy volunteers as a single oral 
dose up to 10 mg or daily oral doses up to 3 mg/day for 7 days.  Following a single oral dose 
of 10 mg, the median maximum drug concentration was 0.2 nmol/L and the area under the 
curve was 0.8 nmol × h/L (n=6).  Pharmacodynamic evaluation of bile acid synthesis 
biomarkers 
(serum 
fibroblast 
growth 
factor 
19 [s-FGF19] 
and 
plasma 
7α Hydroxy 4-cholesten-3-one [p-C4]) identified the expected decrease in s-FGF19 and an 
increase in p-C4.  In addition, total plasma bile acids decreased and total fecal bile acids 
increased in A4250-treated patients compared to placebo. A4250 was generally well 
tolerated.  The only findings were GI-related effects, such as diarrhea at higher doses. 
In the ADME study (A4250-007), healthy subjects were administered a single oral dose of 
3 mg [14C]-A4250 capsule.  The main excretion route was determined to be fecal.  There 
were no quantifiable concentrations of A4250 in plasma or total radioactivity in plasma or 
whole blood.  A4250 was well tolerated.  AEs were reported and most were mild and not 
related to A4250. The 3 AEs experienced by 2 subjects reported as related to study drug were 
GI in nature, including diarrhea (2 AEs; 2 subjects) and abdominal pain (1 AE; 1 subject). 
In an exploratory Phase 2 study in children with cholestatic pruritus (A4250-003), A4250 was 
administered as oral daily doses of 10 to 200 µg/kg/day for 4 weeks.  Twenty 
patients (8 females and 12 males) aged 1 to 17 years were enrolled in this open-label 
dose-escalation study.  Four patients were re-enrolled into a second dose level.  No 


 
 
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treatment-related SAEs were reported, and most AEs, including some increased 
transaminases, were transient.  Four-week, once-daily treatment with A4250 reduced mean 
levels of s-BA in all cohorts.  Substantial s-BA reductions in 11 of 13 PFIC patients (ranging 
from 43% to 98%) were observed.  A4250 also reduced s-BA in the 11 non-PFIC patients, 
but to a lesser extent than in PFIC patients.  Patient-reported diaries documented 
improvement of the visual analogue scale itch score in 17 of 24 patients during treatment.  
The dose with the greatest improvement showed a mean decrease (± standard error of the 
mean) of 2.9 ±1.1 from baseline.  The results from this study form the basis of the current 
(A4250-005) study design. 
5.3 
Rationale 
In this investigational study, A4250 is under development for treatment of PFIC and is also 
being considered for other orphan cholestatic liver diseases. 
There is currently no medical treatment approved for PFIC.  In PFIC patients with severe 
pruritus, biliary diversion surgery is used to decrease systemic bile acids through interruption 
of the enterohepatic circulation and thereby reducing pruritus [Whitington 1988]. A portion 
of patients who have biliary diversion surgery continue to have elevated serum bile acids and 
pruritus. Liver transplantation is typically used as an option when patients have failed 
medical treatment and/or biliary diversion and have liver failure or persistent uncontrolled 
pruritus. 
By inhibiting IBAT with high selectivity and potency, A4250 has the potential to relieve 
cholestasis and improve liver function without surgical intervention in patients with PFIC.  
Since A4250 is minimally absorbed and achieves a substantial reduction of s-BA, it has the 
potential to be a safer and less invasive alternative to surgical biliary drainage.  The rationale 
for using A4250 is to decrease s-BA levels, and to reduce the major morbidity of pruritus, 
improving the health and wellbeing of patients affected with PFIC.  Ultimately, this 
noninvasive therapeutic approach has the potential to not only improve quality of life (QoL), 
but also reduce the need for biliary diversion and subsequent liver failure and liver 
transplantation.  For those patients who have undergone biliary diversion surgery and 
continue to have elevated serum bile acids and symptomatic pruritus, IBAT inhibition has the 
potential to reduce both the serum bile acid levels and the pruritus. 
The doses selected, 40 and 120 µg/kg/day, were based on the efficacy and safety data 
generated from patients with PFIC (n=10 + 3 re-entered) and supporting data from all 
patients (n=20 + 4 re-entered) in the five dose groups dosed in the A4250-003 study.  
Improvements in s-BA and pruritus were observed with doses from 30 µg/kg to 100 µg/kg, 
with a dose-related trend.  The best dose response in all patients was at the 60 µg/kg/day for 
s-BA and 100 µg/kg/day for pruritus.  The best dose response in the PFIC subgroup was at 
30 µg/kg/day for both s-BA and pruritus.  Patients with PFIC responded well to the doses 
given from 30 µg/kg to 200 µg/kg/day; however, a clear dose relationship could not be 


 
 
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Module 5:  Clinical Study Reports 
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established in order to select a single dose for this Phase 3 study.  As efficacy was observed 
at both low and high doses, 40 µg/kg and 120 µg/kg/day are considered to be the most 
optimal for this study.  
This study will be conducted in compliance with the protocol and with the International 
Council for Harmonisation (ICH) guidelines on Good Clinical Practice (GCP). 
Patients completing 24 weeks of double-blind treatment will be offered to enter Study 
A4250-008 and receive open-label treatment with A4250. 
5.3.1 
Risk/Benefit 
A4250 has been evaluated in three Albireo-sponsored clinical studies: a double-blind 
placebo-controlled study in healthy volunteers, a single-dose ADME study, and a Phase 2 
study in children with cholestatic pruritus.  In addition, an investigator-sponsored study has 
been conducted in patients with PBC. 
Healthy subjects have been exposed to up to 10 mg as a single dose and up to 3 mg daily as 
part of a multiple-dose evaluation.  Children with cholestatic liver disease have been treated 
with up to 0.2 mg/kg/day (200 µg/kg/day) for 4 weeks.  In total, 2 SAEs have been reported; 
both were judged by the physician to be not related to the study drug. 
Patients with cholestatic liver diseases suffer from excess bile acids in the liver resulting in 
tissue damage.  A commonly used treatment in these patients is bile diversion surgery, 
whereby approximately 50% to 100% of the enterohepatic circulation of bile acids is 
interrupted.  Inhibition of IBAT with A4250, thereby interrupting the enterohepatic 
circulation of bile acids, is therefore a potential medical alternative to surgery which could be 
of benefit to these patients if shown to be effective and safe.  Data from the Phase 2 study 
showed efficacy of A4250 in reducing s-BA concentrations and pruritus in such patients. 
Based on the mode of action of an IBAT inhibitor, loose stools or diarrhea could be expected.  
However, in the pediatric Phase 2 study with 4 weeks daily treatment, only one patient had 
mild transient diarrhea after a single dose that did not recur on multiple dosing. 


 
 
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6 
STUDY OBJECTIVES 
6.1 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day 
A4250 in children with PFIC Types 1 and 2, as determined by the following: 
• The proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment 
• The proportion of positive pruritus assessments at the subject level over the 24-week 
treatment period.   
6.2 
  Secondary Objectives 
• To evaluate the effect of A4250 on serum ALT concentration 
• To evaluate the effect of A4250 on growth 
• To evaluate the effect of A4250 on sleep disturbance 
• To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or 
liver transplantation) 
• To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 


 
 
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7 
INVESTIGATIONAL PLAN 
7.1 
Overall Study Design and Plan 
7.1.1 
Description 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the 
efficacy and safety of 40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in 
children with PFIC (deficiencies of FIC1 or BSEP). 
Study data will be periodically (approximately on a quarterly basis) reviewed by an 
unblinded Data Safety and Monitoring Board (DSMB). 
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment 
Period and a 4-week Follow-Up Period. 
After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of 
A4250, or a matching placebo. 
Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008; EudraCT Number 
2017-002325-38, subject to a separate study approval) in which all patients will receive 
active treatment.  A patient who prematurely withdraws due to intolerable symptoms and has 
completed at least 12 weeks of the Treatment Period and End of Treatment (EOT) Visit, will 
also be offered the opportunity to enter Study A4250-008. 
Visit windows applicable to each visit are presented in the schedule of assessments (Table 1). 
There will be up to 10 clinic visits during the study (Figure 1) and one scheduled telephone 
contact, as follows: 
• Visit 1: Screening Period start. Patient is considered enrolled after signing the informed 
consent form (ICF) (Days -56 to -35) 
• Visit 2: second Screening Visit (Days -28 to -7) 
• Visit 3: Randomization Visit (Day 0) 
• Telephone Contact 1: (Day 14) 
• Visit 4: Clinic Visit (Day 28) 
• Visit 5: Clinic Visit (Day 56) 
• Visit 6: Clinic Visit (Day 84) 
• Visit 7: Clinic Visit (Day 126) 
• Visit 8: Clinic Visit (Day 154) 
• Visit 9: Clinic Visit, EOT (Day 168) 


 
 
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• Visit 10: Follow-Up Visit, 28 days after last dose of study drug (Day 196).  All patients 
continuing in Study A4250-008 will not have this Follow-up Visit. 
Additional clinic visits may be required for patients who need direct site assistance, e.g., due 
to AE monitoring, to fulfil screening requirements, and/or for safety maintenance. 
Screening Period 
Informed consent must be obtained at Visit 1 prior to performing any study procedures.  After 
signing the ICF, patients will be considered enrolled and evaluated for study eligibility. 
Daily recording of pruritus using an electronic diary (eDiary) will be started to determine 
confirmation of the magnitude and the baseline pruritus symptoms before the study.  After 
consultation with the Medical Monitor, patients failing eligibility criteria may be re-screened 
after 2 months.  Patients not fulfilling inclusion/exclusion criteria after 3 attempts are not 
allowed to re-screen. 
Caregivers/patients will be instructed on the daily use of the eDiary.  The eDiary will include 
patient-reported outcome (PRO) and observer-reported outcome (ObsRO) items for 
evaluation of itching (PRO), scratching (ObsRO), and sleep disturbance (PRO and ObsRO) 
during the Screening Period (study Days -56 to 0), throughout the 24-week Treatment Period 
(study Days 0 to 168), and Follow-up (Day 196). 
The 14 consecutive days prior to randomization will be used for eligibility calculation of the 
diary entries.  If a patient does not meet the minimum number of observations (see 
Section 9.2.2 for scoring and missing data definitions) to determine eligibility, the patient 
may be re-screened.  Observer-reported outcomes in patients of all ages will be recorded by a 
caregiver.  If possible, the same caregiver will complete the Albireo ObsRO items throughout 
the study. 
At a second Screening Visit (Visit 2) additional laboratory samples will be taken for 
eligibility assessments. 
Treatment Period 
Eligibility for randomization will be determined at Visit 3 using the pruritus (scratching 
reports from the Albireo ObsRO) data in the 2 weeks before Visit 3, clinical genetic 
confirmation of diagnosis, and the liver biochemistry evaluations from the previous screening 
visits.  Two weeks after Visit 3, there will be a telephone contact with the caregiver/patient to 
evaluate any AEs. 
The patient will return to the clinic at Visit 4, Visit 5, Visit 6, Visit 7, Visit 8, and Visit 9 for 
assessments.  If a new caregiver will begin to complete the Albireo ObsRO during the course 
of the trial, they will be provided with training during a clinic visit before they begin to use 
the eDiary.  Additionally, caregivers will be requested to use the diary to report the time each 
dose of study drug is administered during the Treatment Period. 


 
 
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The last dose of study drug will be administered the day before Visit 9.  At study Visit 9, the 
patient has the option to enroll in an open-label extension study (A4250-008) should they 
meet the required entry criteria.  If the patient is continuing into Study A4250-008 this will be 
the last visit in this study and the first visit in Study A4250-008.  Visit 9/EOT should also be 
performed at the time patients are prematurely withdrawn from the study. 
Clinic Visit 10 will take place 28 days after Visit 9 for all patients not continuing into Study 
A4250-008. 
Figure 1 
Study Design 
 
T: telephone contact; V: study visit 
End of Study 
The end of the study is defined as follows: 
a) End of study in one country: last patient last visit (LPLV) and sites are closed 
b) End of study globally: LPLV globally and all sites closed 
7.1.2 
Schedule of Assessments 
The schedule of assessments is presented in Table 1.  For information on blood volumes, 
please see Appendix 7. 
 


 
 
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Module 5:  Clinical Study Reports 
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Table 1 
Schedule of Assessments 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Informed consent c 
X 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
Concomitant medicationd 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical and surgical history 
X 
 
X 
 
 
 
 
 
 
 
 
Physical examination including 
voluntary photography 
X 
 
X 
 
 
 
X 
 
 
X 
 
Skin examination 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
Vital signse 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
eDiary: itching, scratching, and 
sleep scoresf 
Daily diary entry 
Clinical chemistryg 
X 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Hematologyg 
 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Urinalysisg 
 
X 
 
 
 
 
X 
 
 
X 
 
Serum bile acidsh 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 


 
 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
INR 
 
X 
 
 
X 
 
X 
 
X 
 
X 
Autotaxini 
 
 
X 
 
X 
 
 
 
 
X 
 
p-C4i 
 
 
X 
 
X 
 
 
 
 
X 
 
AFP 
 
 
X 
 
 
 
 
 
 
X 
 
Vitamins A & Eh 
 
X 
 
 
 
 
X 
 
 
X 
 
25-hydroxy vitamin D 
 
 
X 
 
 
 
X 
 
 
X 
 
Blood sample for A4250 PKi 
 
 
 
 
X 
 
 
 
 
X 
 
Abdominal Ultrasound 
 
 
X 
 
 
 
 
 
 
X 
 
Fibroscan® (where available) 
 
 
X 
 
 
 
 
 
 
X 
 
QoL questionnaire (PedsQL) 
 
 
X 
 
 
 
 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of Change 
 
 
 
 
X 
 
X 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of 
Symptoms 
 
 
X 
 
X 
 
X 
 
 
X 
 
Pregnancy testj 
Xj 
X 
X 
 
X 
X 
X 
X 
X 
Xj 
X 


 
 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Study drug dispensedk 
 
 
X 
 
X 
X 
X 
X 
X 
 
 
Adverse eventsl 
Continuous collection 
Study drug compliance 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
AFP: alfa-fetoprotein; BMI: body mass index; eCRF: electronic case report form; eDiary: electronic diary; EOT: End of Treatment; ICF: informed consent form; 
INR: international normalized ratio; p-C4: plasma 7α Hydroxy 4-cholesten-3-one; PedsQL: Pediatric Quality of Life Inventory; PK: pharmacokinetic; QoL: quality of life; 
RR: respiratory rate; SAE: serious adverse event; s-BA: serum bile acid. 
 
a 
Must also be performed at the time a patient is prematurely withdrawn from the study.  This visit is the same as Visit 1 in the extension protocol (Study A4250-008). 
b 
Assessments must be performed 28 days following the final dose of study drug.  This visit is omitted if patients are entering Study A4250-008. 
c 
The ICF (and assent) must be signed before any study procedures are performed. 
d 
Past (within 1 month prior to Visit 1) and current medication will be documented in the eCRF. 
e 
Includes blood pressure, pulse, RR, temperature, height (or length depending on age), and body weight (using a certified weight scale).  BMI will be calculated. 
f 
Includes training on use of the device at Screening (Visit 1) and review of patient daily entries during 14 days prior to Clinic Visit 3 to determine eligibility.  
g 
See Table 3 for detailed parameters. 
h 
Patients will fast (water intake only is permissible) for at least 4 hours prior to the collection of samples for s-BA and vitamin A.  Exceptions can be made for infants, 
less than 12 months of age, if unable to fast for the full 4 hours.  All s-BA results during the treatment period and follow-up will be blinded. 
i 
Samples will not be collected for patients ≤10 kg.  In the event of an SAE, a PK sample should be collected as close to the onset of the event as possible. 
j 
For girls who have reached menarche.  Serum test at Visit 1; urine test at other visits.  A serum pregnancy test will also be conducted at Visit 9 for patients entering 
Study A4250-008.  If a urine pregnancy test is positive, a serum pregnancy test should be performed to confirm the pregnancy. 
k 
Study drug to be taken once daily on Day 1 through Day 167 as described in Section 8.2. 


 
 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
l 
Adverse event information will be collected from the time of signing of the ICF to study discontinuation. 


 
 
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7.1.3 
Study Procedures and Assessments 
7.1.3.1 
Screening Period (Days -56 to 0) 
Day -56 to Day -35/Clinic Visit 1 
Patients will undergo a Screening Visit up to 56 days prior to the planned first day of study 
treatment.  Screening procedures and assessments are as follows: 
• Obtain written informed consent 
• Assess inclusion/exclusion criteria (Section 7.2) 
• Record demographics (age, full date of birth, gender, race, and ethnicity) 
• Document concomitant medications 
• Medical and surgical history (date of diagnosis of PFIC Types 1 or 2, prior investigational 
medications for PFIC, historical liver function test [LFT] values, any surgery performed 
including biliary diversion (if applicable), any other diagnosis, and historical liver biopsy 
data) 
• Collect, if available, past confirmatory clinical genetic laboratory report for 
PFIC-1/PFIC-2 and send to central reader for review.  If a historical report is equivocal, 
unavailable, or unobtainable, a blood sample for clinical genetic testing will be collected 
to determine eligibility 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• eDiary training and compliance requirements; caregivers and, if applicable, patients begin 
daily recording of pruritus using the eDiary (Appendix 2) 
• Clinical chemistry (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Serum pregnancy test for girls who have reached menarche.  Please see Appendix 6 for 
contraceptive requirements 
• AE monitoring 
Day -28 to Day -7/Clinic Visit 2 
A second Screening Visit will be performed.  Screening procedures and assessments are as 
follows: 
• Review concomitant medications 
• Vital signs (Section 10.2.6) 


 
 
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• Albireo PRO/ObsRO eDiary review for compliance 
• Urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• International normalized ratio (INR) 
• Vitamins A and E 
• Urine pregnancy test for girls of who have reached menarche 
• AE monitoring 
7.1.3.2 
Treatment Period (Day 0 to Day 168) 
Study Day 0/Clinic Visit 3 (Randomization) 
At the randomization visit, the following assessments will be conducted: 
• Review inclusion/exclusion criteria (Section 7.2) 
• Review concomitant medications, medical and surgical history 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance and entrance criteria requirement (see 
Inclusion Criteria 4, Section 7.2.1) 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1)  
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• 25-hydroxy vitamin D 
• Alfa-fetoprotein (AFP) 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer QoL (Pediatric Quality of Life Inventory [PedsQL]) questionnaire 
• Patient/caregiver/clinician complete the patient global impression of symptoms (PGIS) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Study drug is dispensed and patients are instructed to administer daily from Day 1 
Study Day 14 (Telephone Contact) 


 
 
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A study nurse will contact the patient/caregiver by telephone for monitoring of the following:  
• Review concomitant medications 
• Albireo PRO/ObsRO eDiary review for compliance 
• AEs 
Study Day 28 and Day 84/Clinic Visits 4 and 6  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Physical examination, including voluntary photography (at Clinic Visit 6 only; 
Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• Urinalysis (at Clinic Visit 6 only; Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Autotaxin, p-C4, and A4250 pharmacokinetic (PK) assessment (at Clinic Visit 4 only; 
samples will not be taken for patients ≤10 kg) 
• Vitamins A and E, and 25-hydroxy vitamin D (at Clinic Visit 6 only) 
• Patient/caregiver/clinician complete the patient global impression of change (PGIC) and 
PGIS 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 56 and Day 126/Clinic Visits 5 and 7 
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  


 
 
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• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 154/Clinic Visit 8  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 168/Clinic Visit 9/EOT 
The last dose of study drug is taken in the morning the day before Clinic Visit 9, and the 
following procedures and assessments will be performed (also performed at the time a patient 
prematurely withdraws):  
• Review of concomitant medications 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry, hematology, and urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Autotaxin, p-C4, and A4250 PK assessment (samples will not be taken for patients 
≤10 kg) 


 
 
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• AFP  
• Vitamins A, E, and 25-hydroxy vitamin D 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer PedsQL questionnaire 
• Patient/caregiver/clinician complete the PGIC and PGIS 
• Urine pregnancy test for girls who have reached menarche, or serum pregnancy test for 
girls continuing to study A4250-008 
• AE monitoring 
• Evaluation of study drug compliance 
7.1.3.3 
Follow-Up Period 
Study Day 196/Clinic Visit 10 
All patients not continuing into Study A4250-008 will return to the study site 28 days after 
Clinic Visit 9 or the last dose of study drug for the following assessments: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
7.2 
Study Population 
A total of approximately 60 patients with a clinical diagnosis of PFIC Type 1 or 2 that have 
been genetically confirmed will be randomized.  The study will target 15% of enrolled 
patients to be PFIC-1 patients.  If enrollment of all PFIC-2 patients is completed and the 15% 
enrollment of PFIC-1 patients has not yet been achieved, then enrollment of PFIC-2 patients 
will continue in order to reach the total study target enrollment. 
An attempt will be made to randomize a minimum of 10 patients from each age category 
(6 months to 5 years, 6 to 12 years, and 13 to ≤18 years).  


 
 
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A maximum of 5 patients that were treated in Study A4250-003 will be allowed to participate 
in this study. 
7.2.1 
Inclusion Criteria 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages 
of ≥6 months and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have clinical genetic confirmation of PFIC-1 or PFIC-2 through 
identification of biallelic pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, 
taken as the average of 2 samples at least 7 days apart (Visits 1 and 2) prior to 
randomization 
4. Patient must have history of significant pruritus and a caregiver-reported observed 
scratching in the eDiary average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to 
randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  
Patients who turn 18 years of age (or legal age per country) during the study will be 
required to re-consent in order to remain in the study 
6. Patients will be expected to have a consistent caregiver for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use 
an eDiary device as required by the study 
7.2.2 
Exclusion Criteria 
1. Patient with pathologic variations of the ABCB11 gene that predict complete absence of 
the BSEP protein 
2. Patient with past medical history or ongoing presence of other types of liver disease 
including, but not limited to, the following: 
a) Biliary atresia of any kind 
b) Benign recurrent intrahepatic cholestasis, indicated by any history of normal s-BAs 
c) Suspected or proven liver cancer or metastasis to the liver on imaging studies 
d) Histopathology on liver biopsy is suggestive of alternate non-PFIC related etiology of 
cholestasis 
3. Patient with a past medical history or ongoing presence of any other disease or condition 
known to interfere with the absorption, distribution, metabolism (specifically bile acid 
metabolism), or excretion of drugs in the intestine, including but not limited to, 
inflammatory bowel disease 


 
 
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4. Patient with past medical history or ongoing chronic (i.e., >3 months) diarrhea requiring 
intravenous fluid or nutritional intervention for treatment of the diarrhea and/or its 
sequelae 
5. Patient has a confirmed past diagnosis of infection with human immunodeficiency virus 
or other present and active, clinically significant, acute, or chronic infection, or past 
medical history of any major episode of infection requiring hospitalization or treatment 
with parenteral anti-infective treatment within 4 weeks of treatment start (study Day 1) or 
completion of oral anti-infective treatment within 2 weeks prior to start of Screening 
Period 
6. Any patient with suspected or confirmed cancers except for basal cell carcinoma, and 
non-liver cancers treated at least 5 years prior to Screening with no evidence of 
recurrence 
7. Patient with a past medical history of chronic kidney disease with an impaired renal 
function and a glomerular filtration rate <70 mL/min/1.73 m2  
8. Patient with surgical history of disruption of the enterohepatic circulation (biliary 
diversion surgery) within 6 months prior to start of Screening Period. 
9. Patient has had a liver transplant or a liver transplant is planned within 6 months of 
randomization 
10. Decompensated liver disease, coagulopathy, history or presence of clinically significant 
ascites, variceal hemorrhage, and/or encephalopathy 
11. INR >1.4 (the patient may be treated with Vitamin K intravenously, and if INR is ≤1.4 at 
resampling the patient may be randomized) 
12. Serum ALT >10 × upper limit of normal (ULN) at Screening 
13. Serum ALT >15 × ULN at any time point during the last 6 months unless an alternate 
etiology was confirmed for the elevation 
14. Total bilirubin >5 × ULN at Screening 
15. Patient suffers from uncontrolled, recalcitrant pruritic condition other than PFIC.  
Examples include, but not limited to, refractory atopic dermatitis or other primary pruritic 
skin diseases 
16. Any patient who is pregnant or lactating or who is planning to become pregnant within 
24 weeks of randomization 
17. Sexually active males and females who are not using a reliable contraceptive method with 
≤1% failure rate (such as hormonal contraception, intrauterine device, or complete 
abstinence) throughout the duration of the study and 90 days thereafter (from signed 


 
 
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informed consent through 90 days after last dose of study drug).  See Appendix 6 for 
further details 
18. Patient with a past medical history of alcohol or substance abuse will be excluded.  
Patient must agree to refrain from illicit drug and alcohol use during the study 
19. Administration of bile acid or lipid binding resins and medications that slow GI motility 
(Refer to Appendix 1 – Concomitant Medications Guidelines) 
20. Patient has had investigational exposure to a drug, biologic agent, or medical device 
within 30 days prior to Screening, or 5 half-lives of the study agent, whichever is longer 
21. Patient who has been previously treated with an IBAT inhibitor whose pruritus has not 
responded to treatment 
22. Any other conditions or abnormalities which, in the opinion of the investigator or Medical 
Monitor, may compromise the safety of the patient, or interfere with the patient 
participating in or completing the study 
7.2.3 
Withdrawal of Patients 
Patients/caregivers will be informed that they have the right to withdraw from the study at 
any time, without prejudice to their medical care, and that they are not obliged to state their 
reasons. 
Any withdrawal, and reasons for withdrawal, must be fully documented in the electronic case 
report form (eCRF) and source documents and the patient followed by the 
investigator/investigative staff.  Even after the study is completed at the Follow-up Visit, the 
investigator/investigative staff will, regardless of reason for withdrawal, record any patient 
data they receive concerning SAEs, and all drug-related non-serious AEs, and subsequently 
report these according to Section 10.1.  Withdrawn patients will not be replaced. 
Patients will be withdrawn in the following circumstances: 
• A patient’s/caregiver’s desire for withdrawal for any reason 
• Lost to follow-up (every effort must be made to contact the patient/caregiver; a certified 
letter must be sent) 
• An AE that, in the opinion of the investigator, necessitates withdrawal 
• A worsening of the symptoms related to diarrhea occurs 
• Death 
• Patient/caregiver estimation of intolerable symptoms 
• A patient’s/caregiver’s substantial noncompliance (eDiary and study drug compliance) or 
protocol violation 


 
 
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• An investigator’s opinion that continuing the patient in the study is not appropriate.  The 
investigator may withdraw a patient at any time, if it is considered to be in the patient’s 
best interest 
A patient who prematurely withdraws due to intolerable symptoms and has completed at least 
12 weeks treatment in the double-blind study, will be offered the opportunity to enter 
open-label Study A4250-008.  A patient who withdraws from treatment prematurely and does 
not enter Study A4250-008 will have EOT (Visit 9) assessments at the time of withdrawal 
and a follow-up assessment (Visit 10) 28 days following the last dose of study drug, and the 
patient will be recorded as withdrawn. 
7.2.4 
Study Termination by Sponsor 
This study may be terminated at any time by Albireo if serious side effects should occur, if 
the investigator does not adhere to the protocol; or if, in the sponsor's judgment, there are no 
further benefits to be achieved from the study.  In this event, Albireo/designee will inform the 
study investigators, institutions, and all regulatory authorities. 
Albireo may temporarily or permanently discontinue the study at an investigative site at any 
time for safety, ethical, compliance, or other reasons.  If this is necessary, Albireo will 
endeavor to provide advance notification to the site.  If a site or the study is suspended or 
discontinued, the investigator/investigative staff will be responsible for promptly informing 
the Independent Ethics Committee (IEC).  If required by local regulations, Albireo/designee 
will be responsible for informing the IEC and the regulatory authority of study or site 
discontinuation.  In such an event, all study data and unused study drug must be returned to 
Albireo. 


 
 
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8 
TREATMENT OF PATIENTS 
8.1 
Identity of Study Drug 
A4250 and placebo will be supplied as capsules for oral administration.  White opaque 
capsules filled with pellets containing A4250 or placebo will be provided.  Two different 
capsule sizes will be available, as follows: 
• Capsule size 0 that can be opened 
• Capsule size 3 that should be swallowed intact.  These size 3 capsules may be opened 
only under exceptional circumstances, e.g., patient cannot swallow capsule intact 
The content of the A4250 and placebo capsules will be identical in appearance.  Capsule 
filling weight will also be identical for high dose, low dose, and placebo.  Bottles with 
34 capsules will be given to the patient at each visit.  A patient who requires 2 or more 
capsules per day will be given multiple bottles.  Refer to the investigational product manual. 
8.2 
Administration of Study Drug 
Patients will be dosed with A4250 at a dose of 40 µg/kg/day, 120 µg/kg/day, or placebo for 
24 weeks.  Study drug will be dispensed to the patient at Visits 3, 4, 5, 6, 7, and 8, together 
with instructions on how to store and take the drug.  Study drug administration data, 
including whether each patient took each dose or partial doses of study drug, whether there 
were any delayed or missed doses, and whether the capsule was opened or swallowed whole 
will be documented through the eDiary and transferred to the study database. 
A4250 should be taken in the morning, together with food.  On clinic visits days when 
laboratory assessments are conducted, study drug should be taken after the visit and after 
laboratory samples are taken.  Patients should not crush or chew the capsule(s).  When 
swallowing the capsule intact, the patient should administer the dose with a glass of water. 
For a patient unable to swallow capsule intact, the capsule can be opened and the contents 
sprinkled and mixed in a small amount of soft room-temperature yogurt, apple sauce, or fruit 
purée.  A list of food options can be found in the Pharmacy manual. The amount of food 
should be less than what the patient/infant is expected to eat and should be followed by water.  
1. Hold the capsule over the prepared food, gently twist open and allow the content to fall 
into food 
2. Mix the entire capsule content with 1 or 2 tablespoons (15 to 30 mL) of soft food (apple 
sauce, fruit purée, or yogurt) 
3. Administer the mixture immediately after mixing. 
The number of capsules provided to the patient will be based on body weight at Visit 3.  At 
Visit 6, if a patient’s weight has shifted to another weight category (identified in Table 2), the 
number of capsules per day may be adjusted.   


 
 
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Table 2 
Dosing and Capsule Strength 
Body Weight 
(kg) 
Capsule 
Size 
Number of 
Capsules 
per Day 
Capsule 
Strength, 
Low Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Strength, 
High Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Size, 
Placebo 
(µg) 
5.0 to <7.5 
0 
1 
200 
200 
600 
600 
0 
7.5 to <12.5 
0 
2 
200 
400 
600 
1200 
0 
12.5 to <17.5 
0 
3 
200 
600 
600 
1800 
0 
17.5 to <19.5 
0 
4 
200 
800 
600 
2400 
0 
19.5 to <25.5 
3 
2 
400 
800 
1200 
2400 
3 
25.5 to <35.5 
3 
3 
400 
1200 
1200 
3600 
3 
35.5 to <45.5 
3 
4 
400 
1600 
1200 
4800 
3 
45.5 to 55.5 
3 
5 
400 
2000 
1200 
6000 
3 
>55.5 
3 
6 
400 
2400 
1200 
7200 
3 
8.3 
Study Treatment Packaging and Labeling 
8.3.1 
Packaging and Labeling 
The capsules will be packed in high-density polyethylene containers, with child-proof 
polypropylene caps.  Study drug capsules containing A4250 in the strength specified in 
Table 2 above will be manufactured. 
Packaging and labeling will be prepared to comply with applicable regulatory requirements. 
8.3.2 
Storage 
Treatment packs containing A4250 capsules should be stored and dispensed in accordance 
with regulations in their original containers.  The storage facility at the investigative site 
should be locked and the storage temperature should be between 15°C and 25°C. 
Patients/caregivers should be informed of appropriate storage conditions (i.e., room 
temperature, between 15°C and 25°C). 
Any deviations from the recommended storage conditions should be immediately reported to 
Albireo and the study drug should not be used until authorization has been given by Albireo. 
8.3.3 
Blinding and Randomization of Study Drug 
A double-blind design is employed so that both the investigators and the patients will be 
unaware of the treatment assignment during the study.  
After written informed consent is obtained from an eligible patient, an 8-digit patient 
identification number will be assigned.  The first 2 digits will denote the country, followed by 
a 3-digit site number and a 3-digit patient sequence number.  This number will be created and 


 
 
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allocated by the Interactive Web Response System (IWRS), when the patient first enters the 
system at Screening.  The randomization codes will be computer-generated by 
 
Biostatistics and kept by a statistician independent from the project team.  The randomization 
will be done in blocks and stratified according to PFIC class (Type 1 and 2) and age class 
(6 months to 5 years, 6 to 12 years, and 13 to ≤18 years) to ensure approximate balance 
between dose schemes (1:1:1).  A separate randomization list will be prepared for the 
maximum 5 patients from Study A4250-003 regardless of stratification.  Randomization 
codes will be assigned sequentially as patients become eligible for randomization. 
Patients who are eligible for randomization will be assigned a unique 4-digit randomization 
number by IWRS.  This randomization number identifies which treatment will be allocated to 
the patient.  Patients who withdraw from the study after randomization visit are not to be 
replaced and their randomization number will not be reused.   
To ensure blinding, the study drug and the matching placebo have the same shape and size.  
Patients will receive capsule(s) of A4250 according to their dose group or capsule(s) of 
matching placebo once a day during the double-blind Treatment Period.  Labels on the study 
drug containers will not identify the treatment that a patient is randomized to.  Traceability of 
the treatment is ensured by the study drug number. 
The 5-digit study drug number will identify study drug packs and will be detailed on the 
study drug label.  Dispensing of study drug will be coordinated by IWRS.  
The system will assign a study drug number corresponding to the randomization arm.  The 
randomization number will be used in the background only to ensure no unblinding and will 
not be displayed to end-users in the IWRS. 
8.4 
Procedure for Breaking the Randomization Code 
Should a situation arise where unblinding is urgently required, i.e., only where knowledge of 
the study drug is required to adequately manage a life-threatening situation, the investigator 
at that study site may perform immediate unblinding through IWRS.  The responsibility to 
break the treatment code in emergency situations resides solely with the investigator.  The 
investigator should make every effort to discuss the rationale (status and outcome) for 
emergency unblinding with the sponsor Medical Monitor or the 
 Medical Monitor as 
soon as possible to review the individual patient details. 
The study site investigator and appropriate 
 project team members will be authorized to 
access the emergency unblinding functionality within the IWRS.  Emergency unblinding 
takes place in the IWRS via a dedicated emergency unblinding module.  The system will 
require the user to enter their emergency unblinding, which is typically provided at the same 
time login credentials are distributed.  The exact description of the treatment assigned to the 
individual patient then will be accessible to the user.  Once the randomization code is broken 
for a patient, he/she must be withdrawn from the study and all assessments and procedures at 
EOT should be performed.  As a follow-up, Visit 10, will be performed according to the time 




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9 
ASSESSMENT OF EFFICACY 
9.1 
Efficacy Endpoints 
9.1.1 
Primary Efficacy Endpoint 
The primary efficacy endpoints are as follows: 
•
European Union (EU) and rest of the world (RoW): Proportion of patients
experiencing at least a 70% reduction in fasting s-BA concentration from baseline to
the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24
weeks of treatment.
Fasting s-BA baseline will be calculated as the average of the value at randomization
and the previous visit (Clinic Visit 2).  The end value is the average of the values at
Weeks 22 and 24 after the start of double-blind treatment.
•
United States (US): Proportion of positive pruritus assessments at the subject level
over the 24-week Treatment Period. A positive pruritus assessment is defined as a
scratching score of ≤1 or at least a one-point drop from baseline on the Albireo
ObsRO instrument. At each assessment, the AM score will be compared to the AM
baseline, and the PM score will be compared to the PM baseline. Both AM baseline
and PM baseline pruritus assessments will be included in the analysis of this endpoint.
AM scores from the 14 days prior to the first dose of study medications will be
averaged as AM baseline. PM scores from the 14 days prior to the first dose will be
averaged as PM baseline.
9.1.2 
Secondary Efficacy Endpoint 
Secondary efficacy endpoints include the following: 
•
EU and RoW: Proportion of positive pruritus assessments at the subject level over
the 24-week Treatment Period. A positive pruritus assessment is defined as a
scratching score of ≤1 or at least a one-point drop from baseline on the Albireo
ObsRO instrument. At each assessment, the AM score will be compared to the AM
baseline, and the PM score will be compared to the PM baseline. Both AM baseline
and PM baseline pruritus assessments will be included in the analysis of this endpoint.
AM scores from the 14 days prior to the first dose of study medications will be
averaged as AM baseline. PM scores from the 14 days prior to the first dose will be
averaged as PM baseline.
•
US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA
concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L
compared to placebo after 24 weeks of treatment.
•
All Regions:


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All secondary endpoints are compared to placebo. 
o Change from baseline to Week 12 and to Week 24 in fasting serum bile acids 
o Change from baseline to Week 12 and to Week 24 in serum ALT 
concentration 
o Change in growth from baseline to Week 12 and to Week 24 
o Proportion of responders for pruritus scores at Weeks 12 and 24 based on the 
Albireo PRO and ObsRO instruments 
o Change in sleep parameters measured with the Albireo PRO and ObsRO 
instruments from baseline over the 24-week Treatment Period 
o Proportion of individual assessments meeting the definition of a positive 
pruritus assessment at the subject level over the 24-week Treatment Period. A 
positive pruritus assessment includes an itch score ≤1, or at least a one-point 
drop  from  baseline  based  on  the  Albireo  PRO  instrument;  only  patients 
≥8 years of age will complete the Albireo PRO instrument 
o Proportion of individual assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0- 
12, Weeks 0-18, Weeks 0-24, respectively, or the proportion of positive 
pruritus assessments at each 4-week interval. 
o Proportion of individual AM assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0- 
12, Weeks 0-18, Weeks 0-24, respectively, or the proportion of positive 
pruritus assessments at each 4-week interval. 
o Proportion of individual PM assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0- 
12, Weeks 0-18, Weeks 0-24, respectively, or the proportion of positive 
pruritus assessments at each 4-week interval. 
o Number of patients undergoing biliary diversion surgery or liver 
transplantation 
9.1.3 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, 
caregiver, and clinician via the PGIC items (Appendix 3) 
• Change from baseline in fasting s-BA at Week 4 
 
 
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• Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
by each 4 week-interval of the Treatment Period as measured by the Albireo ObsRO 
instrument 
• Change from baseline by each 4-week interval of the Treatment Period in patient-reported 
and observer-reported night time itching and scratching severity scores, respectively  
• Change from baseline by each 4-week interval of the Treatment Period in patient-reported 
and observer-reported morning time itching and scratching severity scores, respectively 
• Change from baseline by each 4-week interval of the Treatment Period in pooled pruritus 
score including observer-reported scratching for patients <8 years of age and 
patient-reported itch severity for patients ≥8 years of age 
• Change from baseline by each 4-week interval of the Treatment Period in additional 
patient-reported and observer-reported sleep parameters (e.g., tiredness and number of 
awakenings) 
• Change from baseline to Week 24 in PedsQL (Appendix 4) 
• Change from baseline in serum ALT concentration at Week 4  
• Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• Change from baseline in AST and GGT at Weeks 4, 12, and 24 
• Change from baseline for other biochemical markers and measures of bile acid synthesis 
(autotaxin, p-C4) at Week 24 
• Change in pediatric end-stage liver disease/model for end-stage liver disease 
(PELD/MELD) score, AST to platelet ratio index (APRI) score, and Fib-4 score from 
baseline to Week 24 
• Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® 
(where available) 
• Liver-related mortality and liver decompensation events 
• All-cause mortality 
9.2 
Efficacy Assessments 
9.2.1 
Serum Bile Acids 
Blood samples for analysis of fasting total s-BA will be drawn at all visits.  Patients will fast 
(water intake only is permissible) for at least 4 hours prior to the collection of samples for 
s-BA.  Exceptions can be made for infants, <12 months of age, if unable to fast for the full 
4 hours.  Any visit at which a bile acid sample result is unreportable, an additional 
unscheduled visit for a repeat sample collection may be scheduled.  All s-BA results during 


 
 
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the treatment period and follow-up will be blinded.  Samples will be processed and 
transported to a central laboratory per instructions in the laboratory manual. 
9.2.2 
Itching, Scratching and Sleep Score 
Itching, observed scratching, and sleep disturbance will be recorded twice each day via 
eDiary.  Patients and/or caregivers will be instructed to complete the eDiary every day in the 
morning after the patient wakes and in the evening just before the patient goes to sleep 
throughout the study.  Patients and caregivers will receive a formal training session during 
Visit 1. 
The eDiary includes Albireo ObsRO and PRO items.  Patients <8 years of age will not be 
asked to complete the Albireo PRO items; only the Albireo ObsRO will be completed by 
caregivers of patients in this age group.  Older patients, 8 to 18 years of age, will complete 
the Albireo PRO items and the caregiver will complete the Albireo ObsRO items.  The 
Albireo PRO items assess severity of itch, aspects of sleep disturbance (morning diary only), 
and tiredness.  For patients 8 to 12 years of age, the caregiver will read the Albireo PRO 
items along with the child and record the child’s response.  A guide will be provided to the 
caregivers that provides standardized explanations of the Albireo PRO items, in case the 
patient is confused or requires clarification about the meaning of a question.  The Albireo 
ObsRO items assess severity of observed scratching, aspects of observed sleep disturbance 
(morning diary only), and observed signs of tiredness (evening diary only).  The Albireo 
ObsRO and PRO scratching and itch severity items use 0 to 4 response scales, where each 
response is distinguished by a unique facial expression, verbal anchor, number, and color 
code (Appendix 2).  To meet eligibility criteria, a scratch score will be calculated from daily 
diary entries (ObsRO items) the 14 consecutive days prior to randomization.  
A daily AM & PM score for the Albireo ObsRO scratching item will be averaged from the 2 
ratings for each day.  A daily score will be considered missing if both of the daily 
assessments are missing.  A weekly score will be calculated by averaging the daily scratching 
scores.  A weekly score will be considered missing if ≥4 out of 7 days a week of data are 
missing.  For AM & PM baseline, a 14-day daily score of AM & PM prior to the first dose of 
study medications will be averaged as the baseline score. 
For AM baseline, a 14-day AM score prior to the first dose of study medications will be 
averaged as the AM baseline score.  The same approach will be used for PM baseline and to 
calculate a patient-reported itch severity score.  The handling of missing data is described in 
Section 11.2.2.2. 
9.2.3 
Growth 
Growth will be measured as height (velocity) and weight (Z-score) using a certified weight 
scale.  BMI will be calculated by weight (kg) / height (m)2.  Change will be defined as linear 
growth deficit (weight and BMI for age) compared to a standard growth curve (Z-score, SD 
from P50). 


 
 
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9.2.4 
Quality of Life Questionnaire (PedsQL) 
Patients and caregivers will be asked to complete a QoL questionnaire (PedsQL) at Visit 3 
and Visit 9.  Details of the questions included on the questionnaire are provided in 
Appendix 4. 
9.2.5 
Biomarkers and PK Samples 
Blood samples for autotaxin and p-C4 will be drawn at Visits 3, 4, and 9 only for children 
with body weight >10 kg.  Blood for A4250 PK assessment will only be drawn at Visits 4 
and 9 (only for children with body weight >10 kg), and as close to the onset of an SAE as 
possible, should an SAE occur.  Samples will be processed and transported to a laboratory 
per instructions in the laboratory manual. 
9.2.6 
PELD/MELD Score 
At randomization and end of treatment, PELD score will be calculated for children up to 
12 years of age.  For children 13 years or older, the MELD score will be calculated. 
PELD Score = 4.80 * ln(total bilirubin) + 18.57 * ln(INR) - 6.87 * ln(albumin) + 4.36 (if 
patient <1 year: scores for patients listed for liver transplantation before the patient's first 
birthday continue to include the value assigned for age (<1 year) until the patient reaches the 
age of 24 months) + 6.67 (if the patient has growth failure [≤2 SD]) 
Laboratory values less than 1.0 will be set to 1.0 for the calculation of the PELD score. 
MELD Score = 9.57 * ln(creatinine) + 3.78 * ln(total bilirubin) + 11.2 * ln(INR) + 6.43 
Laboratory values less than 1.0 will be set to 1.0 and serum creatinine values greater than 
4.0 mg/dL (equivalent to 353.6 µmol/L) will be set to 4.0 for calculation of the MELD score. 
9.2.7 
Fibroscan® 
Where available, Fibroscan® will be performed as per institution standard practice at Visits 3 
and 9. 
9.2.8 
Markers of Fibrosis 
APRI score and Fib-4 score will be calculated at randomization and at end of treatment. 
APRI = [(AST in U/L) / (AST ULN in U/L)] / (Platelets in 109/L) 
Fibrosis 4 Score = (Age*AST in U/L) / (Platelets in 109/L * √(ALT in U/L)). 
9.2.9 
Global Impression of Change and Global Impression of Symptom Measures 
Patients (≥8 years of age), caregivers, and clinicians will complete the PGIC and the PGIS 
measures (Appendix 3) at randomization (Visit 3; PGIS only), Visits 4 and 6, and at EOT 
(Visit 9). 
The PGIC items assess change in itch (patient version), scratching (caregiver and clinician 
versions), and sleep (all versions) since starting the study drug.  The PGIS items assess itch 


 
 
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(patient version), scratching (caregiver and clinician versions), and sleep (all versions) in the 
past week.  Caregivers and clinicians will complete the PGIC and PGIS for all patients; those 
patients ≥8 years of age will complete the patient version. 


 
 
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10 
ASSESSMENT OF SAFETY 
The timing and frequency of safety assessments are described in Section 7.1.3 and in Table 1 
above. 
The primary safety analysis for this study will include the incidence of total 
treatment-emergent adverse events (TEAE) and TEAEs categorized by causality, severity, 
and seriousness assessments made by the investigator by comparing study drug exposure to 
placebo. 
Trends in safety will also be evaluated for the following assessments: 
• Physical examinations 
• Concomitant medications 
• Vital signs 
• Laboratory test results (including clinical chemistry, hematology, urinalysis, AFP, and 
vitamins A and E, 25-hydroxy vitamin D, and INR) 
• Abdominal ultrasound 
• Discontinuations due to AEs 
10.1 
Adverse Events 
10.1.1 
Definitions and Investigator Assessments 
An AE is defined as any untoward medical occurrence in an enrolled patient regardless of 
causal relationship with study drug.  An AE can therefore be any clinically significant 
unfavorable and unintended sign, symptom, or disease that occurs once a patient is enrolled 
(ICF is signed) in the study until the patient is discharged from the study, whether or not 
related to the study drug. 
10.1.1.1 
Clinical Significance 
Clinical significance is defined as any abnormal finding that results in further clinical 
investigation(s), treatment(s), or the diagnosis of new condition.  Patient-reported events and 
protocol-mandated laboratory values, vital signs, and physical examination findings can be 
considered clinically significant (i.e., an AE) if there is a deterioration as compared to 
baseline.  Examples of clinically significant worsening from baseline could include, but is not 
limited to, events causing withdrawal from the study and events requiring medical 
intervention outside of the study causing apparent clinical manifestations, or judged relevant 
by the investigator.   
10.1.1.2 
Serious Adverse Events 
Serious criteria are applied by the investigator to each AE as specifically defined below.  
These criteria are used to determine whether an AE is serious or non-serious.  The assessment 


 
 
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is made independently of severity assessment (Section 10.1.1.3).  For example, the 
development of a severe rash that occurs after signing of informed consent may not meet 
serious criteria as defined below and therefore would be considered a severe, non-serious AE. 
Any AE that meets any 1 of the following 6 criteria is considered an SAE: 
• The outcome of the AE is death 
• The AE is immediately life-threatening.  Life-threatening means that the patient is, in the 
opinion of the investigator, at immediate risk of death from the reaction as it occurred.  
This does not include an AE that, if more severe, might have caused death 
• The AE results in persistent or significant disability/incapacity.  Disability means a 
substantial disruption of a person's ability to conduct normal life functions 
• The AE requires or prolongs hospitalization  
• The AE results in a congenital anomaly/birth defect 
• The AE is an important medical event.  Important medical events may meet serious 
criteria should the investigator assess that they may significantly jeopardize the patient, 
represent a significant hazard, or requires medical/surgical intervention to prevent one of 
the outcomes listed above.  Examples of potential SAEs based on this criteria include, but 
are not limited to, allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions even if they do not result in inpatient 
hospitalization, or the development of drug dependency and drug abuse. 
10.1.1.3 
Severity Assessment 
Severity assessments are based on the intensity of the event in relation to expectation.  The 
investigator will assess the intensity of AEs based on the following definitions: 
• Mild (awareness of sign or symptom, but easily tolerated) 
• Moderate (discomfort sufficient to cause interference with normal activities) 
• Severe (incapacitating, with inability to perform normal activities) 
Severity is a measure of intensity where seriousness is defined by the criteria outlined in 
Section 10.1.1.2.  An AE of severe intensity need not necessarily be considered serious.  For 
example, nausea that persists for several hours may be considered severe nausea, but not meet 
serious criteria, and therefore would be assessed as a severe AE but not an SAE. 
10.1.1.4 
Causality Assessment 
The investigator determines the causality of all AEs to the study drug using medical judgment 
and considering all relevant factors such as (but not limited to) the underlying study 
indication, coexisting disease, concomitant medication, relevant history, pattern of the AE, 
temporal relationship to the study drug, and de-challenge or re-challenge.  The causality 
assessment of the AE/SAE is to be made as follows. 


 
 
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Related to study drug (possibly, probably, or definitely related) 
Based on medical judgment, there is at least a reasonable possibility that the study drug 
caused the event; one or more of the following criteria apply: 
• The event follows a reasonable temporal sequence from administration of study drug 
• The event could not be reasonably attributed to the known characteristics of the patient's 
clinical state, environmental or toxic factors, or other modes of therapy administered to 
the patient 
• The event follows a known pattern of response to study drug 
• The event disappears or decreases on cessation or reduction in dose of the study drug.  (It 
should be noted that in some situations an AE will not disappear or decrease in intensity 
upon discontinuation of study drug despite other clear indications of relatedness) 
• The event reappears or worsens when the study drug is re-administered 
Unrelated to study drug (unlikely or unrelated) 
Based on medical judgment there is no reasonable possibility that the study drug caused the 
event; one or more of the following criteria apply: 
• The event does not follow a reasonable temporal sequence from administration of study 
drug 
• The event could be reasonably attributed to the known characteristics of the patient's 
clinical state, concurrent illness, environment or toxic factors, or other modes of therapy 
administered to the patient 
• The event does not follow a known pattern of response to study drug 
• The event does not disappear or decrease on cessation or reduction in dose of the study 
drug, and it does not reappear or worsen when the study drug is re-administered 
10.1.2 
Recording of Adverse Events 
It is the investigator’s responsibility to assess whether each untoward event is a clinically 
significant worsening from baseline, thereby considered an AE.  For all AEs, the severity, 
seriousness, and causality to study drug for each AE as outlined in Section 10.1.1 will be 
assessed and recorded in the eCRF. 
All serious and non-serious AEs are collected once the caregiver/patient has signed the ICF 
and until the post-treatment follow-up (Visit 10) or 28 days after the last dose of study drug. 
Any AEs or SAEs that are unresolved at the patient’s last AE assessment in the study are 
followed up by the investigator until resolution or stabilization up to the database lock and 
recorded in the eCRF.  Albireo retains the right to request additional information for any 
patient with ongoing AE(s) or SAE(s) at the end of the study. 


 
 
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TEAEs are defined as any AE that occurs after randomization (Day 0).  All AEs that occur in 
the Screening Period, i.e., after enrollment and prior to randomization, will be collected on 
the eCRF as non-treatment-emergent AEs. 
If there is a clinically significant deterioration of a laboratory value/vital sign or other routine 
study assessment that is associated with a diagnosis, the clinical diagnosis will be reported as 
an AE and the associated signs and symptoms will be considered additional information 
unless the sign or symptom is more severe than expected given the diagnosis.  For example, if 
an investigator diagnoses a study patient with hepatitis during the study period, hepatitis 
would be considered the AE and the concomitant signs and symptoms of abdominal pain, 
vomiting, and elevated ALT and AST would not be reported separately unless, in the opinion 
of the investigator, one of these signs or symptoms is more severe than expected and 
therefore a separate AE assessment is indicated. 
10.1.3 
Recording and Reporting of Serious Adverse Events 
Every SAE (regardless of severity and causality) that occurs once the caregiver/patient has 
signed the ICF and until the post-treatment follow-up (Visit 10), and 28 days after the final 
dose of study drug, should immediately and not later than within 24 hours of knowledge of 
the event, be reported by the investigator or delegate in the SAE report form.    
Report of a SAE must include at least the following information: 
• Patient identification information (study number, site number, initials, and date of birth 
[as per local country requirements for data protection]) 
• The last study drug administration date 
• The diagnosis of the event with the description (or a brief description of 
signs/symptoms/clinical course if the diagnosis is not available) and the date of onset 
• The action used to treat the event (i.e., treatment medications, temporary discontinuation) 
• The reason(s) for considering the event serious 
• The relationship of the event to the study drug or to the study procedure (e.g., the 
investigator's assessment of causality) 
• A brief narrative of the SAE 
Follow-up reports including all new information obtained of the subsequent course of the 
SAE must be prepared and the information collected in the SAE submitted to 
 
 (Pharmacovigilance and Safety Services Department) by e-mail within 24 hours of 
knowledge of the event. 
The pharmacovigilance manager (PVM) may contact the investigator to obtain further 
information on a reported SAE.  The investigator/investigative staff must respond to any 


 
 
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request for follow-up information or answers to questions regarding the SAE within the same 
timelines as for initial reports. 
The PVM reports the occurrence of the SAE and follow-up to the Albireo medical monitor 
for medical assessment of the case. 
Should an outcome of death occur within the study period or within 28 days after the last 
administration of study drug, an SAE form should be completed including the main and 
contributory causes of death. 
All SAE reports must be e-mailed to the following e-mail address within 24 hours: 
 
 
If email is unavailable, SAEs may be transmitted via fax to the following number: 
 
In a study-related medical emergency situation, when the assigned medical monitors for the 
study cannot be reached by a caller, an on call physician can be reached 24 hours per day, 
7 days per week, via the 
 call center.  The following number is a chargeable telephone 
number allowing a global reach from both landlines and mobile phones and the internet page 
indicated below contains a list of country-specific toll-free telephone numbers that is 
provided.  It should be noted that not all countries globally have access to toll-free numbers 
as indicated on the “24/7 Medical Help desk” index.  Countries without toll-free numbers 
need to dial the chargeable number as indicated below.  Furthermore, toll-free numbers might 
not be available from mobile phones. 
 
 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions 
Suspected unexpected serious adverse reaction (SUSAR) is an SAE that occurs in a patient, 
the nature or severity of which is not expected per the applicable product information (e.g., 
the IB for an unauthorized investigational product or summary of product characteristics for 
an authorized product).  
Reporting and tracking of SUSARs will be in accordance to all applicable competent 
authority regulations.  The Ethics Committee and all investigators involved in this study will 
be informed according to national and international requirements. 


 
 
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10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments 
10.2.1 
Laboratory Assessments 
Samples will be collected for clinical chemistry, hematology, and urinalysis testing at the 
time points specified in Table 1. The parameters assessed are presented in Table 3. 
Blood for AFP will be drawn at Visits 3 and 9.  Fat-soluble vitamin levels including vitamins 
A and E, and 25-hydroxy vitamin D will be drawn at Visit 2 (vitamins A & E only), 
Visit 3 (25-hydroxy vitamin D only), Visit 6, and Visit 9.  Blood for INR (surrogate for 
vitamin K) will be drawn at Visits 2, 4, 6, 8, and 10.  Patients will fast (water intake only is 
permissible) for at least 4 hours prior to the collection of samples for vitamin A.  Exceptions 
can be made for infants, <12 months of age, if unable to fast for the full 4 hours.  If a patient 
has any vitamin level(s) that are outside of the reference range, vitamin supplementation 
adjustments may be required.  Target ranges and supplementation strategy guidelines can be 
found in Appendix 5. 
A serum pregnancy test will be performed at Visit 1 and Visit 9 if the patient is entering the 
A4250-008 study. A urine pregnancy test will be collected at all other visits for girls who 
have reached menarche.  If a urine pregnancy test is positive, a serum pregnancy test should 
be performed to confirm the pregnancy.  Study drug administration will be held pending the 
confirmatory results.  If the serum pregnancy test is negative, the patient can resume dosing.  
If the serum pregnancy test is positive, the patient should be discontinued (see 
Section 10.2.9). 
All samples will be processed and transported to a laboratory per instructions in the 
laboratory manual. 
The observed values will be recorded and assessed as “normal” or “abnormal not clinically 
significant” or “abnormal clinically significant”. 
Additional safety blood samples may be needed due to follow-up of an abnormal value or 
analysis failure.  The blood samples collected for safety laboratory analysis will be destroyed 
after the analyses have been completed.  


 
 
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Table 3 
Routine Laboratory Parameters 
Clinical Chemistry 
Hematology 
Urinalysis 
• 
Albumin 
• 
ALT 
• 
Alkaline phosphatase  
• 
AST 
• 
Bilirubin – total and direct 
• 
Calcium 
• 
Chloride 
• 
Creatinine 
• 
Creatine kinase 
• 
Gamma-glutamyl transferase 
• 
Potassium 
• 
Sodium 
• 
Hematocrit 
• 
Hemoglobin 
• 
Platelet count 
• 
Red blood cell count 
• 
White blood cell count and 
differential (neutrophils, 
eosinophils, basophils, 
lymphocytes, and monocytes) 
• 
Blood 
• 
Glucose 
• 
Ketones 
• 
Leukocytes 
• 
Nitrites 
• 
pH 
• 
Protein 
 
10.2.2 
Individual Patient Safety Monitoring 
10.2.2.1 
Liver Monitoring 
Strategies to monitor markers of liver disease throughout the study are outlined below where 
the ULN will be based on central laboratory reference values for age and gender. 
If isolated transaminase elevations are observed, defined as: 
1. Normal bilirubin AND absence of clinical hepatitis symptoms AND 
• ALT or AST ≥5 × ULN (if normal at baseline) or an absolute threshold of 800 U/L, 
whichever comes first  
• OR ALT or AST ≥3 × baseline (if abnormal at baseline) or an absolute threshold of 
800 U/L, whichever comes first 
Then: 
a) Repeat liver profile (AST, ALT, bilirubin, and prothrombin time [PT] or INR) within 
2 to 3 days 
b) Evaluate creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) 
c) As needed (for example, persistent ALT/total bilirubin elevation or worsening of liver 
function), consider evaluation for alternative etiologies 
Monitor the patient using close observation found in Section 10.2.2.2 
If any ONE of the following criteria are met: 
1. Transaminases (ALT or AST ≥3 × baseline or 800 U/L, whichever comes first) AND 
bilirubin (total bilirubin > 2 × ULN) elevations 


 
 
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2. Transaminase elevations alone (ALT or AST >10 × ULN or 5 × baseline or absolute 
threshold of 800 U/L, whichever comes first) in presence of normal LDH and CPK 
3. Total bilirubin increased, unrelated to hemolysis (elevated reticulocyte count) or 
established genetic diseases, such as Gilbert’s syndrome 
a. Doubling if total bilirubin was <3 mg/dL (equivalent to 51.3 µmol/L) at baseline  
b. OR Increase by >3 mg/dL (equivalent to 51.3 µmol/L) if total bilirubin was ≥3 mg/dL 
(equivalent to 51.3 µmol/L) at baseline 
4. INR increase refractory to vitamin K administration 
a. INR >1.5 if INR was normal at baseline  
b. OR Increase by >0.4 if INR was abnormal at baseline 
5. Any increase in total bilirubin and transaminases if accompanied by EITHER a symptom 
of clinical hepatitis (e.g., vomiting, nausea, right upper quadrant pain) OR immunological 
reaction (rash or >5% eosinophilia) 
Then: 
a) Interrupt study medication 
b) Initiate drug-induced liver injury work-up for alternative etiologies 
c) Repeat liver profile (AST, ALT, total bilirubin, direct bilirubin) and PT or INR within 
48 to 72 hours 
d) Monitor the patient using close observation found in Section 10.2.2.2 
e) If a patient lives in a remote area, they may be tested locally and the results 
communicated to the investigator site promptly 
10.2.2.2 
Close Observation 
• Repeat liver enzyme and serum bilirubin tests two or three times weekly.  Frequency 
of re-testing can decrease to once a week or less if abnormalities stabilize or the trial 
drug has been discontinued and the subject is asymptomatic  
• Obtain a more detailed history of symptoms and prior or concurrent diseases 
• Obtain a history of concomitant drug use (including non-prescription medications and 
herbal and dietary supplement preparations), alcohol use, recreational drug use, and 
special diets  
• Consider ruling out alternate etiology including acute viral hepatitis types A, B, C, D, 
and 
E; 
autoimmune 
or 
alcoholic 
hepatitis; 
non-alcoholic 
steatohepatitis; 
hypoxic/ischemic hepatopathy; and biliary tract disease 
• Obtain a history of exposure to environmental chemical agents.  


 
 
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• Obtain additional tests to evaluate liver function, as appropriate (e.g., INR, direct 
bilirubin) 
• Consider gastroenterology or hepatology consultations. 
10.2.2.3 
De-challenge/Re-challenge for Liver and Clinical Hepatitis Monitoring 
1. Re-challenge is not recommended: 
a. If a patient has had possible/probable drug-induced liver injury 
b. If a decompensation event has occurred (i.e., variceal hemorrhage, ascites, hepatic 
encephalopathy, etc.). 
2. If due to underlying cholestatic liver disease variability or another alternative etiology is 
identified AND liver tests returned to baseline, re-challenge may be considered after 
consultation with the sponsor Medical Monitor. 
3. If the ALT/total bilirubin elevations are observed after re-challenge, then repeat 
re-challenge is discouraged. 
If patient is permanently discontinued, monitoring should be continued as outlined in 
Section 10.2.2.5.  
10.2.2.4 
Diarrhea 
Study drug should be discontinued if a patient develops diarrhea with at least 1 of the 
following concomitant signs or symptoms: grossly bloody stools, vomiting, dehydration 
requiring treatment with oral or intravenous rehydration and/or electrolyte imbalances, fever 
(≥38 C) and/or the diarrhea persists for 7 or more days. 
If there is gross blood in stool, INR and platelets should be measured.  
Study drug will be reintroduced (re-challenge) when the symptoms have resolved.  If the 
diarrhea re-occurs within 1 week after the re-challenge with no alternate etiology, the patient 
will be permanently discontinued and monitored as outlined in Section 10.2.2.5. 
10.2.2.5 
Monitoring after Permanent Discontinuation of Study Drug Due to Safety 
Once study drug is permanently discontinued for liver, hepatitis, diarrhea, or other severe AE 
related to study drug, the patient will be monitored weekly until the laboratory and clinical 
parameters have normalized/stabilized. 
10.2.3 
Demographics/Medical and Surgical History 
Demographic information per country regulations (age, full date of birth, gender, race, and 
ethnicity), along with medical and surgical history, will be obtained and recorded in the eCRF 
at Visit 1. 
Medical and surgical history will be entered in the eCRF at Visit 1.  This includes date of 
diagnosis of PFIC, prior investigational medications for PFIC, historical (3 months prior to 


 
 
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Screening) LFT values (AST, ALT, and total bilirubin), ongoing medication, any surgery 
performed, any other diagnosis, and historical biopsy data.   
If a liver biopsy has been performed within 1 year prior to Screening, or during the study, the 
results will be recorded in the eCRF. 
10.2.4 
Clinical Genetic Testing 
For previous confirmatory clinical genetic testing results for PFIC Type 1 or 2 performed 
prior to Screening Visit 1, the confirmatory clinical genetic testing laboratory report will be 
verified to determine eligibility. 
If the historical clinical genetic result is equivocal, unavailable, or unobtainable, clinical 
genetic analysis to confirm pathologic biallelic variations in the ATP8B1 or ABCB11 genes 
will be performed and verified to determine eligibility.  No other diagnostic genetic testing 
will be offered. 
10.2.5 
Physical Examination 
A physician or suitably trained qualified assistant will perform a complete physical 
examination, including voluntary photography of skin lesions due to scratching, at Visits 1, 3, 
6, and 9.  A skin examination will be conducted at Visits 1, 3, 4, 6, 9, and 10 (see Table 1).  
A complete physical examination will include assessment of general appearance, eyes, ears, 
nose, throat, head/neck/thyroid, lymph nodes, cardiovascular, lungs/chest, abdomen, 
genitourinary, extremities, skin, musculoskeletal, neurologic, and other.  
Skin will be examined thoroughly and excoriations/scratch marks recorded.  
10.2.6 
Vital Signs 
Evaluation of vital signs will be performed at all visits.  This includes blood pressure (systolic 
and diastolic), pulse, respiratory rate, temperature, height (or length depending on age), and 
body weight (using a certified weight scale) at clinic visits.  BMI will be calculated. 
10.2.7 
Abdominal Ultrasound 
Ultrasound of the liver and spleen will be performed at randomization (Visit 3) and at EOT 
(Visit 9).  Liver size, echogenicity, and presence of masses/nodules as well as spleen size will 
be recorded. 
10.2.8 
Overdose 
A4250 is minimally absorbed and has a very low systemic availability.  Based on toxicology 
data, for study purposes any dose exceeding a total of >3 mg/kg body weight of A4250 taken 
as a single dose or as a cumulative dose within 24 hours is defined as an overdose.   
The no-adverse-effect dose level in the most sensitive species (defined as 20 mg/kg/day) in 
the rat 1 month toxicity study gives a human equivalent dose of approximately 194 mg/day in 


 
 
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a 60 kg person.  This dose is approximately 10-fold higher than the maximum possible dose 
predicted in human studies (20 mg).  
The investigator/investigative staff should immediately and not later than within 24 hours of 
knowledge of the overdose, be reported by the investigator or delegate on the SAE report 
form.  In the event of an A4250 overdose, the patient should be monitored closely. 
10.2.9 
Pregnancy 
If a pregnancy is discovered in a female patient enrolled in the study before the end of dosing, 
the patient will be immediately discontinued from the study and will attend the same visits as 
a prematurely withdrawn patient.  If the pregnancy is discovered in a female patient enrolled 
in the study after the end of dosing, the patient will continue in the study per protocol.  If a 
pregnancy occurs in a male patient’s partner at any time during the study, the pregnancy 
should also be reported and followed. 
Pregnancy is not considered to be an AE.  However, if the patient has been dosed with the 
study drug, the pregnancy must be reported on the Paper Pregnancy Notification Form 
immediately within 24 hours after recognition to Safety Manager (please refer to Section 
10.1.3).  Date of exposure and as far as possible, details of the period of gestation at the time 
of exposure must be given. 
The pregnancy should be followed to determine outcome, including spontaneous termination, 
details of birth, and presence of any birth defects, congenital anomalies or newborn or 
maternal complication.  The infant who was exposed in-utero will be followed for up to 
2 years after delivery.  Individual cases with an abnormal outcome in association with the 
study drug should be reported on an expedited basis, i.e., reported rapidly to a competent 
authority. 


 
 
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11 
STATISTICAL EVALUATION 
All personnel involved with the analysis of the study will remain blinded until database lock 
for the double-blind Treatment Period and until major protocol violations have been 
identified. 
All statistical analyses will be performed using SAS version 9.3 or higher. 
11.1 
Sample Size and Power 
For the EU and RoW, the primary analysis will relate to the serum bile acid responder 
endpoint.  For the US, the primary analysis will relate to the pruritus endpoint. For each 
primary endpoint, a pooled analysis for the closed testing procedure will be applied to control 
the 1-sided overall type I error rate for two treatment comparisons vs. the placebo at the 0.025 
level (Kane et al, 2015, Lehmacher et al, 1991, and Marcus et al, 1976). The study will enroll 
60 to 70 patients in order to obtain at least least 20 evaluable patients in each arm. For each 
primary endpoint, simulations with 5000 iterations using 20 patients per arm were conducted 
to estimate the power after multiplicity adjustment, resulting in a standard error of <0.7% for 
each estimated power.   
Based on the Phase 2 A4250-003 study data both dose groups are predicted to have similar 
treatment effects for both serum bile acids and pruritus. For serum bile acids, simulated 
proportions were analyzed using the CMH test to generate the following 1-sided p-values: 
both A4250 arms pooled vs. placebo, low dose vs. placebo, and high dose vs. placebo. 
Assuming 60% responders in the A4250 arms and 10% responders in the placebo arm, the 
power to claim significance for a particular A4250 arm after multiplicity adjustment is 
approximately 94%. The power to claim significance for at least one arm or both arms are 
approximately 99% and 91%, respectively.  
For the proportion of positive pruritus assessments at the subject level in pruritus scores, 
simulated proportions were analyzed using ANCOVA to generate the following 1-sided p-
values: both A4250 arms pooled vs. placebo, low dose vs. placebo, and high dose vs. 
placebo. With an effect size of 1.0526, the simulated power to claim significance for a 
particular arm after multiplicity adjustment is approximately 89%. The probability to claim 
significance for at least one arm or both arms are approximately 95% and 83%, respectively.   
11.2 
Statistical Methods 
11.2.1 
Statistical Analysis Sets 
Safety Analysis Set 
The safety analysis set will consist of all patients who received at least 1 dose of study drug.  
Patients will be analyzed according to the treatment they actually received.  The safety 
analysis set will be the primary analysis set for safety analyses.  
Full Analysis Set 


 
 
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The full analysis set (FAS) will consist of all randomized patients who received at least 
1 dose of assigned treatment.  Patients will be analyzed in the treatment group they were 
randomized to even if they received incorrect study drug.  The FAS will be the primary 
analysis set for efficacy analyses. 
Per Protocol Analysis Set 
The per protocol (PP) analysis set is a subset of the FAS, and will consist of all randomized 
patients for whom no major protocol violation (no violation which may affect the study 
efficacy outcome) is documented.  The PP analysis set will provide supportive data for the 
efficacy analyses. 
Allocation of patients to the PP analysis set will be performed before unblinding of the study. 
11.2.2 
Methods of Statistical Analyses 
11.2.2.1 
General Principles 
The primary objective of this study is to demonstrate the efficacy of repeated daily doses of 
40 µg/kg/day and 120 µg/kg/day A4250 in children with PFIC Types 1 and 2.  
In the EU and RoW, for the primary efficacy variable of fasting bile acid responder endpoint 
(at least a 70% reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the 
end of treatment), the Cochran Mantel Haenszel (CMH) test stratified by PFIC class and age 
class will be performed to compare the proportion in fasting bile acid responder endpoint at 
the end of treatment (Week 22 and 24) in the two A4250 dose groups to placebo group. To 
ascertain that all data are used in the CMH analysis, neighboring strata will be pooled if all 
subjects in a stratum have the same response. Dropouts will be treated as non-responders. The 
proportion together with corresponding 95% CI, odds ratio and corresponding 95% Clopper-
Pearson exact CI and p-value for the CMH test will be presented.   
In the US, for the primary efficacy variable of the proportion of positive pruritus assessments 
at subject level over the 24-week treatment period, an analysis of covariance (ANCOVA) will 
be used. The model will include treatment arm, AM baseline pruritus score, PM baseline 
pruritus score, and randomization stratification factors, i.e. PFIC class and age class. LS 
mean(SE) by treatment arm and LS mean difference (SE), 95% confidence intervals, and p-
values (where applicable) between treatments (120 µg/kg/day and 40 µg/kg/day, respectively) 
vs. placebo will be provided. If there are concerns on model assumptions, i.e. normality and 
homogeneity of variances, a non-parametric ANCOVA based on the rank scores will be used 
as a sensitivity analysis. LS mean/SE on the outcome by treatment arm and LS mean 
difference/SE between active dose and placebo will be provided.  
The procedure for multiplicity adjustment is specified as follows:    
In the closed testing procedure, the low and high dose groups will be pooled to compare with 
the placebo group first. If the 1-sided p-value is ≤0.025, the 1-sided p-values for low dose vs. 


 
 
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placebo and high dose vs. placebo will be calculated, respectively. If both individual p-values 
are ≤0.025, a significant treatment effect will be declared on both dose groups. If only one of 
them is ≤0.025, a significant treatment effect will be declared on the corresponding dose 
group.  
Other secondary and exploratory outcome variables will provide supportive efficacy and 
safety information regarding the differences between the treatment groups. No adjustments 
will be performed for multiple comparisons when testing secondary and exploratory efficacy 
variables. 
If not otherwise specified, the baseline value of a parameter is defined as the last assessment 
of that parameter prior to the first intake of study treatment. 
11.2.2.2 
Missing Data 
In the primary analysis of the pruritus endpoint, all intermittently missing assessments will be 
classified as negative pruritus assessments. Similarly, all planned assessments after the 
intercurrent events (premature treatment discontinuation, death or initiation of rescue 
treatments such as biliary diversion surgery or liver transplantation) will be counted as 
negative pruritus assessments. However, as described above, all 336 assessments planned 
over the 24-week treatment period will be included in the denominator of the endpoint 
definition.  
In the primary analysis of fasting bile acid responder endpoint, dropouts and patients with a 
missing average at the end of the treatment will be treated as non-responders. Patients in need 
of surgical rescue, i.e. biliary diversion and listing for liver transplant will be classified as 
non-responders. Continued collection of efficacy data for patients who discontinue treatment 
will be made as far as possible. 
11.2.2.3 
Demographic and Baseline Characteristics 
Descriptive summaries of demographics and other baseline characteristics (including medical 
and surgical history) will be presented by treatment group and overall using the FAS.  
Prior and concomitant medication use during the study will be summarized by treatment 
group and overall using the safety analysis set. 
11.2.2.4 
Subject Disposition 
The following will be summarized (by treatment group and overall where applicable): 
• Patients enrolled (who signed the informed consent) 
• Patients randomized 
• Patients treated 
• Patients completing the study 
• Patients withdrawing early (including withdrawal reason) 


 
 
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Additionally, patients enrolled, included in the safety, in the FAS, and in the PP analysis set 
will be summarized by region. 
 
11.2.2.5 
Evaluation of Primary Efficacy Variables 
For EU and RoW, the primary responder endpoint based on change in fasting bile acids will 
be analyzed in terms of the proportion of patients achieving the event of interest. Patients in 
need of surgical rescue, i.e. biliary diversion and listing for liver transplant, will be classified 
as non-responders. Dropouts will be treated as non-responders.  
CMH stratified by PFIC class and age class will be performed to compare the proportion in 
fasting bile acid responders at the end of treatment (Week 22 and 24) in the two A4250 dose 
groups to the placebo group. To ascertain that all data are used in the CMH analysis, 
neighboring strata will be pooled if all subjects in a stratum have the same response. The 
proportion together with corresponding 95% CI, odds ratio and corresponding 95% Clopper-
Pearson exact CI and p-value for the CMH test will be presented 
Logistic regression will be performed as a supportive analysis to compare the proportion in 
fasting bile acid responders at the end of treatment (Week 22 and 24) in the two A4250 dose 
groups to the placebo group. The model will include treatment arm, baseline value, and 
randomization stratification factors, i.e. PFIC class and age class. Baseline value will be 
calculated as the average of serum bile acid values at randomization and the previous visit 
(Clinic Visit 2).   
In the US, for the primary efficacy variable of the proportion of positive pruritus assessments 
at the subject level over the 24-week treatment period, ANCOVA will be used to analyze the 
pruritus endpoint. The model will include treatment arm, AM baseline pruritus score, PM 
baseline pruritus score, and randomization stratification factors, i.e. PFIC class and age class. 
LS mean(SE) by treatment arm and LS mean difference (SE), 95% confidence intervals, and 
p-values (where applicable) between treatments (120 µg/kg/day and 40 µg/kg/day, 
respectively) vs. placebo will be provided. If there are concerns on model assumptions, i.e. 
normality and homogeneity of variances, a non-parametric ANCOVA based on the rank 
scores can be used. LS mean/SE on the outcome by treatment arm and LS mean 
difference/SE between active dose and placebo will be provided.  
Sensitivity analysis will be conducted to assess the impact of intermittently missing data as 
well as intercurrent events and will be specified in the SAP. Subgroup analyses will also be 
performed for each of the 3 age groups (6 months to 5 years, 6 to 12 years, and 13 to ≤18 
years) and for patients who were not included in the previous Study A4250-003. 
11.2.2.6 
Evaluation of Secondary and Exploratory Efficacy Variables 
The change in the secondary endpoints such as serum bile acids, total bilirubin, ALT, growth, 
Albireo PRO and ObsRO sleep parameters, and patient-reported itch severity will be 


 
 
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summarized by visit using descriptive statistics. Change in growth (weight for age and BMI 
for age) will also be displayed using graphical presentations.  
Proportion of responders at Weeks 12 and 24 based on the PRO/ObsRO instruments 
estimated from both distribution and anchor-based approaches will be analyzed using the 
same model as specified for the primary analysis for EU and RoW. 
Proportion of individual assessments meeting the definition of a positive pruritus assessment 
at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, 
respectively, or the proportion of positive pruritus assessments at each 4-week interval over 
the 24-week treatment period for AM or PM, respectively will be analyzed using the same 
model as specified for the primary analysis for US.   
Undergoing biliary diversion surgery and/or liver transplant, all-cause mortality, and liver 
decompensation events will mainly be analyzed by using descriptive statistics. Kaplan-Meier 
curves will be used when appropriate for time to event data.  
Comparisons of the change from baseline at Week 24 in PedsQL between the treatment 
groups will be conducted using ANCOVA. The model will include terms for baseline, PFIC 
class, age class, and treatment. 
Other exploratory variables (Global Symptom Relief, biochemical markers and measures of 
bile acid synthesis, additional Albireo PRO and ObsRO sleep parameters, PELD/MELD 
score, APRI score, and Fib-4 score) will be analyzed descriptively for categorical and 
continuous data, as applicable. For continuous data, the change from baseline will be 
analyzed in addition to the actual visit values. For categorical data, shift tables or frequency 
and percentages of patients will be presented as appropriate. Additional details on secondary 
and exploratory efficacy variables and subgroup analyses by age class (6 months to 5 years, 6 
to 12 years, and 13 to ≤18 years) will be specified in the SAP.  
11.2.2.7 
Evaluation of Safety Variables 
Safety data will be analyzed using descriptive statistics and summaries by treatment group of 
SAEs, AEs, vital signs, clinical laboratory tests (hematology, clinical chemistry and 
urinalysis), and concomitant medication.  Analyses will be performed using the safety 
analysis set.  
Summaries of AEs (coded according to the Medical Dictionary for Regulatory Activities 
[MedDRA] system organ class [SOC] and MedDRA preferred term) will include the 
following:  
• Overview of the incidence of TEAEs (TEAEs, drug-related TEAEs, TEAEs leading to 
study discontinuation, and treatment-emergent SAEs) 
• TEAEs by SOC and preferred term 
• Intensity of TEAEs by SOC and preferred term 


 
 
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• Drug-related TEAEs by SOC and preferred term 
• TEAEs leading to study discontinuation by SOC and preferred term 
• Treatment-emergent SAEs by SOC and preferred term 
Concomitant medication use during the Treatment Period will be summarized by Anatomical 
Therapeutic Chemical (ATC) class and World Health Organization (WHO) preferred name.  
Summaries of vital signs will be presented.  For each visit, the actual results and the change 
from baseline, and the number and percentage of patients with potentially clinically 
significant (PCS) values observed post-baseline will be presented. 
Summaries of clinical safety laboratory data will be presented.  For each visit, the actual 
result and the change from baseline, and the number and percentage of patients with PCS 
values observed post-baseline will be presented. 
Data listings will be provided for each patient for all safety parameters. 
11.2.2.8 
Compliance and Exposure 
Exposure will be analyzed by calculating the number of days with exposure to study drug.  
Results will be presented by treatment group using the safety analysis set.  
The percentage compliance will be described by treatment group and the number of patients 
with a compliance <80%, between 80% and 120%, and >120% will be presented based on the 
safety analysis set. 
11.2.3 
Blinded Analyses to Confirm Responder Definitions 
A blinded analysis of Albireo ObsRO and PRO eDiary data will be performed after 50% or 
more of the planned sample has completed the Week 24 Follow-Up Period.  The blinded 
analysis will be used to estimate a threshold of clinically meaningful change (i.e., responder 
definition) in Albireo ObsRO and PRO pruritus scores.  The analysis will be performed by a 
group independent from both the study team and Albireo.  Blinded data will be used for this 
analysis; data will be collapsed across treatment groups. 
The analyses will include distribution- and anchor-based analyses to examine a responder 
definition for the Albireo PRO/ObsRO pruritus scores.  Distribution-based analyses will 
include calculation of the SD and standard error of measurement for the baseline Albireo 
PRO and ObsRO scores.  Anchor-based analyses will involve examining the degree of 
change on the Albireo PRO and ObsRO scores from baseline to Week 24 for patients who 
experience change in pruritus according to PGIC and symptom items.  Details of this analysis 
will be included in a stand-alone SAP for assessing the measurement characteristics 
(reliability, validity, sensitivity to change, responder definition) of the Albireo PRO and 
ObsRO items. 


 
 
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11.2.4 
Data Safety Monitoring Board 
A DSMB consisting of sponsor-independent clinical and statistical experts will be 
established.  The DSMB will periodically (approximately on a quarterly basis) meet for the 
review of accumulating study data, including safety (AE and laboratory data) and liver 
values.  
The DSMB will have access to unblinded data.  The DSMB will make recommendations for 
the remaining part of the study (further details will be provided in the DSMB charter).  The 
DSMB may recommend continuing with the study as planned or stopping the study early for 
safety reasons.  The DSMB will submit its recommendations in writing to Albireo 
Medical/
 who are responsible for responding to the recommendations of the DSMB and 
taking appropriate action.  The investigators will only be informed by Albireo Medical/
 
if the study requires a protocol amendment or is stopped.  The DSMB may choose to make 
additional evaluations at any time if they feel this is warranted from a safety point of view.  
The DSMB will act according to its own written standard operating procedure described in a 
charter and will prepare written minutes of its meetings.  The charter of the DSMB will be 
stored in the trial master file.  The DSMB will maintain records of its meetings and these will 
become part of the study file when the study is complete.  In order not to disseminate 
unblinded data and to ensure that all staff involved in the conduct and final analysis of the 
study remain blind to the data, only the members of the DSMB and the unblinded DSMB 
statistician will have access to these data. 
 


 
 
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12 
DIRECT ACCESS TO SOURCE DATA/NOTES 
The investigator/institution shall provide direct access to source data/documents for 
study-related monitoring, audits, IEC/Institutional Review Board (IRB) review and 
regulatory inspection. 


 
 
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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
13.1 
Conduct of the Study 
Albireo/
 shall implement and maintain quality control and quality assurance procedures 
with written standard operating procedures to ensure that the study is conducted and data are 
generated, documented, and reported in compliance with the protocol, ICH GCP and 
applicable regulatory requirements. 
This study shall be conducted in accordance with the provisions of the Declaration of 
Helsinki (October 1996) and all revisions thereof, and in accordance with United States Food 
and Drug Administration (FDA) regulations (Code of Federal Regulations, Sections 312.50 
and 312.56) and with ICH GCP (CPMP 135/95). 
The investigator may not deviate from the protocol without a formal protocol amendment 
having been established and approved by an appropriate competent authority and IEC/IRB, 
except when necessary to eliminate immediate hazards to the patient or when the change(s) 
involve(s) only logistical or administrative aspects of the study.  Any deviations may result in 
the patient having to be withdrawn from the study and render that patient non-evaluable. 
13.2 
Study Monitoring 
Before study initiation, at a site initiation visit or at an investigator’s meeting, an Albireo 
representative or designee will review the protocol and eCRF with the investigators and the 
investigative staff.  During the study, the clinical monitor (clinical research associate [CRA]) 
will visit the site regularly to check the completeness of patient records, the accuracy of 
entries on the eCRFs, the adherence to the protocol and to GCP, the progress of enrollment, 
and to ensure that study drug is being stored, dispensed, and accounted for according to 
specifications. 
The investigator must ensure that eCRFs are completed within a timely period of patient 
visits, as per individual site agreements, and must allow the CRA and Albireo representative 
or designee periodic access to patient records and all study-related materials, including 
relevant hospital or clinical records, to confirm their consistency with the eCRF entries.  No 
information in these records about the identity of the patients will leave the study center.  
Albireo monitoring standards require full verification for the presence of the signed ICF, 
adherence to the inclusion/exclusion criteria, documentation of SAEs, and recording of 
primary efficacy and safety variables.  The CRA will review source data compared with the 
eCRFs and will verify source data according to the study-specific monitoring plan.  The 
design of the study, the frequency of patient visits, and the site enrollment rate will determine 
the frequency of monitoring visits.  Upon study completion, the CRA will visit the site to 
conduct a study termination visit, which will include collection of any outstanding 
documentation. 


 
 
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It is recommended that the investigator/investigative staff log into the eCRF system every 
working day or at minimum twice weekly in order to provide a prompt response to queries.  
The investigator/investigative staff should respond to queries and make any relevant changes 
to the study data within 3 working days. 


 
 
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14 
ETHICS 
14.1 
Independent Ethics Committee/Institutional Review Board 
Prior to the start of the study, the investigator is responsible for ensuring that the protocol and 
consent form have been reviewed and approved by a relevant IEC/IRB.  The IEC/IRB shall 
be appropriately constituted and perform its functions in accordance with FDA ICH GCP and 
local requirements as applicable. 
The IEC/IRB shall approve all protocol amendments (except for logistical or administrative 
changes), written informed consent documents and document updates, patient recruitment 
procedures (e.g., advertisements), written information to be provided to the patients and 
caregivers, IB, available safety information, information about payment and compensation 
available to patients, the investigator's curriculum vitae and/or other evidence of 
qualifications and any other documents requested by the IEC/IRB and regulatory authority 
(competent authority) as applicable. 
14.2 
Written Informed Consents and Assents 
The investigator (physician) or investigative staff, in accordance with local regulations, will 
explain to each patient (or legally authorized representative) the nature of the study, its 
purpose, the procedures involved, the expected duration, alternative treatment, the potential 
risks and benefits involved, and any discomfort that may occur.  Each caregiver/patient will 
be informed that participation in the study is voluntary and that he/she or their child may 
withdraw from the study at any time and that withdrawal of consent will not affect his/her 
subsequent medical treatment or relationship with the treating physician. 
Caregivers/patients will be informed that they/their children may experience side-effects or 
be at risk for symptoms, illnesses, or complications that cannot be foreseen by Albireo.  As 
with other medications, people treated with A4250 may be at risk of developing allergic 
reactions or anaphylaxis.  Caregivers/patients will be advised that study procedures include 
regular blood sampling for measurement of safety parameters and biological markers and that 
some minor risks are associated with these procedures. 
This informed consent should be given by means of a signed ICF, written in non-technical 
language in accordance with ICH GCP, the Declaration of Helsinki, and regulatory 
authorities.  The caregiver(s)/patient should read and consider the statements before signing 
and dating them and should be given a copy of each signed document.  If written consent is 
not possible, oral consent can be obtained if witnessed and followed by a signed statement 
from one or more persons not involved in the study, indicating why the patient was unable to 
sign the form.  No patient can enter the study before his/her or caregiver(s) informed consent 
has been obtained, as required by country regulations. 
The ICF is part of the protocol and must be submitted by the investigator/investigative staff 
with the protocol for IEC approval.  Albireo will supply an ICF which complies with 


 
 
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regulatory requirements and country laws and is considered appropriate for the study.  Any 
changes to the ICF suggested by the investigator must be agreed to by Albireo before 
submission to the IEC and a copy of the approved version must be provided to the clinical 
monitor after IEC approval. 


 
 
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15 
DATA HANDLING AND RECORD KEEPING 
15.1 
Case Report Forms/Source Data Handling 
The investigator shall be provided with standardized eCRFs and shall ensure that all data 
from patient visits are promptly entered into the eCRFs in accordance with the specific 
instructions given.  The investigator must sign each eCRF to verify the integrity of the data 
recorded. 
A list of the normal ranges for all laboratory tests to be undertaken forms part of the 
documentation to be collated prior to study start.  As a central laboratory has been selected to 
conduct any or all tests, it is essential that all samples be analyzed at that laboratory. 
The investigator must maintain source documents such as laboratory reports, consultation 
reports, and complete medical history and physical examination reports. 
As no ethnicity data can be collected in the source document for study sites in France, the 
eCRF will be the Source Document for “origines ethniques”,  
15.2 
Retention of Essential Documents 
Essential documents, as defined by ICH GCP, include: the signed protocol and any 
amendment(s); copies of the completed eCRFs (for site archiving, compact discs of eCRF 
data for specific patients will be provided); signed ICFs; hospital records and other source 
documents; IEC approvals and all related correspondence including approved documents; 
drug accountability records; study correspondence; and a list of patients’ names and 
addresses. 
The investigator/investigative staff must retain copies of these essential documents for the 
period specified by ICH GCP and by applicable regulatory requirements.  The 
investigator/investigative staff will inform Albireo of the location where the essential 
documents are stored and must contact Albireo for approval before disposing of any essential 
documents.  The investigator/investigative staff should take measures to prevent accidental or 
premature destruction of these documents. 


 
 
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16 
FINANCING AND INSURANCE 
Financing and insurance are addressed in a separate agreement. 
 


 
 
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17 
PUBLICATION POLICY 
Albireo will retain the ownership of all data.  When the study is complete, Albireo shall 
arrange the analysis, tabulation of data and preparation of a clinical study report. Albireo may 
also use the data for publication, presentation at scientific meetings and submission to 
regulatory authorities.  All proposed publications based on this study must be subject to the 
sponsor's approval requirements. 
 


 
 
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18 
REFERENCE LIST 
1. A4250 Investigator’s Brochure. 
2. Alissa FT, Jaffe R, Shneider BL. Update on progressive familial intrahepatic cholestasis. 
J Pediatr Gastroenterol Nutr. 2008;46(3):241-52. 
3. Davit-Spraul A, Gonzales E, Baussan C, et al. Progressive familial intrahepatic 
cholestasis. Orphanet J Rare Disease 2009;4:1-12. 
4. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and 
functions. Front Biosci (Landmark Ed). 2009 Jan 1;14:2584-98. 
5. Hori T, Justin H, Nguyen JH and Uemoto S. Progressive familial intrahepatic cholestasis. 
Hepatobiliary pancreat Dis Int. 2010; 9(6):570-8. 
6. Jacquemin E. Progressive familial intrahepatic cholestasis. Genetic basis and treatment. 
Clin Liver Dis. 2000;4(4):753-63. 
7. Miethke AG, Zhang W, Simmons J, et al. Pharmacological inhibition of ASBT changes 
bile composition and blocks progression of sclerosing cholangitis in mdr2 knockout mice. 
Hepatology. 2016; 63(2):512–23. 
8. Murray CS, Rees JL. Are subjective accounts of itch to be relied on? The lack of relation 
between visual analogue itch scores and actigraphic measures of scratch. Acta Derm 
Venereol. 2011;91(1):18-23. 
9. Pawlikowska L, Strautnieks S, Jankowska I, et al. Differences in presentation and 
progression between severe FIC1 and BSEP deficiencies. J Hepatol. 2010;53(1):170-8. 
10. Shneider BL, Magee JC, Bezerra JA, et al. Efficacy of fat-soluble vitamin 
supplementation in infants with biliary atresia. Pediatrics, 2012;130(3):e607-14.  
11. Suchy FJ, Sokol RJ, Balistreri WF, eds. Liver Disease in Children. 3rd Edition. New 
York, NY: Cambridge University Press; 2007:310–4. 
12. Venkat VL, Shneider BL, Magee JC, et al. Total Serum Bilirubin Predicts Fat-Soluble 
Vitamin Deficiency Better Than Serum Bile Acids in Infants with Biliary Atresia. J 
Pediatr Gastroenterol Nutr. 2014;59(6):702-7.  
13. Whitington PF, Whitington GL. Partial external diversion of bile for the treatment of 
intractable 
pruritus 
associated 
with 
intrahepatic 
cholestasis. 
Gastroenterology. 
1988;95(1):130-6. 
 
 


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19 
APPENDICES 
Appendix 1 
Concomitant Medication Guidelines 


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EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Final Amendment 04, 01 March 2019 
Appendix 2 
Diary Questions including Albireo Patient-Reported 
Outcomes/Observer-Reported Outcome Instrument 
Page 77


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Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) 
Page 80


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Final Amendment 04, 01 March 2019 
Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaire 
Page 85


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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Final Amendment 04, 01 March 2019 
Appendix 5 
Guideline for Fat-Soluble Vitamin Supplementation 
Page 106


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M5.3.5.1 – Protocol A4250-005 
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Appendix 6 
Contraceptive Requirements 
Page 109


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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
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Final Amendment 04, 01 March 2019 
Appendix 7 
Blood Volumes 
Page 112


 
 
 
CLINICAL STUDY PROTOCOL A4250-005 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
 
IND Number:   
130591 
EudraCT Number 
2017-002338-21 
Test Product: 
A4250 
Indication: 
Progressive Familial Intrahepatic Cholestasis (PFIC) 
Sponsor: 
Albireo AB 
Development Phase: 
Phase 3 
Sponsor Signatory: 
 
Sponsor Medical Monitor: 
 
Principal Investigator: 
 
Date of the Protocol: 
22 November 2018 
Version of the Protocol: 
Final Amendment 03 
 
 
 
 
Confidential 
The information in this document contains trade secrets and commercial information that are 
privileged or confidential and may not be used, divulged, published, or otherwise disclosed 
without the written consent of Albireo AB.  These restrictions on disclosure apply equally to 
all future information supplied to you or generated by you in connection with the study. 




 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 3 
Final Amendment 03, 22 November 2018 
 
INVESTIGATOR SIGNATURE PAGE 
 
PROTOCOL TITLE:  A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children with Progressive 
Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
 
PROTOCOL NUMBER:  A4250-005 
I have read this protocol and agree that it contains all necessary details for performing this 
study.  I will conduct the study as outlined herein and will complete the study within the time 
designated, in accordance with all stipulations of the protocol and in accordance with Good 
Clinical Practice (GCP), local regulatory requirements, and the Declaration of Helsinki. 
I will provide copies of the protocol and all pertinent information to all individuals 
responsible to me who assist in the conduct of this study.  I will discuss this material with 
them to ensure that they are fully informed regarding the study drug and the conduct of the 
study. 
I will use only the informed consent approved by the Independent Ethics Committee (IEC) 
and will fulfil all responsibilities for submitting pertinent information to the IEC responsible 
for this study. 
I agree that the sponsor (Albireo AB) shall have access to any source documents from which 
case report form information may have been generated. 
I further agree not to originate or use the name of Albireo AB, A4250 in any publicity, news 
release, or other public announcement, written or oral, whether to the public, press, or 
otherwise, relating to this protocol, to any amendment to the protocol, or to the performance 
of this protocol without the prior written consent of Albireo AB. 
 
 
 
 
 
 
Name of Investigator 
 
 
 
 
 
 
Signature 
 
Date (day/month/year) 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 4 
Final Amendment 03, 22 November 2018 
 
1 
ADMINISTRATIVE INFORMATION 
A Double-blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
Protocol No.: 
A4250-005 
Date of the initial Protocol: 
20 September 2017 
Date and Number of Amendment(s): 
Amendment 01, 06 December 2017 
 
Amendment 02, 10 May 2018 
 
Amendment 03, 22 November 2018 
Sponsor: 
Albireo AB 
 
Arvid Wallgrens backe 20 
 
413 46 Gӧteborg 
Sweden 
Clinical Research Organization: 
 
 
 
 
 
 
 
Sponsor Signatory: 
 
 
  
 
  
 
  
 
 
  
Sponsor Medical Monitor: 
 
 
 
 
Principal Investigator: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Page 5 
Final Amendment 03, 22 November 2018 
2 
STUDY SYNOPSIS 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Title of Study: 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate Efficacy and Safety of A4250 
in Children with Progressive Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
Principal Investigator: 
 
Study Centers: 
Up to 50 sites will be initiated for this study in the United States (US), Canada, European Union (EU), Middle 
East, and Australia. 
Publication(s): 
None. 
Planned Study Period: 
Q2 2018 to Q4 2019 
Development Phase: 
Phase 3 
Objectives: 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day A4250 in children with 
progressive familial intrahepatic cholestasis (PFIC) Types 1 and 2, as determined by the following: 
•
Proportion of patients experiencing at least a 70% reduction in fasting serum bile acid (s-BA)
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L compared to placebo 
after 24 weeks of treatment 
•
The proportion of positive pruritus assessments at the subject level over the 24-week Treatment Period
Secondary Objectives 
•
To evaluate the effect of A4250 on serum alanine aminotransferase (ALT) concentration
•
To evaluate the effect of A4250 on growth
•
To evaluate the effect of A4250 on sleep disturbance
•
To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or liver
transplantation)
•
To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks
Methodology: 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the efficacy and safety of 
40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in children with PFIC (deficiencies of familial 
intrahepatic cholestasis-1 or bile salt export pump).  
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment Period and a 4-week 
Follow-up Period.  After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of A4250, or a 
matching placebo.  Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008) in which all patients will receive 
active treatment. 
There will be up to 10 clinic visits during the study and one scheduled telephone contact. 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
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Final Amendment 03, 22 November 2018 
 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Number of Patients: 
Approximately 60 patients diagnosed with PFIC-1 or PFIC-2 will be randomized.  The study will target 15% of 
enrolled patients to be PFIC-1 patients.  If enrollment of all PFIC-2 patients is completed and the 15% 
enrollment of PFIC-1 patients has not yet been achieved, then enrollment of PFIC-2 patients will continue in 
order to reach the total study target enrollment. 
Diagnosis and Main Criteria for Inclusion: 
1. 
A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages of ≥6 months 
and ≤18 years at Visit 1 with a body weight above 5 kg 
2. 
Patient must have clinical genetic confirmation of PFIC-1 or PFIC-2 through identification of biallelic 
pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. 
Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, taken as the 
average of 2 samples at least 7 days apart (Visits 1 and 2) prior to randomization  
4. 
Patient must have history of significant pruritus and a caregiver-reported observed scratching in the 
electronic diary (eDiary) average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to randomization 
5. 
Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  Patients who 
turn 18 years of age (or legal age per country) during the study will be required to re-consent in order 
to remain in the study 
6. 
Patients are expected to have a consistent caregiver for the duration of the study 
7. 
Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use an eDiary 
device as required by the study 
Test Product, Dose and Mode of Administration: 
A4250, 40 µg/kg/day or 120 µg/kg/day, orally administered 
Reference Therapy, Dose and Duration of Administration: 
Matching placebo, orally administered 
Duration of Treatment: 
24 weeks 
Variables 
Efficacy:  
Primary Efficacy Endpoints 
The primary efficacy endpoints are: 
• 
EU and rest of world (RoW): Proportion of patients experiencing at least a 70% reduction in fasting 
s-BA concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L compared to 
placebo after 24 weeks of treatment. 
• 
US: Proportion of positive pruritus assessments at the subject level over the 24-week Treatment Period. 
Secondary Efficacy Endpoints 
Secondary efficacy endpoints include the following: 
• 
EU and RoW: Proportion of positive pruritus assessments at the subject level over the 24-week 
Treatment Period.  
• 
US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA concentration from 
baseline to the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 weeks of 
treatment. 
 
All Regions: 
All secondary endpoints will be compared to placebo. 
• 
Change from baseline to Week 12 and to Week 24 in fasting s-BA 
• 
Change from baseline to Week 12 and to Week 24 in serum ALT concentration 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 7 
Final Amendment 03, 22 November 2018 
 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
• 
Change in growth from baseline to Week 12 and Week 24 
• 
Proportion of responders for pruritus scores at Weeks 12 and 24 based on the Albireo PRO and ObsRO 
instruments  
• 
Change in sleep parameters measured with the Albireo PRO and ObsRO instruments from baseline 
over the 24-week Treatment Period  
• 
Proportion of individual assessments meeting the definition of a positive pruritus assessment at the 
subject level over the 24-week Treatment Period. A positive pruritus assessment includes an itch score 
≤1, or at least a one-point drop from baseline based on the Albireo PRO instrument; only patients ≥8 
years of age will complete the Albireo PRO instrument 
• 
Proportion of individual assessments meeting the definition of a positive pruritus assessment at the 
subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, respectively, or the 
proportion of positive pruritus assessments at each 4-week interval.  
• 
Proportion of individual AM assessments meeting the definition of a positive pruritus assessment at the 
subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, respectively, or the 
proportion of positive pruritus assessments at each 4-week interval.  
• 
Proportion of individual PM assessments meeting the definition of a positive pruritus assessment at the 
subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, respectively, or the 
proportion of positive pruritus assessments at each 4-week interval.  
• 
Number of patients undergoing biliary diversion surgery or being listed for liver transplantation 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• 
Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, caregiver, and 
clinician via the patient global impression of change (PGIC) items 
• 
Change from baseline in fasting s-BA at Week 4 Change in pruritus, as indexed by caregiver-reported 
observed scratching, from baseline by each 4 week interval of the Treatment Period as measured by the 
Albireo ObsRO instrument 
• 
Change from baseline by each 4-week interval of the Treatment Period in patient-reported and 
observer-reported night time itching and scratching severity scores, respectively Change from baseline 
by each 4-week interval of the Treatment Period in patient-reported and observer-reported morning 
time itching and scratching severity scores, respectively 
• 
Change from baseline by each 4-week interval of the Treatment Period in pooled pruritus score 
including observer-reported scratching for patients <8 years of age and patient-reported itch severity 
for patients ≥8 years of age 
• 
Change from baseline by each 4-week interval of the Treatment Period in additional patient-reported 
and observer-reported sleep parameters (e.g., tiredness and number of awakenings) 
• 
Change from baseline to Week 24 in Pediatric Quality of Life Inventory (PedsQL) 
• 
Change from baseline in serum ALT concentration at Week 4  
• 
Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• 
Change from baseline in AST and gamma-glutamyl transferase at Weeks 4, 12, and 24 
• 
Change from baseline for other biochemical markers and measures of bile acid synthesis (autotaxin, 
plasma 7α-hydroxy-4-cholesten-3-one concentration [p-C4]) at Week 24 
• 
Change in pediatric end-stage liver disease/model for end-stage liver disease (PELD/MELD) score, 
AST to platelet ratio index (APRI) score, and Fib-4 score from baseline to Week 24 
• 
Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® (where available) 
• 
Liver-related mortality and liver decompensation events. 
• 
All-cause mortality 
Safety:   


 
 
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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 03, 22 November 2018 
 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Safety criteria are as follows: 
• 
Occurrence of treatment-emergent adverse events including severity and relatedness to study drug at all 
visits 
• 
The incidence of treatment-emergent serious adverse events (SAEs), based upon information from 
patient reports, including the description, incidence, and severity of an SAE 
• 
Trends evaluated for the following assessments: physical examinations, concomitant medications, vital 
signs, laboratory test results (including clinical chemistry, hematology, urinalysis, alfa-fetoprotein, 
vitamins A and E, 25-hydroxy vitamin D and INR), and abdominal ultrasound 
Statistical Methods: 
The primary objective of this study is to show superiority in efficacy of A4250 (120 µg/kg/day and 
40 µg/kg/day) compared to placebo in patients with PFIC Types 1 and 2. 
The Cochran Mantel Haenzel (CMH) test stratified by PFIC class and age class will be performed to compare 
the proportion in fasting bile acid responders at the end of treatment (Week 22 and 24) in the two A4250 dose 
groups to the placebo group. To ascertain that all data are used in the CMH analysis, neighboring strata will be 
pooled if all subjects in a stratum have the same response. The proportion together with the corresponding 95% 
CI, odds ratio and corresponding 95% Clopper-Pearson exact CI and p-value for the CMH test will be presented.  
 
For the primary efficacy variable of the proportion of positive pruritus assessments at subject level over the 24-
week treatment period, an ANCOVA model will be used to analyse the comparisons between the treatment 
groups. The model will include treatment arm, the averaged AM and PM baseline pruritus scores, and 
randomization stratification factors, i.e. PFIC class and age class. LS mean(SE) by treatment arm and LS mean 
difference (SE), 95% confidence intervals, and p-values (where applicable) between treatments (120 µg/kg/day 
and 40 µg/kg/day, respectively) vs. placebo will be provided. If there are concerns on model assumptions, i.e. 
normality and homogeneity of variances, a non-parametric ANCOVA based on the rank scores can be used as a 
sensitivity analysis. LS mean/SE on the outcome by treatment arm and LS mean difference/SE between active 
dose and placebo will be provided.  
 
For each primary endpoint by region (EU & RoW and US), a pooled analysis for the closed testing procedure 
will be applied to control the 1-sided overall type I error rate for two treatment comparisons vs. the placebo at 
the 0.025 level, as specified below: 
 
In the closed testing procedure, the low and high dose groups will be pooled to compare with the placebo group 
first. If the 1-sided p-value is ≤0.025, the 1-sided p-values for low dose vs. placebo and high dose vs. placebo 
will be calculated respectively. If both individual p-values are ≤0.025, a significant treatment effect will be 
declared on both dose groups. If only one of them is ≤0.025, a significant treatment effect will be declared on 
the corresponding dose group.  
 
Other secondary and exploratory outcome variables will provide supportive efficacy and safety information 
regarding the differences between the treatment groups. No adjustments will be performed for multiple 
comparisons when testing secondary and exploratory efficacy variables. 
 
Date of the Protocol:  22 November 2018 


A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Page 9 
Final Amendment 03, 22 November 2018 
3 
TABLE OF CONTENTS 
1 
ADMINISTRATIVE INFORMATION ........................................................................ 4 
2 
STUDY SYNOPSIS ...................................................................................................... 5 
3 
TABLE OF CONTENTS .............................................................................................. 9 
3.1 
List of Tables .................................................................................................. 13 
3.2 
List of Figures ................................................................................................ 13 
3.3 
List of Appendices .......................................................................................... 13 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ................................ 14 
5 
INTRODUCTION ....................................................................................................... 16 
5.1 
Investigational Medicinal Product .................................................................. 16 
5.2 
Background .................................................................................................... 16 
5.2.1 
Progressive Familial Intrahepatic Cholestasis ........................................... 16 
5.2.2 
Summary of Clinical and Nonclinical Studies ........................................... 18 
5.3 
Rationale ......................................................................................................... 19 
5.3.1 
Risk/Benefit ............................................................................................... 20 
6 
STUDY OBJECTIVES ............................................................................................... 21 
6.1 
Primary Objectives ......................................................................................... 21 
6.2 
Secondary Objectives ..................................................................................... 21 
7 
INVESTIGATIONAL PLAN ...................................................................................... 22 
7.1 
Overall Study Design and Plan....................................................................... 22 
7.1.1 
Description ................................................................................................. 22 
7.1.2 
Schedule of Assessments ........................................................................... 24 
7.1.3 
Study Procedures and Assessments ........................................................... 29 
7.1.3.1 
Screening Period (Days -56 to 0) ............................................... 29 
7.1.3.2 
Treatment Period (Day 0 to Day 168) ........................................ 30 
7.1.3.3 
Follow-Up Period ....................................................................... 33 
7.2 
Study Population ............................................................................................ 33 
7.2.1 
Inclusion Criteria ....................................................................................... 34 
7.2.2 
Exclusion Criteria ...................................................................................... 34 
7.2.3 
Withdrawal of Patients............................................................................... 36 
7.2.4 
Study Termination by Sponsor .................................................................. 37 
8 
TREATMENT OF PATIENTS ................................................................................... 38 
8.1 
Identity of Study Drug .................................................................................... 38 
8.2 
Administration of Study Drug ........................................................................ 38 
8.3 
Study Treatment Packaging and Labeling ...................................................... 39 


 
 
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M5.3.5.1 – Protocol A4250-005 
 
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8.3.1 
Packaging and Labeling ............................................................................. 39 
8.3.2 
Storage ....................................................................................................... 39 
8.3.3 
Blinding and Randomization of Study Drug ............................................. 39 
8.4 
Procedure for Breaking the Randomization Code .......................................... 40 
8.5 
Patient Compliance ......................................................................................... 41 
8.6 
Study Drug Accountability ............................................................................. 41 
8.7 
Concomitant Therapy and Prohibited Medications ........................................ 41 
9 
ASSESSMENT OF EFFICACY ................................................................................. 42 
9.1 
Efficacy Endpoints ......................................................................................... 42 
9.1.1 
Primary Efficacy Endpoint ........................................................................ 42 
9.1.2 
Secondary Efficacy Endpoint .................................................................... 42 
9.1.3 
Exploratory Efficacy Endpoints ................................................................. 43 
9.2 
Efficacy Assessments ..................................................................................... 44 
9.2.1 
Serum Bile Acids ....................................................................................... 44 
9.2.2 
Itching, Scratching and Sleep Score .......................................................... 45 
9.2.3 
Growth ....................................................................................................... 45 
9.2.4 
Quality of Life Questionnaire (PedsQL).................................................... 46 
9.2.5 
Biomarkers and PK Samples ..................................................................... 46 
9.2.6 
PELD/MELD Score ................................................................................... 46 
9.2.7 
Fibroscan® .................................................................................................. 46 
9.2.8 
Markers of Fibrosis .................................................................................... 46 
9.2.9 
Global Impression of Change and Global Impression of Symptom 
Measures .................................................................................................... 46 
10 
ASSESSMENT OF SAFETY ..................................................................................... 48 
10.1 
Adverse Events ............................................................................................... 48 
10.1.1 
Definitions and Investigator Assessments ................................................. 48 
10.1.1.1 Clinical Significance .................................................................. 48 
10.1.1.2 Serious Adverse Events .............................................................. 48 
10.1.1.3 Severity Assessment ................................................................... 49 
10.1.1.4 Causality Assessment ................................................................. 49 
10.1.2 
Recording of Adverse Events .................................................................... 50 
10.1.3 
Recording and Reporting of Serious Adverse Events ................................ 51 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions .............. 52 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments .................................................................................................... 53 
10.2.1 
Laboratory Assessments ............................................................................ 53 


 
 
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10.2.2 
Individual Patient Safety Monitoring ........................................................ 54 
10.2.2.1 Liver Monitoring ........................................................................ 54 
10.2.2.2 Close Observation ...................................................................... 55 
10.2.2.3 De-challenge/Re-challenge for Liver and Clinical Hepatitis 
Monitoring .................................................................................. 56 
10.2.2.4 Diarrhea ...................................................................................... 56 
10.2.2.5 Monitoring after Permanent Discontinuation of Study Drug 
Due to Safety .............................................................................. 56 
10.2.3 
Demographics/Medical and Surgical History ............................................ 56 
10.2.4 
Clinical Genetic Testing ............................................................................ 57 
10.2.5 
Physical Examination ................................................................................ 57 
10.2.6 
Vital Signs ................................................................................................. 57 
10.2.7 
Abdominal Ultrasound ............................................................................... 57 
10.2.8 
Overdose .................................................................................................... 57 
10.2.9 
Pregnancy .................................................................................................. 58 
11 
STATISTICAL EVALUATION ................................................................................. 59 
11.1 
Sample Size and Power .................................................................................. 59 
11.2 
Statistical Methods ......................................................................................... 59 
11.2.1 
Statistical Analysis Sets ............................................................................. 59 
11.2.2 
Methods of Statistical Analyses ................................................................. 60 
11.2.2.1 General Principles ...................................................................... 60 
11.2.2.2 Missing Data .............................................................................. 61 
11.2.2.3 Demographic and Baseline Characteristics ................................ 61 
11.2.2.4 Subject Disposition..................................................................... 61 
11.2.2.5 Evaluation of Primary Efficacy Variables .................................. 62 
11.2.2.6 Evaluation 
of 
Secondary 
and 
Exploratory 
Efficacy 
Variables ..................................................................................... 63 
11.2.2.7 Evaluation of Safety Variables ................................................... 63 
11.2.2.8 Compliance and Exposure .......................................................... 64 
11.2.3 
Blinded Analyses to Confirm Responder Definitions ............................... 64 
11.2.4 
Data Safety Monitoring Board ................................................................... 65 
12 
DIRECT ACCESS TO SOURCE DATA/NOTES ..................................................... 66 
13 
QUALITY CONTROL AND QUALITY ASSURANCE .......................................... 67 
13.1 
Conduct of the Study ...................................................................................... 67 
13.2 
Study Monitoring ........................................................................................... 67 


 
 
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14 
ETHICS ....................................................................................................................... 69 
14.1 
Independent Ethics Committee/Institutional Review Board .......................... 69 
14.2 
Written Informed Consents and Assents ........................................................ 69 
15 
DATA HANDLING AND RECORD KEEPING ....................................................... 71 
15.1 
Case Report Forms/Source Data Handling..................................................... 71 
15.2 
Retention of Essential Documents .................................................................. 71 
16 
FINANCING AND INSURANCE .............................................................................. 72 
17 
PUBLICATION POLICY ........................................................................................... 73 
18 
REFERENCE LIST ..................................................................................................... 74 
19 
APPENDICES ............................................................................................................. 75 
 




 
 
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4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
ADME 
absorption, distribution, metabolism, and excretion 
AE(s) 
adverse event(s) 
AFP 
alfa-fetoprotein 
ALT 
alanine aminotransferase 
APRI 
AST to platelet ratio index 
ASBT 
apical sodium-dependent bile transporter (also known as IBAT) 
AST 
aspartate aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BMI 
body mass index 
BSEP 
bile salt export pump 
CPK 
creatine phosphokinase 
CRA 
clinical research associate 
CYP 
cytochrome P450 
DSMB 
Data and Safety Monitoring Board 
eCRF 
electronic case report form 
eDiary 
electronic diary 
EOT 
End of Treatment 
EU 
European Union 
EudraCT 
European Union drug regulatory agency clinical trial 
FAS 
full analysis set 
FDA 
United States Food and Drug Administration 
FIC1 
familial intrahepatic cholestasis-1 
GCP 
Good Clinical Practice 
GGT 
gamma-glutamyl transferase 
GI 
gastrointestinal 
GLMM 
generalized linear mixed modelling 
IB 
Investigator’s Brochure 
IBAT 
ileal bile acid transporter (also known as ASBT) 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
 
 
IEC 
Independent Ethics Committee 
INR 
international normalized ratio 
IRB 
Institutional Review Board 


 
 
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IWRS 
Interactive Web Response System 
LDH 
lactate dehydrogenase 
LFT 
liver function test 
LPLV 
last patient last visit 
MDR3 
multidrug-resistance protein 3 
MedDRA 
Medical Dictionary for Regulatory Activities 
MELD 
model for end-stage liver disease 
MMRM 
mixed model repeated measures 
ObsRO 
observer-reported outcome 
PBC 
primary biliary cholangitis 
p-C4 
plasma 7α-hydroxy-4-cholesten-3-one concentration 
PCS 
potentially clinically significant 
PedsQL 
Pediatric Quality of Life Inventory 
PELD 
pediatric end-stage liver disease 
PFIC 
progressive familial intrahepatic cholestasis 
PGIC 
patient global impression of change 
PGIS 
patient global impression of symptoms 
PK 
pharmacokinetic(s) 
PP 
per protocol 
PRO 
patient-reported outcome 
PT 
prothrombin time 
PVM 
pharmacovigilance manager 
QoL 
quality of life 
RoW 
rest of world 
RR 
respiratory rate 
SAE(s) 
serious adverse event(s) 
SAP 
statistical analysis plan 
s-BA 
serum bile acid 
SD 
standard deviation 
s-FGF19 
serum fibroblast growth factor 19 concentration 
SOC 
system organ class 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE(s) 
treatment-emergent adverse event(s) 
ULN 
upper limit of normal 
US 
United States 
WHO 
World Health Organization 


 
 
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5 
INTRODUCTION 
5.1 
Investigational Medicinal Product 
A4250 is a small molecule and a potent selective inhibitor of the ileal bile acid transporter 
(IBAT), also known as the apical sodium-dependent bile acid transporter (ASBT).  IBAT is 
an integral brush border membrane glycoprotein that co-transports sodium and bile acids and 
appears to be a critical component in the regulation of the bile acid pool size in animals and 
man.  This transporter, expressed in the distal ileum, is a key element in the enterohepatic 
circulation of bile acids since it facilitates the high-affinity, high-capacity reabsorption of bile 
acids.  Indeed, 95% of secreted bile acids are reabsorbed via IBAT [Hofmann 2009; 
Miethke 2016].  A4250 is orally administered and acts locally in the gut where it binds 
reversibly to IBAT to decrease the reuptake of bile acids.  A4250 has minimal systemic 
exposure at the predicted therapeutic dose ranges. 
No safety signals were found with A4250 in preclinical safety and toxicity studies.  In the 
first clinical study of A4250 in healthy volunteers, A4250 was given as a single oral dose up 
to 10 mg or daily oral doses up to 3 mg/day for 7 days.  A4250 was well tolerated and the 
only findings were gastrointestinal (GI) related effects, such as diarrhea, at higher doses. 
In an exploratory Phase 2 study in children with cholestatic pruritus, A4250 was given as oral 
doses of 10 to 200 µg/kg/day for 4 weeks.  A4250 reduced mean levels of serum bile acids 
(s-BA) in all cohorts.  In 11 of 13 progressive familial intrahepatic cholestasis 
(PFIC) patients, substantial reductions (ranging from 43% to 98%) were observed.  
Patient-reported diary data documented improvement of pruritus during treatment.  A4250 
was well tolerated.  No serious adverse events (SAEs) were deemed to be related to study 
drug, and most adverse events (AEs), including some increased transaminases, were 
considered unrelated to study drug and transient in nature. 
5.2 
Background 
5.2.1 
Progressive Familial Intrahepatic Cholestasis 
PFIC is a rare autosomal recessive cholestatic liver disease estimated to affect between 1 in 
every 50,000 to 100,000 children born worldwide.  PFIC represents 10% to 15% of causes of 
cholestasis in children and 10% to 15% of liver transplantation indications in children.  All 
types of PFIC exist worldwide and both sexes appear to be equally affected. 
The common underlying pathogenesis of PFIC is disruption of bile formation and bile 
transport through the liver [Jacquemin 2000].  The classification of PFIC has evolved over 
the years.  The most commonly used sub-classifications are PFIC Types 1, 2, and 3, which 
are based on the associated affected gene and described in more detail below. 
• PFIC, Type 1:  also referred to as “Byler disease” or “familial intrahepatic 
cholestasis-1 (FIC1) protein deficiency.” FIC1 protein is located on the canalicular 


 
 
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membrane of hepatocytes and facilitates movement of aminophospholipids from the outer 
to inner leaflet of the plasma membrane of the hepatocyte.  The ATP8B1 gene encodes 
FIC1 protein.  Biallelic pathologic variants in the ATP8B1 gene are associated with FIC1 
dysfunction and classified clinically as PFIC Type 1 disease. 
• PFIC, Type 2:  also referred to as “Byler syndrome” or “bile salt export pump (BSEP) 
deficiency.”  BSEP is a transporter protein that is expressed at the canalicular membrane 
of hepatocytes, and is the primary exporter of bile acids.  The ABCB11 gene encodes the 
BSEP protein.  Biallelic pathologic variations in the ABCB11 gene are associated with 
BSEP dysfunction and classified clinically as PFIC Type 2 disease. 
• PFIC, Type 3:  is caused by a deficiency of the multidrug-resistance protein 3 (MDR3) 
due to mutations in the ABCB4 gene.  MDR3 is a phospholipid translocase critical for 
phospholipid secretion. 
Severe pruritus is common in children diagnosed with PFIC.  Itching (and subsequent 
scratching) is a significant morbidity for these patients and their families [Suchy 2007].  A 
more severe degree of pruritus is experienced compared to patients with other forms of liver 
disease and to other pruritic conditions such as atopic dermatitis [Murray 2011].  In patients 
with PFIC, liver biopsy reveals canalicular cholestasis and, later, the appearance of portal 
fibrosis.  Serum biochemistry indicates cholestasis with hyperbilirubinemia, elevated alanine 
aminotransferase (ALT) and aspartate aminotransferase (AST).  The concentrations of bile 
acids in serum are very high, while serum gamma-glutamyl transferase (GGT) activity 
(exception being MDR3 variants) and cholesterol are normal [Hori 2010].  Symptoms of 
portal hypertension and liver failure will develop during the course of the disease [Davit-
Spraul 2009; Alissa 2008].  Symptoms develop early; median age at onset of symptoms is 
2 months, and 78% of PFIC patients present with jaundice [Pawlikowska 2010].  The 
life-threatening and debilitating nature of PFIC is reflected by the fact that survival in those 
not resorting to surgery is 50% at 10 years of age and almost zero at 20 years of age.  
Approximately half of PFIC patients undergo liver transplantation [Davit-Spraul 2009] and 
treatment-resistant pruritus is the leading indication for the surgical procedure partial external 
biliary diversion, mostly in PFIC Type 1 and 2 patients. 
PFIC is life-threatening and debilitating.  Currently, the treatment for PFIC is palliative and 
there is no pharmaceutical treatment approved for use in PFIC.  The therapeutic choices are 
restricted to non-specific therapy of the symptoms and signs of the disease such as nutritional 
support, preventing vitamin deficiencies, and treatment of extrahepatic features.  Medical 
treatment options include off-label use of ursodeoxycholic acid, rifampin, antihistamines, and 
naltrexone.  A minority of patients respond nominally and transiently to these interventions.  
Biliary diversion is used to decrease systemic bile acids through interruption of the 
enterohepatic circulation and so avoid transplantation.  Liver transplantation is typically only 
viewed as an option when patients have failed medical treatment and/or biliary diversion and 
have liver failure or persistent uncontrolled pruritus. 


 
 
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5.2.2 
Summary of Clinical and Nonclinical Studies 
This is a summary of nonclinical and clinical studies.  More detail is provided in the 
Investigator’s Brochure ([IB] see A4250 IB).  
A4250 is a potent, selective IBAT inhibitor with no safety signals seen in the safety 
pharmacological studies.  In addition, no safety signals were seen in repeat-dose oral toxicity 
studies in rats with up to 26 weeks of daily dosing and in dogs with up to 39 weeks of daily 
dosing.  No safety signals were identified when A4250 was administered daily to juvenile 
rats from age 14 days to 63 days. 
A4250 is minimally absorbed with very low systemic exposure in both humans and other 
species.  In blood, protein binding is high (>99.7%).  Systemically absorbed A4250 is slowly 
metabolized in humans and in preclinical studies by hydroxylation only, a metabolite that 
retains IBAT activity.  A4250 did not show any significant inhibition of cytochrome 
P450 (CYP) 
enzymes 
in 
the 
rat. 
 
A4250 
inhibited 
human 
CYP3A4 
and 
CYP2D6 (IC50: 16 µmol/L for both enzymes), and CYP2C9 (IC50:1.2 µmol/L).  
To date, A4250 has been studied in 3 Albireo-sponsored clinical studies: a study in healthy 
volunteers evaluating single and multiple administration of A4250; a single-dose absorption, 
distribution, metabolism, and excretion (ADME) study; and a Phase 2 study in children with 
cholestatic pruritus.  In addition, an investigator-sponsored study has been conducted in 
patients with primary biliary cholangitis (PBC).  In total, 98 subjects have been exposed to 
A4250. 
In the first clinical study (A4250-001), A4250 was given to healthy volunteers as a single oral 
dose up to 10 mg or daily oral doses up to 3 mg/day for 7 days.  Following a single oral dose 
of 10 mg, the median maximum drug concentration was 0.2 nmol/L and the area under the 
curve was 0.8 nmol × h/L (n=6).  Pharmacodynamic evaluation of bile acid synthesis 
biomarkers 
(serum 
fibroblast 
growth 
factor 
19 [s-FGF19] 
and 
plasma 
7α Hydroxy 4-cholesten-3-one [p-C4]) identified the expected decrease in s-FGF19 and an 
increase in p-C4.  In addition, total plasma bile acids decreased and total fecal bile acids 
increased in A4250-treated patients compared to placebo. A4250 was generally well 
tolerated.  The only findings were GI-related effects, such as diarrhea at higher doses. 
In the ADME study (A4250-007), healthy subjects were administered a single oral dose of 
3 mg [14C]-A4250 capsule.  The main excretion route was determined to be fecal.  There 
were no quantifiable concentrations of A4250 in plasma or total radioactivity in plasma or 
whole blood.  A4250 was well tolerated.  AEs were reported and most were mild and not 
related to A4250. The 3 AEs experienced by 2 subjects reported as related to study drug were 
GI in nature, including diarrhea (2 AEs; 2 subjects) and abdominal pain (1 AE; 1 subject). 
In an exploratory Phase 2 study in children with cholestatic pruritus (A4250-003), A4250 was 
administered as oral daily doses of 10 to 200 µg/kg/day for 4 weeks.  Twenty 
patients (8 females and 12 males) aged 1 to 17 years were enrolled in this open-label 


 
 
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dose-escalation study.  Four patients were re-enrolled into a second dose level.  No 
treatment-related SAEs were reported, and most AEs, including some increased 
transaminases, were transient.  Four-week, once-daily treatment with A4250 reduced mean 
levels of s-BA in all cohorts.  Substantial s-BA reductions in 11 of 13 PFIC patients (ranging 
from 43% to 98%) were observed.  A4250 also reduced s-BA in the 11 non-PFIC patients, 
but to a lesser extent than in PFIC patients.  Patient-reported diaries documented 
improvement of the visual analogue scale itch score in 17 of 24 patients during treatment.  
The dose with the greatest improvement showed a mean decrease (± standard error of the 
mean) of 2.9 ±1.1 from baseline.  The results from this study form the basis of the current 
(A4250-005) study design. 
5.3 
Rationale 
In this investigational study, A4250 is under development for treatment of PFIC and is also 
being considered for other orphan cholestatic liver diseases. 
There is currently no medical treatment approved for PFIC.  In PFIC patients with severe 
pruritus, biliary diversion surgery is used to decrease systemic bile acids through interruption 
of the enterohepatic circulation and thereby reducing pruritus [Whitington 1988].  Liver 
transplantation is typically used as an option when patients have failed medical treatment 
and/or biliary diversion and have liver failure or persistent uncontrolled pruritus. 
By inhibiting IBAT with high selectivity and potency, A4250 has the potential to relieve 
cholestasis and improve liver function without surgical intervention in patients with PFIC.  
Since A4250 is minimally absorbed and achieves a substantial reduction of s-BA, it has the 
potential to be a safer and less invasive alternative to surgical biliary drainage.  The rationale 
for using A4250 is to decrease s-BA levels, and to reduce the major morbidity of pruritus, 
improving the health and wellbeing of patients affected with PFIC.  Ultimately, this 
noninvasive therapeutic approach has the potential to not only improve quality of life (QoL), 
but also reduce the need for biliary diversion and subsequent liver failure and liver 
transplantation. 
The doses selected, 40 and 120 µg/kg/day, were based on the efficacy and safety data 
generated from patients with PFIC (n=10 + 3 re-entered) and supporting data from all 
patients (n=20 + 4 re-entered) in the five dose groups dosed in the A4250-003 study.  
Improvements in s-BA and pruritus were observed with doses from 30 µg/kg to 100 µg/kg, 
with a dose-related trend.  The best dose response in all patients was at the 60 µg/kg/day for 
s-BA and 100 µg/kg/day for pruritus.  The best dose response in the PFIC subgroup was at 
30 µg/kg/day for both s-BA and pruritus.  Patients with PFIC responded well to the doses 
given from 30 µg/kg to 200 µg/kg/day; however, a clear dose relationship could not be 
established in order to select a single dose for this Phase 3 study.  As efficacy was observed 
at both low and high doses, 40 µg/kg and 120 µg/kg/day are considered to be the most 
optimal for this study.  


 
 
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This study will be conducted in compliance with the protocol and with the International 
Council for Harmonisation (ICH) guidelines on Good Clinical Practice (GCP). 
Patients completing 24 weeks of double-blind treatment will be offered to enter Study 
A4250-008 and receive open-label treatment with A4250. 
5.3.1 
Risk/Benefit 
A4250 has been evaluated in three Albireo-sponsored clinical studies: a double-blind 
placebo-controlled study in healthy volunteers, a single-dose ADME study, and a Phase 2 
study in children with cholestatic pruritus.  In addition, an investigator-sponsored study has 
been conducted in patients with PBC. 
A total of 98 subjects/patients have been exposed to A4250.  Healthy subjects have been 
exposed to up to 10 mg as a single dose and up to 3 mg daily as part of a multiple-dose 
evaluation.  Children with cholestatic liver disease have been treated with up to 
0.2 mg/kg/day (200 µg/kg/day) for 4 weeks.  In total, 2 SAEs have been reported; both were 
judged by the physician to be not related to the study drug. 
Patients with cholestatic liver diseases suffer from excess bile acids in the liver resulting in 
tissue damage.  A commonly used treatment in these patients is bile diversion surgery, 
whereby approximately 50% to 100% of the enterohepatic circulation of bile acids is 
interrupted.  Inhibition of IBAT with A4250, thereby interrupting the enterohepatic 
circulation of bile acids, is therefore a potential medical alternative to surgery which could be 
of benefit to these patients if shown to be effective and safe.  Data from the Phase 2 study 
showed efficacy of A4250 in reducing s-BA concentrations and pruritus in such patients. 
Based on the mode of action of an IBAT inhibitor, loose stools or diarrhea could be expected.  
However, in the pediatric Phase 2 study with 4 weeks daily treatment, only one patient had 
mild transient diarrhea after a single dose that did not recur on multiple dosing. 


 
 
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6 
STUDY OBJECTIVES 
6.1 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day 
A4250 in children with PFIC Types 1 and 2, as determined by the following: 
• The proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment 
• The proportion of positive pruritus assessments at the subject level over the 24-week 
treatment period.   
6.2 
  Secondary Objectives 
• To evaluate the effect of A4250 on serum ALT concentration 
• To evaluate the effect of A4250 on growth 
• To evaluate the effect of A4250 on sleep disturbance 
• To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or 
liver transplantation) 
• To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 


 
 
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7 
INVESTIGATIONAL PLAN 
7.1 
Overall Study Design and Plan 
7.1.1 
Description 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the 
efficacy and safety of 40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in 
children with PFIC (deficiencies of FIC1 or BSEP). 
Study data will be periodically (approximately on a quarterly basis) reviewed by an 
unblinded Data Safety and Monitoring Board (DSMB). 
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment 
Period and a 4-week Follow-Up Period. 
After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of 
A4250, or a matching placebo. 
Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008; EudraCT Number 
2017-002325-38, subject to a separate study approval) in which all patients will receive 
active treatment.  A patient who prematurely withdraws due to intolerable symptoms, and has 
completed at least 12 weeks of the Treatment Period and End of Treatment (EOT) Visit, will 
also be offered the opportunity to enter Study A4250-008. 
Visit windows applicable to each visit are presented in the schedule of assessments (Table 1). 
There will be up to 10 clinic visits during the study (Figure 1) and one scheduled telephone 
contact, as follows: 
• Visit 1: Screening Period start. Patient is considered enrolled after signing the informed 
consent form (ICF) (Days -56 to -35) 
• Visit 2: second Screening Visit (Days -28 to -7) 
• Visit 3: Randomization Visit (Day 0) 
• Telephone Contact 1: (Day 14) 
• Visit 4: Clinic Visit (Day 28) 
• Visit 5: Clinic Visit (Day 56) 
• Visit 6: Clinic Visit (Day 84) 
• Visit 7: Clinic Visit (Day 126) 
• Visit 8: Clinic Visit (Day 154) 
• Visit 9: Clinic Visit, EOT (Day 168) 


 
 
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• Visit 10: Follow-Up Visit, 28 days after last dose of study drug (Day 196).  All patients 
continuing in Study A4250-008 will not have this Follow-up Visit. 
Additional clinic visits may be required for patients who need direct site assistance, e.g., due 
to AE monitoring, to fulfil screening requirements, and/or for safety maintenance. 
Screening Period 
Informed consent must be obtained at Visit 1 prior to performing any study procedures.  After 
signing the ICF, patients will be considered enrolled and evaluated for study eligibility. 
Daily recording of pruritus using an electronic diary (eDiary) will be started to determine 
confirmation of the magnitude and the baseline pruritus symptoms before the study.  After 
consultation with the Medical Monitor, patients failing eligibility criteria may be re-screened 
after 2 months.  Patients not fulfilling inclusion/exclusion criteria after 3 attempts are not 
allowed to re-screen. 
Caregivers/patients will be instructed on the daily use of the eDiary.  The eDiary will include 
patient-reported outcome (PRO) and observer-reported outcome (ObsRO) items for 
evaluation of itching (PRO), scratching (ObsRO), and sleep disturbance (PRO and ObsRO) 
during the Screening Period (study Days -56 to 0), throughout the 24-week Treatment Period 
(study Days 0 to 168), and Follow-up (Day 196). 
The 14 consecutive days prior to randomization will be used for eligibility calculation of the 
diary entries.  If a patient does not meet the minimum number of observations (see 
Section 9.2.2 for scoring and missing data definitions) to determine eligibility, the patient 
may be re-screened.  Observer-reported outcomes in patients of all ages will be recorded by a 
caregiver.  If possible, the same caregiver will complete the Albireo ObsRO items throughout 
the study. 
At a second Screening Visit (Visit 2) additional laboratory samples will be taken for 
eligibility assessments. 
Treatment Period 
Eligibility for randomization will be determined at Visit 3 using the pruritus (scratching 
reports from the Albireo ObsRO) data in the 2 weeks before Visit 3, clinical genetic 
confirmation of diagnosis, and the liver biochemistry evaluations from the previous screening 
visits.  Two weeks after Visit 3, there will be a telephone contact with the caregiver/patient to 
evaluate any AEs. 
The patient will return to the clinic at Visit 4, Visit 5, Visit 6, Visit 7, Visit 8, and Visit 9 for 
assessments.  If a new caregiver will begin to complete the Albireo ObsRO during the course 
of the trial, they will be provided with training during a clinic visit before they begin to use 
the eDiary.  Additionally, caregivers will be requested to use the diary to report the time each 
dose of study drug is administered during the Treatment Period. 


 
 
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The last dose of study drug will be administered the day before Visit 9.  At study Visit 9, the 
patient has the option to enroll in an open-label extension study (A4250-008) should they 
meet the required entry criteria.  If the patient is continuing into Study A4250-008 this will be 
the last visit in this study and the first visit in Study A4250-008.  Visit 9/EOT should also be 
performed at the time patients are prematurely withdrawn from the study. 
Clinic Visit 10 will take place 28 days after Visit 9 for all patients not continuing into Study 
A4250-008. 
Figure 1 
Study Design 
 
T: telephone contact; V: study visit 
End of Study 
The end of the study is defined as follows: 
a) End of study in one country: last patient last visit (LPLV) and sites are closed 
b) End of study globally: LPLV globally and all sites closed 
7.1.2 
Schedule of Assessments 
The schedule of assessments is presented in Table 1.  For information on blood volumes, 
please see Appendix 7. 
 


 
 
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Table 1 
Schedule of Assessments 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Informed consent c 
X 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
Concomitant medicationd 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical and surgical history 
X 
 
X 
 
 
 
 
 
 
 
 
Physical examination including 
voluntary photography 
X 
 
X 
 
 
 
X 
 
 
X 
 
Skin examination 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
Vital signse 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
eDiary: itching, scratching, and 
sleep scoresf 
Daily diary entry 
Clinical chemistryg 
X 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Hematologyg 
 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Urinalysisg 
 
X 
 
 
 
 
X 
 
 
X 
 
Serum bile acidsh 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 


 
 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
INR 
 
X 
 
 
X 
 
X 
 
X 
 
X 
Autotaxini 
 
 
X 
 
X 
 
 
 
 
X 
 
p-C4i 
 
 
X 
 
X 
 
 
 
 
X 
 
AFP 
 
 
X 
 
 
 
 
 
 
X 
 
Vitamins A & Eh 
 
X 
 
 
 
 
X 
 
 
X 
 
25-hydroxy vitamin D 
 
 
X 
 
 
 
X 
 
 
X 
 
Blood sample for A4250 PKi 
 
 
 
 
X 
 
 
 
 
X 
 
Abdominal Ultrasound 
 
 
X 
 
 
 
 
 
 
X 
 
Fibroscan® (where available) 
 
 
X 
 
 
 
 
 
 
X 
 
QoL questionnaire (PedsQL) 
 
 
X 
 
 
 
 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of Change 
 
 
 
 
X 
 
X 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of 
Symptoms 
 
 
X 
 
X 
 
X 
 
 
X 
 
Pregnancy testj 
Xj 
X 
X 
 
X 
X 
X 
X 
X 
Xj 
X 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
 
Page 27 
Final Amendment 03, 22 November 2018 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Study drug dispensedk 
 
 
X 
 
X 
X 
X 
X 
X 
 
 
Adverse eventsl 
Continuous collection 
Study drug compliance 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
AFP: alfa-fetoprotein; BMI: body mass index; eCRF: electronic case report form; eDiary: electronic diary; EOT: End of Treatment; ICF: informed consent form; 
INR: international normalized ratio; p-C4: plasma 7α Hydroxy 4-cholesten-3-one; PedsQL: Pediatric Quality of Life Inventory; PK: pharmacokinetic; QoL: quality of life; 
RR: respiratory rate; SAE: serious adverse event; s-BA: serum bile acid. 
 
a 
Must also be performed at the time a patient is prematurely withdrawn from the study.  This visit is the same as Visit 1 in the extension protocol (Study A4250-008). 
b 
Assessments must be performed 28 days following the final dose of study drug.  This visit is omitted if patients are entering Study A4250-008. 
c 
The ICF (and assent) must be signed before any study procedures are performed. 
d 
Past (within 1 month prior to Visit 1) and current medication will be documented in the eCRF. 
e 
Includes blood pressure, pulse, RR, temperature, height (or length depending on age), and body weight (using a certified weight scale).  BMI will be calculated. 
f 
Includes training on use of the device at Screening (Visit 1) and review of patient daily entries during 14 days prior to Clinic Visit 3 to determine eligibility.  
g 
See Table 3 for detailed parameters. 
h 
Patients will fast (water intake only is permissible) for at least 4 hours prior to the collection of samples for s-BA and vitamin A.  Exceptions can be made for infants, 
less than 12 months of age, if unable to fast for the full 4 hours.  All s-BA results during the treatment period and follow-up will be blinded. 
i 
Samples will not be collected for patients ≤10 kg.  In the event of an SAE, a PK sample should be collected as close to the onset of the event as possible. 
j 
For girls who have reached menarche.  Serum test at Visit 1; urine test at other visits.  A serum pregnancy test will also be conducted at Visit 9 for patients entering 
Study A4250-008.  If a urine pregnancy test is positive, a serum pregnancy test should be performed to confirm the pregnancy. 
k 
Study drug to be taken once daily on Day 1 through Day 167 as described in Section 8.2. 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
 
Page 28 
Final Amendment 03, 22 November 2018 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
l 
Adverse event information will be collected from the time of signing of the ICF to study discontinuation. 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 29 
Final Amendment 03, 22November 2018 
 
7.1.3 
Study Procedures and Assessments 
7.1.3.1 
Screening Period (Days -56 to 0) 
Day -56 to Day -35/Clinic Visit 1 
Patients will undergo a Screening Visit up to 56 days prior to the planned first day of study 
treatment.  Screening procedures and assessments are as follows: 
• Obtain written informed consent 
• Assess inclusion/exclusion criteria (Section 7.2) 
• Record demographics (age, full date of birth, gender, race, and ethnicity) 
• Document concomitant medications 
• Medical and surgical history (date of diagnosis of PFIC Types 1 or 2, prior investigational 
medications for PFIC, historical liver function test [LFT] values, any surgery performed, 
any other diagnosis, and historical liver biopsy data) 
• Collect, if available, past confirmatory clinical genetic laboratory report for 
PFIC-1/PFIC-2 and send to central reader for review.  If a historical report is equivocal, 
unavailable, or unobtainable, a blood sample for clinical genetic testing will be collected 
to determine eligibility 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• eDiary training and compliance requirements; caregivers and, if applicable, patients begin 
daily recording of pruritus using the eDiary (Appendix 2) 
• Clinical chemistry (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Serum pregnancy test for girls who have reached menarche.  Please see Appendix 6 for 
contraceptive requirements 
• AE monitoring 
Day -28 to Day -7/Clinic Visit 2 
A second Screening Visit will be performed.  Screening procedures and assessments are as 
follows: 
• Review concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 30 
Final Amendment 03, 22November 2018 
 
• Urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• International normalized ratio (INR) 
• Vitamins A and E 
• Urine pregnancy test for girls of who have reached menarche 
• AE monitoring 
7.1.3.2 
Treatment Period (Day 0 to Day 168) 
Study Day 0/Clinic Visit 3 (Randomization) 
At the randomization visit, the following assessments will be conducted: 
• Review inclusion/exclusion criteria (Section 7.2) 
• Review concomitant medications, medical and surgical history 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance and entrance criteria requirement (see 
Inclusion Criteria 4, Section 7.2.1) 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1)  
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• 25-hydroxy vitamin D 
• Alfa-fetoprotein (AFP) 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer QoL (Pediatric Quality of Life Inventory [PedsQL]) questionnaire 
• Patient/caregiver/clinician complete the patient global impression of symptoms (PGIS) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Study drug is dispensed and patients are instructed to administer daily from Day 1 
Study Day 14 (Telephone Contact) 
A study nurse will contact the patient/caregiver by telephone for monitoring of the following:  


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 31 
Final Amendment 03, 22November 2018 
 
• Review concomitant medications 
• Albireo PRO/ObsRO eDiary review for compliance 
• AEs 
Study Day 28 and Day 84/Clinic Visits 4 and 6  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Physical examination, including voluntary photography (at Clinic Visit 6 only; 
Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• Urinalysis (at Clinic Visit 6 only; Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Autotaxin, p-C4, and A4250 pharmacokinetic (PK) assessment (at Clinic Visit 4 only; 
samples will not be taken for patients ≤10 kg) 
• Vitamins A and E, and 25-hydroxy vitamin D (at Clinic Visit 6 only) 
• Patient/caregiver/clinician complete the patient global impression of change (PGIC) and 
PGIS 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 56 and Day 126/Clinic Visits 5 and 7 
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 32 
Final Amendment 03, 22November 2018 
 
• s-BA (Section 9.2.1) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 154/Clinic Visit 8  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 168/Clinic Visit 9/EOT 
The last dose of study drug is taken in the morning the day before Clinic Visit 9, and the 
following procedures and assessments will be performed (also performed at the time a patient 
prematurely withdraws):  
• Review of concomitant medications 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry, hematology, and urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Autotaxin, p-C4, and A4250 PK assessment (samples will not be taken for patients 
≤10 kg) 
• AFP  


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 33 
Final Amendment 03, 22November 2018 
 
• Vitamins A, E, and 25-hydroxy vitamin D 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer PedsQL questionnaire 
• Patient/caregiver/clinician complete the PGIC and PGIS 
• Urine pregnancy test for girls who have reached menarche, or serum pregnancy test for 
girls continuing to study A4250-008 
• AE monitoring 
• Evaluation of study drug compliance 
7.1.3.3 
Follow-Up Period 
Study Day 196/Clinic Visit 10 
All patients not continuing into Study A4250-008 will return to the study site 28 days after 
Clinic Visit 9 or the last dose of study drug for the following assessments: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
7.2 
Study Population 
A total of approximately 60 patients with a clinical diagnosis of PFIC Type 1 or 2 that have 
been genetically confirmed will be randomized.  The study will target 15% of enrolled 
patients to be PFIC-1 patients.  If enrollment of all PFIC-2 patients is completed and the 15% 
enrollment of PFIC-1 patients has not yet been achieved, then enrollment of PFIC-2 patients 
will continue in order to reach the total study target enrollment. 
An attempt will be made to randomize a minimum of 10 patients from each age category 
(6 months to 5 years, 6 to 12 years, and 13 to ≤18 years).  
A maximum of 5 patients that were treated in Study A4250-003 will be allowed to participate 
in this study. 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 34 
Final Amendment 03, 22November 2018 
 
7.2.1 
Inclusion Criteria 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages 
of ≥6 months and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have clinical genetic confirmation of PFIC-1 or PFIC-2 through 
identification of biallelic pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, 
taken as the average of 2 samples at least 7 days apart (Visits 1 and 2) prior to 
randomization 
4. Patient must have history of significant pruritus and a caregiver-reported observed 
scratching in the eDiary average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to 
randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  
Patients who turn 18 years of age (or legal age per country) during the study will be 
required to re-consent in order to remain in the study 
6. Patients will be expected to have a consistent caregiver for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use 
an eDiary device as required by the study 
7.2.2 
Exclusion Criteria 
1. Patient with pathologic variations of the ABCB11 gene that predict complete absence of 
the BSEP protein 
2. Patient with past medical history or ongoing presence of other types of liver disease 
including, but not limited to, the following: 
a) Biliary atresia of any kind 
b) Benign recurrent intrahepatic cholestasis, indicated by any history of normal s-BAs 
c) Suspected or proven liver cancer or metastasis to the liver on imaging studies 
d) Histopathology on liver biopsy is suggestive of alternate non-PFIC related etiology of 
cholestasis 
3. Patient with a past medical history or ongoing presence of any other disease or condition 
known to interfere with the absorption, distribution, metabolism (specifically bile acid 
metabolism), or excretion of drugs in the intestine, including but not limited to, 
inflammatory bowel disease 
4. Patient with past medical history or ongoing chronic (i.e., >3 months) diarrhea requiring 
intravenous fluid or nutritional intervention for treatment of the diarrhea and/or its 
sequelae 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 35 
Final Amendment 03, 22November 2018 
 
5. Patient has a confirmed past diagnosis of infection with human immunodeficiency virus 
or other present and active, clinically significant, acute, or chronic infection, or past 
medical history of any major episode of infection requiring hospitalization or treatment 
with parenteral anti-infective treatment within 4 weeks of treatment start (study Day 1) or 
completion of oral anti-infective treatment within 2 weeks prior to start of Screening 
Period 
6. Any patient with suspected or confirmed cancers except for basal cell carcinoma, and 
non-liver cancers treated at least 5 years prior to Screening with no evidence of 
recurrence 
7. Patient with a past medical history of chronic kidney disease with an impaired renal 
function and a glomerular filtration rate <70 mL/min/1.73 m2  
8. Patient with surgical history of disruption of the enterohepatic circulation excluding those 
who have undergone a successful reversal procedure that has permanently restored flow 
of bile acids from the liver to the duodenum 
9. Patient has had a liver transplant or a liver transplant is planned within 6 months of 
randomization 
10. Decompensated liver disease, coagulopathy, history or presence of clinically significant 
ascites, variceal hemorrhage, and/or encephalopathy 
11. INR >1.4 (the patient may be treated with Vitamin K intravenously, and if INR is ≤1.4 at 
resampling the patient may be randomized) 
12. Serum ALT >10 × upper limit of normal (ULN) at Screening 
13. Serum ALT >15 × ULN at any time point during the last 6 months unless an alternate 
etiology was confirmed for the elevation 
14. Total bilirubin >5 × ULN at Screening 
15. Patient suffers from uncontrolled, recalcitrant pruritic condition other than PFIC.  
Examples include, but not limited to, refractory atopic dermatitis or other primary pruritic 
skin diseases 
16. Any patient who is pregnant or lactating or who is planning to become pregnant within 
24 weeks of randomization 
17. Sexually active males and females who are not using a reliable contraceptive method with 
≤1% failure rate (such as hormonal contraception, intrauterine device, or complete 
abstinence) throughout the duration of the study and 90 days thereafter (from signed 
informed consent through 90 days after last dose of study drug).  See Appendix 6 for 
further details 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 36 
Final Amendment 03, 22November 2018 
 
18. Patient with a past medical history of alcohol or substance abuse will be excluded.  
Patient must agree to refrain from illicit drug and alcohol use during the study 
19. Administration of bile acid or lipid binding resins and medications that slow GI motility 
(Refer to Appendix 1 – Concomitant Medications Guidelines) 
20. Patient has had investigational exposure to a drug, biologic agent, or medical device 
within 30 days prior to Screening, or 5 half-lives of the study agent, whichever is longer 
21. Patient who has been previously treated with an IBAT inhibitor whose pruritus has not 
responded to treatment 
22. Any other conditions or abnormalities which, in the opinion of the investigator or Medical 
Monitor, may compromise the safety of the patient, or interfere with the patient 
participating in or completing the study 
7.2.3 
Withdrawal of Patients 
Patients/caregivers will be informed that they have the right to withdraw from the study at 
any time, without prejudice to their medical care, and that they are not obliged to state their 
reasons. 
Any withdrawal, and reasons for withdrawal, must be fully documented in the electronic case 
report form (eCRF) and source documents and the patient followed by the 
investigator/investigative staff.  Even after the study is completed at the Follow-up Visit, the 
investigator/investigative staff will, regardless of reason for withdrawal, record any patient 
data they receive concerning SAEs, and all drug-related non-serious AEs, and subsequently 
report these according to Section 10.1.  Withdrawn patients will not be replaced. 
Patients will be withdrawn in the following circumstances: 
• A patient’s/caregiver’s desire for withdrawal for any reason 
• Lost to follow-up (every effort must be made to contact the patient/caregiver; a certified 
letter must be sent) 
• An AE that, in the opinion of the investigator, necessitates withdrawal 
• A worsening of the symptoms related to diarrhea occurs 
• Death 
• Patient/caregiver estimation of intolerable symptoms 
• A patient’s/caregiver’s substantial noncompliance (eDiary and study drug compliance) or 
protocol violation 
• An investigator’s opinion that continuing the patient in the study is not appropriate.  The 
investigator may withdraw a patient at any time, if it is considered to be in the patient’s 
best interest 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 37 
Final Amendment 03, 22November 2018 
 
A patient who prematurely withdraws due to intolerable symptoms, and has completed at 
least 12 weeks treatment in the double-blind study, will be offered the opportunity to enter 
open-label Study A4250-008.  A patient who withdraws from treatment prematurely and does 
not enter Study A4250-008 will have EOT (Visit 9) assessments at the time of withdrawal 
and a follow-up assessment (Visit 10) 28 days following the last dose of study drug, and the 
patient will be recorded as withdrawn. 
7.2.4 
Study Termination by Sponsor 
This study may be terminated at any time by Albireo if serious side effects should occur, if 
the investigator does not adhere to the protocol; or if, in the sponsor's judgment, there are no 
further benefits to be achieved from the study.  In this event, Albireo/designee will inform the 
study investigators, institutions, and all regulatory authorities. 
Albireo may temporarily or permanently discontinue the study at an investigative site at any 
time for safety, ethical, compliance, or other reasons.  If this is necessary, Albireo will 
endeavor to provide advance notification to the site.  If a site or the study is suspended or 
discontinued, the investigator/investigative staff will be responsible for promptly informing 
the Independent Ethics Committee (IEC).  If required by local regulations, Albireo/designee 
will be responsible for informing the IEC and the regulatory authority of study or site 
discontinuation.  In such an event, all study data and unused study drug must be returned to 
Albireo. 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 38 
Final Amendment 03, 22November 2018 
 
8 
TREATMENT OF PATIENTS 
8.1 
Identity of Study Drug 
A4250 and placebo will be supplied as capsules for oral administration.  White opaque 
capsules filled with pellets containing A4250 or placebo will be provided.  Two different 
capsule sizes will be available, as follows: 
• Capsule size 0 that can be opened 
• Capsule size 3 that should be swallowed intact.  These size 3 capsules may be opened 
only under exceptional circumstances, e.g., patient cannot swallow capsule intact 
The content of the A4250 and placebo capsules will be identical in appearance.  Capsule 
filling weight will also be identical for high dose, low dose, and placebo.  Bottles with 
34 capsules will be given to the patient at each visit.  A patient who requires 2 or more 
capsules per day will be given multiple bottles.  Refer to the investigational product manual. 
8.2 
Administration of Study Drug 
Patients will be dosed with A4250 at a dose of 40 µg/kg/day, 120 µg/kg/day, or placebo for 
24 weeks.  Study drug will be dispensed to the patient at Visits 3, 4, 5, 6, 7, and 8, together 
with instructions on how to store and take the drug.  Study drug administration data, 
including whether each patient took each dose or partial doses of study drug, whether there 
were any delayed or missed doses, and whether the capsule was opened or swallowed whole 
will be documented through the eDiary and transferred to the study database. 
A4250 should be taken in the morning, together with food.  On clinic visits days when 
laboratory assessments are conducted, study drug should be taken after the visit and after 
laboratory samples are taken.  Patients should not crush or chew the capsule(s).  When 
swallowing the capsule intact, the patient should administer the dose with a glass of water. 
For a patient unable to swallow capsule intact, the capsule can be opened and the contents 
sprinkled and mixed in a small amount of soft room-temperature yogurt, apple sauce, or fruit 
purée.  A list of food options can be found in the Pharmacy manual. The amount of food 
should be less than what the patient/infant is expected to eat and should be followed by water.  
1. Hold the capsule over the prepared food, gently twist open and allow the content to fall 
into food 
2. Mix the entire capsule content with 1 or 2 tablespoons (15 to 30 mL) of soft food (apple 
sauce, fruit purée, or yogurt) 
3. Administer the mixture immediately after mixing. 
The number of capsules provided to the patient will be based on body weight at Visit 3.  At 
Visit 6, if a patient’s weight has shifted to another weight category (identified in Table 2), the 
number of capsules per day may be adjusted.   


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 39 
Final Amendment 03, 22November 2018 
 
 
Table 2 
Dosing and Capsule Strength 
Body Weight 
(kg) 
Capsule 
Size 
Number of 
Capsules 
per Day 
Capsule 
Strength, 
Low Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Strength, 
High Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Size, 
Placebo 
(µg) 
5.0 to <7.5 
0 
1 
200 
200 
600 
600 
0 
7.5 to <12.5 
0 
2 
200 
400 
600 
1200 
0 
12.5 to <17.5 
0 
3 
200 
600 
600 
1800 
0 
17.5 to <19.5 
0 
4 
200 
800 
600 
2400 
0 
19.5 to <25.5 
3 
2 
400 
800 
1200 
2400 
3 
25.5 to <35.5 
3 
3 
400 
1200 
1200 
3600 
3 
35.5 to <45.5 
3 
4 
400 
1600 
1200 
4800 
3 
45.5 to 55.5 
3 
5 
400 
2000 
1200 
6000 
3 
>55.5 
3 
6 
400 
2400 
1200 
7200 
3 
8.3 
Study Treatment Packaging and Labeling 
8.3.1 
Packaging and Labeling 
The capsules will be packed in high-density polyethylene containers, with child-proof 
polypropylene caps.  Study drug capsules containing A4250 in the strength specified in 
Table 2 above will be manufactured. 
Packaging and labeling will be prepared to comply with applicable regulatory requirements. 
8.3.2 
Storage 
Treatment packs containing A4250 capsules should be stored and dispensed in accordance 
with regulations in their original containers.  The storage facility at the investigative site 
should be locked and the storage temperature should be between 15°C and 25°C. 
Patients/caregivers should be informed of appropriate storage conditions (i.e., room 
temperature, between 15°C and 25°C). 
Any deviations from the recommended storage conditions should be immediately reported to 
Albireo and the study drug should not be used until authorization has been given by Albireo. 
8.3.3 
Blinding and Randomization of Study Drug 
A double-blind design is employed so that both the investigators and the patients will be 
unaware of the treatment assignment during the study.  
After written informed consent is obtained from an eligible patient, an 8-digit patient 
identification number will be assigned.  The first 2 digits will denote the country, followed by 
a 3-digit site number and a 3-digit patient sequence number.  This number will be created and 




 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 41 
Final Amendment 03, 22November 2018 
 
schedule.  Once a randomization code has been broken, the investigator must inform the 
 and sponsor Medical Monitor in writing within 24 hours.  In case of technical issues 
accessing the system online, please see the IWRS site user manual for country-specific 
contact telephone numbers for the 
 HelpDesk 24/7 system support. 
8.5 
Patient Compliance 
The study nurse will monitor eDiary compliance by routine review of the CRF health 
website.  If both diary entries on a day are missing, the study nurse will call the 
patient/caregiver to remind them to complete all scheduled entries.  Any noncompliance will 
be documented and explained in the source documents. 
Study drug compliance will also be checked through diary responses.  Additionally, patients 
will return all unused study drug at Visits 4, 5, 6, 7, 8, and 9.  The study site staff will count 
all returned drug, assess compliance, and record details in the eCRF. 
Treatment Compliance = 100 × ((Number of study drug dispensed – number of study drug 
returned)/number of study drug that should be taken). 
Treatment compliance between 80% and 120% will be acceptable. 
8.6 
Study Drug Accountability 
Records shall be maintained of the delivery of study treatment(s) to the study site(s), of the 
inventory at the study site(s), of each use of the study treatment(s) for each patient, and of the 
return of unused study treatment(s) to Albireo. 
These records shall include dates, quantities, batch numbers, expiry dates, and the unique 
code numbers assigned to the study drug and to the study patients. 
The investigator will be responsible for ensuring that the records adequately document that 
the patients were provided the quantities specified in the protocol and that all study drug 
received from Albireo is reconciled.  
8.7 
Concomitant Therapy and Prohibited Medications 
The investigator will note all ongoing medication and any medication recently stopped 
(within 1 month prior to Visit 1) in the eCRF.  At Visits 2 through 10, all changes in 
medication (stopping or starting new medication or changes in dose) will be recorded in the 
eCRF.  All medications (prescribed or over the counter) for pruritus and sleep will be 
recorded. 
All medications taken by a patient within 3 months prior to the first intake of study drug are 
regarded as prior medication. 
All medications taken by a patient on or after the first intake of study drug, and which 
continue to be taken during the study, are regarded as concomitant medication.  
Prohibited medications are listed in Appendix 1. 


 
 
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9 
ASSESSMENT OF EFFICACY 
9.1 
Efficacy Endpoints 
9.1.1 
Primary Efficacy Endpoint 
The primary efficacy endpoints are as follows: 
• European Union (EU) and rest of the world (RoW): Proportion of patients 
experiencing at least a 70% reduction in fasting s-BA concentration from baseline to 
the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 
weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization 
and the previous visit (Clinic Visit 2).  The end value is the average of the values at 
Weeks 22 and 24 after the start of double-blind treatment. 
• United States (US): Proportion of positive pruritus assessments at the subject level 
over the 24-week Treatment Period. A positive pruritus assessment is defined as a 
scratching score of ≤1 or at least a one-point drop from baseline on the Albireo 
ObsRO instrument. At each assessment, the AM score will be compared to the 
baseline AM average, and the PM score will be compared to the baseline PM average. 
Both AM and PM pruritus assessments will be included in the analysis of this 
endpoint. AM scores from the 14 days prior to the first dose of study medications will 
be averaged as AM baseline. PM scores from the 14 days prior to the first dose will be 
averaged as PM baseline.  
9.1.2 
Secondary Efficacy Endpoint 
Secondary efficacy endpoints include the following:  
• EU and RoW: Proportion of positive pruritus assessments at the subject level over 
the 24-week Treatment Period. A positive pruritus assessment is defined as a 
scratching score of ≤1 or at least a one-point drop from baseline on the Albireo 
ObsRO instrument. At each assessment, the AM score will be compared to the 
baseline AM average, and the PM score will be compared to the baseline PM average. 
Both AM and PM pruritus assessments will be included in the analysis of this 
endpoint. AM scores from the 14 days prior to the first dose of study medications will 
be averaged as AM baseline. PM scores from the 14 days prior to the first dose will be 
averaged as PM baseline.   
• US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment. 
• All Regions: 


 
 
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All secondary endpoints are compared to placebo. 
o Change from baseline to Week 12 and to Week 24 in fasting serum bile acids 
o Change from baseline to Week 12 and to Week 24 in serum ALT 
concentration 
o Change in growth from baseline to Week 12 and to Week 24Proportion of 
responders for pruritus scores at Weeks 12 and 24 based on the Albireo PRO 
and ObsRO instruments  
o Change in sleep parameters measured with the Albireo PRO and ObsRO 
instruments from baseline over the 24-week Treatment Period  
o Proportion of individual assessments meeting the definition of a positive 
pruritus assessment at the subject level over the 24-week Treatment Period. A 
positive pruritus assessment includes an itch score ≤1, or at least a one-point 
drop from baseline based on the Albireo PRO instrument; only patients 
≥8 years of age will complete the Albireo PRO instrument  
o Proportion of individual assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-
12, Weeks 0-18, Weeks 0-24, respectively, or the proportion of positive 
pruritus assessments at each 4-week interval.  
o Proportion of individual AM assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-
12, Weeks 0-18, Weeks 0-24, respectively, or the proportion of positive 
pruritus assessments at each 4-week interval.  
o Proportion of individual PM assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-
12, Weeks 0-18, Weeks 0-24, respectively, or the proportion of positive 
pruritus assessments at each 4-week interval.  
o Number of patients undergoing biliary diversion surgery or being listed for 
liver transplantation 
9.1.3 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, 
caregiver, and clinician via the PGIC items (Appendix 3) 
• Change from baseline in fasting s-BA at Week 4  


 
 
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• Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
by each 4 week-interval of the Treatment Period as measured by the Albireo ObsRO 
instrument 
• Change from baseline by each 4-week interval of the Treatment Period in patient-reported 
and observer-reported night time itching and scratching severity scores, respectively  
• Change from baseline by each 4-week interval of the Treatment Period in patient-reported 
and observer-reported morning time itching and scratching severity scores, respectively 
• Change from baseline by each 4-week interval of the Treatment Period in pooled pruritus 
score including observer-reported scratching for patients <8 years of age and 
patient-reported itch severity for patients ≥8 years of age 
• Change from baseline by each 4-week interval of the Treatment Period in additional 
patient-reported and observer-reported sleep parameters (e.g., tiredness and number of 
awakenings) 
• Change from baseline to Week 24 in PedsQL (Appendix 4) 
• Change from baseline in serum ALT concentration at Week 4  
• Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• Change from baseline in AST and GGT at Weeks 4, 12, and 24 
• Change from baseline for other biochemical markers and measures of bile acid synthesis 
(autotaxin, p-C4) at Week 24 
• Change in pediatric end-stage liver disease/model for end-stage liver disease 
(PELD/MELD) score, AST to platelet ratio index (APRI) score, and Fib-4 score from 
baseline to Week 24 
• Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® 
(where available) 
• Liver-related mortality and liver decompensation events 
• All-cause mortality 
9.2 
Efficacy Assessments 
9.2.1 
Serum Bile Acids 
Blood samples for analysis of fasting total s-BA will be drawn at all visits.  Patients will fast 
(water intake only is permissible) for at least 4 hours prior to the collection of samples for 
s-BA.  Exceptions can be made for infants, <12 months of age, if unable to fast for the full 
4 hours.  Any visit at which a bile acid sample result is unreportable, an additional 
unscheduled visit for a repeat sample collection may be scheduled.  All s-BA results during 


 
 
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the treatment period and follow-up will be blinded.  Samples will be processed and 
transported to a central laboratory per instructions in the laboratory manual. 
9.2.2 
Itching, Scratching and Sleep Score 
Itching, observed scratching, and sleep disturbance will be recorded twice each day via 
eDiary.  Patients and/or caregivers will be instructed to complete the eDiary every day in the 
morning after the patient wakes and in the evening just before the patient goes to sleep 
throughout the study.  Patients and caregivers will receive a formal training session during 
Visit 1. 
The eDiary includes Albireo ObsRO and PRO items.  Patients <8 years of age will not be 
asked to complete the Albireo PRO items; only the Albireo ObsRO will be completed by 
caregivers of patients in this age group.  Older patients, 8 to 18 years of age, will complete 
the Albireo PRO items and the caregiver will complete the Albireo ObsRO items.  The 
Albireo PRO items assess severity of itch, aspects of sleep disturbance (morning diary only), 
and tiredness.  For patients 8 to 12 years of age, the caregiver will read the Albireo PRO 
items along with the child and record the child’s response.  A guide will be provided to the 
caregivers that provides standardized explanations of the Albireo PRO items, in case the 
patient is confused or requires clarification about the meaning of a question.  The Albireo 
ObsRO items assess severity of observed scratching, aspects of observed sleep disturbance 
(morning diary only), and observed signs of tiredness (evening diary only).  The Albireo 
ObsRO and PRO scratching and itch severity items use 0 to 4 response scales, where each 
response is distinguished by a unique facial expression, verbal anchor, number, and color 
code (Appendix 2).  To meet eligibility criteria, a scratch score will be calculated from daily 
diary entries (ObsRO items) the 14 consecutive days prior to randomization.  
A daily AM & PM score for the Albireo ObsRO scratching item will be averaged from the 2 
ratings for each day.  A daily score will be considered missing if both of the daily 
assessments are missing.  A weekly score will be calculated by averaging the daily scratching 
scores.  A weekly score will be considered missing if ≥4 out of 7 days a week of data are 
missing.  For AM & PM baseline, a 14-day daily score of AM & PM prior to the first dose of 
study medications will be averaged as the baseline score. 
For AM baseline, a 14-day AM score prior to the first dose of study medications will be 
averaged as the baseline AM score.  The same approach will be used for PM baseline and to 
calculate a patient-reported itch severity score.  The handling of missing data is described in 
Section 11.2.2.2. 
9.2.3 
Growth 
Growth will be measured as height (velocity) and weight (Z-score) using a certified weight 
scale.  BMI will be calculated by weight (kg) / height (m)2.  Change will be defined as linear 
growth deficit (weight and BMI for age) compared to a standard growth curve (Z-score, SD 
from P50). 


 
 
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9.2.4 
Quality of Life Questionnaire (PedsQL) 
Patients and caregivers will be asked to complete a QoL questionnaire (PedsQL) at Visit 3 
and Visit 9.  Details of the questions included on the questionnaire are provided in 
Appendix 4. 
9.2.5 
Biomarkers and PK Samples 
Blood samples for autotaxin and p-C4 will be drawn at Visits 3, 4, and 9 only for children 
with body weight >10 kg.  Blood for A4250 PK assessment will only be drawn at Visits 4 
and 9 (only for children with body weight >10 kg), and as close to the onset of an SAE as 
possible, should an SAE occur.  Samples will be processed and transported to a laboratory 
per instructions in the laboratory manual. 
9.2.6 
PELD/MELD Score 
At randomization and end of treatment, PELD score will be calculated for children up to 
12 years of age.  For children 13 years or older, the MELD score will be calculated. 
PELD Score = 4.80 * ln(total bilirubin) + 18.57 * ln(INR) - 6.87 * ln(albumin) + 4.36 (if 
patient <1 year: scores for patients listed for liver transplantation before the patient's first 
birthday continue to include the value assigned for age (<1 year) until the patient reaches the 
age of 24 months) + 6.67 (if the patient has growth failure [≤2 SD]) 
Laboratory values less than 1.0 will be set to 1.0 for the calculation of the PELD score. 
MELD Score = 9.57 * ln(creatinine) + 3.78 * ln(total bilirubin) + 11.2 * ln(INR) + 6.43 
Laboratory values less than 1.0 will be set to 1.0 and serum creatinine values greater than 
4.0 mg/dL (equivalent to 353.6 µmol/L)will be set to 4.0 for calculation of the MELD score. 
9.2.7 
Fibroscan® 
Where available, Fibroscan® will be performed as per institution standard practice at Visits 3 
and 9. 
9.2.8 
Markers of Fibrosis 
APRI score and Fib-4 score will be calculated at randomization and at end of treatment. 
APRI = [(AST in U/L) / (AST ULN in U/L)] / (Platelets in 109/L) 
Fibrosis 4 Score = (Age*AST in U/L) / (Platelets in 109/L * √(ALT in U/L)). 
9.2.9 
Global Impression of Change and Global Impression of Symptom Measures 
Patients (≥8 years of age), caregivers, and clinicians will complete the PGIC and the PGIS 
measures (Appendix 3) at randomization (Visit 3; PGIS only), Visits 4 and 6, and at EOT 
(Visit 9). 
The PGIC items assess change in itch (patient version), scratching (caregiver and clinician 
versions), and sleep (all versions) since starting the study drug.  The PGIS items assess itch 


 
 
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(patient version), scratching (caregiver and clinician versions), and sleep (all versions) in the 
past week.  Caregivers and clinicians will complete the PGIC and PGIS for all patients; those 
patients ≥8 years of age will complete the patient version. 


 
 
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10 
ASSESSMENT OF SAFETY 
The timing and frequency of safety assessments are described in Section 7.1.3 and in Table 1 
above. 
The primary safety analysis for this study will include the incidence of total 
treatment-emergent adverse events (TEAE) and TEAEs categorized by causality, severity, 
and seriousness assessments made by the investigator by comparing study drug exposure to 
placebo. 
Trends in safety will also be evaluated for the following assessments: 
• Physical examinations 
• Concomitant medications 
• Vital signs 
• Laboratory test results (including clinical chemistry, hematology, urinalysis, AFP, and 
vitamins A and E, 25-hydroxy vitamin D, and INR) 
• Abdominal ultrasound 
• Discontinuations due to AEs 
10.1 
Adverse Events 
10.1.1 
Definitions and Investigator Assessments 
An AE is defined as any untoward medical occurrence in an enrolled patient regardless of 
causal relationship with study drug.  An AE can therefore be any clinically significant 
unfavorable and unintended sign, symptom, or disease that occurs once a patient is enrolled 
(ICF is signed) in the study until the patient is discharged from the study, whether or not 
related to the study drug. 
10.1.1.1 
Clinical Significance 
Clinical significance is defined as any abnormal finding that results in further clinical 
investigation(s), treatment(s), or the diagnosis of new condition.  Patient-reported events and 
protocol-mandated laboratory values, vital signs, and physical examination findings can be 
considered clinically significant (i.e., an AE) if there is a deterioration as compared to 
baseline.  Examples of clinically significant worsening from baseline could include, but is not 
limited to, events causing withdrawal from the study and events requiring medical 
intervention outside of the study causing apparent clinical manifestations, or judged relevant 
by the investigator.   
10.1.1.2 
Serious Adverse Events 
Serious criteria are applied by the investigator to each AE as specifically defined below.  
These criteria are used to determine whether an AE is serious or non-serious.  The assessment 


 
 
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is made independently of severity assessment (Section 10.1.1.3).  For example, the 
development of a severe rash that occurs after signing of informed consent may not meet 
serious criteria as defined below and therefore would be considered a severe, non-serious AE. 
Any AE that meets any 1 of the following 6 criteria is considered an SAE: 
• The outcome of the AE is death 
• The AE is immediately life-threatening.  Life-threatening means that the patient is, in the 
opinion of the investigator, at immediate risk of death from the reaction as it occurred.  
This does not include an AE that, if more severe, might have caused death 
• The AE results in persistent or significant disability/incapacity.  Disability means a 
substantial disruption of a person's ability to conduct normal life functions 
• The AE requires or prolongs hospitalization  
• The AE results in a congenital anomaly/birth defect 
• The AE is an important medical event.  Important medical events may meet serious 
criteria should the investigator assess that they may significantly jeopardize the patient, 
represent a significant hazard, or requires medical/surgical intervention to prevent one of 
the outcomes listed above.  Examples of potential SAEs based on this criteria include, but 
are not limited to, allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions even if they do not result in inpatient 
hospitalization, or the development of drug dependency and drug abuse. 
10.1.1.3 
Severity Assessment 
Severity assessments are based on the intensity of the event in relation to expectation.  The 
investigator will assess the intensity of AEs based on the following definitions: 
• Mild (awareness of sign or symptom, but easily tolerated) 
• Moderate (discomfort sufficient to cause interference with normal activities) 
• Severe (incapacitating, with inability to perform normal activities) 
Severity is a measure of intensity where seriousness is defined by the criteria outlined in 
Section 10.1.1.2.  An AE of severe intensity need not necessarily be considered serious.  For 
example, nausea that persists for several hours may be considered severe nausea, but not meet 
serious criteria, and therefore would be assessed as a severe AE but not an SAE. 
10.1.1.4 
Causality Assessment 
The investigator determines the causality of all AEs to the study drug using medical judgment 
and considering all relevant factors such as (but not limited to) the underlying study 
indication, coexisting disease, concomitant medication, relevant history, pattern of the AE, 
temporal relationship to the study drug, and de-challenge or re-challenge.  The causality 
assessment of the AE/SAE is to be made as follows. 


 
 
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Related to study drug (possibly, probably, or definitely related) 
Based on medical judgment, there is at least a reasonable possibility that the study drug 
caused the event; one or more of the following criteria apply: 
• The event follows a reasonable temporal sequence from administration of study drug 
• The event could not be reasonably attributed to the known characteristics of the patient's 
clinical state, environmental or toxic factors, or other modes of therapy administered to 
the patient 
• The event follows a known pattern of response to study drug 
• The event disappears or decreases on cessation or reduction in dose of the study drug.  (It 
should be noted that in some situations an AE will not disappear or decrease in intensity 
upon discontinuation of study drug despite other clear indications of relatedness) 
• The event reappears or worsens when the study drug is re-administered 
Unrelated to study drug (unlikely or unrelated) 
Based on medical judgment there is no reasonable possibility that the study drug caused the 
event; one or more of the following criteria apply: 
• The event does not follow a reasonable temporal sequence from administration of study 
drug 
• The event could be reasonably attributed to the known characteristics of the patient's 
clinical state, concurrent illness, environment or toxic factors, or other modes of therapy 
administered to the patient 
• The event does not follow a known pattern of response to study drug 
• The event does not disappear or decrease on cessation or reduction in dose of the study 
drug, and it does not reappear or worsen when the study drug is re-administered 
10.1.2 
Recording of Adverse Events 
It is the investigator’s responsibility to assess whether each untoward event is a clinically 
significant worsening from baseline, thereby considered an AE.  For all AEs, the severity, 
seriousness, and causality to study drug for each AE as outlined in Section 10.1.1 will be 
assessed and recorded in the eCRF. 
All serious and non-serious AEs are collected once the caregiver/patient has signed the ICF 
and until the post-treatment follow-up (Visit 10) or 28 days after the last dose of study drug. 
Any AEs or SAEs that are unresolved at the patient’s last AE assessment in the study are 
followed up by the investigator until resolution or stabilization up to the database lock and 
recorded in the eCRF.  Albireo retains the right to request additional information for any 
patient with ongoing AE(s) or SAE(s) at the end of the study. 


 
 
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TEAEs are defined as any AE that occurs after randomization (Day 0).  All AEs that occur in 
the Screening Period, i.e., after enrollment and prior to randomization, will be collected on 
the eCRF as non-treatment-emergent AEs. 
If there is a clinically significant deterioration of a laboratory value/vital sign or other routine 
study assessment that is associated with a diagnosis, the clinical diagnosis will be reported as 
an AE and the associated signs and symptoms will be considered additional information 
unless the sign or symptom is more severe than expected given the diagnosis.  For example, if 
an investigator diagnoses a study patient with hepatitis during the study period, hepatitis 
would be considered the AE and the concomitant signs and symptoms of abdominal pain, 
vomiting, and elevated ALT and AST would not be reported separately unless, in the opinion 
of the investigator, one of these signs or symptoms is more severe than expected and 
therefore a separate AE assessment is indicated. 
10.1.3 
Recording and Reporting of Serious Adverse Events 
Every SAE (regardless of severity and causality) that occurs once the caregiver/patient has 
signed the ICF and until the post-treatment follow-up (Visit 10), and 28 days after the final 
dose of study drug, should immediately and not later than within 24 hours of knowledge of 
the event, be reported by the investigator or delegate in the SAE report form.    
Report of a SAE must include at least the following information: 
• Patient identification information (study number, site number, initials, and date of birth 
[as per local country requirements for data protection]) 
• The last study drug administration date 
• The diagnosis of the event with the description (or a brief description of 
signs/symptoms/clinical course if the diagnosis is not available) and the date of onset 
• The action used to treat the event (i.e., treatment medications, temporary discontinuation) 
• The reason(s) for considering the event serious 
• The relationship of the event to the study drug or to the study procedure (e.g., the 
investigator's assessment of causality) 
• A brief narrative of the SAE 
Follow-up reports including all new information obtained of the subsequent course of the 
SAE must be prepared and the information collected in the SAE submitted to 
 
 (Pharmacovigilance and Safety Services Department) by e-mail within 24 hours of 
knowledge of the event. 
The pharmacovigilance manager (PVM) may contact the investigator to obtain further 
information on a reported SAE.  The investigator/investigative staff must respond to any 


 
 
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request for follow-up information or answers to questions regarding the SAE within the same 
timelines as for initial reports. 
The PVM reports the occurrence of the SAE and follow-up to the Albireo medical monitor 
for medical assessment of the case. 
Should an outcome of death occur within the study period or within 28 days after the last 
administration of study drug, an SAE form should be completed including the main and 
contributory causes of death. 
All SAE reports must be e-mailed to the following e-mail address within 24 hours: 
 
 
If email is unavailable, SAEs may be transmitted via fax to the following number: 
 
In a study-related medical emergency situation, when the assigned medical monitors for the 
study cannot be reached by a caller, an on call physician can be reached 24 hours per day, 
7 days per week, via the 
 call center.  The following number is a chargeable telephone 
number allowing a global reach from both landlines and mobile phones and the internet page 
indicated below contains a list of country-specific toll-free telephone numbers that is 
provided.  It should be noted that not all countries globally have access to toll-free numbers 
as indicated on the “24/7 Medical Help desk” index.  Countries without toll-free numbers 
need to dial the chargeable number as indicated below.  Furthermore, toll-free numbers might 
not be available from mobile phones. 
 
 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions 
Suspected unexpected serious adverse reaction (SUSAR) is an SAE that occurs in a patient, 
the nature or severity of which is not expected per the applicable product information (e.g., 
the IB for an unauthorized investigational product or summary of product characteristics for 
an authorized product).  
Reporting and tracking of SUSARs will be in accordance to all applicable competent 
authority regulations.  The Ethics Committee and all investigators involved in this study will 
be informed according to national and international requirements. 


 
 
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10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments 
10.2.1 
Laboratory Assessments 
Samples will be collected for clinical chemistry, hematology, and urinalysis testing at the 
time points specified in Table 1. The parameters assessed are presented in Table 3. 
Blood for AFP will be drawn at Visits 3 and 9.  Fat-soluble vitamin levels including vitamins 
A and E, and 25-hydroxy vitamin D will be drawn at Visit 2 (vitamins A & E only), 
Visit 3 (25-hydroxy vitamin D only), Visit 6, and Visit 9.  Blood for INR (surrogate for 
vitamin K) will be drawn at Visits 2, 4, 6, 8, and 10.  Patients will fast (water intake only is 
permissible) for at least 4 hours prior to the collection of samples for vitamin A.  Exceptions 
can be made for infants, <12 months of age, if unable to fast for the full 4 hours.  If a patient 
has any vitamin level(s) that are outside of the reference range, vitamin supplementation 
adjustments may be required.  Target ranges and supplementation strategy guidelines can be 
found in Appendix 5. 
A serum pregnancy test will be performed at Visit 1 and Visit 9 if the patient is entering the 
A4250-008 study. A urine pregnancy test will be collected at all other visits for girls who 
have reached menarche.  If a urine pregnancy test is positive, a serum pregnancy test should 
be performed to confirm the pregnancy.  Study drug administration will be held pending the 
confirmatory results.  If the serum pregnancy test is negative, the patient can resume dosing.  
If the serum pregnancy test is positive, the patient should be discontinued (see 
Section 10.2.9). 
All samples will be processed and transported to a laboratory per instructions in the 
laboratory manual. 
The observed values will be recorded and assessed as “normal” or “abnormal not clinically 
significant” or “abnormal clinically significant”. 
Additional safety blood samples may be needed due to follow-up of an abnormal value or 
analysis failure.  The blood samples collected for safety laboratory analysis will be destroyed 
after the analyses have been completed.  


 
 
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Table 3 
Routine Laboratory Parameters 
Clinical Chemistry 
Hematology 
Urinalysis 
• 
Albumin 
• 
ALT 
• 
Alkaline phosphatase  
• 
AST 
• 
Bilirubin – total and direct 
• 
Calcium 
• 
Chloride 
• 
Creatinine 
• 
Creatine kinase 
• 
Gamma-glutamyl transferase 
• 
Potassium 
• 
Sodium 
• 
Hematocrit 
• 
Hemoglobin 
• 
Platelet count 
• 
Red blood cell count 
• 
White blood cell count and 
differential (neutrophils, 
eosinophils, basophils, 
lymphocytes, and monocytes) 
• 
Blood 
• 
Glucose 
• 
Ketones 
• 
Leukocytes 
• 
Nitrites 
• 
pH 
• 
Protein 
 
10.2.2 
Individual Patient Safety Monitoring 
10.2.2.1 
Liver Monitoring 
Strategies to monitor markers of liver disease throughout the study are outlined below where 
the ULN will be based on central laboratory reference values for age and gender. 
If isolated transaminase elevations are observed, defined as: 
1. Normal bilirubin AND absence of clinical hepatitis symptoms AND 
• ALT or AST ≥5 × ULN (if normal at baseline) or an absolute threshold of 800 U/L, 
whichever comes first  
• OR ALT or AST ≥3 × baseline (if abnormal at baseline) or an absolute threshold of 
800 U/L, whichever comes first 
Then: 
a) Repeat liver profile (AST, ALT, bilirubin, and prothrombin time [PT] or INR) within 
2 to 3 days 
b) Evaluate creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) 
c) As needed (for example, persistent ALT/total bilirubin elevation or worsening of liver 
function), consider evaluation for alternative etiologies 
Monitor the patient using close observation found in Section 10.2.2.2 
If any ONE of the following criteria are met: 
1. Transaminases (ALT or AST ≥3 × baseline or 800 U/L, whichever comes first) AND 
bilirubin (total bilirubin > 2 × ULN) elevations 


 
 
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2. Transaminase elevations alone (ALT or AST >10 × ULN or 5 × baseline or absolute 
threshold of 800 U/L, whichever comes first) in presence of normal LDH and CPK 
3. Total bilirubin increased, unrelated to hemolysis (elevated reticulocyte count) or 
established genetic diseases, such as Gilbert’s syndrome 
a. Doubling if total bilirubin was <3 mg/dL (equivalent to 51.3 µmol/L) at baseline  
b. OR Increase by >3 mg/dL (equivalent to 51.3 µmol/L) if total bilirubin was ≥3 mg/dL 
(equivalent to 51.3 µmol/L) at baseline 
4. INR increase refractory to vitamin K administration 
a. INR >1.5 if INR was normal at baseline  
b. OR Increase by >0.4 if INR was abnormal at baseline 
5. Any increase in total bilirubin and transaminases if accompanied by EITHER a symptom 
of clinical hepatitis (e.g., vomiting, nausea, right upper quadrant pain) OR immunological 
reaction (rash or >5% eosinophilia) 
Then: 
a) Interrupt study medication 
b) Initiate drug-induced liver injury work-up for alternative etiologies 
c) Repeat liver profile (AST, ALT, total bilirubin, direct bilirubin) and PT or INR within 
48 to 72 hours 
d) Monitor the patient using close observation found in Section 10.2.2.2 
e) If a patient lives in a remote area, they may be tested locally and the results 
communicated to the investigator site promptly 
10.2.2.2 
Close Observation 
• Repeat liver enzyme and serum bilirubin tests two or three times weekly.  Frequency 
of re-testing can decrease to once a week or less if abnormalities stabilize or the trial 
drug has been discontinued and the subject is asymptomatic  
• Obtain a more detailed history of symptoms and prior or concurrent diseases 
• Obtain a history of concomitant drug use (including non-prescription medications and 
herbal and dietary supplement preparations), alcohol use, recreational drug use, and 
special diets  
• Consider ruling out alternate etiology including acute viral hepatitis types A, B, C, D, 
and 
E; 
autoimmune 
or 
alcoholic 
hepatitis; 
non-alcoholic 
steatohepatitis; 
hypoxic/ischemic hepatopathy; and biliary tract disease 
• Obtain a history of exposure to environmental chemical agents.  


 
 
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• Obtain additional tests to evaluate liver function, as appropriate (e.g., INR, direct 
bilirubin) 
• Consider gastroenterology or hepatology consultations. 
10.2.2.3 
De-challenge/Re-challenge for Liver and Clinical Hepatitis Monitoring 
1. Re-challenge is not recommended: 
a. If a patient has had possible/probable drug-induced liver injury 
b. If a decompensation event has occurred (i.e., variceal hemorrhage, ascites, hepatic 
encephalopathy, etc.). 
2. If due to underlying cholestatic liver disease variability or another alternative etiology is 
identified AND liver tests returned to baseline, re-challenge may be considered after 
consultation with the sponsor Medical Monitor. 
3. If the ALT/total bilirubin elevations are observed after re-challenge, then repeat 
re-challenge is discouraged. 
If patient is permanently discontinued, monitoring should be continued as outlined in 
Section 10.2.2.5.  
10.2.2.4 
Diarrhea 
Study drug should be discontinued if a patient develops diarrhea with at least 1 of the 
following concomitant signs or symptoms: grossly bloody stools, vomiting, dehydration 
requiring treatment with oral or intravenous rehydration and/or electrolyte imbalances, fever 
(≥38 C) and/or the diarrhea persists for 7 or more days. 
If there is gross blood in stool, INR and platelets should be measured.  
Study drug will be reintroduced (re-challenge) when the symptoms have resolved.  If the 
diarrhea re-occurs within 1 week after the re-challenge with no alternate etiology, the patient 
will be permanently discontinued and monitored as outlined in Section 10.2.2.5. 
10.2.2.5 
Monitoring after Permanent Discontinuation of Study Drug Due to Safety 
Once study drug is permanently discontinued for liver, hepatitis, diarrhea, or other severe AE 
related to study drug, the patient will be monitored weekly until the laboratory and clinical 
parameters have normalized/stabilized. 
10.2.3 
Demographics/Medical and Surgical History 
Demographic information per country regulations (age, full date of birth, gender, race, and 
ethnicity), along with medical and surgical history, will be obtained and recorded in the eCRF 
at Visit 1. 
Medical and surgical history will be entered in the eCRF at Visit 1.  This includes date of 
diagnosis of PFIC, prior investigational medications for PFIC, historical (3 months prior to 


 
 
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Screening) LFT values (AST, ALT, and total bilirubin), ongoing medication, any surgery 
performed, any other diagnosis, and historical biopsy data.   
If a liver biopsy has been performed within 1 year prior to Screening, or during the study, the 
results will be recorded in the eCRF. 
10.2.4 
Clinical Genetic Testing 
For previous confirmatory clinical genetic testing results for PFIC Type 1 or 2 performed 
prior to Screening Visit 1, the confirmatory clinical genetic testing laboratory report will be 
verified to determine eligibility. 
If the historical clinical genetic result is equivocal, unavailable, or unobtainable, clinical 
genetic analysis to confirm pathologic biallelic variations in the ATP8B1 or ABCB11 genes 
will be performed and verified to determine eligibility.  No other diagnostic genetic testing 
will be offered. 
10.2.5 
Physical Examination 
A physician or suitably trained qualified assistant will perform a complete physical 
examination, including voluntary photography of skin lesions due to scratching, at Visits 1, 3, 
6, and 9.  A skin examination will be conducted at Visits 1, 3, 4, 6, 9, and 10 (see Table 1).  
A complete physical examination will include assessment of general appearance, eyes, ears, 
nose, throat, head/neck/thyroid, lymph nodes, cardiovascular, lungs/chest, abdomen, 
genitourinary, extremities, skin, musculoskeletal, neurologic, and other.  
Skin will be examined thoroughly and excoriations/scratch marks recorded.  
10.2.6 
Vital Signs 
Evaluation of vital signs will be performed at all visits.  This includes blood pressure (systolic 
and diastolic), pulse, respiratory rate, temperature, height (or length depending on age), and 
body weight (using a certified weight scale) at clinic visits.  BMI will be calculated. 
10.2.7 
Abdominal Ultrasound 
Ultrasound of the liver and spleen will be performed at randomization (Visit 3) and at EOT 
(Visit 9).  Liver size, echogenicity, and presence of masses/nodules as well as spleen size will 
be recorded. 
10.2.8 
Overdose 
A4250 is minimally absorbed and has a very low systemic availability.  Based on toxicology 
data, for study purposes any dose exceeding a total of >3 mg/kg body weight of A4250 taken 
as a single dose or as a cumulative dose within 24 hours is defined as an overdose.   
The no-adverse-effect dose level in the most sensitive species (defined as 20 mg/kg/day) in 
the rat 1 month toxicity study gives a human equivalent dose of approximately 194 mg/day in 


 
 
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a 60 kg person.  This dose is approximately 10-fold higher than the maximum possible dose 
predicted in human studies (20 mg).  
The investigator/investigative staff should immediately and not later than within 24 hours of 
knowledge of the overdose, be reported by the investigator or delegate on the SAE report 
form.  In the event of an A4250 overdose, the patient should be monitored closely. 
10.2.9 
Pregnancy 
If a pregnancy is discovered in a female patient enrolled in the study before the end of dosing, 
the patient will be immediately discontinued from the study and will attend the same visits as 
a prematurely withdrawn patient.  If the pregnancy is discovered in a female patient enrolled 
in the study after the end of dosing, the patient will continue in the study per protocol.  If a 
pregnancy occurs in a male patient’s partner at any time during the study, the pregnancy 
should also be reported and followed. 
Pregnancy is not considered to be an AE.  However, if the patient has been dosed with the 
study drug, the pregnancy must be reported on the Paper Pregnancy Notification Form 
immediately within 24 hours after recognition to Safety Manager (please refer to Section 
10.1.3).  Date of exposure and as far as possible, details of the period of gestation at the time 
of exposure must be given. 
The pregnancy should be followed to determine outcome, including spontaneous termination, 
details of birth, and presence of any birth defects, congenital anomalies or newborn or 
maternal complication.  The infant who was exposed in-utero will be followed for up to 
2 years after delivery.  Individual cases with an abnormal outcome in association with the 
study drug should be reported on an expedited basis, i.e., reported rapidly to a competent 
authority. 


 
 
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11 
STATISTICAL EVALUATION 
All personnel involved with the analysis of the study will remain blinded until database lock 
for the double-blind Treatment Period and until major protocol violations have been 
identified. 
All statistical analyses will be performed using SAS version 9.3 or higher. 
11.1 
Sample Size and Power 
For the EU and RoW, the primary analysis will relate to the serum bile acid responder 
endpoint.  For the US, the primary analysis will relate to the pruritus endpoint. For each 
primary endpoint, a pooled analysis for the closed testing procedure will be applied to control 
the 1-sided overall type I error rate for two treatment comparisons vs. the placebo at the 0.025 
level (Kane et al, 2015, Lehmacher et al, 1991, and Marcus et al, 1976). The study will enroll 
60 to 70 patients in order to obtain at least least 20 evaluable patients in each arm. For each 
primary endpoint, simulations with 5000 iterations using 20 patients per arm were conducted 
to estimate the power after multiplicity adjustment, resulting in a standard error of <0.7% for 
each estimated power.   
Based on the Phase 2 A4250-003 study data both dose groups are predicted to have similar 
treatment effects for both serum bile acids and pruritus. For serum bile acids, simulated 
proportions were analyzed using the CMH test to generate the following 1-sided p-values: 
both A4250 arms pooled vs. placebo, low dose vs. placebo, and high dose vs. placebo. 
Assuming 60% responders in the A4250 arms and 10% responders in the placebo arm, the 
power to claim significance for a particular A4250 arm after multiplicity adjustment is 
approximately 94%. The power to claim significance for at least one arm or both arms are 
approximately 99% and 91%, respectively.  
For the proportion of positive pruritus assessments at the subject level in pruritus scores, 
simulated proportions were analyzed using ANCOVA to generate the following 1-sided p-
values: both A4250 arms pooled vs. placebo, low dose vs. placebo, and high dose vs. 
placebo. With an effect size of 1.0526, the simulated power to claim significance for a 
particular arm after multiplicity adjustment is approximately 89%. The probability to claim 
significance for at least one arm or both arms are approximately 95% and 83%, respectively.   
11.2 
Statistical Methods 
11.2.1 
Statistical Analysis Sets 
Safety Analysis Set 
The safety analysis set will consist of all patients who received at least 1 dose of study drug.  
Patients will be analyzed according to the treatment they actually received.  The safety 
analysis set will be the primary analysis set for safety analyses.  
Full Analysis Set 


 
 
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The full analysis set (FAS) will consist of all randomized patients who received at least 
1 dose of assigned treatment.  Patients will be analyzed in the treatment group they were 
randomized to even if they received incorrect study drug.  The FAS will be the primary 
analysis set for efficacy analyses. 
Per Protocol Analysis Set 
The per protocol (PP) analysis set is a subset of the FAS, and will consist of all randomized 
patients for whom no major protocol violation (no violation which may affect the study 
efficacy outcome) is documented.  The PP analysis set will provide supportive data for the 
efficacy analyses. 
Allocation of patients to the PP analysis set will be performed before unblinding of the study. 
11.2.2 
Methods of Statistical Analyses 
11.2.2.1 
General Principles 
The primary objective of this study is to show superiority in efficacy of 
A4250 (120 µg/kg/day and 40 µg/kg/day) compared to placebo in patients with PFIC Types 1 
and 2. 
In the EU and RoW, for the primary efficacy variable of fasting bile acid responder endpoint 
(at least a 70% reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the 
end of treatment), the Cochran Mantel Haenszel (CMH) test stratified by PFIC class and age 
class will be performed to compare the proportion in fasting bile acid responder endpoint at 
the end of treatment (Week 22 and 24) in the two A4250 dose groups to placebo group. To 
ascertain that all data are used in the CMH analysis, neighboring strata will be pooled if all 
subjects in a stratum have the same response. Dropouts will be treated as non-responders. The 
proportion together with corresponding 95% CI, odds ratio and corresponding 95% Clopper-
Pearson exact CI and p-value for the CMH test will be presented.   
In the US, for the primary efficacy variable of the proportion of positive pruritus assessments 
at subject level over the 24-week treatment period, an analysis of covariance (ANCOVA) will 
be used. The model will include treatment arm, AM and PM baseline pruritus scores, and 
randomization stratification factors, i.e. PFIC class and age class. LS mean(SE) by treatment 
arm and LS mean difference (SE), 95% confidence intervals, and p-values (where applicable) 
between treatments (120 µg/kg/day and 40 µg/kg/day, respectively) vs. placebo will be 
provided. If there are concerns on model assumptions, i.e. normality and homogeneity of 
variances, a non-parametric ANCOVA based on the rank scores will be used as a sensitivity 
analysis. LS mean/SE on the outcome by treatment arm and LS mean difference/SE between 
active dose and placebo will be provided.  
The procedure for multiplicity adjustment is specified as follows:    


 
 
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In the closed testing procedure, the low and high dose groups will be pooled to compare with 
the placebo group first. If the 1-sided p-value is ≤0.025, the 1-sided p-values for low dose vs. 
placebo and high dose vs. placebo will be calculated, respectively. If both individual p-values 
are ≤0.025, a significant treatment effect will be declared on both dose groups. If only one of 
them is ≤0.025, a significant treatment effect will be declared on the corresponding dose 
group.  
Other secondary and exploratory outcome variables will provide supportive efficacy and 
safety information regarding the differences between the treatment groups. No adjustments 
will be performed for multiple comparisons when testing secondary and exploratory efficacy 
variables. 
If not otherwise specified, the baseline value of a parameter is defined as the last assessment 
of that parameter prior to the first intake of study treatment. 
11.2.2.2 
Missing Data 
In the primary analysis of the pruritus endpoint, all intermittently missing assessments will be 
classified as negative pruritus assessments. Similarly, all planned assessments after the 
intercurrent events (premature treatment discontinuation, death or initiation of rescue 
treatments such as biliary diversion surgery or liver transplantation) will be counted as 
negative pruritus assessments. However, as described above, all 336 assessments planned 
over the 24-week treatment period will be included in the denominator of the endpoint 
definition.  
In the primary analysis of fasting bile acid responder endpoint, dropouts and patients with a 
missing average at the end of the treatment will be treated as non-responders. Patients in need 
of surgical rescue, i.e. biliary diversion and listing for liver transplant will be classified as 
non-responders. Continued collection of efficacy data for patients who discontinue treatment 
will be made as far as possible. 
11.2.2.3 
Demographic and Baseline Characteristics 
Descriptive summaries of demographics and other baseline characteristics (including medical 
and surgical history) will be presented by treatment group and overall using the FAS.  
Prior and concomitant medication use during the study will be summarized by treatment 
group and overall using the safety analysis set. 
11.2.2.4 
Subject Disposition 
The following will be summarized (by treatment group and overall where applicable): 
• Patients enrolled (who signed the informed consent) 
• Patients randomized 
• Patients treated 


 
 
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• Patients completing the study 
• Patients withdrawing early (including withdrawal reason) 
Additionally, patients enrolled, included in the safety, in the FAS, and in the PP analysis set 
will be summarized by region. 
 
11.2.2.5 
Evaluation of Primary Efficacy Variables 
For EU and RoW, the primary responder endpoint based on change in fasting bile acids will 
be analyzed in terms of the proportion of patients achieving the event of interest. Patients in 
need of surgical rescue, i.e. biliary diversion and listing for liver transplant, will be classified 
as non-responders. Dropouts will be treated as non-responders.  
CMH stratified by PFIC class and age class will be performed to compare the proportion in 
fasting bile acid responders at the end of treatment (Week 22 and 24) in the two A4250 dose 
groups to the placebo group. To ascertain that all data are used in the CMH analysis, 
neighboring strata will be pooled if all subjects in a stratum have the same response. The 
proportion together with corresponding 95% CI, odds ratio and corresponding 95% Clopper-
Pearson exact CI and p-value for the CMH test will be presented 
Logistic regression will be performed as a supportive analysis to compare the proportion in 
fasting bile acid responders at the end of treatment (Week 22 and 24) in the two A4250 dose 
groups to the placebo group. The model will include treatment arm, baseline value, and 
randomization stratification factors, i.e. PFIC class and age class. Baseline value will be 
calculated as the average of serum bile acid values at randomization and the previous visit 
(Clinic Visit 2).   
In the US, for the primary efficacy variable of the proportion of positive pruritus assessments 
at the subject level over the 24-week treatment period, ANCOVA will be used to analyze the 
pruritus endpoint. The model will include treatment arm, the averaged AM and PM baseline 
pruritus scores, and randomization stratification factors, i.e. PFIC class and age class. LS 
mean(SE) by treatment arm and LS mean difference (SE), 95% confidence intervals, and p-
values (where applicable) between treatments (120 µg/kg/day and 40 µg/kg/day, respectively) 
vs. placebo will be provided. If there are concerns on model assumptions, i.e. normality and 
homogeneity of variances, a non-parametric ANCOVA based on the rank scores can be used. 
LS mean/SE on the outcome by treatment arm and LS mean difference/SE between active 
dose and placebo will be provided.  
Sensitivity analysis will be conducted to assess the impact of intermittently missing data as 
well as intercurrent events and will be specified in the SAP. Subgroup analyses will also be 
performed for each of the 3 age groups (6 months to 5 years, 6 to 12 years, and 13 to ≤18 
years) and for patients who were not included in the previous Study A4250-003. 


 
 
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11.2.2.6 
Evaluation of Secondary and Exploratory Efficacy Variables 
The change in the secondary endpoints such as serum bile acids, total bilirubin, ALT, growth, 
Albireo PRO and ObsRO sleep parameters, and patient-reported itch severity will be 
summarized by visit using descriptive statistics. Change in growth (weight for age and BMI 
for age) will also be displayed using graphical presentations.  
Proportion of responders at Weeks 12 and 24 based on the PRO/ObsRO instruments 
estimated from both distribution and anchor-based approaches will be analyzed using the 
same model as specified for the primary analysis for EU and RoW. 
Proportion of individual assessments meeting the definition of a positive pruritus assessment 
at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, 
respectively, or the proportion of positive pruritus assessments at each 4-week interval over 
the 24-week treatment period for AM or PM, respectively will be analyzed using the same 
model as specified for the primary analysis for US.   
Undergoing biliary diversion surgery and/or listed for liver transplant, all-cause mortality, 
and liver decompensation events will mainly be analyzed by using descriptive statistics. 
Kaplan-Meier curves will be used when appropriate for time to event data.  
Comparisons of the change from baseline at Week 24 in PedsQL between the treatment 
groups will be conducted using ANCOVA. The model will include terms for baseline, PFIC 
class, age class, and treatment. 
Other exploratory variables (Global Symptom Relief, biochemical markers and measures of 
bile acid synthesis, additional Albireo PRO and ObsRO sleep parameters, PELD/MELD 
score, APRI score, and Fib-4 score) will be analyzed descriptively for categorical and 
continuous data, as applicable. For continuous data, the change from baseline will be 
analyzed in addition to the actual visit values. For categorical data, shift tables or frequency 
and percentages of patients will be presented as appropriate. Additional details on secondary 
and exploratory efficacy variables and subgroup analyses by age class (6 months to 5 years, 6 
to 12 years, and 13 to ≤18 years) will be specified in the SAP.  
11.2.2.7 
Evaluation of Safety Variables 
Safety data will be analyzed using descriptive statistics and summaries by treatment group of 
SAEs, AEs, vital signs, clinical laboratory tests (hematology, clinical chemistry and 
urinalysis), and concomitant medication.  Analyses will be performed using the safety 
analysis set.  
Summaries of AEs (coded according to the Medical Dictionary for Regulatory Activities 
[MedDRA] system organ class [SOC] and MedDRA preferred term) will include the 
following:  


 
 
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• Overview of the incidence of TEAEs (TEAEs, drug-related TEAEs, TEAEs leading to 
study discontinuation, and treatment-emergent SAEs) 
• TEAEs by SOC and preferred term 
• Intensity of TEAEs by SOC and preferred term 
• Drug-related TEAEs by SOC and preferred term 
• TEAEs leading to study discontinuation by SOC and preferred term 
• Treatment-emergent SAEs by SOC and preferred term 
Concomitant medication use during the Treatment Period will be summarized by Anatomical 
Therapeutic Chemical (ATC) class and World Health Organization (WHO) preferred name.  
Summaries of vital signs will be presented.  For each visit, the actual results and the change 
from baseline, and the number and percentage of patients with potentially clinically 
significant (PCS) values observed post-baseline will be presented. 
Summaries of clinical safety laboratory data will be presented.  For each visit, the actual 
result and the change from baseline, and the number and percentage of patients with PCS 
values observed post-baseline will be presented. 
Data listings will be provided for each patient for all safety parameters. 
11.2.2.8 
Compliance and Exposure 
Exposure will be analyzed by calculating the number of days with exposure to study drug.  
Results will be presented by treatment group using the safety analysis set.  
The percentage compliance will be described by treatment group and the number of patients 
with a compliance <80%, between 80% and 120%, and >120% will be presented based on the 
safety analysis set. 
11.2.3 
Blinded Analyses to Confirm Responder Definitions 
A blinded analysis of Albireo ObsRO and PRO eDiary data will be performed after 50% or 
more of the planned sample has completed the Week 24 Follow-Up Period.  The blinded 
analysis will be used to estimate a threshold of clinically meaningful change (i.e., responder 
definition) in Albireo ObsRO and PRO pruritus scores.  The analysis will be performed by a 
group independent from both the study team and Albireo.  Blinded data will be used for this 
analysis; data will be collapsed across treatment groups. 
The analyses will include distribution- and anchor-based analyses to examine a responder 
definition for the Albireo PRO/ObsRO pruritus scores.  Distribution-based analyses will 
include calculation of the SD and standard error of measurement for the baseline Albireo 
PRO and ObsRO scores.  Anchor-based analyses will involve examining the degree of 
change on the Albireo PRO and ObsRO scores from baseline to Week 24 for patients who 
experience change in pruritus according to PGIC and symptom items.  Details of this analysis 


 
 
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will be included in a stand-alone SAP for assessing the measurement characteristics 
(reliability, validity, sensitivity to change, responder definition) of the Albireo PRO and 
ObsRO items. 
11.2.4 
Data Safety Monitoring Board 
A DSMB consisting of sponsor-independent clinical and statistical experts will be 
established.  The DSMB will periodically (approximately on a quarterly basis) meet for the 
review of accumulating study data, including safety (AE and laboratory data) and liver 
values.  
The DSMB will have access to unblinded data.  The DSMB will make recommendations for 
the remaining part of the study (further details will be provided in the DSMB charter).  The 
DSMB may recommend continuing with the study as planned or stopping the study early for 
safety reasons.  The DSMB will submit its recommendations in writing to Albireo 
Medical
 who are responsible for responding to the recommendations of the DSMB and 
taking appropriate action.  The investigators will only be informed by Albireo Medical/
 
if the study requires a protocol amendment or is stopped.  The DSMB may choose to make 
additional evaluations at any time if they feel this is warranted from a safety point of view.  
The DSMB will act according to its own written standard operating procedure described in a 
charter and will prepare written minutes of its meetings.  The charter of the DSMB will be 
stored in the trial master file.  The DSMB will maintain records of its meetings and these will 
become part of the study file when the study is complete.  In order not to disseminate 
unblinded data and to ensure that all staff involved in the conduct and final analysis of the 
study remain blind to the data, only the members of the DSMB and the unblinded DSMB 
statistician will have access to these data. 
 


 
 
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12 
DIRECT ACCESS TO SOURCE DATA/NOTES 
The investigator/institution shall provide direct access to source data/documents for 
study-related monitoring, audits, IEC/Institutional Review Board (IRB) review and 
regulatory inspection. 


 
 
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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
13.1 
Conduct of the Study 
Albireo/
 shall implement and maintain quality control and quality assurance procedures 
with written standard operating procedures to ensure that the study is conducted and data are 
generated, documented, and reported in compliance with the protocol, ICH GCP and 
applicable regulatory requirements. 
This study shall be conducted in accordance with the provisions of the Declaration of 
Helsinki (October 1996) and all revisions thereof, and in accordance with United States Food 
and Drug Administration (FDA) regulations (Code of Federal Regulations, Sections 312.50 
and 312.56) and with ICH GCP (CPMP 135/95). 
The investigator may not deviate from the protocol without a formal protocol amendment 
having been established and approved by an appropriate competent authority and IEC/IRB, 
except when necessary to eliminate immediate hazards to the patient or when the change(s) 
involve(s) only logistical or administrative aspects of the study.  Any deviations may result in 
the patient having to be withdrawn from the study and render that patient non-evaluable. 
13.2 
Study Monitoring 
Before study initiation, at a site initiation visit or at an investigator’s meeting, an Albireo 
representative or designee will review the protocol and eCRF with the investigators and the 
investigative staff.  During the study, the clinical monitor (clinical research associate [CRA]) 
will visit the site regularly to check the completeness of patient records, the accuracy of 
entries on the eCRFs, the adherence to the protocol and to GCP, the progress of enrollment, 
and to ensure that study drug is being stored, dispensed, and accounted for according to 
specifications. 
The investigator must ensure that eCRFs are completed within a timely period of patient 
visits, as per individual site agreements, and must allow the CRA and Albireo representative 
or designee periodic access to patient records and all study-related materials, including 
relevant hospital or clinical records, to confirm their consistency with the eCRF entries.  No 
information in these records about the identity of the patients will leave the study center.  
Albireo monitoring standards require full verification for the presence of the signed ICF, 
adherence to the inclusion/exclusion criteria, documentation of SAEs, and recording of 
primary efficacy and safety variables.  The CRA will review source data compared with the 
eCRFs and will verify source data according to the study-specific monitoring plan.  The 
design of the study, the frequency of patient visits, and the site enrollment rate will determine 
the frequency of monitoring visits.  Upon study completion, the CRA will visit the site to 
conduct a study termination visit, which will include collection of any outstanding 
documentation. 


 
 
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It is recommended that the investigator/investigative staff log into the eCRF system every 
working day or at minimum twice weekly in order to provide a prompt response to queries.  
The investigator/investigative staff should respond to queries and make any relevant changes 
to the study data within 3 working days. 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 69 
Final Amendment 03, 22November 2018 
 
14 
ETHICS 
14.1 
Independent Ethics Committee/Institutional Review Board 
Prior to the start of the study, the investigator is responsible for ensuring that the protocol and 
consent form have been reviewed and approved by a relevant IEC/IRB.  The IEC/IRB shall 
be appropriately constituted and perform its functions in accordance with FDA ICH GCP and 
local requirements as applicable. 
The IEC/IRB shall approve all protocol amendments (except for logistical or administrative 
changes), written informed consent documents and document updates, patient recruitment 
procedures (e.g., advertisements), written information to be provided to the patients and 
caregivers, IB, available safety information, information about payment and compensation 
available to patients, the investigator's curriculum vitae and/or other evidence of 
qualifications and any other documents requested by the IEC/IRB and regulatory authority 
(competent authority) as applicable. 
14.2 
Written Informed Consents and Assents 
The investigator (physician) or investigative staff, in accordance with local regulations, will 
explain to each patient (or legally authorized representative) the nature of the study, its 
purpose, the procedures involved, the expected duration, alternative treatment, the potential 
risks and benefits involved, and any discomfort that may occur.  Each caregiver/patient will 
be informed that participation in the study is voluntary and that he/she or their child may 
withdraw from the study at any time and that withdrawal of consent will not affect his/her 
subsequent medical treatment or relationship with the treating physician. 
Caregivers/patients will be informed that they/their children may experience side-effects or 
be at risk for symptoms, illnesses, or complications that cannot be foreseen by Albireo.  As 
with other medications, people treated with A4250 may be at risk of developing allergic 
reactions or anaphylaxis.  Caregivers/patients will be advised that study procedures include 
regular blood sampling for measurement of safety parameters and biological markers and that 
some minor risks are associated with these procedures. 
This informed consent should be given by means of a signed ICF, written in non-technical 
language in accordance with ICH GCP, the Declaration of Helsinki, and regulatory 
authorities.  The caregiver(s)/patient should read and consider the statements before signing 
and dating them and should be given a copy of each signed document.  If written consent is 
not possible, oral consent can be obtained if witnessed and followed by a signed statement 
from one or more persons not involved in the study, indicating why the patient was unable to 
sign the form.  No patient can enter the study before his/her or caregiver(s) informed consent 
has been obtained, as required by country regulations. 
The ICF is part of the protocol and must be submitted by the investigator/investigative staff 
with the protocol for IEC approval.  Albireo will supply an ICF which complies with 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 70 
Final Amendment 03, 22November 2018 
 
regulatory requirements and country laws, and is considered appropriate for the study.  Any 
changes to the ICF suggested by the investigator must be agreed to by Albireo before 
submission to the IEC and a copy of the approved version must be provided to the clinical 
monitor after IEC approval. 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 71 
Final Amendment 03, 22November 2018 
 
15 
DATA HANDLING AND RECORD KEEPING 
15.1 
Case Report Forms/Source Data Handling 
The investigator shall be provided with standardized eCRFs and shall ensure that all data 
from patient visits are promptly entered into the eCRFs in accordance with the specific 
instructions given.  The investigator must sign each eCRF to verify the integrity of the data 
recorded. 
A list of the normal ranges for all laboratory tests to be undertaken forms part of the 
documentation to be collated prior to study start.  As a central laboratory has been selected to 
conduct any or all tests, it is essential that all samples be analyzed at that laboratory. 
The investigator must maintain source documents such as laboratory reports, consultation 
reports, and complete medical history and physical examination reports. 
As no ethnicity data can be collected in the source document for study sites in France, the 
eCRF will be the Source Document for “origines ethniques”,  
15.2 
Retention of Essential Documents 
Essential documents, as defined by ICH GCP, include: the signed protocol and any 
amendment(s); copies of the completed eCRFs (for site archiving, compact discs of eCRF 
data for specific patients will be provided); signed ICFs; hospital records and other source 
documents; IEC approvals and all related correspondence including approved documents; 
drug accountability records; study correspondence; and a list of patients’ names and 
addresses. 
The investigator/investigative staff must retain copies of these essential documents for the 
period specified by ICH GCP and by applicable regulatory requirements.  The 
investigator/investigative staff will inform Albireo of the location where the essential 
documents are stored and must contact Albireo for approval before disposing of any essential 
documents.  The investigator/investigative staff should take measures to prevent accidental or 
premature destruction of these documents. 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 72 
Final Amendment 03, 22November 2018 
 
16 
FINANCING AND INSURANCE 
Financing and insurance are addressed in a separate agreement. 
 


 
 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 73 
Final Amendment 03, 22November 2018 
 
17 
PUBLICATION POLICY 
Albireo will retain the ownership of all data.  When the study is complete, Albireo shall 
arrange the analysis, tabulation of data and preparation of a clinical study report. Albireo may 
also use the data for publication, presentation at scientific meetings and submission to 
regulatory authorities.  All proposed publications based on this study must be subject to the 
sponsor's approval requirements. 
 


A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Page 74 
Final Amendment 03, 22November 2018 
18 
REFERENCE LIST 
1. A4250 Investigator’s Brochure.
2. Alissa FT, Jaffe R, Shneider BL. Update on progressive familial intrahepatic cholestasis.
J Pediatr Gastroenterol Nutr. 2008;46(3):241-52.
3. Davit-Spraul A, Gonzales E, Baussan C, et al. Progressive familial intrahepatic
cholestasis. Orphanet J Rare Disease 2009;4:1-12.
4. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and
functions. Front Biosci (Landmark Ed). 2009 Jan 1;14:2584-98.
5. Hori T, Justin H, Nguyen JH and Uemoto S. Progressive familial intrahepatic cholestasis.
Hepatobiliary pancreat Dis Int. 2010; 9(6):570-8.
6. Jacquemin E. Progressive familial intrahepatic cholestasis. Genetic basis and treatment.
Clin Liver Dis. 2000;4(4):753-63.
7. Miethke AG, Zhang W, Simmons J, et al. Pharmacological inhibition of ASBT changes
bile composition and blocks progression of sclerosing cholangitis in mdr2 knockout mice.
Hepatology. 2016; 63(2):512–23.
8. Murray CS, Rees JL. Are subjective accounts of itch to be relied on? The lack of relation
between visual analogue itch scores and actigraphic measures of scratch. Acta Derm
Venereol. 2011;91(1):18-23.
9. Pawlikowska L, Strautnieks S, Jankowska I, et al. Differences in presentation and
progression between severe FIC1 and BSEP deficiencies. J Hepatol. 2010;53(1):170-8.
10. Shneider BL, Magee JC, Bezerra JA, et al. Efficacy of fat-soluble vitamin
supplementation in infants with biliary atresia. Pediatrics, 2012;130(3):e607-14.
11. Suchy FJ, Sokol RJ, Balistreri WF, eds. Liver Disease in Children. 3rd Edition. New
York, NY: Cambridge University Press; 2007:310–4.
12. Venkat VL, Shneider BL, Magee JC, et al. Total Serum Bilirubin Predicts Fat-Soluble
Vitamin Deficiency Better Than Serum Bile Acids in Infants with Biliary Atresia. J
Pediatr Gastroenterol Nutr. 2014;59(6):702-7.
13. Whitington PF, Whitington GL. Partial external diversion of bile for the treatment of
intractable 
pruritus 
associated 
with 
intrahepatic 
cholestasis. 
Gastroenterology.
1988;95(1):130-6.


A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Page 75 
Final Amendment 03, 22November 2018 
19 
APPENDICES 
Appendix 1 
Concomitant Medication Guidelines 


A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Final Amendment 03, 22November 2018 
Appendix 2 
Diary Questions including Albireo Patient-Reported 
Outcomes/Observer-Reported Outcome Instrument 
77


A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Final Amendment 03, 22November 2018 
Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) 
80


A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Final Amendment 03, 22November 2018 
Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaire 
85






PedsQL  3 
PedsQL 4.0 – Parent (2-4)       Not to be reproduced without permission         Copyright © 1998 JW Varni, Ph.D. All rights reserved      
01/00    
PedsQL-4.0-Core-PT - United States/English – Original version 
PedsQL-4.0-Core-PT_AU4 0_eng-USori 
3.  Missing school/daycare to go to the doctor or 
     hospital 
0 
1 
2 
3 
4 
 
88






PedsQL 3 
PedsQL 4.0 - (5-7) 
 Not to be reproduced without permission    Copyright © 1998 JW Varni, Ph.D. All rights reserved 
01/00       
PedsQL-4.0-Core-YC – United States/English – Original version 
PedsQL-4 0-Core-YC_AU4.0_eng-USori 
91


 
PedsQL 4.0 - (5-7)       
 
  
   Not to be reproduced without permission    
 
  Copyright © 1998 JW Varni, Ph.D. All rights reserved.                                 
01/00                                  
PedsQL-4.0-Core – United States/English – Original version 
PedsQL-4 0-Core-YC_AU4 0_eng-USori 
 
 
 
 
How much of a problem is this for you? 
 
 
 
 
         Not at all  
 
 
   Sometimes    
 
 
  A lot 
☺

 
 
 
92




























A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Final Amendment 03, 22November 2018 
Appendix 5 
Guideline for Fat-Soluble Vitamin Supplementation 
106


A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Page 1 of 1 
Final Amendment 03, 22 November 2018 
Appendix 5: Guideline for Fat-Soluble Vitamin Supplementation 
Cholestasis predisposes to fat-soluble vitamin deficiencies.  Fat-soluble vitamin levels, 
i.e., vitamins A and E, 25-hydroxy vitamin D, and INR (surrogate for vitamin K) are
measured routinely during the study.  If a patient has any fat-soluble vitamin level(s) that 
are out of range, vitamin supplementation adjustments may be required.  Below are 
suggested guidelines for fat-soluble vitamin deficiency treatment [Venkat 2014; 
Shneider 2012].  Additional patient monitoring and/or treatment strategies may be 
warranted at the discretion of the investigator. 
Target Fat-soluble Vitamin Levels and Replacement Regimens 
Vitamin 
Target Range 
(Serum Level) 
Supplementation Strategy 
A (retinol) 
19 to 77 µg/dL* 
retinol:retinol-binding 
protein molar ratio > 0.8 
Increments of 5000 IU (up to 25,000 
to 50,000 IU/day) orally or monthly 
intramuscular administration of 
50,000 IU** 
D (25-hydroxy 
vitamin D) 
15 to 45 ng/mL* 
Increments of 1200 to 8000 IU 
orally daily of cholecalciferol or 
ergocalciferol; alternatively 
calcitriol at 0.05 to 0.2 µg/kg/day* 
E (α tocopherol) 
3.8 to 20.3 µg/mL 
vitamin E:total serum 
lipids ratio >0.6 mg/g 
Increments of 25 IU/kg of 
d-α-tocopheryl polyethylene glycol 
– 1000 succinate (TPGS) orally
daily (to 100 IU/kg/day) 
K (phytonadione) 
INR ≤1.2 
•
1.2 <INR ≤1.5: 2.5 mg
vitamin K orally daily
•
1.5 <INR ≤1.8: 2.0 to 5.0 mg
vitamin K intramuscular and
2.5 mg vitamin K orally daily
•
INR >1.8: 2.0 to 5.0 mg vitamin
K intramuscular and 5.0 mg
vitamin K orally daily
*Clinical practice may vary.  This is meant as a guidance only and does not override local standard of care. Investigators
should provide best case practices for management and treatment 
** For further detail regarding age related Tolerable Upper Intake (UL) for preformed vitamin A please refer to page 163 in 
the European Food Safety Authority guidance: Tolerable Upper Intake Levels for Vitamins and Minerals; Scientific Committee 
on food Scientific Panel on Dietetic Products, Nutrition and Allergies 
http://www.efsa.europa.eu/sites/default/files/efsa rep/blobserver assets/ndatolerableuil.pdf 
107


A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Final Amendment 03, 22November 2018 
Appendix 6 
Contraceptive Requirements 
108


 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
 
Confidential 
Page 1 of 2 
Final Amendment 03, 22 November 2018 
Appendix 6: Contraceptive Requirements 
A woman is considered of childbearing potential (WOCBP), i.e. fertile, following 
menarche and until becoming post-menopausal unless permanently sterile.  Permanent 
sterilisation methods include hysterectomy, bilateral salpingectomy and bilateral 
oophorectomy. 
Contraceptive methods, or combinations of contraceptive methods, for males and females 
that can achieve a failure rate of less than 1% per year when used consistently and 
correctly are considered as highly effective birth control methods and must be used at 
least to up to 90 days following the last day of treatment. 
Such methods include: 
• Combined (estrogen and progestogen containing) hormonal contraception 
associated with inhibition of ovulation: 
o oral 
o intravaginal 
o transdermal 
• Progestogen-only hormonal contraception associated with inhibition of ovulation: 
o oral 
o injectable 
o implantable 
• Intrauterine device (IUD) 
• Intrauterine hormone-releasing system (IUS) 
• Bilateral tubal occlusion 
• Vasectomised partner 
• Sexual abstinence 
Sexual abstinence is considered a highly effective method only if defined as refraining 
from heterosexual intercourse during the entire study period up to at least 90 days after 
the last day of treatment. The reliability of sexual abstinence needs to be evaluated in 
relation to the duration of the clinical trial and the preferred and usual lifestyle of the 
subject. Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation 
methods) and withdrawal are not acceptable methods of contraception. 
109


 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
 
Confidential 
Page 2 of 2 
Final Amendment 03, 22 November 2018 
Requirements according to “Recommendations related to contraception and pregnancy 
testing in clinical trials”, HMA CTFG (Clinical Trial Facilitation Group), 2014 
110


A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Final Amendment 03, 22November 2018 
Appendix 7 
Blood Volumes 
111


Page 1 of 2
Scientific Affairs
Estimated Blood Volume Request
Version:
11.0
SPONSOR: Albireo
Protocol#:  A4250-005
6 mon h to 18 yrs of age
Visit^
Testing^1-8
Tube 
volume 
(mL)
Tube 
Type
# of 
Collection 
Tubes
# of 
Aliquots
Volume 
(mLs)
Volume/ 
visit 
(mL)
Volume
/visit 
(tsp)
Volume
/visit 
(TBS)
Visit 1
Screening
Study Day -56 
- (-35)  ± 2
Chemistry, hCG
1 1 SST 
1
1
1 1
Bile Acids, Total1
1 1 SST 
1
2
1 1
DNA2
3 0 EDTA
1
0
3 0
5 2
1.05
0.35
Visit 2
Screen-28D
Study Day -28 
- (-7)  ± 2
Coagulation (INR/PT)
1 4 NaCit
1
1
1 4
Vitamins, A, E1
2 5 SST 
1
1
2 5
Bile Acids, Total1
1 1 SST 
1
2
1 1
5 0
1.01
0.34
Visit 3
Study Day 0
Chemistry, AFP
1 1 SST
1
1
1 1
Hematology
1 0 EDTA
1
0
1 0
Autotaxin7
1 2 LiHep
1
2
1 2
C4 (LC-MS/MS)4
1 2 LiHep
1
2
1 2
Vitamin D,25OH
1 1 SST
1
1
1 1
Bile Acids, Total1
1 1 SST 
1
2
1 1
6 7
1.36
0.45
Visit 4
Study Day 28 ± 3
Chemistry
1 1 SST
1
1
1 1
Hematology
1 0 EDTA
1
0
1 0
Coagulation (INR/PT)
1 4 NaCit
1
1
1 4
Autotaxin7
1 2 LiHep
1
2
1 2
C4 (LC-MS/MS)4
1 2 LiHep
1
2
1 2
Bile Acids, Total1
1 1 SST 
1
2
1 1
PK
1 1 Plasma
1
2
1 1
8 1
1.64
0.55
Visit 5
Study Day 56 ± 3
Chemistry
1 1 SST
1
1
1 1
Hematology
1 0 EDTA
1
0
1 0
Bile Acids, Total1
1 1 SST 
1
2
1 1
3 2
0.65
0.22
Visit 6
Study Day 84 ± 3
Chemistry 
1 1 SST
1
1
1 1
Hematology
1 0 EDTA
1
0
1 0
Coagulation (INR/PT)
1 4 NaCit
1
1
1 4
Bile Acids, Total1
1 1 SST 
1
2
1 1
Vitamins, A, E1
2 5 SST 
1
1
2 5
Vitamin D,25OH
1 1 SST
1
1
1 1
8 2
1.66
0.55
Visit 7
Study Day 126 ± 3
Chemistry
1 1 SST
1
1
1 1
Hematology
1 0 EDTA
1
0
1 0
Bile Acids, Total1
1 1 SST 
1
2
1 1
3 2
0.65
0.22
Visit 8
Study Day 154 ± 3
Coagulation (INR/PT)
1 4 NaCit
1
1
1 4
Bile Acids, Total1
1 1 SST
1
2
1 1
2 5
0.51
0.17
Visit 9/V9A 
EOT
Study Day 168 ± 3
Chemistry, AFP, hCG3
1 1 SST
1
1
1 1
Hematology
1 0 EDTA
1
0
1 0
Autotaxin7
1 2 LiHep
1
2
1 2
C4 (LC-MS/MS)4
1 2 LiHep
1
2
1 2
112


Page 2 of 2
Scientific Affairs
Estimated Blood Volume Request
Version:
11.0
SPONSOR: Albireo
Protocol#:  A4250-005
6 mon h to 18 yrs of age
Visit^
Testing^1-8
Tube 
volume 
(mL)
Tube 
Type
# of 
Collection 
Tubes
# of 
Aliquots
Volume 
(mLs)
Volume/ 
visit 
(mL)
Volume
/visit 
(tsp)
Volume
/visit 
(TBS)
Bile Acids, Total1
1 1 SST 
1
2
1 1
Vitamins, A, E1
2 5 SST 
1
1
2 5
Vitamin D,25OH
1 1 SST
1
1
1 1
PK
1 1 Plasma
1
2
1 1
10 3
2.09
0.70
Visit 10
Follow-Up 
Study Day 196 ± 3
Chemistry 
1 1 SST
1
1
1 1
Hematology
1 0 EDTA
1
0
1 0
Coagulation (INR/PT)
1 4 NaCit
1
1
1 4
Bile Acids, Total1
1 1 SST
1
2
1 1
4 6
0.93
0.31
Study Total Volume*
57.0
11.6
3.9
^ Visit Schedule and testing based on CLW v1 09
*Total Volume reflects the volumes required for testing by ICL and 3rd Parties as defined in the test list above
Notes: #1  Bile Acids, Total, Vitamin A, and Vitamin E testing at ARUP Lab, USA 
#2  Optional sample  Only collected if required
#3  hCG is an optional sample, collected if patient is female and of child bearing potential
#4  C4 will be performed on plasma sample at LGC   
#5  The following absolute 'minimum' volume does not allow for repeat testing:      
Chemistry, AFP requires minimum – 500uL serum         
Bile Acids requires minimum – 500uL serum         
Coagulation (PT/INR only) requires minimum – 900uL plasma
#6  Sites must collect Full Draw and measure exact aliquot of sample into a  "False Bottom"  transport tube    
#7  Autotaxin serum samples will go to LGC lab for testing  Optional sample  Collected if subject meets weight/age/medical history criteria for collection
#8  For subjects requiring optional pregnancy testing, collect 1 1mL SST
#9  PK will be performed on plasma sample at LGC   
Created by:
Jennifer Rodriguez, Protocol Review
Date:
14-May-18
113


 
 
 
CLINICAL STUDY PROTOCOL A4250-005 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
 
IND Number:   
130591 
EudraCT Number 
2017-002338-21 
Test Product: 
A4250 
Indication: 
Progressive Familial Intrahepatic Cholestasis (PFIC) 
Sponsor: 
Albireo AB 
Development Phase: 
Phase 3 
Sponsor Signatory: 
 
Sponsor Medical Monitor: 
 
Principal Investigator: 
 
Date of the Protocol: 
10 May 2018 
Version of the Protocol: 
Final Amendment 02 
 
 
 
 
Confidential 
The information in this document contains trade secrets and commercial information that are 
privileged or confidential and may not be used, divulged, published, or otherwise disclosed 
without the written consent of Albireo AB.  These restrictions on disclosure apply equally to 
all future information supplied to you or generated by you in connection with the study. 






 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
  
Final Amendment 02, 10 May 2018 
 
INVESTIGATOR SIGNATURE PAGE 
 
PROTOCOL TITLE:  A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children with Progressive 
Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
 
PROTOCOL NUMBER:  A4250-005 
I have read this protocol and agree that it contains all necessary details for performing this 
study.  I will conduct the study as outlined herein and will complete the study within the time 
designated, in accordance with all stipulations of the protocol and in accordance with Good 
Clinical Practice (GCP), local regulatory requirements, and the Declaration of Helsinki. 
I will provide copies of the protocol and all pertinent information to all individuals 
responsible to me who assist in the conduct of this study.  I will discuss this material with 
them to ensure that they are fully informed regarding the study drug and the conduct of the 
study. 
I will use only the informed consent approved by the Independent Ethics Committee (IEC) 
and will fulfil all responsibilities for submitting pertinent information to the IEC responsible 
for this study. 
I agree that the sponsor (Albireo AB) shall have access to any source documents from which 
case report form information may have been generated. 
I further agree not to originate or use the name of Albireo AB, A4250 in any publicity, news 
release, or other public announcement, written or oral, whether to the public, press, or 
otherwise, relating to this protocol, to any amendment to the protocol, or to the performance 
of this protocol without the prior written consent of Albireo AB. 
 
 
 
 
 
 
Name of Investigator 
 
 
 
 
 
 
Signature 
 
Date (day/month/year) 
Page 4


 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
  
Final Amendment 02, 10 May 2018 
 
1 
ADMINISTRATIVE INFORMATION 
A Double-blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
Protocol No.: 
A4250-005 
Date of the initial Protocol: 
20 September 2017 
Date and Number of Amendment(s): 
Amendment 01, 06 December 2017 
 
Amendment 02, 10 May 2018 
Sponsor: 
Albireo AB 
 
Arvid Wallgrens backe 20 
 
413 46 Gӧteborg 
Sweden 
Clinical Research Organization: 
 
 
 
 
 
 
 
 
Sponsor Signatory: 
 
 
  
 
  
 
  
 
 
 
  
Sponsor Medical Monitor: 
 
 
 
 
Principal Investigator: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Page 5


 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
  
Final Amendment 02, 10 May 2018 
 
2 
STUDY SYNOPSIS 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Title of Study: 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate Efficacy and Safety of A4250 
in Children with Progressive Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
Principal Investigator: 
 
Study Centers: 
Up to 50 sites will be initiated for this study in the United States (US), Canada, European Union (EU), Middle 
East, and Australia. 
Publication(s): 
None. 
Planned Study Period: 
Q2 2018 to Q4 2019 
Development Phase: 
Phase 3 
Objectives: 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day A4250 in children with 
progressive familial intrahepatic cholestasis (PFIC) Types 1 and 2, as determined by the following: 
• 
Proportion of patients experiencing at least a 70% reduction in fasting serum bile acid (s-BA) 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L compared to placebo 
after 24 weeks of treatment 
• 
Change in pruritus compared to placebo over 24 weeks of treatment 
Secondary Objectives 
• 
To evaluate the effect of A4250 on serum alanine aminotransferase (ALT) concentration 
• 
To evaluate the effect of A4250 on growth 
• 
To evaluate the effect of A4250 on sleep disturbance 
• 
To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or liver 
transplantation) 
• 
To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 
Methodology: 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the efficacy and safety of 
40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in children with PFIC (deficiencies of familial 
intrahepatic cholestasis-1 or bile salt export pump).  
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment Period and a 4-week 
Follow-up Period.  After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of A4250, or a 
matching placebo.  Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008) in which all patients will receive 
active treatment. 
There will be up to 10 clinic visits during the study and one scheduled telephone contact. 
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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 02, 10 May 2018 
 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Number of Patients: 
Approximately 60 patients diagnosed with PFIC-1 or PFIC-2 will be randomized.  The study will target 15% of 
enrolled patients to be PFIC-1 patients.  If enrollment of all PFIC-2 patients is completed and the 15% 
enrollment of PFIC-1 patients has not yet been achieved, then enrollment of PFIC-2 patients will continue in 
order to reach the total study target enrollment. 
Diagnosis and Main Criteria for Inclusion: 
1. 
A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages of ≥6 months 
and ≤18 years at Visit 1 with a body weight above 5 kg 
2. 
Patient must have clinical genetic confirmation of PFIC-1 or PFIC-2 through identification of biallelic 
pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. 
Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, taken as the 
average of 2 samples 7 to 28 days apart (Visits 1 and 2) prior to randomization  
4. 
Patient must have history of significant pruritus and a caregiver-reported observed scratching in the 
electronic diary (eDiary) average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to randomization 
5. 
Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  Patients who 
turn 18 years of age (or legal age per country) during the study will be required to re-consent in order 
to remain in the study 
6. 
Patients are expected to have a consistent caregiver for the duration of the study 
7. 
Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use an eDiary 
device as required by the study 
Test Product, Dose and Mode of Administration: 
A4250, 40 µg/kg/day or 120 µg/kg/day, orally administered 
Reference Therapy, Dose and Duration of Administration: 
Matching placebo, orally administered 
Duration of Treatment: 
24 weeks 
Variables 
Efficacy:  
Primary Efficacy Endpoints 
The primary efficacy endpoints are: 
• 
EU and rest of world (RoW): Proportion of patients experiencing at least a 70% reduction in fasting 
s-BA concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L compared to 
placebo after 24 weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization and the previous 
visit (Clinic Visit 2).  The end value is the average of the values at Weeks 22 and 24 after the start of 
double-blind treatment. 
• 
US: Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline over the 
last 5 months of the Treatment Period as measured by an overall mean of weekly averages of the worst 
daily scratch score using the Albireo observer-reported outcome (ObsRO) instrument. 
Secondary Efficacy Endpoints 
Secondary efficacy endpoints include the following: 
• 
EU and RoW: Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
over the last 5 months of the Treatment Period as measured by an overall mean of weekly averages of 
the worst daily scratch score using the Albireo ObsRO instrument. 
• 
US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA concentration from 
baseline to the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 weeks of 
treatment. 
 
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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
  
Final Amendment 02, 10 May 2018 
 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
All Regions: 
All secondary endpoints will be compared to placebo. 
• 
Change from baseline to Week 24 in fasting s-BA 
• 
Change from baseline to Week 24 in serum ALT concentration 
• 
Change in growth from baseline to Week 24, defined as the linear deficit (weight for age and body 
mass index) compared to a standard growth curve (Z-score, standard deviation from P50) 
• 
Proportion of patients achieving meaningful reduction in caregiver-reported observed scratching over 
the Treatment Period 
• 
Change from baseline over the last 5 months of the Treatment Period as measured by an overall mean 
of weekly averages of the sleep parameters measured with the Albireo patient-reported outcome (PRO) 
and ObsRO instruments 
• 
Change from baseline over the last 5 months of the Treatment Period as measured by an overall mean 
of weekly averages of patient-reported itch severity as measured by the average daily score of the 
Albireo PRO instrument; only patients ≥8 years of age will complete the Albireo PRO instrument 
• 
Number of patients undergoing biliary diversion surgery or being listed for liver transplantation 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• 
Proportion of patients achieving meaningful reduction in patient-reported itch severity over the 
Treatment Period 
• 
Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, caregiver, and 
clinician via the patient global impression of change (PGIC) items 
• 
Change from baseline in fasting s-BA at Weeks 4 and 12 
• 
Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline to Week 4 of 
the Treatment Period as measured by the Albireo ObsRO instrument 
• 
Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline over the last 
3 months of the Treatment Period as measured by an overall mean of weekly averages of the worst 
daily scratch of the Albireo ObsRO instrument 
• 
Change from baseline over the last 5 months of the Treatment Period in patient-reported and 
observer-reported night time itching and scratching severity scores, respectively 
• 
Change from baseline over the last 5 months of the Treatment Period in pooled pruritus score including 
observer-reported scratching for patients <8 years of age and patient-reported itch severity for patients 
≥8 years of age 
• 
Change from baseline over the last 5 months of the Treatment Period in additional patient-reported and 
observer-reported sleep parameters (e.g., tiredness and number of awakenings) 
• 
Change from baseline to Week 24 in Pediatric Quality of Life Inventory (PedsQL) 
• 
Change from baseline in serum ALT concentration at Weeks 4 and 12 
• 
Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• 
Change from baseline in AST and gamma-glutamyl transferase at Weeks 4, 12, and 24 
• 
Change from baseline for other biochemical markers and measures of bile acid synthesis (autotaxin, 
plasma 7α-hydroxy-4-cholesten-3-one concentration [p-C4]) at Week 24 
• 
Change in pediatric end-stage liver disease/model for end-stage liver disease (PELD/MELD) score, 
AST to platelet ratio index (APRI) score, and Fib-4 score from baseline to Week 24 
• 
Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® (where available) 
• 
Liver-related mortality and liver decompensation events. 
• 
All-cause mortality 
Safety:   
Safety criteria are as follows: 
• 
Occurrence of treatment-emergent adverse events including severity and relatedness to study drug at all 
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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 02, 10 May 2018 
 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
visits 
• 
The incidence of treatment-emergent serious adverse events (SAEs), based upon information from 
patient reports, including the description, incidence, and severity of an SAE 
• 
Trends evaluated for the following assessments: physical examinations, concomitant medications, vital 
signs, laboratory test results (including clinical chemistry, hematology, urinalysis, alfa-fetoprotein, 
vitamins A and E, 25-hydroxy vitamin D and INR), and abdominal ultrasound 
Statistical Methods: 
The primary objective of this study is to show superiority in efficacy of A4250 (120 µg/kg/day and 
40 µg/kg/day) compared to placebo in patients with PFIC Types 1 and 2. 
In the EU and RoW, for the primary efficacy variable fasting bile acid responder endpoint (at least a 70% 
reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the end of treatment), the null 
hypothesis will be tested against the alternative hypothesis (α=0.0125, 1-sided).  Generalized linear mixed 
modelling will be used with a logistic link, to allow for repeated assessments over time.  The model will include 
baseline value, treatment group, PFIC class, age class, region, visit and visit*treatment group interaction.  The 
contrasts of primary interest will be the treatment difference at Week 24 but also the difference at Week 4, 
Week 8, Week 12, and Week 18 will be estimated and analyzed. 
In the US, for the primary efficacy variable pruritus score endpoint (change from baseline over the treatment 
period in observer-reported scratching), the null hypothesis will be tested against the alternative hypothesis 
(α=0.0125, 1-sided).  The primary analysis of the change from baseline in pruritus score (indexed by 
observer-reported scratching) will be analyzed using a mixed model repeated measures analysis.  The 
comparison of primary interest will be the treatment differences over the last 5 months of the treatment 
period estimated by averaging the worst daily itch score means from five 14 day post-treatment intervals (Week 
7 to 8, Week 11 to 12, Week 15 to 16, Week 19 to 20, and Week 23 to 24).  The treatment difference at each of 
these post-treatment time intervals will also be estimated and analyzed.  
To strictly control overall Type I error rate across the dose groups and also across the primary and the main 
secondary endpoint to a 1-sided 2.5% level, a Bonferroni-based parallel gate-keeping procedure will be used. 
Other secondary and exploratory outcome variables shall provide supportive efficacy and safety information 
regarding the differences between the treatment groups.  No adjustments will be performed for multiple-dose 
comparisons when testing the other secondary and exploratory efficacy variables. 
Date of the Protocol:  10 May 2018 
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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
  
Final Amendment 02, 10 May 2018 
 
3 
TABLE OF CONTENTS 
1 
ADMINISTRATIVE INFORMATION ........................................................................ 5 
2 
STUDY SYNOPSIS ...................................................................................................... 6 
3 
TABLE OF CONTENTS ............................................................................................ 10 
3.1 
List of Tables .................................................................................................. 14 
3.2 
List of Figures................................................................................................. 14 
3.3 
List of Appendices .......................................................................................... 14 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ................................ 15 
5 
INTRODUCTION ....................................................................................................... 17 
5.1 
Investigational Medicinal Product .................................................................. 17 
5.2 
Background..................................................................................................... 17 
5.2.1 
Progressive Familial Intrahepatic Cholestasis ........................................... 17 
5.2.2 
Summary of Clinical and Nonclinical Studies ........................................... 19 
5.3 
Rationale ......................................................................................................... 20 
5.3.1 
Risk/Benefit ............................................................................................... 21 
6 
STUDY OBJECTIVES ............................................................................................... 22 
6.1 
Primary Objectives ......................................................................................... 22 
6.2 
Secondary Objectives ..................................................................................... 22 
7 
INVESTIGATIONAL PLAN ...................................................................................... 23 
7.1 
Overall Study Design and Plan....................................................................... 23 
7.1.1 
Description ................................................................................................. 23 
7.1.2 
Schedule of Assessments ........................................................................... 25 
7.1.3 
Study Procedures and Assessments ........................................................... 29 
7.1.3.1 
Screening Period (Days -56 to 0) ............................................... 29 
7.1.3.2 
Treatment Period (Day 0 to Day 168) ........................................ 30 
7.1.3.3 
Follow-Up Period ....................................................................... 33 
7.2 
Study Population ............................................................................................ 33 
7.2.1 
Inclusion Criteria ....................................................................................... 33 
7.2.2 
Exclusion Criteria ...................................................................................... 34 
7.2.3 
Withdrawal of Patients............................................................................... 36 
7.2.4 
Study Termination by Sponsor .................................................................. 37 
8 
TREATMENT OF PATIENTS ................................................................................... 38 
8.1 
Identity of Study Drug .................................................................................... 38 
8.2 
Administration of Study Drug ........................................................................ 38 
8.3 
Study Treatment Packaging and Labeling ...................................................... 39 
8.3.1 
Packaging and Labeling ............................................................................. 39 
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8.3.2 
Storage ....................................................................................................... 39 
8.3.3 
Blinding and Randomization of Study Drug ............................................. 39 
8.4 
Procedure for Breaking the Randomization Code .......................................... 40 
8.5 
Patient Compliance ......................................................................................... 41 
8.6 
Study Drug Accountability ............................................................................. 41 
8.7 
Concomitant Therapy and Prohibited Medications ........................................ 41 
9 
ASSESSMENT OF EFFICACY ................................................................................. 42 
9.1 
Efficacy Endpoints ......................................................................................... 42 
9.1.1 
Primary Efficacy Endpoint ........................................................................ 42 
9.1.2 
Secondary Efficacy Endpoint .................................................................... 42 
9.1.3 
Exploratory Efficacy Endpoints ................................................................. 43 
9.2 
Efficacy Assessments ..................................................................................... 44 
9.2.1 
Serum Bile Acids ....................................................................................... 44 
9.2.2 
Itching, Scratching and Sleep Score .......................................................... 44 
9.2.3 
Growth ....................................................................................................... 45 
9.2.4 
Quality of Life Questionnaire (PedsQL) ................................................... 45 
9.2.5 
Biomarkers and PK Samples ..................................................................... 45 
9.2.6 
PELD/MELD Score ................................................................................... 45 
9.2.7 
Fibroscan® .................................................................................................. 46 
9.2.8 
Markers of Fibrosis .................................................................................... 46 
9.2.9 
Global Impression of Change and Global Impression of Symptom 
Measures .................................................................................................... 46 
10 
ASSESSMENT OF SAFETY...................................................................................... 47 
10.1 
Adverse Events ............................................................................................... 47 
10.1.1 
Definitions and Investigator Assessments ................................................. 47 
10.1.1.1 Clinical Significance .................................................................. 47 
10.1.1.2 Serious Adverse Events .............................................................. 47 
10.1.1.3 Severity Assessment ................................................................... 48 
10.1.1.4 Causality Assessment ................................................................. 48 
10.1.2 
Recording of Adverse Events .................................................................... 49 
10.1.3 
Recording and Reporting of Serious Adverse Events ................................ 50 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions .............. 51 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments .................................................................................................... 51 
10.2.1 
Laboratory Assessments ............................................................................ 51 
10.2.2 
Individual Patient Safety Monitoring ........................................................ 53 
10.2.2.1 Liver Monitoring ........................................................................ 53 
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Final Amendment 02, 10 May 2018 
 
10.2.2.2 Close Observation ...................................................................... 54 
10.2.2.3 De-challenge/Re-challenge for Liver and Clinical Hepatitis 
Monitoring .................................................................................. 54 
10.2.2.4 Diarrhea ...................................................................................... 55 
10.2.2.5 Monitoring after Permanent Discontinuation of Study Drug 
Due to Safety .............................................................................. 55 
10.2.3 
Demographics/Medical and Surgical History ............................................ 55 
10.2.4 
Clinical Genetic Testing ............................................................................ 55 
10.2.5 
Physical Examination ................................................................................ 56 
10.2.6 
Vital Signs ................................................................................................. 56 
10.2.7 
Abdominal Ultrasound ............................................................................... 56 
10.2.8 
Overdose .................................................................................................... 56 
10.2.9 
Pregnancy .................................................................................................. 56 
11 
STATISTICAL EVALUATION ................................................................................. 58 
11.1 
Sample Size and Power .................................................................................. 58 
11.2 
Statistical Methods ......................................................................................... 58 
11.2.1 
Statistical Analysis Sets ............................................................................. 58 
11.2.2 
Methods of Statistical Analyses ................................................................. 59 
11.2.2.1 General Principles ...................................................................... 59 
11.2.2.2 Missing Data............................................................................... 60 
11.2.2.3 Demographic and Baseline Characteristics ................................ 60 
11.2.2.4 Subject Disposition..................................................................... 61 
11.2.2.5 Evaluation of Primary Efficacy Variables .................................. 61 
11.2.2.6 Evaluation 
of 
Secondary 
and 
Exploratory 
Efficacy 
Variables ..................................................................................... 62 
11.2.2.7 Evaluation of Safety Variables ................................................... 63 
11.2.2.8 Compliance and Exposure .......................................................... 64 
11.2.3 
Blinded Analyses to Confirm Responder Definitions ............................... 64 
11.2.4 
Data Safety Monitoring Board ................................................................... 64 
12 
DIRECT ACCESS TO SOURCE DATA/NOTES ..................................................... 66 
13 
QUALITY CONTROL AND QUALITY ASSURANCE .......................................... 67 
13.1 
Conduct of the Study ...................................................................................... 67 
13.2 
Study Monitoring............................................................................................ 67 
14 
ETHICS ....................................................................................................................... 69 
14.1 
Independent Ethics Committee/Institutional Review Board .......................... 69 
14.2 
Written Informed Consents and Assents ........................................................ 69 
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Final Amendment 02, 10 May 2018 
 
15 
DATA HANDLING AND RECORD KEEPING ....................................................... 71 
15.1 
Case Report Forms/Source Data Handling..................................................... 71 
15.2 
Retention of Essential Documents .................................................................. 71 
16 
FINANCING AND INSURANCE .............................................................................. 72 
17 
PUBLICATION POLICY ........................................................................................... 73 
18 
REFERENCE LIST ..................................................................................................... 74 
19 
APPENDICES ............................................................................................................. 75 
 
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Final Amendment 02, 10 May 2018 
 
3.1 
List of Tables 
Table 1 
Schedule of Assessments ................................................................................... 26 
Table 2 
Dosing and Capsule Strength ............................................................................. 39 
Table 3 
Routine Laboratory Parameters .......................................................................... 52 
 
3.2 
List of Figures 
Figure 1 
Study Design ...................................................................................................... 25 
Figure 2 
Bonferroni Holm Multiple Comparison Procedure ............................................ 59 
 
3.3 
List of Appendices 
Appendix 1 
Concomitant Medication Guidelines .................................................................. 75 
Appendix 2 
Diary Questions including Albireo Patient-Reported 
Outcomes/Observer-Reported Outcome Instrument .......................................... 76 
Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) ....................................................................... 77 
Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaire .............................. 78 
Appendix 5 
Guideline for Fat-Soluble Vitamin Supplementation......................................... 79 
Appendix 6 
Contraceptive Requirements .............................................................................. 80 
Appendix 7 
Blood Volumes ................................................................................................... 81 
 
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M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 02, 10 May 2018 
 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
ADME 
absorption, distribution, metabolism, and excretion 
AE(s) 
adverse event(s) 
AFP 
alfa-fetoprotein 
ALT 
alanine aminotransferase 
APRI 
AST to platelet ratio index 
ASBT 
apical sodium-dependent bile transporter (also known as IBAT) 
AST 
aspartate aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BMI 
body mass index 
BSEP 
bile salt export pump 
CPK 
creatine phosphokinase 
CRA 
clinical research associate 
CYP 
cytochrome P450 
DSMB 
Data and Safety Monitoring Board 
eCRF 
electronic case report form 
eDiary 
electronic diary 
EOT 
End of Treatment 
EU 
European Union 
EudraCT 
European Union drug regulatory agency clinical trial 
FAS 
full analysis set 
FDA 
United States Food and Drug Administration 
FIC1 
familial intrahepatic cholestasis-1 
GCP 
Good Clinical Practice 
GGT 
gamma-glutamyl transferase 
GI 
gastrointestinal 
GLMM 
generalized linear mixed modelling 
IB 
Investigator’s Brochure 
IBAT 
ileal bile acid transporter (also known as ASBT) 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
 
 
IEC 
Independent Ethics Committee 
INR 
international normalized ratio 
IRB 
Institutional Review Board 
IWRS 
Interactive Web Response System 
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Final Amendment 02, 10 May 2018 
 
LDH 
lactate dehydrogenase 
LFT 
liver function test 
LPLV 
last patient last visit 
MDR3 
multidrug-resistance protein 3 
MedDRA 
Medical Dictionary for Regulatory Activities 
MELD 
model for end-stage liver disease 
MMRM 
mixed model repeated measures 
ObsRO 
observer-reported outcome 
PBC 
primary biliary cholangitis 
p-C4 
plasma 7α-hydroxy-4-cholesten-3-one concentration 
PCS 
potentially clinically significant 
PedsQL 
Pediatric Quality of Life Inventory 
PELD 
pediatric end-stage liver disease 
PFIC 
progressive familial intrahepatic cholestasis 
PGIC 
patient global impression of change 
PGIS 
patient global impression of symptoms 
PK 
pharmacokinetic(s) 
PP 
per protocol 
PRO 
patient-reported outcome 
PT 
prothrombin time 
PVM 
pharmacovigilance manager 
QoL 
quality of life 
RoW 
rest of world 
RR 
respiratory rate 
SAE(s) 
serious adverse event(s) 
SAP 
statistical analysis plan 
s-BA 
serum bile acid 
SD 
standard deviation 
s-FGF19 
serum fibroblast growth factor 19 concentration 
SOC 
system organ class 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE(s) 
treatment-emergent adverse event(s) 
ULN 
upper limit of normal 
US 
United States 
WHO 
World Health Organization 
 
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Final Amendment 02, 10 May 2018 
 
5 
INTRODUCTION 
5.1 
Investigational Medicinal Product 
A4250 is a small molecule and a potent selective inhibitor of the ileal bile acid transporter 
(IBAT), also known as the apical sodium-dependent bile acid transporter (ASBT).  IBAT is 
an integral brush border membrane glycoprotein that co-transports sodium and bile acids and 
appears to be a critical component in the regulation of the bile acid pool size in animals and 
man.  This transporter, expressed in the distal ileum, is a key element in the enterohepatic 
circulation of bile acids since it facilitates the high-affinity, high-capacity reabsorption of bile 
acids.  Indeed, 95% of secreted bile acids are reabsorbed via IBAT [Hofmann 2009; 
Miethke 2016].  A4250 is orally administered and acts locally in the gut where it binds 
reversibly to IBAT to decrease the reuptake of bile acids.  A4250 has minimal systemic 
exposure at the predicted therapeutic dose ranges. 
No safety signals were found with A4250 in preclinical safety and toxicity studies.  In the 
first clinical study of A4250 in healthy volunteers, A4250 was given as a single oral dose up 
to 10 mg or daily oral doses up to 3 mg/day for 7 days.  A4250 was well tolerated and the 
only findings were gastrointestinal (GI) related effects, such as diarrhea, at higher doses. 
In an exploratory Phase 2 study in children with cholestatic pruritus, A4250 was given as oral 
doses of 10 to 200 µg/kg/day for 4 weeks.  A4250 reduced mean levels of serum bile acids 
(s-BA) in all cohorts.  In 11 of 13 progressive familial intrahepatic cholestasis 
(PFIC) patients, substantial reductions (ranging from 43% to 98%) were observed.  
Patient-reported diary data documented improvement of pruritus during treatment.  A4250 
was well tolerated.  No serious adverse events (SAEs) were deemed to be related to study 
drug, and most adverse events (AEs), including some increased transaminases, were 
considered unrelated to study drug and transient in nature. 
5.2 
Background 
5.2.1 
Progressive Familial Intrahepatic Cholestasis 
PFIC is a rare autosomal recessive cholestatic liver disease estimated to affect between 1 in 
every 50,000 to 100,000 children born worldwide.  PFIC represents 10% to 15% of causes of 
cholestasis in children and 10% to 15% of liver transplantation indications in children.  All 
types of PFIC exist worldwide and both sexes appear to be equally affected. 
The common underlying pathogenesis of PFIC is disruption of bile formation and bile 
transport through the liver [Jacquemin 2000].  The classification of PFIC has evolved over 
the years.  The most commonly used sub-classifications are PFIC Types 1, 2, and 3, which 
are based on the associated affected gene and described in more detail below. 
• PFIC, Type 1:  also referred to as “Byler disease” or “familial intrahepatic 
cholestasis-1 (FIC1) protein deficiency.” FIC1 protein is located on the canalicular 
membrane of hepatocytes and facilitates movement of aminophospholipids from the outer 
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to inner leaflet of the plasma membrane of the hepatocyte.  The ATP8B1 gene encodes 
FIC1 protein.  Biallelic pathologic variants in the ATP8B1 gene are associated with FIC1 
dysfunction and classified clinically as PFIC Type 1 disease. 
• PFIC, Type 2:  also referred to as “Byler syndrome” or “bile salt export pump (BSEP) 
deficiency.”  BSEP is a transporter protein that is expressed at the canalicular membrane 
of hepatocytes, and is the primary exporter of bile acids.  The ABCB11 gene encodes the 
BSEP protein.  Biallelic pathologic variations in the ABCB11 gene are associated with 
BSEP dysfunction and classified clinically as PFIC Type 2 disease. 
• PFIC, Type 3:  is caused by a deficiency of the multidrug-resistance protein 3 (MDR3) 
due to mutations in the ABCB4 gene.  MDR3 is a phospholipid translocase critical for 
phospholipid secretion. 
Severe pruritus is common in children diagnosed with PFIC.  Itching (and subsequent 
scratching) is a significant morbidity for these patients and their families [Suchy 2007].  A 
more severe degree of pruritus is experienced compared to patients with other forms of liver 
disease and to other pruritic conditions such as atopic dermatitis [Murray 2011].  In patients 
with PFIC, liver biopsy reveals canalicular cholestasis and, later, the appearance of portal 
fibrosis.  Serum biochemistry indicates cholestasis with hyperbilirubinemia, elevated alanine 
aminotransferase (ALT) and aspartate aminotransferase (AST).  The concentrations of bile 
acids in serum are very high, while serum gamma-glutamyl transferase (GGT) activity 
(exception being MDR3 variants) and cholesterol are normal [Hori 2010].  Symptoms of 
portal hypertension and liver failure will develop during the course of the disease [Davit-
Spraul 2009; Alissa 2008].  Symptoms develop early; median age at onset of symptoms is 
2 months, and 78% of PFIC patients present with jaundice [Pawlikowska 2010].  The 
life-threatening and debilitating nature of PFIC is reflected by the fact that survival in those 
not resorting to surgery is 50% at 10 years of age and almost zero at 20 years of age.  
Approximately half of PFIC patients undergo liver transplantation [Davit-Spraul 2009] and 
treatment-resistant pruritus is the leading indication for the surgical procedure partial external 
biliary diversion, mostly in PFIC Type 1 and 2 patients. 
PFIC is life-threatening and debilitating.  Currently, the treatment for PFIC is palliative and 
there is no pharmaceutical treatment approved for use in PFIC.  The therapeutic choices are 
restricted to non-specific therapy of the symptoms and signs of the disease such as nutritional 
support, preventing vitamin deficiencies, and treatment of extrahepatic features.  Medical 
treatment options include off-label use of ursodeoxycholic acid, rifampin, antihistamines, and 
naltrexone.  A minority of patients respond nominally and transiently to these interventions.  
Biliary diversion is used to decrease systemic bile acids through interruption of the 
enterohepatic circulation and so avoid transplantation.  Liver transplantation is typically only 
viewed as an option when patients have failed medical treatment and/or biliary diversion and 
have liver failure or persistent uncontrolled pruritus. 
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A4250 – IND 130591 
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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
  
Final Amendment 02, 10 May 2018 
 
5.2.2 
Summary of Clinical and Nonclinical Studies 
This is a summary of nonclinical and clinical studies.  More detail is provided in the 
Investigator’s Brochure ([IB] see A4250 IB).  
A4250 is a potent, selective IBAT inhibitor with no safety signals seen in the safety 
pharmacological studies.  In addition, no safety signals were seen in repeat-dose oral toxicity 
studies in rats with up to 26 weeks of daily dosing and in dogs with up to 39 weeks of daily 
dosing.  No safety signals were identified when A4250 was administered daily to juvenile 
rats from age 14 days to 63 days. 
A4250 is minimally absorbed with very low systemic exposure in both humans and other 
species.  In blood, protein binding is high (>99.7%).  Systemically absorbed A4250 is slowly 
metabolized in humans and in preclinical studies by hydroxylation only, a metabolite that 
retains IBAT activity.  A4250 did not show any significant inhibition of cytochrome 
P450 (CYP) 
enzymes 
in 
the 
rat. 
 
A4250 
inhibited 
human 
CYP3A4 
and 
CYP2D6 (IC50: 16 µmol/L for both enzymes), and CYP2C9 (IC50:1.2 µmol/L).  
To date, A4250 has been studied in 3 Albireo-sponsored clinical studies: a study in healthy 
volunteers evaluating single and multiple administration of A4250; a single-dose absorption, 
distribution, metabolism, and excretion (ADME) study; and a Phase 2 study in children with 
cholestatic pruritus.  In addition, an investigator-sponsored study has been conducted in 
patients with primary biliary cholangitis (PBC).  In total, 98 subjects have been exposed to 
A4250. 
In the first clinical study (A4250-001), A4250 was given to healthy volunteers as a single oral 
dose up to 10 mg or daily oral doses up to 3 mg/day for 7 days.  Following a single oral dose 
of 10 mg, the median maximum drug concentration was 0.2 nmol/L and the area under the 
curve was 0.8 nmol × h/L (n=6).  Pharmacodynamic evaluation of bile acid synthesis 
biomarkers 
(serum 
fibroblast 
growth 
factor 
19 [s-FGF19] 
and 
plasma 
7α Hydroxy 4-cholesten-3-one [p-C4]) identified the expected decrease in s-FGF19 and an 
increase in p-C4.  In addition, total plasma bile acids decreased and total fecal bile acids 
increased in A4250-treated patients compared to placebo. A4250 was generally well 
tolerated.  The only findings were GI-related effects, such as diarrhea at higher doses. 
In the ADME study (A4250-007), healthy subjects were administered a single oral dose of 
3 mg [14C]-A4250 capsule.  The main excretion route was determined to be fecal.  There 
were no quantifiable concentrations of A4250 in plasma or total radioactivity in plasma or 
whole blood.  A4250 was well tolerated.  AEs were reported and most were mild and not 
related to A4250. The 3 AEs experienced by 2 subjects reported as related to study drug were 
GI in nature, including diarrhea (2 AEs; 2 subjects) and abdominal pain (1 AE; 1 subject). 
In an exploratory Phase 2 study in children with cholestatic pruritus (A4250-003), A4250 was 
administered as oral daily doses of 10 to 200 µg/kg/day for 4 weeks.  Twenty 
patients (8 females and 12 males) aged 1 to 17 years were enrolled in this open-label 
dose-escalation study.  Four patients were re-enrolled into a second dose level.  No 
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A4250 – IND 130591 
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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
  
Final Amendment 02, 10 May 2018 
 
treatment-related SAEs were reported, and most AEs, including some increased 
transaminases, were transient.  Four-week, once-daily treatment with A4250 reduced mean 
levels of s-BA in all cohorts.  Substantial s-BA reductions in 11 of 13 PFIC patients (ranging 
from 43% to 98%) were observed.  A4250 also reduced s-BA in the 11 non-PFIC patients, 
but to a lesser extent than in PFIC patients.  Patient-reported diaries documented 
improvement of the visual analogue scale itch score in 17 of 24 patients during treatment.  
The dose with the greatest improvement showed a mean decrease (± standard error of the 
mean) of 2.9 ±1.1 from baseline.  The results from this study form the basis of the current 
(A4250-005) study design. 
5.3 
Rationale 
In this investigational study, A4250 is under development for treatment of PFIC and is also 
being considered for other orphan cholestatic liver diseases. 
There is currently no medical treatment approved for PFIC.  In PFIC patients with severe 
pruritus, biliary diversion surgery is used to decrease systemic bile acids through interruption 
of the enterohepatic circulation and thereby reducing pruritus [Whitington 1988].  Liver 
transplantation is typically used as an option when patients have failed medical treatment 
and/or biliary diversion and have liver failure or persistent uncontrolled pruritus. 
By inhibiting IBAT with high selectivity and potency, A4250 has the potential to relieve 
cholestasis and improve liver function without surgical intervention in patients with PFIC.  
Since A4250 is minimally absorbed and achieves a substantial reduction of s-BA, it has the 
potential to be a safer and less invasive alternative to surgical biliary drainage.  The rationale 
for using A4250 is to decrease s-BA levels, and to reduce the major morbidity of pruritus, 
improving the health and wellbeing of patients affected with PFIC.  Ultimately, this 
noninvasive therapeutic approach has the potential to not only improve quality of life (QoL), 
but also reduce the need for biliary diversion and subsequent liver failure and liver 
transplantation. 
The doses selected, 40 and 120 µg/kg/day, were based on the efficacy and safety data 
generated from patients with PFIC (n=10 + 3 re-entered) and supporting data from all 
patients (n=20 + 4 re-entered) in the five dose groups dosed in the A4250-003 study.  
Improvements in s-BA and pruritus were observed with doses from 30 µg/kg to 100 µg/kg, 
with a dose-related trend.  The best dose response in all patients was at the 60 µg/kg/day for 
s-BA and 100 µg/kg/day for pruritus.  The best dose response in the PFIC subgroup was at 
30 µg/kg/day for both s-BA and pruritus.  Patients with PFIC responded well to the doses 
given from 30 µg/kg to 200 µg/kg/day; however, a clear dose relationship could not be 
established in order to select a single dose for this Phase 3 study.  As efficacy was observed 
at both low and high doses, 40 µg/kg and 120 µg/kg/day are considered to be the most 
optimal for this study.  
This study will be conducted in compliance with the protocol and with the International 
Council for Harmonisation (ICH) guidelines on Good Clinical Practice (GCP). 
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A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
  
Final Amendment 02, 10 May 2018 
 
Patients completing 24 weeks of double-blind treatment will be offered to enter Study 
A4250-008 and receive open-label treatment with A4250. 
5.3.1 
Risk/Benefit 
A4250 has been evaluated in three Albireo-sponsored clinical studies: a double-blind 
placebo-controlled study in healthy volunteers, a single-dose ADME study, and a Phase 2 
study in children with cholestatic pruritus.  In addition, an investigator-sponsored study has 
been conducted in patients with PBC. 
A total of 98 subjects/patients have been exposed to A4250.  Healthy subjects have been 
exposed to up to 10 mg as a single dose and up to 3 mg daily as part of a multiple-dose 
evaluation.  Children with cholestatic liver disease have been treated with up to 
0.2 mg/kg/day (200 µg/kg/day) for 4 weeks.  In total, 2 SAEs have been reported; both were 
judged by the physician to be not related to the study drug. 
Patients with cholestatic liver diseases suffer from excess bile acids in the liver resulting in 
tissue damage.  A commonly used treatment in these patients is bile diversion surgery, 
whereby approximately 50% to 100% of the enterohepatic circulation of bile acids is 
interrupted.  Inhibition of IBAT with A4250, thereby interrupting the enterohepatic 
circulation of bile acids, is therefore a potential medical alternative to surgery which could be 
of benefit to these patients if shown to be effective and safe.  Data from the Phase 2 study 
showed efficacy of A4250 in reducing s-BA concentrations and pruritus in such patients. 
Based on the mode of action of an IBAT inhibitor, loose stools or diarrhea could be expected.  
However, in the pediatric Phase 2 study with 4 weeks daily treatment, only one patient had 
mild transient diarrhea after a single dose that did not recur on multiple dosing. 
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A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
  
Final Amendment 02, 10 May 2018 
 
6 
STUDY OBJECTIVES 
6.1 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day 
A4250 in children with PFIC Types 1 and 2, as determined by the following: 
• The proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment 
• Change in pruritus compared to placebo over 24 weeks of treatment 
6.2 
Secondary Objectives 
• To evaluate the effect of A4250 on serum ALT concentration 
• To evaluate the effect of A4250 on growth 
• To evaluate the effect of A4250 on sleep disturbance 
• To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or 
liver transplantation) 
• To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 
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A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
  
Final Amendment 02, 10 May 2018 
 
7 
INVESTIGATIONAL PLAN 
7.1 
Overall Study Design and Plan 
7.1.1 
Description 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the 
efficacy and safety of 40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in 
children with PFIC (deficiencies of FIC1 or BSEP). 
Study data will be periodically reviewed by an unblinded Data Safety and Monitoring Board 
(DSMB). 
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment 
Period and a 4-week Follow-Up Period. 
After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of 
A4250, or a matching placebo. 
Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008; EudraCT Number 
2017-002325-38, subject to a separate study approval) in which all patients will receive 
active treatment.  A patient who prematurely withdraws due to no improvement/intolerable 
symptoms, and has completed at least 12 weeks of the Treatment Period and End of 
Treatment (EOT) Visit, will also be offered the opportunity to enter Study A4250-008. 
Visit windows applicable to each visit are presented in the schedule of assessments (Table 1). 
There will be up to 10 clinic visits during the study (Figure 1) and one scheduled telephone 
contact, as follows: 
• Visit 1: Screening Period start. Patient is considered enrolled after signing the informed 
consent form (ICF) (Days -56 to -35) 
• Visit 2: second Screening Visit (Days -28 to -7) 
• Visit 3: Randomization Visit (Day 0) 
• Telephone Contact 1: (Day 14) 
• Visit 4: Clinic Visit (Day 28) 
• Visit 5: Clinic Visit (Day 56) 
• Visit 6: Clinic Visit (Day 84) 
• Visit 7: Clinic Visit (Day 126) 
• Visit 8: Clinic Visit (Day 154) 
• Visit 9: Clinic Visit, EOT (Day 168) 
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A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
  
Final Amendment 02, 10 May 2018 
 
• Visit 10: Follow-Up Visit, 28 days after last dose of study drug (Day 196).  All patients 
continuing in Study A4250-008 will not have this Follow-up Visit. 
Additional clinic visits may be required for patients who need direct site assistance, e.g., due 
to AE monitoring, to fulfil screening requirements, and/or for safety maintenance. 
Screening Period 
Informed consent must be obtained at Visit 1 prior to performing any study procedures.  After 
signing the ICF, patients will be considered enrolled and evaluated for study eligibility. 
Daily recording of pruritus using an electronic diary (eDiary) will be started to determine 
confirmation of the magnitude and the baseline pruritus symptoms before the study.  After 
consultation with the Medical Monitor, patients failing eligibility criteria may be re-screened 
after 2 months.  Patients not fulfilling inclusion/exclusion criteria after 3 attempts are not 
allowed to re-screen. 
Caregivers/patients will be instructed on the daily use of the eDiary.  The eDiary will include 
patient-reported outcome (PRO) and observer-reported outcome (ObsRO) items for 
evaluation of itching (PRO), scratching (ObsRO), and sleep disturbance (PRO and ObsRO) 
during the Screening Period (study Days -56 to 0), throughout the 24-week Treatment Period 
(study Days 0 to 168), and Follow-up (Day 196). 
The 14 consecutive days prior to randomization will be used for eligibility calculation of the 
diary entries.  If a patient does not meet the minimum number of observations (see 
Section 9.2.2 for scoring and missing data definitions) to determine eligibility, the patient 
may be re-screened.  Observer-reported outcomes in patients of all ages will be recorded by a 
caregiver.  If possible, the same caregiver will complete the Albireo ObsRO items throughout 
the study. 
At a second Screening Visit (Visit 2) additional laboratory samples will be taken for 
eligibility assessments. 
Treatment Period 
Eligibility for randomization will be determined at Visit 3 using the pruritus (scratching 
reports from the Albireo ObsRO) data in the 2 weeks before Visit 3, clinical genetic 
confirmation of diagnosis, and the liver biochemistry evaluations from the previous screening 
visits.  Two weeks after Visit 3, there will be a telephone contact with the caregiver/patient to 
evaluate any AEs. 
The patient will return to the clinic at Visit 4, Visit 5, Visit 6, Visit 7, Visit 8, and Visit 9 for 
assessments.  If a new caregiver will begin to complete the Albireo ObsRO during the course 
of the trial, they will be provided with training during a clinic visit before they begin to use 
the eDiary.  Additionally, caregivers will be requested to use the diary to report the time each 
dose of study drug is administered during the Treatment Period. 
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A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
  
Final Amendment 02, 10 May 2018 
 
The last dose of study drug will be administered the day before Visit 9.  At study Visit 9, the 
patient has the option to enroll in an open-label extension study (A4250-008) should they 
meet the required entry criteria.  If the patient is continuing into Study A4250-008 this will be 
the last visit in this study and the first visit in Study A4250-008.  Visit 9/EOT should also be 
performed at the time patients are prematurely withdrawn from the study. 
Clinic Visit 10 will take place 28 days after Visit 9 for all patients not continuing into Study 
A4250-008. 
Figure 1 
Study Design 
 
T: telephone contact; V: study visit 
End of Study 
The end of the study is defined as follows: 
a) End of study in one country: last patient last visit (LPLV) and sites are closed 
b) End of study globally: LPLV globally and all sites closed 
7.1.2 
Schedule of Assessments 
The schedule of assessments is presented in Table 1.  For information on blood volumes, 
please see Appendix 7. 
 
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A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Final Amendment 02, 10 May 2018 
Table 1 
Schedule of Assessments 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone 
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35) 
±2 
-28 – (-7) 
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic 
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic 
Visit 4 
Clinic 
Visit 5 
Clinic 
Visit 6 
Clinic 
Visit 7
Clinic 
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Informed consent c 
X 
Inclusion/exclusion criteria 
X 
X 
Demographics 
X 
Concomitant medicationd 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical and surgical history 
X 
X 
Physical examination including 
voluntary photography 
X 
X 
X 
X 
Skin examination 
X 
X 
X 
X 
X 
X 
Vital signse
X 
X 
X
X 
X 
X 
X 
X 
X 
X 
eDiary: itching, scratching, and 
sleep scoresf 
Daily diary entry 
Clinical chemistryg 
X 
X 
X 
X 
X 
X 
X 
X 
Hematologyg 
X 
X 
X 
X 
X 
X 
X 
Urinalysisg 
X 
X 
X 
Serum bile acidsh 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
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A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Final Amendment 02, 10 May 2018 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone 
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35) 
±2 
-28 – (-7) 
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic 
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic 
Visit 4 
Clinic 
Visit 5 
Clinic 
Visit 6 
Clinic 
Visit 7
Clinic 
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
INR 
X 
X 
X 
X 
X 
Autotaxini 
X 
X 
X 
p-C4i 
X 
X 
X 
AFP 
X 
X 
Vitamins A & Eh 
X 
X 
X 
25-hydroxy vitamin D 
X 
X 
X 
Blood sample for A4250 PKi 
X 
X 
Abdominal Ultrasound 
X 
X 
Fibroscan® (where available) 
X 
X 
QoL questionnaire (PedsQL) 
X 
X 
Patient/Caregiver/Clinician 
Global Impression of Change 
X 
X 
X 
Patient/Caregiver/Clinician 
Global Impression of 
Symptoms 
X 
X 
X 
X 
Pregnancy testj
Xj 
X 
X 
X 
X 
X 
X 
X 
Xj 
X 
Study drug dispensedk
X 
X 
X 
X 
X 
X 
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A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
Final Amendment 02, 10 May 2018 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone 
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35) 
±2 
-28 – (-7) 
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic 
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic 
Visit 4 
Clinic 
Visit 5 
Clinic 
Visit 6 
Clinic 
Visit 7
Clinic 
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Adverse eventsl 
Continuous collection 
Study drug compliance 
X 
X 
X 
X 
X 
X 
AFP: alfa-fetoprotein; BMI: body mass index; eCRF: electronic case report form; eDiary: electronic diary; EOT: End of Treatment; ICF: informed consent form; 
INR: international normalized ratio; p-C4: plasma 7α Hydroxy 4-cholesten-3-one; PedsQL: Pediatric Quality of Life Inventory; PK: pharmacokinetic; QoL: quality of life; 
RR: respiratory rate; SAE: serious adverse event; s-BA: serum bile acid. 
a 
Must also be performed at the time a patient is prematurely withdrawn from the study.  This visit is the same as Visit 1 in the extension protocol (Study A4250-008). 
b 
Assessments must be performed 28 days following the final dose of study drug.  This visit is omitted if patients are entering Study A4250-008. 
c 
The ICF (and assent) must be signed before any study procedures are performed. 
d 
Past (within 1 month prior to Visit 1) and current medication will be documented in the eCRF. 
e 
Includes blood pressure, pulse, RR, temperature, height (or length depending on age), and body weight (using a certified weight scale).  BMI will be calculated. 
f 
Includes training on use of the device at Screening (Visit 1) and review of patient daily entries during 14 days prior to Clinic Visit 3 to determine eligibility.  
g 
See Table 3 for detailed parameters. 
h 
Patients will fast (water intake only is permissible) for at least 4 hours prior to the collection of samples for s-BA and vitamin A.  Exceptions can be made for infants, 
less than 12 months of age, if unable to fast for the full 4 hours.  All s-BA results during the treatment period and follow-up will be blinded. 
i 
Samples will not be collected for patients ≤10 kg.  In the event of an SAE, a PK sample should be collected as close to the onset of the event as possible. 
j 
For girls who have reached menarche.  Serum test at Visit 1; urine test at other visits.  A serum pregnancy test will also be conducted at Visit 9 for patients entering 
Study A4250-008.  If a urine pregnancy test is positive, a serum pregnancy test should be performed to confirm the pregnancy. 
k 
Study drug to be taken once daily on Day 1 through Day 167 as described in Section 8.2. 
l 
Adverse event information will be collected from the time of signing of the ICF to study discontinuation. 
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A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
Confidential 
 
Final Amendment 02, 10 May 2018 
7.1.3 
Study Procedures and Assessments 
7.1.3.1 
Screening Period (Days -56 to 0) 
Day -56 to Day -35/Clinic Visit 1 
Patients will undergo a Screening Visit up to 56 days prior to the planned first day of study 
treatment.  Screening procedures and assessments are as follows: 
•
Obtain written informed consent
•
Assess inclusion/exclusion criteria (Section 7.2)
•
Record demographics (age, full date of birth, gender, race, and ethnicity)
•
Document concomitant medications
•
Medical and surgical history (date of diagnosis of PFIC Types 1 or 2, prior investigational
medications for PFIC, historical liver function test [LFT] values, any surgery performed,
any other diagnosis, and historical liver biopsy data)
•
Collect, if available, past confirmatory clinical genetic laboratory report for
PFIC-1/PFIC-2 and send to central reader for review.  If a historical report is equivocal,
unavailable, or unobtainable, a blood sample for clinical genetic testing will be collected
to determine eligibility
•
Physical examination, including voluntary photography (Section 10.2.5)
•
Skin examination (Section 10.2.5)
•
Vital signs (Section 10.2.6)
•
eDiary training and compliance requirements; caregivers and, if applicable, patients begin
daily recording of pruritus using the eDiary (Appendix 2)
•
Clinical chemistry (Section 10.2.1)
•
s-BA (Section 9.2.1)
•
Serum pregnancy test for girls who have reached menarche.  Please see Appendix 6 for
contraceptive requirements
•
AE monitoring
Day -28 to Day -7/Clinic Visit 2 
A second Screening Visit will be performed.  Screening procedures and assessments are as 
follows: 
•
Review concomitant medications
•
Vital signs (Section 10.2.6)
•
Albireo PRO/ObsRO eDiary review for compliance
•
Urinalysis (Section 10.2.1)
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A4250 – IND 130591 
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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
 
Final Amendment 02, 10 May 2018 
 
• s-BA (Section 9.2.1) 
• International normalized ratio (INR) 
• Vitamins A and E 
• Urine pregnancy test for girls of who have reached menarche 
• AE monitoring 
7.1.3.2 
Treatment Period (Day 0 to Day 168) 
Study Day 0/Clinic Visit 3 (Randomization) 
At the randomization visit, the following assessments will be conducted: 
• Review inclusion/exclusion criteria (Section 7.2) 
• Review concomitant medications, medical and surgical history 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance and entrance criteria requirement (see 
Inclusion Criteria 4, Section 7.2.1) 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1)  
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• 25-hydroxy vitamin D 
• Alfa-fetoprotein (AFP) 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer QoL (Pediatric Quality of Life Inventory [PedsQL]) questionnaire 
• Patient/caregiver/clinician complete the patient global impression of symptoms (PGIS) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Study drug is dispensed and patients are instructed to administer daily from Day 1 
Study Day 14 (Telephone Contact) 
A study nurse will contact the patient/caregiver by telephone for monitoring of the following:  
• Review concomitant medications 
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A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
 
Final Amendment 02, 10 May 2018 
 
• Albireo PRO/ObsRO eDiary review for compliance 
• AEs 
Study Day 28 and Day 84/Clinic Visits 4 and 6  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Physical examination, including voluntary photography (at Clinic Visit 6 only; 
Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• Urinalysis (at Clinic Visit 6 only; Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Autotaxin, p-C4, and A4250 pharmacokinetic (PK) assessment (at Clinic Visit 4 only; 
samples will not be taken for patients ≤10 kg) 
• Vitamins A and E, and 25-hydroxy vitamin D (at Clinic Visit 6 only) 
• Patient/caregiver/clinician complete the patient global impression of change (PGIC) and 
PGIS 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 56 and Day 126/Clinic Visits 5 and 7 
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Urine pregnancy test for girls who have reached menarche 
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• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 154/Clinic Visit 8  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 168/Clinic Visit 9/EOT 
The last dose of study drug is taken in the morning the day before Clinic Visit 9, and the 
following procedures and assessments will be performed (also performed at the time a patient 
prematurely withdraws):  
• Review of concomitant medications 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry, hematology, and urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Autotaxin, p-C4, and A4250 PK assessment (samples will not be taken for patients 
≤10 kg) 
• AFP  
• Vitamins A, E, and 25-hydroxy vitamin D 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
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• Administer PedsQL questionnaire 
• Patient/caregiver/clinician complete the PGIC and PGIS 
• Urine pregnancy test for girls who have reached menarche, or serum pregnancy test for 
girls continuing to study A4250-008 
• AE monitoring 
• Evaluation of study drug compliance 
7.1.3.3 
Follow-Up Period 
Study Day 196/Clinic Visit 10 
All patients not continuing into Study A4250-008 will return to the study site 28 days after 
Clinic Visit 9 or the last dose of study drug for the following assessments: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
7.2 
Study Population 
A total of approximately 60 patients with a clinical diagnosis of PFIC Type 1 or 2 that have 
been genetically confirmed will be randomized.  The study will target 15% of enrolled 
patients to be PFIC-1 patients.  If enrollment of all PFIC-2 patients is completed and the 15% 
enrollment of PFIC-1 patients has not yet been achieved, then enrollment of PFIC-2 patients 
will continue in order to reach the total study target enrollment. 
An attempt will be made to randomize a minimum of 10 patients from each age category 
(6 months to 5 years, 6 to 12 years, and 13 to ≤18 years).  
A maximum of 5 patients that were treated in Study A4250-003 will be allowed to participate 
in this study. 
7.2.1 
Inclusion Criteria 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages 
of ≥6 months and ≤18 years at Visit 1 with a body weight above 5 kg 
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2. Patient must have clinical genetic confirmation of PFIC-1 or PFIC-2 through 
identification of biallelic pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, 
taken as the average of 2 samples 7 to 28 days apart (Visits 1 and 2) prior to 
randomization 
4. Patient must have history of significant pruritus and a caregiver-reported observed 
scratching in the eDiary average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to 
randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  
Patients who turn 18 years of age (or legal age per country) during the study will be 
required to re-consent in order to remain in the study 
6. Patients will be expected to have a consistent caregiver for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use 
an eDiary device as required by the study 
7.2.2 
Exclusion Criteria 
1. Patient with pathologic variations of the ABCB11 gene that predict complete absence of 
the BSEP protein 
2. Patient with past medical history or ongoing presence of other types of liver disease 
including, but not limited to, the following: 
a) Biliary atresia of any kind 
b) Benign recurrent intrahepatic cholestasis, indicated by any history of normal s-BAs 
c) Suspected or proven liver cancer or metastasis to the liver on imaging studies 
d) Histopathology on liver biopsy is suggestive of alternate non-PFIC related etiology of 
cholestasis 
3. Patient with a past medical history or ongoing presence of any other disease or condition 
known to interfere with the absorption, distribution, metabolism (specifically bile acid 
metabolism), or excretion of drugs in the intestine, including but not limited to, 
inflammatory bowel disease 
4. Patient with past medical history or ongoing chronic (i.e., >3 months) diarrhea requiring 
intravenous fluid or nutritional intervention for treatment of the diarrhea and/or its 
sequelae 
5. Patient has a confirmed past diagnosis of infection with human immunodeficiency virus 
or other present and active, clinically significant, acute, or chronic infection, or past 
medical history of any major episode of infection requiring hospitalization or treatment 
with parenteral anti-infective treatment within 4 weeks of treatment start (study Day 1) or 
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completion of oral anti-infective treatment within 2 weeks prior to start of Screening 
Period 
6. Any patient with suspected or confirmed cancers except for basal cell carcinoma, and 
non-liver cancers treated at least 5 years prior to Screening with no evidence of 
recurrence 
7. Patient with a past medical history of chronic kidney disease with an impaired renal 
function and a glomerular filtration rate <70 mL/min/1.73 m2  
8. Patient with surgical history of disruption of the enterohepatic circulation excluding those 
who have undergone a successful reversal procedure that has permanently restored flow 
of bile acids from the liver to the duodenum 
9. Patient has had a liver transplant or listed for liver transplant, excluding those who have 
been listed for transplant for pruritus who have since been removed from the transplant 
list 
10. Decompensated liver disease, coagulopathy, history or presence of clinically significant 
ascites, variceal hemorrhage, and/or encephalopathy 
11. INR >1.4 (the patient may be treated with Vitamin K intravenously, and if INR is ≤1.4 at 
resampling the patient may be randomized) 
12. Serum ALT >10 × upper limit of normal (ULN) at Screening 
13. Serum ALT >15 × ULN at any time point during the last 6 months unless an alternate 
etiology was confirmed for the elevation 
14. Total bilirubin >5 × ULN at Screening 
15. Patient suffers from uncontrolled, recalcitrant pruritic condition other than PFIC.  
Examples include, but not limited to, refractory atopic dermatitis or other primary pruritic 
skin diseases 
16. Any patient who is pregnant or lactating or who is planning to become pregnant within 
24 weeks of randomization 
17. Sexually active males and females who are not using a reliable contraceptive method with 
≤1% failure rate (such as barrier protection, hormonal contraception, intrauterine device, 
or complete abstinence) throughout the duration of the study and 90 days thereafter (from 
signed informed consent through 90 days after last dose of study drug).  See Appendix 6 
for further details 
18. Patient with a past medical history of alcohol or substance abuse will be excluded.  
Patient must agree to refrain from illicit drug and alcohol use during the study 
19. Administration of bile acid or lipid binding resins and medications that slow GI motility 
(Refer to Appendix 1 – Concomitant Medications Guidelines) 
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20. Patient has had investigational exposure to a drug, biologic agent, or medical device 
within 30 days prior to Screening, or 5 half-lives of the study agent, whichever is longer 
21. Patient who has been previously treated with an IBAT inhibitor whose pruritus has not 
responded to treatment 
22. Any other conditions or abnormalities which, in the opinion of the investigator or Medical 
Monitor, may compromise the safety of the patient, or interfere with the patient 
participating in or completing the study 
7.2.3 
Withdrawal of Patients 
Patients/caregivers will be informed that they have the right to withdraw from the study at 
any time, without prejudice to their medical care, and that they are not obliged to state their 
reasons. 
Any withdrawal, and reasons for withdrawal, must be fully documented in the electronic case 
report form (eCRF) and source documents and the patient followed by the 
investigator/investigative staff.  Even after the study is completed at the Follow-up Visit, the 
investigator/investigative staff will, regardless of reason for withdrawal, record any patient 
data they receive concerning SAEs, and all drug-related non-serious AEs, and subsequently 
report these to Albireo.  Withdrawn patients will not be replaced. 
Patients will be withdrawn in the following circumstances: 
• A patient’s/caregiver’s desire for withdrawal for any reason 
• Lost to follow-up (every effort must be made to contact the patient/caregiver; a certified 
letter must be sent) 
• An AE that, in the opinion of the investigator, necessitates withdrawal 
• A worsening of the symptoms related to diarrhea occurs 
• Death 
• Patient/caregiver estimation of lack of improvement/intolerable symptoms 
• A patient’s/caregiver’s substantial noncompliance (eDiary and study drug compliance) or 
protocol violation 
• An investigator’s opinion that continuing the patient in the study is not appropriate.  The 
investigator may withdraw a patient at any time, if it is considered to be in the patient’s 
best interest 
A patient who prematurely withdraws due to no improvement/intolerable symptoms, and has 
completed at least 12 weeks treatment in the double-blind study, will be offered the 
opportunity to enter open-label Study A4250-008.  A patient who withdraws from treatment 
prematurely and does not enter Study A4250-008 will have EOT (Visit 9) assessments at the 
time of withdrawal and a follow-up assessment (Visit 10) 28 days following the last dose of 
study drug, and the patient will be recorded as withdrawn. 
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7.2.4 
Study Termination by Sponsor 
This study may be terminated at any time by Albireo if serious side effects should occur, if 
the investigator does not adhere to the protocol; or if, in the sponsor's judgment, there are no 
further benefits to be achieved from the study.  In this event, Albireo/designee will inform the 
study investigators, institutions, and all regulatory authorities. 
Albireo may temporarily or permanently discontinue the study at an investigative site at any 
time for safety, ethical, compliance, or other reasons.  If this is necessary, Albireo will 
endeavor to provide advance notification to the site.  If a site or the study is suspended or 
discontinued, the investigator/investigative staff will be responsible for promptly informing 
the Independent Ethics Committee (IEC).  If required by local regulations, Albireo/designee 
will be responsible for informing the IEC and the regulatory authority of study or site 
discontinuation.  In such an event, all study data and unused study drug must be returned to 
Albireo. 
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8 
TREATMENT OF PATIENTS 
8.1 
Identity of Study Drug 
A4250 and placebo will be supplied as capsules for oral administration.  White opaque 
capsules filled with pellets containing A4250 or placebo will be provided.  Two different 
capsule sizes will be available, as follows: 
• Capsule size 0 that can be opened 
• Capsule size 3 that should be swallowed intact.  These size 3 capsules may be opened 
only under exceptional circumstances, e.g., patient cannot swallow capsule intact 
The content of the A4250 and placebo capsules will be identical in appearance.  Capsule 
filling weight will also be identical for high dose, low dose, and placebo.  Bottles with 
34 capsules will be given to the patient at each visit.  A patient who requires 2 or more 
capsules per day will be given multiple bottles.  Refer to the investigational product manual. 
8.2 
Administration of Study Drug 
Patients will be dosed with A4250 at a dose of 40 µg/kg/day, 120 µg/kg/day, or placebo for 
24 weeks.  Study drug will be dispensed to the patient at Visits 3, 4, 5, 6, 7, and 8, together 
with instructions on how to store and take the drug.  Study drug administration data, 
including whether each patient took each dose or partial doses of study drug, whether there 
were any delayed or missed doses, and whether the capsule was opened or swallowed whole 
will be documented through the eDiary and transferred to the study database. 
A4250 should be taken in the morning, together with food.  On clinic visits days when 
laboratory assessments are conducted, study drug should be taken after the visit and after 
laboratory samples are taken.  Patients should not crush or chew the capsule(s).  When 
swallowing the capsule intact, the patient should administer the dose with a glass of water. 
For a patient unable to swallow capsule intact, the capsule can be opened and the contents 
sprinkled and mixed in a small amount of soft room-temperature yogurt, apple sauce, or fruit 
purée.  The amount of food should be less than what the patient/infant is expected to eat and 
should be followed by water.  
1. Hold the capsule over the prepared food, gently twist open and allow the content to fall 
into food 
2. Mix the entire capsule content with 1 or 2 tablespoons (15 to 30 mL) of soft food (apple 
sauce, fruit purée, or yogurt) 
3. Administer the mixture immediately after mixing. 
The number of capsules provided to the patient will be based on body weight at Visit 3.  At 
Visit 6, if a patient’s weight has shifted to another weight category (identified in Table 2), the 
number of capsules per day may be adjusted.   
 
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Table 2 
Dosing and Capsule Strength 
Body Weight 
(kg) 
Capsule 
Size 
Number of 
Capsules 
per Day 
Capsule 
Strength, 
Low Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Strength, 
High Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Size, 
Placebo 
(µg) 
5.0 to <7.5 
0 
1 
200 
200 
600 
600 
0 
7.5 to <12.5 
0 
2 
200 
400 
600 
1200 
0 
12.5 to <17.5 
0 
3 
200 
600 
600 
1800 
0 
17.5 to <19.5 
0 
4 
200 
800 
600 
2400 
0 
19.5 to <25.5 
3 
2 
400 
800 
1200 
2400 
3 
25.5 to <35.5 
3 
3 
400 
1200 
1200 
3600 
3 
35.5 to <45.5 
3 
4 
400 
1600 
1200 
4800 
3 
45.5 to 55.5 
3 
5 
400 
2000 
1200 
6000 
3 
>55.5 
3 
6 
400 
2400 
1200 
7200 
3 
8.3 
Study Treatment Packaging and Labeling 
8.3.1 
Packaging and Labeling 
The capsules will be packed in high-density polyethylene containers, with child-proof 
polypropylene caps.  Study drug capsules containing A4250 in the strength specified in 
Table 2 above will be manufactured. 
Packaging and labeling will be prepared to comply with applicable regulatory requirements. 
8.3.2 
Storage 
Treatment packs containing A4250 capsules should be stored and dispensed in accordance 
with regulations in their original containers.  The storage facility at the investigative site 
should be locked and the storage temperature should be between 15°C and 25°C. 
Patients/caregivers should be informed of appropriate storage conditions (i.e., room 
temperature, between 15°C and 25°C). 
Any deviations from the recommended storage conditions should be immediately reported to 
Albireo and the study drug should not be used until authorization has been given by Albireo. 
8.3.3 
Blinding and Randomization of Study Drug 
A double-blind design is employed so that both the investigators and the patients will be 
unaware of the treatment assignment during the study.  
After written informed consent is obtained from an eligible patient, an 8-digit patient 
identification number will be assigned.  The first 2 digits will denote the country, followed by 
a 3-digit site number and a 3-digit patient sequence number.  This number will be created and 
allocated by the Interactive Web Response System (IWRS), when the patient first enters the 
system at Screening.  The randomization codes will be computer-generated by 
 
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Biostatistics, and kept by a statistician independent from the project team.  The 
randomization will be done in blocks and stratified according to PFIC class (Type 1 and 2) 
and age class (6 months to 5 years, 6 to 12 years, and 13 to ≤18 years) to ensure approximate 
balance between dose schemes (1:1:1).  A separate randomization list will be prepared for the 
maximum 5 patients from Study A4250-003 regardless of stratification.  Randomization 
codes will be assigned sequentially as patients become eligible for randomization. 
Patients who are eligible for randomization will be assigned a unique 4-digit randomization 
number by IWRS.  This randomization number identifies which treatment will be allocated to 
the patient.  Patients who withdraw from the study after randomization visit are not to be 
replaced and their randomization number will not be reused.   
To ensure blinding, the study drug and the matching placebo have the same shape and size.  
Patients will receive capsule(s) of A4250 according to their dose group or capsule(s) of 
matching placebo once a day during the double-blind Treatment Period.  Labels on the study 
drug containers will not identify the treatment that a patient is randomized to.  Traceability of 
the treatment is ensured by the study drug number. 
The 5-digit study drug number will identify study drug packs and will be detailed on the 
study drug label.  Dispensing of study drug will be coordinated by IWRS.  
The system will assign a study drug number corresponding to the randomization arm.  The 
randomization number will be used in the background only to ensure no unblinding and will 
not be displayed to end-users in the IWRS. 
8.4 
Procedure for Breaking the Randomization Code 
Should a situation arise where unblinding is urgently required, i.e., only where knowledge of 
the study drug is required to adequately manage a life-threatening situation, the investigator 
at that study site may perform immediate unblinding through IWRS.  The responsibility to 
break the treatment code in emergency situations resides solely with the investigator.  The 
investigator should make every effort to discuss the rationale (status and outcome) for 
emergency unblinding with the sponsor Medical Monitor or the 
 Medical Monitor as 
soon as possible to review the individual patient details. 
The study site investigator and appropriate 
 project team members will be authorized to 
access the emergency unblinding functionality within the IWRS.  Emergency unblinding 
takes place in the IWRS via a dedicated emergency unblinding module.  The system will 
require the user to enter their emergency unblinding, which is typically provided at the same 
time login credentials are distributed.  The exact description of the treatment assigned to the 
individual patient then will be accessible to the user.  Once the randomization code is broken 
for a patient, he/she must be withdrawn from the study and all assessments and procedures at 
EOT should be performed.  As a follow-up, Visit 10 will be performed according to the time 
schedule.  Once a randomization code has been broken, the investigator must inform the 
 and sponsor Medical Monitor in writing within 24 hours.  In case of technical issues 
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accessing the system online, please see the IWRS site user manual for country-specific 
contact telephone numbers for the 
 HelpDesk 24/7 system support. 
8.5 
Patient Compliance 
The study nurse will monitor eDiary compliance by routine review of the CRF health 
website.  If both diary entries on a day are missing, the study nurse will call the 
patient/caregiver to remind them to complete all scheduled entries.  Any noncompliance will 
be documented and explained in the source documents. 
Study drug compliance will also be checked through diary responses.  Additionally, patients 
will return all unused study drug at Visits 4, 5, 6, 7, 8, and 9.  The study site staff will count 
all returned drug, assess compliance, and record details in the eCRF. 
Treatment Compliance = 100 × ((Number of study drug dispensed – number of study drug 
returned)/number of study drug that should be taken). 
Treatment compliance between 80% and 120% will be acceptable. 
8.6 
Study Drug Accountability 
Records shall be maintained of the delivery of study treatment(s) to the study site(s), of the 
inventory at the study site(s), of each use of the study treatment(s) for each patient, and of the 
return of unused study treatment(s) to Albireo. 
These records shall include dates, quantities, batch numbers, expiry dates, and the unique 
code numbers assigned to the study drug and to the study patients. 
The investigator will be responsible for ensuring that the records adequately document that 
the patients were provided the quantities specified in the protocol and that all study drug 
received from Albireo is reconciled.  
8.7 
Concomitant Therapy and Prohibited Medications 
The investigator will note all ongoing medication and any medication recently stopped 
(within 1 month prior to Visit 1) in the eCRF.  At Visits 2 through 10, all changes in 
medication (stopping or starting new medication or changes in dose) will be recorded in the 
eCRF.  All medications (prescribed or over the counter) for pruritus and sleep will be 
recorded. 
All medications taken by a patient within 3 months prior to the first intake of study drug are 
regarded as prior medication. 
All medications taken by a patient on or after the first intake of study drug, and which 
continue to be taken during the study, are regarded as concomitant medication.  
Prohibited medications are listed in Appendix 1. 
 
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9 
ASSESSMENT OF EFFICACY 
9.1 
Efficacy Endpoints 
9.1.1 
Primary Efficacy Endpoint 
The primary efficacy endpoints are as follows: 
• European Union (EU) and rest of the world (RoW): Proportion of patients 
experiencing at least a 70% reduction in fasting s-BA concentration from baseline to 
the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 
weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization 
and the previous visit (Clinic Visit 2).  The end value is the average of the values at 
Weeks 22 and 24 after the start of double-blind treatment. 
• United States (US): Change in pruritus, as indexed by caregiver-reported observed 
scratching, from baseline over the last 5 months of the Treatment Period as measured 
by an overall mean of weekly averages of the worst daily scratch score using the 
Albireo ObsRO instrument (Appendix 2). 
9.1.2 
Secondary Efficacy Endpoint 
Secondary efficacy endpoints include the following:  
• EU and RoW: Change in pruritus, as indexed by caregiver-reported observed 
scratching, from baseline over the last 5 months of the Treatment Period as measured 
by an overall mean of weekly averages of the worst daily scratch score using the 
Albireo ObsRO instrument (Appendix 2). 
• US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment. 
• All Regions: 
• All secondary endpoints are compared to placebo. 
o Change from baseline to Week 24 in fasting s-BA 
o Change from baseline to Week 24 in serum ALT concentration 
o Change in growth from baseline to Week 24, defined as the linear deficit 
(weight for age, and body mass index [BMI]) compared to a standard growth 
curve (Z-score, standard deviation [SD] from P50)  
o Proportion of patients achieving meaningful reduction in caregiver-reported 
observed scratching over the Treatment Period 
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o Change from baseline over the last 5 months of the Treatment Period as 
measured by an overall mean of weekly averages of the sleep parameters 
measured with the Albireo PRO and ObsRO instruments 
o Change from baseline over the last 5 months of the Treatment Period as 
measured by an overall mean of weekly averages of patient-reported itch 
severity as measured by the average daily score of the Albireo PRO instrument 
(Appendix 2); only patients ≥8 years of age will complete the Albireo PRO 
instrument  
o Number of patients undergoing biliary diversion surgery or being listed for 
liver transplantation 
9.1.3 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• Proportion of patients achieving meaningful reduction in patient-reported itch severity 
over the Treatment Period 
• Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, 
caregiver, and clinician via the PGIC items (Appendix 3) 
• Change from baseline in fasting s-BA at Weeks 4 and 12 
• Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
to Week 4 of the Treatment Period as measured by the Albireo ObsRO instrument 
• Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
over the last 3 months of the Treatment Period as measured by an overall mean of weekly 
averages of the worst daily scratch of the Albireo ObsRO instrument 
• Change from baseline over the last 5 months of the Treatment Period in patient-reported 
and observer-reported night time itching and scratching severity scores, respectively 
• Change from baseline over the last 5 months of the Treatment Period in pooled pruritus 
score including observer-reported scratching for patients <8 years of age and 
patient-reported itch severity for patients ≥8 years of age 
• Change from baseline over the last 5 months of the Treatment Period in additional 
patient-reported and observer-reported sleep parameters (e.g., tiredness and number of 
awakenings) 
• Change from baseline to Week 24 in PedsQL (Appendix 4) 
• Change from baseline in serum ALT concentration at Weeks 4 and 12 
• Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• Change from baseline in AST and GGT at Weeks 4, 12, and 24 
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• Change from baseline for other biochemical markers and measures of bile acid synthesis 
(autotaxin, p-C4) at Week 24 
• Change in pediatric end-stage liver disease/model for end-stage liver disease 
(PELD/MELD) score, AST to platelet ratio index (APRI) score, and Fib-4 score from 
baseline to Week 24 
• Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® 
(where available) 
• Liver-related mortality and liver decompensation events 
• All-cause mortality 
9.2 
Efficacy Assessments 
9.2.1 
Serum Bile Acids 
Blood samples for analysis of fasting total s-BA will be drawn at all visits.  Patients will fast 
(water intake only is permissible) for at least 4 hours prior to the collection of samples for 
s-BA.  Exceptions can be made for infants, <12 months of age, if unable to fast for the full 
4 hours.  Any visit at which a bile acid sample result is unreportable, an additional 
unscheduled visit for a repeat sample collection may be scheduled.  All s-BA results during 
the treatment period and follow-up will be blinded.  Samples will be processed and 
transported to a central laboratory per instructions in the laboratory manual. 
9.2.2 
Itching, Scratching and Sleep Score 
Itching, observed scratching, and sleep disturbance will be recorded twice each day via 
eDiary.  Patients and/or caregivers will be instructed to complete the eDiary every day in the 
morning after the patient wakes and in the evening just before the patient goes to sleep 
throughout the study.  Patients and caregivers will receive a formal training session during 
Visit 1. 
The eDiary includes Albireo ObsRO and PRO items.  Patients <8 years of age will not be 
asked to complete the Albireo PRO items; only the Albireo ObsRO will be completed by 
caregivers of patients in this age group.  Older patients, 8 to 18 years of age, will complete 
the Albireo PRO items and the caregiver will complete the Albireo ObsRO items.  The 
Albireo PRO items assess severity of itch, aspects of sleep disturbance (morning diary only), 
and tiredness.  For patients 8 to 12 years of age, the caregiver will read the Albireo PRO 
items along with the child and record the child’s response.  A guide will be provided to the 
caregivers that provides standardized explanations of the Albireo PRO items, in case the 
patient is confused or requires clarification about the meaning of a question.  The Albireo 
ObsRO items assess severity of observed scratching, aspects of observed sleep disturbance 
(morning diary only), and observed signs of tiredness (evening diary only).  The Albireo 
ObsRO and PRO scratching and itch severity items use 0 to 4 response scales, where each 
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response is distinguished by a unique facial expression, verbal anchor, number, and color 
code (Appendix 2).  
A daily score for the Albireo ObsRO scratching item will be calculated by selecting the 
maximum scratching severity score from the 2 ratings for each day.  A daily score will be 
considered missing if both of the daily assessments are missing.  A weekly score will be 
calculated by averaging the daily scratching scores.  A weekly score will be considered 
missing if ≥4 out of 7 days a week of data are missing.  For baseline, a 14-day score will be 
calculated by averaging the 2 weekly scores prior to randomization and will be considered 
missing if one or both weekly scores for that 14-day period are missing.  For each treatment 
month, a 14-day score will be calculated by averaging the 2 last weekly scores and will be 
considered missing if both weekly scores for that 14-day period are missing.  The same 
approach will be used to calculate a patient-reported itch severity score.  The handling of 
missing data is described in Section 11.2.2.2. 
9.2.3 
Growth 
Growth will be measured as height (velocity) and weight (Z-score) using a certified weight 
scale.  BMI will be calculated by weight (kg) / height (m)2.  Change will be defined as linear 
growth deficit (weight and BMI for age) compared to a standard growth curve (Z-score, SD 
from P50). 
9.2.4 
Quality of Life Questionnaire (PedsQL) 
Patients and caregivers will be asked to complete a QoL questionnaire (PedsQL) at Visit 3 
and Visit 9.  Details of the questions included on the questionnaire are provided in 
Appendix 4. 
9.2.5 
Biomarkers and PK Samples 
Blood samples for autotaxin and p-C4 will be drawn at Visits 3, 4, and 9 only for children 
with body weight >10 kg.  Blood for A4250 PK assessment will only be drawn at Visits 4 
and 9 (only for children with body weight >10 kg), and as close to the onset of an SAE as 
possible, should an SAE occur.  Samples will be processed and transported to a laboratory 
per instructions in the laboratory manual. 
9.2.6 
PELD/MELD Score 
At randomization and end of treatment, PELD score will be calculated for children up to 
12 years of age.  For children 13 years or older, the MELD score will be calculated. 
PELD Score = 4.80 * ln(total bilirubin) + 18.57 * ln(INR) - 6.87 * ln(albumin) + 4.36 (if 
patient <1 year: scores for patients listed for liver transplantation before the patient's first 
birthday continue to include the value assigned for age (<1 year) until the patient reaches the 
age of 24 months) + 6.67 (if the patient has growth failure [≤2 SD]) 
Laboratory values less than 1.0 will be set to 1.0 for the calculation of the PELD score. 
MELD Score = 9.57 * ln(creatinine) + 3.78 * ln(total bilirubin) + 11.2 * ln(INR) + 6.43 
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Laboratory values less than 1.0 will be set to 1.0 and serum creatinine values greater than 
4.0 mg/dL (equivalent to 353.6 µmol/L)will be set to 4.0 for calculation of the MELD score. 
9.2.7 
Fibroscan® 
Where available, Fibroscan® will be performed as per institution standard practice at Visits 3 
and 9. 
9.2.8 
Markers of Fibrosis 
APRI score and Fib-4 score will be calculated at randomization and at end of treatment. 
APRI = [(AST in U/L) / (AST ULN in U/L)] / (Platelets in 109/L) 
Fibrosis 4 Score = (Age*AST in U/L) / (Platelets in 109/L * √(ALT in U/L)). 
9.2.9 
Global Impression of Change and Global Impression of Symptom Measures 
Patients (≥8 years of age), caregivers, and clinicians will complete the PGIC and the PGIS 
measures (Appendix 3) at randomization (Visit 3; PGIS only), Visits 4 and 6, and at EOT 
(Visit 9). 
The PGIC items assess change in itch (patient version), scratching (caregiver and clinician 
versions), and sleep (all versions) since starting the study drug.  The PGIS items assess itch 
(patient version), scratching (caregiver and clinician versions), and sleep (all versions) in the 
past week.  Caregivers and clinicians will complete the PGIC and PGIS for all patients; those 
patients ≥8 years of age will complete the patient version. 
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10 
ASSESSMENT OF SAFETY 
The timing and frequency of safety assessments are described in Section 7.1.3 and in Table 1 
above. 
The primary safety analysis for this study will include the incidence of total 
treatment-emergent adverse events (TEAE) and TEAEs categorized by causality, severity, 
and seriousness assessments made by the investigator by comparing study drug exposure to 
placebo. 
Trends in safety will also be evaluated for the following assessments: 
• Physical examinations 
• Concomitant medications 
• Vital signs 
• Laboratory test results (including clinical chemistry, hematology, urinalysis, AFP, and 
vitamins A and E, 25-hydroxy vitamin D, and INR) 
• Abdominal ultrasound 
• Discontinuations due to AEs 
10.1 
Adverse Events 
10.1.1 
Definitions and Investigator Assessments 
An AE is defined as any untoward medical occurrence in an enrolled patient regardless of 
causal relationship with study drug.  An AE can therefore be any clinically significant 
unfavorable and unintended sign, symptom, or disease that occurs once a patient is enrolled 
(ICF is signed) in the study until the patient is discharged from the study, whether or not 
related to the study drug. 
10.1.1.1 
Clinical Significance 
Clinical significance is defined as any abnormal finding that results in further clinical 
investigation(s), treatment(s), or the diagnosis of new condition.  Patient-reported events and 
protocol-mandated laboratory values, vital signs, and physical examination findings can be 
considered clinically significant (i.e., an AE) if there is a deterioration as compared to 
baseline.  Examples of clinically significant worsening from baseline could include, but is not 
limited to, events causing withdrawal from the study and events requiring medical 
intervention outside of the study causing apparent clinical manifestations, or judged relevant 
by the investigator.   
10.1.1.2 
Serious Adverse Events 
Serious criteria are applied by the investigator to each AE as specifically defined below.  
These criteria are used to determine whether an AE is serious or non-serious.  The assessment 
is made independently of severity assessment (Section 10.1.1.3).  For example, the 
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development of a severe rash that occurs after signing of informed consent may not meet 
serious criteria as defined below and therefore would be considered a severe, non-serious AE. 
Any AE that meets any 1 of the following 6 criteria is considered an SAE: 
• The outcome of the AE is death 
• The AE is immediately life-threatening.  Life-threatening means that the patient is, in the 
opinion of the investigator, at immediate risk of death from the reaction as it occurred.  
This does not include an AE that, if more severe, might have caused death 
• The AE results in persistent or significant disability/incapacity.  Disability means a 
substantial disruption of a person's ability to conduct normal life functions 
• The AE requires or prolongs hospitalization  
• The AE results in a congenital anomaly/birth defect 
• The AE is an important medical event.  Important medical events may meet serious 
criteria should the investigator assess that they may significantly jeopardize the patient, 
represent a significant hazard, or requires medical/surgical intervention to prevent one of 
the outcomes listed above.  Examples of potential SAEs based on this criteria include, but 
are not limited to, allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions even if they do not result in inpatient 
hospitalization, or the development of drug dependency and drug abuse. 
10.1.1.3 
Severity Assessment 
Severity assessments are based on the intensity of the event in relation to expectation.  The 
investigator will assess the intensity of AEs based on the following definitions: 
• Mild (awareness of sign or symptom, but easily tolerated) 
• Moderate (discomfort sufficient to cause interference with normal activities) 
• Severe (incapacitating, with inability to perform normal activities) 
Severity is a measure of intensity where seriousness is defined by the criteria outlined in 
Section 10.1.1.2.  An AE of severe intensity need not necessarily be considered serious.  For 
example, nausea that persists for several hours may be considered severe nausea, but not meet 
serious criteria, and therefore would be assessed as a severe AE but not an SAE. 
10.1.1.4 
Causality Assessment 
The investigator determines the causality of all AEs to the study drug using medical judgment 
and considering all relevant factors such as (but not limited to) the underlying study 
indication, coexisting disease, concomitant medication, relevant history, pattern of the AE, 
temporal relationship to the study drug, and de-challenge or re-challenge.  The causality 
assessment of the AE/SAE is to be made as follows. 
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Related to study drug (possibly, probably, or definitely related) 
Based on medical judgment, there is at least a reasonable possibility that the study drug 
caused the event; one or more of the following criteria apply: 
• The event follows a reasonable temporal sequence from administration of study drug 
• The event could not be reasonably attributed to the known characteristics of the patient's 
clinical state, environmental or toxic factors, or other modes of therapy administered to 
the patient 
• The event follows a known pattern of response to study drug 
• The event disappears or decreases on cessation or reduction in dose of the study drug.  (It 
should be noted that in some situations an AE will not disappear or decrease in intensity 
upon discontinuation of study drug despite other clear indications of relatedness) 
• The event reappears or worsens when the study drug is re-administered 
Unrelated to study drug (unlikely or unrelated) 
Based on medical judgment there is no reasonable possibility that the study drug caused the 
event; one or more of the following criteria apply: 
• The event does not follow a reasonable temporal sequence from administration of study 
drug 
• The event could be reasonably attributed to the known characteristics of the patient's 
clinical state, concurrent illness, environment or toxic factors, or other modes of therapy 
administered to the patient 
• The event does not follow a known pattern of response to study drug 
• The event does not disappear or decrease on cessation or reduction in dose of the study 
drug, and it does not reappear or worsen when the study drug is re-administered 
10.1.2 
Recording of Adverse Events 
It is the investigator’s responsibility to assess whether each untoward event is a clinically 
significant worsening from baseline, thereby considered an AE.  For all AEs, the severity, 
seriousness, and causality to study drug for each AE as outlined in Section 10.1.1 will be 
assessed and recorded in the eCRF. 
All serious and non-serious AEs are collected once the caregiver/patient has signed the ICF 
and until the post-treatment follow-up (Visit 10) or 28 days after the last dose of study drug. 
Any AEs or SAEs that are unresolved at the patient’s last AE assessment in the study are 
followed up by the investigator until resolution or stabilization up to the database lock and 
recorded in the eCRF.  Albireo retains the right to request additional information for any 
patient with ongoing AE(s) or SAE(s) at the end of the study. 
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TEAEs are defined as any AE that occurs after randomization (Day 0).  All AEs that occur in 
the Screening Period, i.e., after enrollment and prior to randomization, will be collected on 
the eCRF as non-treatment-emergent AEs. 
If there is a clinically significant deterioration of a laboratory value/vital sign or other routine 
study assessment that is associated with a diagnosis, the clinical diagnosis will be reported as 
an AE and the associated signs and symptoms will be considered additional information 
unless the sign or symptom is more severe than expected given the diagnosis.  For example, if 
an investigator diagnoses a study patient with hepatitis during the study period, hepatitis 
would be considered the AE and the concomitant signs and symptoms of abdominal pain, 
vomiting, and elevated ALT and AST would not be reported separately unless, in the opinion 
of the investigator, one of these signs or symptoms is more severe than expected and 
therefore a separate AE assessment is indicated. 
10.1.3 
Recording and Reporting of Serious Adverse Events 
Every SAE (regardless of severity and causality) that occurs once the caregiver/patient has 
signed the ICF and until the post-treatment follow-up (Visit 10), and 28 days after the final 
dose of study drug, should immediately and not later than within 24 hours of knowledge of 
the event, be reported by the investigator or delegate in the SAE report form.    
Report of a SAE must include at least the following information: 
• Patient identification information (study number, site number, initials, and date of birth 
[as per local country requirements for data protection]) 
• The last study drug administration date 
• The diagnosis of the event with the description (or a brief description of 
signs/symptoms/clinical course if the diagnosis is not available) and the date of onset 
• The action used to treat the event (i.e., treatment medications, temporary discontinuation) 
• The reason(s) for considering the event serious 
• The relationship of the event to the study drug or to the study procedure (e.g., the 
investigator's assessment of causality) 
• A brief narrative of the SAE 
Follow-up reports including all new information obtained of the subsequent course of the 
SAE must be prepared and the information collected in the SAE submitted to 
 
 (Pharmacovigilance and Safety Services Department) by e-mail within 24 hours of 
knowledge of the event. 
The pharmacovigilance manager (PVM) may contact the investigator to obtain further 
information on a reported SAE.  The investigator/investigative staff must respond to any 
request for follow-up information or answers to questions regarding the SAE within the same 
timelines as for initial reports. 
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The PVM reports the occurrence of the SAE and follow-up to the Albireo medical monitor 
for medical assessment of the case. 
Should an outcome of death occur within the study period or within 28 days after the last 
administration of study drug, an SAE form should be completed including the main and 
contributory causes of death. 
All SAE reports must be e-mailed to the following e-mail address within 24 hours: 
 
 
If email is unavailable, SAEs may be transmitted via fax to the following number: 
 
In a study-related medical emergency situation, when the assigned medical monitors for the 
study cannot be reached by a caller, an on call physician can be reached 24 hours per day, 
7 days per week, via the 
 call center.  The following number is a chargeable telephone 
number allowing a global reach from both landlines and mobile phones and the internet page 
indicated below contains a list of country-specific toll-free telephone numbers that is 
provided.  It should be noted that not all countries globally have access to toll-free numbers 
as indicated on the “24/7 Medical Help desk” index.  Countries without toll-free numbers 
need to dial the chargeable number as indicated below.  Furthermore, toll-free numbers might 
not be available from mobile phones. 
 
 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions 
Suspected unexpected serious adverse reaction (SUSAR) is an SAE that occurs in a patient, 
the nature or severity of which is not expected per the applicable product information (e.g., 
the IB for an unauthorized investigational product or summary of product characteristics for 
an authorized product).  
Reporting and tracking of SUSARs will be in accordance to all applicable competent 
authority regulations.  The Ethics Committee and all investigators involved in this study will 
be informed according to national and international requirements. 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments 
10.2.1 
Laboratory Assessments 
Samples will be collected for clinical chemistry, hematology, and urinalysis testing at the 
time points specified in Table 1. The parameters assessed are presented in Table 3. 
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Blood for AFP will be drawn at Visits 3 and 9.  Fat-soluble vitamin levels including vitamins 
A and E, and 25-hydroxy vitamin D will be drawn at Visit 2 (vitamins A & E only), 
Visit 3 (25-hydroxy vitamin D only), Visit 6, and Visit 9.  Blood for INR (surrogate for 
vitamin K) will be drawn at Visits 2, 4, 6, 8, and 10.  Patients will fast (water intake only is 
permissible) for at least 4 hours prior to the collection of samples for vitamin A.  Exceptions 
can be made for infants, <12 months of age, if unable to fast for the full 4 hours.  If a patient 
has any vitamin level(s) that are outside of the reference range, vitamin supplementation 
adjustments may be required.  Target ranges and supplementation strategy guidelines can be 
found in Appendix 5. 
A serum pregnancy test will be performed at Visit 1 and Visit 9 if the patient is entering the 
A4250-008 study. A urine pregnancy test will be collected at all other visits for girls who 
have reached menarche.  If a urine pregnancy test is positive, a serum pregnancy test should 
be performed to confirm the pregnancy.  Study drug administration will be held pending the 
confirmatory results.  If the serum pregnancy test is negative, the patient can resume dosing.  
If the serum pregnancy test is positive, the patient should be discontinued (see 
Section 10.2.9). 
All samples will be processed and transported to a laboratory per instructions in the 
laboratory manual. 
The observed values will be recorded and assessed as “normal” or “abnormal not clinically 
significant” or “abnormal clinically significant”. 
Additional safety blood samples may be needed due to follow-up of an abnormal value or 
analysis failure.  The blood samples collected for safety laboratory analysis will be destroyed 
after the analyses have been completed.  
Table 3 
Routine Laboratory Parameters 
Clinical Chemistry 
Hematology 
Urinalysis 
• 
Albumin 
• 
ALT 
• 
Alkaline phosphatase  
• 
AST 
• 
Bilirubin – total and direct 
• 
Calcium 
• 
Chloride 
• 
Creatinine 
• 
Creatine kinase 
• 
Gamma-glutamyl transferase 
• 
Potassium 
• 
Sodium 
• 
Hematocrit 
• 
Hemoglobin 
• 
Platelet count 
• 
Red blood cell count 
• 
White blood cell count and 
differential (neutrophils, 
eosinophils, basophils, 
lymphocytes, and monocytes) 
• 
Blood 
• 
Glucose 
• 
Ketones 
• 
Leukocytes 
• 
Nitrites 
• 
pH 
• 
Protein 
 
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10.2.2 
Individual Patient Safety Monitoring 
10.2.2.1 
Liver Monitoring 
Strategies to monitor markers of liver disease throughout the study are outlined below where 
the ULN will be based on central laboratory reference values for age and gender. 
If isolated transaminase elevations are observed, defined as: 
1. Normal bilirubin AND absence of clinical hepatitis symptoms AND 
• ALT or AST ≥5 × ULN (if normal at baseline) or an absolute threshold of 800 U/L, 
whichever comes first  
• OR ALT or AST ≥3 × baseline (if abnormal at baseline) or an absolute threshold of 
800 U/L, whichever comes first 
Then: 
a) Repeat liver profile (AST, ALT, bilirubin, and prothrombin time [PT] or INR) within 
2 to 3 days 
b) Evaluate creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) 
c) As needed (for example, persistent ALT/total bilirubin elevation or worsening of liver 
function), consider evaluation for alternative etiologies 
Monitor the patient using close observation found in Section 10.2.2.2 
If any ONE of the following criteria are met: 
1. Transaminases (ALT or AST ≥3 × baseline or 800 U/L, whichever comes first) AND 
bilirubin (total bilirubin > 2 × ULN) elevations 
2. Transaminase elevations alone (ALT or AST >10 × ULN or 5 × baseline or absolute 
threshold of 800 U/L, whichever comes first) in presence of normal LDH and CPK 
3. Total bilirubin increased, unrelated to hemolysis (elevated reticulocyte count) or 
established genetic diseases, such as Gilbert’s syndrome 
a. Doubling if total bilirubin was <3 mg/dL (equivalent to 51.3 µmol/L) at baseline  
b. OR Increase by >3 mg/dL (equivalent to 51.3 µmol/L) if total bilirubin was ≥3 mg/dL 
(equivalent to 51.3 µmol/L) at baseline 
4. INR increase refractory to vitamin K administration 
a. INR >1.5 if INR was normal at baseline  
b. OR Increase by >0.4 if INR was abnormal at baseline 
5. Any increase in total bilirubin and transaminases if accompanied by EITHER a symptom 
of clinical hepatitis (e.g., vomiting, nausea, right upper quadrant pain) OR immunological 
reaction (rash or >5% eosinophilia) 
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Then: 
a) Interrupt study medication 
b) Initiate drug-induced liver injury work-up for alternative etiologies 
c) Repeat liver profile (AST, ALT, total bilirubin, direct bilirubin) and PT or INR within 
48 to 72 hours 
d) Monitor the patient using close observation found in Section 10.2.2.2 
e) If a patient lives in a remote area, they may be tested locally and the results 
communicated to the investigator site promptly 
10.2.2.2 
Close Observation 
• Repeat liver enzyme and serum bilirubin tests two or three times weekly.  Frequency 
of re-testing can decrease to once a week or less if abnormalities stabilize or the trial 
drug has been discontinued and the subject is asymptomatic  
• Obtain a more detailed history of symptoms and prior or concurrent diseases 
• Obtain a history of concomitant drug use (including non-prescription medications and 
herbal and dietary supplement preparations), alcohol use, recreational drug use, and 
special diets  
• Consider ruling out alternate etiology including acute viral hepatitis types A, B, C, D, 
and 
E; 
autoimmune 
or 
alcoholic 
hepatitis; 
non-alcoholic 
steatohepatitis; 
hypoxic/ischemic hepatopathy; and biliary tract disease 
• Obtain a history of exposure to environmental chemical agents.  
• Obtain additional tests to evaluate liver function, as appropriate (e.g., INR, direct 
bilirubin) 
• Consider gastroenterology or hepatology consultations. 
10.2.2.3 
De-challenge/Re-challenge for Liver and Clinical Hepatitis Monitoring 
1. Re-challenge is not recommended: 
a. If a patient has had possible/probable drug-induced liver injury 
b. If a decompensation event has occurred (i.e., variceal hemorrhage, ascites, hepatic 
encephalopathy, etc.). 
2. If due to underlying cholestatic liver disease variability or another alternative etiology is 
identified AND liver tests returned to baseline, re-challenge may be considered after 
consultation with the sponsor Medical Monitor. 
3. If the ALT/total bilirubin elevations are observed after re-challenge, then repeat 
re-challenge is discouraged. 
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If patient is permanently discontinued, monitoring should be continued as outlined in 
Section 10.2.2.5.  
10.2.2.4 
Diarrhea 
Study drug should be discontinued if a patient develops diarrhea with at least 1 of the 
following concomitant signs or symptoms: grossly bloody stools, vomiting, dehydration 
requiring treatment with oral or intravenous rehydration and/or electrolyte imbalances, fever 
(≥38 C) and/or the diarrhea persists for 7 or more days. 
If there is gross blood in stool, INR and platelets should be measured.  
Study drug will be reintroduced (re-challenge) when the symptoms have resolved.  If the 
diarrhea re-occurs within 1 week after the re-challenge with no alternate etiology, the patient 
will be permanently discontinued and monitored as outlined in Section 10.2.2.5. 
10.2.2.5 
Monitoring after Permanent Discontinuation of Study Drug Due to Safety 
Once study drug is permanently discontinued for liver, hepatitis, diarrhea, or other severe AE 
related to study drug, the patient will be monitored weekly until the laboratory and clinical 
parameters have normalized/stabilized. 
10.2.3 
Demographics/Medical and Surgical History 
Demographic information per country regulations (age, full date of birth, gender, race, and 
ethnicity), along with medical and surgical history, will be obtained and recorded in the eCRF 
at Visit 1. 
Medical and surgical history will be entered in the eCRF at Visit 1.  This includes date of 
diagnosis of PFIC, prior investigational medications for PFIC, historical (3 months prior to 
Screening) LFT values (AST, ALT, and total bilirubin), ongoing medication, any surgery 
performed, any other diagnosis, and historical biopsy data.   
If a liver biopsy has been performed within 1 year prior to Screening, or during the study, the 
results will be recorded in the eCRF. 
10.2.4 
Clinical Genetic Testing 
For previous confirmatory clinical genetic testing results for PFIC Type 1 or 2 performed 
prior to Screening Visit 1, the confirmatory clinical genetic testing laboratory report will be 
verified to determine eligibility. 
If the historical clinical genetic result is equivocal, unavailable, or unobtainable, clinical 
genetic analysis to confirm pathologic biallelic variations in the ATP8B1 or ABCB11 genes 
will be performed and verified to determine eligibility.  No other diagnostic genetic testing 
will be offered. 
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10.2.5 
Physical Examination 
A physician or suitably trained qualified assistant will perform a complete physical 
examination, including voluntary photography of skin lesions due to scratching, at Visits 1, 3, 
6, and 9.  A skin examination will be conducted at Visits 1, 3, 4, 6, 9, and 10 (see Table 1).  
A complete physical examination will include assessment of general appearance, eyes, ears, 
nose, throat, head/neck/thyroid, lymph nodes, cardiovascular, lungs/chest, abdomen, 
genitourinary, extremities, skin, musculoskeletal, neurologic, and other.  
Skin will be examined thoroughly and excoriations/scratch marks recorded.  
10.2.6 
Vital Signs 
Evaluation of vital signs will be performed at all visits.  This includes blood pressure (systolic 
and diastolic), pulse, respiratory rate, temperature, height (or length depending on age), and 
body weight (using a certified weight scale) at clinic visits.  BMI will be calculated. 
10.2.7 
Abdominal Ultrasound 
Ultrasound of the liver and spleen will be performed at randomization (Visit 3) and at EOT 
(Visit 9).  Liver size, echogenicity, and presence of masses/nodules as well as spleen size will 
be recorded. 
10.2.8 
Overdose 
A4250 is minimally absorbed and has a very low systemic availability.  Based on toxicology 
data, for study purposes any dose exceeding a total of >3 mg/kg body weight of A4250 taken 
as a single dose or as a cumulative dose within 24 hours is defined as an overdose.   
The no-adverse-effect dose level in the most sensitive species (defined as 20 mg/kg/day) in 
the rat 1 month toxicity study gives a human equivalent dose of approximately 194 mg/day in 
a 60 kg person.  This dose is approximately 10-fold higher than the maximum possible dose 
predicted in human studies (20 mg).  
The investigator/investigative staff should immediately and not later than within 24 hours of 
knowledge of the overdose, be reported by the investigator or delegate on the SAE report 
form.  In the event of an A4250 overdose, the patient should be monitored closely. 
10.2.9 
Pregnancy 
If a pregnancy is discovered in a female patient enrolled in the study before the end of dosing, 
the patient will be immediately discontinued from the study and will attend the same visits as 
a prematurely withdrawn patient.  If the pregnancy is discovered in a female patient enrolled 
in the study after the end of dosing, the patient will continue in the study per protocol.  If a 
pregnancy occurs in a male patient’s partner at any time during the study, the pregnancy 
should also be reported and followed. 
Pregnancy is not considered to be an AE.  However, if the patient has been dosed with the 
study drug, the pregnancy must be reported on the Paper Pregnancy Notification Form 
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immediately within 24 hours after recognition to Albireo/Safety Manager (please refer to 
Section 10.1.3).  Date of exposure and as far as possible, details of the period of gestation at 
the time of exposure must be given. 
The pregnancy should be followed to determine outcome, including spontaneous termination, 
details of birth, and presence of any birth defects, congenital anomalies or newborn or 
maternal complication.  The infant who was exposed in-utero will be followed for up to 
2 years after delivery.  Individual cases with an abnormal outcome in association with the 
study drug should be reported on an expedited basis, i.e., reported rapidly to a competent 
authority. 
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11 
STATISTICAL EVALUATION 
All personnel involved with the analysis of the study will remain blinded until database lock 
for the double-blind Treatment Period and until major protocol violations have been 
identified. 
All statistical analyses will be performed using SAS version 9.3 or higher. 
11.1 
Sample Size and Power 
For the EU and RoW, the primary analysis will relate to the bile acid responder endpoint (at 
the 1-sided 1.25% level).  For the US, the primary analysis will relate to the pruritus endpoint 
(at the 1-sided 1.25% level).  The aim is to enroll 60 to 70 patients in order to get at least 
60 evaluable patients. 
Assuming 60% responders (patients experiencing at least 70% reduction in fasting s-BA 
concentration or reaching ≤70 µmol/L) in the A4250 dose group and 10% responders in the 
placebo group, approximately 20 patients per group are required to achieve a power of 84% 
with a 1.25% 1-sided Type-1 error. 
For pruritus, with approximately 20 patients per treatment group, the power is 83% given an 
assumed treatment effect difference of -1.0 in change from baseline (-1.6 for A4250 
and -0.6 for placebo) on the Albireo ObsRO scratching score with a SD of 0.95 and a Type I 
error of 1.25% (1-sided). 
11.2 
Statistical Methods 
11.2.1 
Statistical Analysis Sets 
Safety Analysis Set 
The safety analysis set will consist of all patients who received at least 1 dose of study drug.  
Patients will be analyzed according to the treatment they actually received.  The safety 
analysis set will be the primary analysis set for safety analyses.  
Full Analysis Set 
The full analysis set (FAS) will consist of all randomized patients who received at least 
1 dose of assigned treatment.  Patients will be analyzed in the treatment group they were 
randomized to even if they received incorrect study drug.  The FAS will be the primary 
analysis set for efficacy analyses. 
Per Protocol Analysis Set 
The per protocol (PP) analysis set is a subset of the FAS, and will consist of all randomized 
patients for whom no major protocol violation (no violation which may affect the study 
efficacy outcome) is documented.  The PP analysis set will provide supportive data for the 
efficacy analyses. 
Allocation of patients to the PP analysis set will be performed before unblinding of the study. 
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11.2.2 
Methods of Statistical Analyses 
11.2.2.1 
General Principles 
The primary objective of this study is to show superiority in efficacy of 
A4250 (120 µg/kg/day and 40 µg/kg/day) compared to placebo in patients with PFIC Types 1 
and 2. 
In the EU and RoW, for the primary efficacy variable fasting bile acid responder endpoint (at 
least a 70% reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the end 
of treatment), the null hypothesis will be tested against the alternative hypothesis (α = 0.0125, 
1-sided). 
The test will be performed at a Type I error rate of ≤0.0125 1-sided, as described above, 
based on the least squares means of the treatment difference in mean probability scale. 
To strictly control overall Type I error rate across the dose groups and also across the primary 
and the main secondary endpoint to a 1-sided 2.5% level, a Bonferroni-based parallel 
gate-keeping procedure will be used.  In this procedure the total significance level of 2.5% 
(1-sided) will be split equally between the dose groups.  Only a dose group that is significant 
for the primary endpoint will be tested, at the nominal significance level of 1.25% (1-sided), 
for the main secondary endpoint (here the pruritus endpoint).  If the previous step was not 
successful, the analysis of the following step will be considered descriptive.  If the procedure 
reaches the secondary endpoint for both dose groups, these will be evaluated using the 
Bonferroni Holm procedure.  The multiple comparison procedure is presented in Figure 2. 
Figure 2 
Bonferroni Holm Multiple Comparison Procedure 
 
In the US, for the primary efficacy variable pruritus score endpoint (change from baseline 
over the Treatment Period in observer-reported scratching), the null hypothesis will be tested 
against the alternative hypothesis (α=0.0125, 1-sided). 
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The test will be performed at a Type I error rate of ≤0.0125 1-sided, as described above, 
based on the least squares means of the treatment difference in mean change from week 7 to 
week 24 (i.e., the average of five 14 day post-treatment intervals). 
To strictly control overall Type I error rate across the dose groups and also across the primary 
and the main secondary endpoint to a one-sided 2.5% level, a Bonferroni-based parallel 
gate-keeping procedure will be used.  In this procedure the total significance level of 2.5% 
(1-sided) will be split equally between the dose groups.  Only a dose group that is significant 
for the primary endpoint will be tested, at the nominal significance level of 1.25% (1-sided), 
for the main secondary endpoint (here the bile acid responder endpoint).  If the previous step 
was not successful, the analysis of the following step will be considered descriptive.  If the 
procedure reaches the secondary endpoint for both dose groups, these will be evaluated using 
the Bonferroni Holm procedure. 
Other secondary and exploratory outcome variables shall provide supportive efficacy and 
safety information regarding the differences between the treatment groups.  No adjustments 
will be performed for multiple dose comparisons when testing the other secondary and 
exploratory efficacy variables. 
A more detailed statistical analysis plan (SAP) will be prepared and finalized before study 
data are locked and unblinded.  
If not otherwise specified, the baseline value of a parameter is defined as the last assessment 
of that parameter prior to the first intake of study treatment. 
11.2.2.2 
Missing Data 
For efficacy variables, with repeated measures, a mixed model will be employed with no 
additional imputation for missing values, i.e., based on “observed cases”, except where 
otherwise specified.  All patients with at least one measurement of change from baseline will 
be included and contribute to the analysis.  The mixed model will handle the missing data, 
but assumes Missing At Random. 
Analyses to investigate the influence of missing values, for example, by using pattern mixture 
models using multiple imputations and tipping point analysis will be made and detailed in the 
SAP. The sensitivity analyses addressing the robustness of results to missing data, will 
consider different approaches imputing data based on Missing Not At Random. Any 
additional sensitivity analysis that may be done using other data imputation will also be 
described in the SAP. 
Continued collection of efficacy data for patients who discontinue treatment will be made as 
far as possible. 
11.2.2.3 
Demographic and Baseline Characteristics 
Descriptive summaries of demographics and other baseline characteristics (including medical 
and surgical history) will be presented by treatment group and overall using the FAS.  
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Prior medication will be summarized by treatment group and overall using the safety analysis 
set. 
11.2.2.4 
Subject Disposition 
The following will be summarized (by treatment group and overall where applicable): 
• Patients enrolled (who signed the informed consent) 
• Patients randomized 
• Patients treated 
• Patients completing the study/withdrawing early (including withdrawal reason) 
Additionally, patients enrolled, included in the safety, in the FAS, and in the PP analysis set 
will be summarized by region. 
11.2.2.5 
Evaluation of Primary Efficacy Variables 
The responder endpoint based on change in fasting bile acids will be analyzed in terms of the 
proportion of patients achieving the event of interest.  Patients in need of rescue therapy, i.e., 
biliary diversion will be classified as non-responders after the biliary diversion.  
Generalized linear mixed modelling (GLMM) will be used with a logistic link, to allow for 
repeated assessments over time.  The model will include baseline value, treatment group, 
PFIC class, age class, region, visit, and visit*treatment group interaction.  The treatment by 
visit interaction will remain in the model regardless of significance.  Region will be included 
in the model as a random effect.  The contrasts of primary interest will be the treatment 
differences at Week 24; also, the difference at Week 4, Week 8, Week 12, and Week 18 will 
be estimated and analyzed.  An unstructured covariance matrix will be used to model the 
within-subject error.  Another covariance structure may be chosen if the fit of this structure 
fails to converge.  Model diagnostics checks will be made to ensure appropriate fit. 
The primary analysis of the change from baseline in pruritus score (indexed by 
observer-reported scratching) will be analyzed using a mixed model repeated measures 
(MMRM) analysis.  The comparison of primary interest will be treatment differences over the 
last 5 months of the Treatment Period estimated by averaging the worst daily itch score 
means from five 14 day post-treatment intervals (Weeks 7 to 8, Weeks 11 to 12, Weeks 15 to 
16, Weeks 19 to 20, and Weeks 23 to 24).  The treatment difference at each of these 
post-treatment time intervals will also be analyzed.  Baseline pruritus score will be included 
as covariate.  The model will include baseline value, treatment group, PFIC class, age class, 
region, time, and time*treatment group interaction.  The treatment by time interaction will 
remain in the model regardless of significance.  There will be six time points defined by the 
last 14 days interval each month (i.e., Weeks 3 to 4, Weeks 7 to 8, Weeks 11 to 12, Weeks 15 
to 16, Weeks 19 to 20, and Weeks 23 to 24), but the first of the six time points (Weeks 3 to 4) 
will not be part of the primary efficacy analysis.  Region will be included in the model as a 
random effect and will be defined on country level.  An unstructured covariance matrix will 
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be used to model the within-subject error.  Another covariance structure may be chosen if the 
fit of this structure fails to converge. 
Non-parametric approach may be considered if more appropriate. 
The primary analyses will be based on the FAS. 
To assess the robustness of the primary efficacy analysis, the following analyses will be 
performed: 
• Primary efficacy analysis using the PP set  
• Analyses to investigate the influence of missing values, for example, by using pattern 
mixture models and tipping point analysis 
Subgroup analyses will also be performed for each of the 3 age groups (6 months to 5 years, 
6 to 12 years, and 13 to ≤18 years) and for patients who were not included in the previous 
Study A4250-003. 
Several sensitivity analyses will be performed, such as the following: 
• The change from baseline in pruritus morning diary scores and evening diary scores, 
respectively, will also be analyzed using the MMRM approach. 
• Cochran Mantel Haenzsel test stratified on PFIC class to compare the proportion in 
fasting bile acid responder endpoint at the end of treatment in the two A4250 dose groups 
to placebo group.  Dropouts will be treated as non-responders 
• Based on the primary analysis including the interaction age class*treatment 
• Same as primary analysis in which the random factor region is considered as fixed factor.  
The region*treatment interaction will also be explored 
• Same as primary analysis including the interaction PFIC*treatment  
Other sensitivity analyses may be performed and will be detailed in the SAP. 
11.2.2.6 
Evaluation of Secondary and Exploratory Efficacy Variables 
The change in the secondary and explorative endpoints such as s-BA, total bilirubin, ALT, 
growth and patient-reported itch severity will also be analyzed using the MMRM model as 
specified for the primary analysis.  Nonparametric methods may be applied as an alternative 
approach for some endpoints, if appropriate. Change in growth (weight for age and BMI for 
age) will also be displayed using graphical presentations. 
Proportions of responders (e.g., meaningful reduction in itch severity and scratching at 
Week 24) will be analyzed using the GLMM model as specified for the primary analysis. 
Comparisons of the change from baseline at Week 24 in PedsQL between the treatment 
groups will be conducted using analysis of covariance.  The model will include terms for 
baseline, PFIC class, age class, region, and treatment.  
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Undergoing biliary diversion surgery and/or listed for liver transplant, all-cause mortality, 
and liver decompensation events will mainly be analyzed by using descriptive statistics.  
Kaplan-Meier curves will be used when appropriate for time to event data. 
Other exploratory variables (Global Symptom Relief, biochemical markers and measures of 
bile acid synthesis, Albireo PRO and ObsRO sleep parameters, PELD/MELD score, APRI 
score, and Fib-4 score) will be analyzed descriptively for categorical and continuous data, as 
applicable.  For continuous data, the change from baseline will be analyzed in addition to the 
actual visit values.  For categorical data, shift tables or frequency and percentages of patients 
will be presented as appropriate. 
In addition, subgroup analyses will be performed for each of the 3 age classes (6 months to 
5 years, 6 to 12 years, and 13 to ≤18 years). 
11.2.2.7 
Evaluation of Safety Variables 
Safety data will be analyzed using descriptive statistics and summaries by treatment group of 
SAEs, AEs, vital signs, clinical laboratory tests (hematology, clinical chemistry and 
urinalysis), and concomitant medication.  Analyses will be performed using the safety 
analysis set.  
Summaries of AEs (coded according to the Medical Dictionary for Regulatory Activities 
[MedDRA] system organ class [SOC] and MedDRA preferred term) will include the 
following:  
• Overview of the incidence of TEAEs (TEAEs, drug-related TEAEs, TEAEs leading to 
study discontinuation, and treatment-emergent SAEs) 
• TEAEs by SOC and preferred term 
• Intensity of TEAEs by SOC and preferred term 
• Drug-related TEAEs by SOC and preferred term 
• TEAEs leading to study discontinuation by SOC and preferred term 
• Treatment-emergent SAEs by SOC and preferred term 
Concomitant medication use during the Treatment Period will be summarized by Anatomical 
Therapeutic Chemical (ATC) class and World Health Organization (WHO) preferred name.  
Summaries of vital signs will be presented.  For each visit, the actual results and the change 
from baseline, and the number and percentage of patients with potentially clinically 
significant (PCS) values observed post-baseline will be presented. 
Summaries of clinical safety laboratory data will be presented.  For each visit, the actual 
result and the change from baseline, and the number and percentage of patients with PCS 
values observed post-baseline will be presented. 
Data listings will be provided for each patient for all safety parameters. 
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11.2.2.8 
Compliance and Exposure 
Exposure will be analyzed by calculating the number of days with exposure to study drug.  
Results will be presented by treatment group using the safety analysis set.  
The percentage compliance will be described by treatment group and the number of patients 
with a compliance <80%, between 80% and 120%, and >120% will be presented based on the 
safety analysis set. 
11.2.3 
Blinded Analyses to Confirm Responder Definitions 
A blinded analysis of Albireo ObsRO and PRO eDiary data will be performed after 50% or 
more of the planned sample has completed the Week 24 Follow-Up Period.  The blinded 
analysis will be used to estimate a threshold of clinically meaningful change (i.e., responder 
definition) in Albireo ObsRO and PRO pruritus scores.  The analysis will be performed by a 
group independent from both the study team and Albireo.  Blinded data will be used for this 
analysis; data will be collapsed across treatment groups. 
The analyses will include distribution- and anchor-based analyses to examine a responder 
definition for the Albireo PRO/ObsRO pruritus scores.  Distribution-based analyses will 
include calculation of the SD and standard error of measurement for the baseline Albireo 
PRO and ObsRO scores.  Anchor-based analyses will involve examining the degree of 
change on the Albireo PRO and ObsRO scores from baseline to Week 24 for patients who 
experience change in pruritus according to PGIC and symptom items.  Details of this analysis 
will be included in a stand-alone SAP for assessing the measurement characteristics 
(reliability, validity, sensitivity to change, responder definition) of the Albireo PRO and 
ObsRO items. 
11.2.4 
Data Safety Monitoring Board 
A DSMB consisting of sponsor-independent clinical and statistical experts will be 
established.  The DSMB will periodically meet for the review of accumulating study data, 
including safety (AE and laboratory data) and liver values.  
The DSMB will have access to unblinded data.  The DSMB will make recommendations for 
the remaining part of the study (further details will be provided in the DSMB charter).  The 
DSMB may recommend continuing with the study as planned or stopping the study early for 
safety reasons.  The DSMB will submit its recommendations in writing to Albireo 
Medical/
 who are responsible for responding to the recommendations of the DSMB and 
taking appropriate action.  The investigators will only be informed by Albireo Medical/
 
if the study requires a protocol amendment or is stopped.  The DSMB may choose to make 
additional evaluations at any time if they feel this is warranted from a safety point of view.  
The DSMB will act according to its own written standard operating procedure described in a 
charter and will prepare written minutes of its meetings.  The charter of the DSMB will be 
stored in the trial master file.  The DSMB will maintain records of its meetings and these will 
become part of the study file when the study is complete.  In order not to disseminate 
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unblinded data and to ensure that all staff involved in the conduct and final analysis of the 
study remain blind to the data, only the members of the DSMB and the unblinded DSMB 
statistician will have access to these data. 
 
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12 
DIRECT ACCESS TO SOURCE DATA/NOTES 
The investigator/institution shall provide direct access to source data/documents for 
study-related monitoring, audits, IEC/Institutional Review Board (IRB) review and 
regulatory inspection. 
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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
13.1 
Conduct of the Study 
Albireo/
 shall implement and maintain quality control and quality assurance procedures 
with written standard operating procedures to ensure that the study is conducted and data are 
generated, documented, and reported in compliance with the protocol, ICH GCP and 
applicable regulatory requirements. 
This study shall be conducted in accordance with the provisions of the Declaration of 
Helsinki (October 1996) and all revisions thereof, and in accordance with United States Food 
and Drug Administration (FDA) regulations (Code of Federal Regulations, Sections 312.50 
and 312.56) and with ICH GCP (CPMP 135/95). 
The investigator may not deviate from the protocol without a formal protocol amendment 
having been established and approved by an appropriate competent authority and IEC/IRB, 
except when necessary to eliminate immediate hazards to the patient or when the change(s) 
involve(s) only logistical or administrative aspects of the study.  Any deviations may result in 
the patient having to be withdrawn from the study and render that patient non-evaluable. 
13.2 
Study Monitoring 
Before study initiation, at a site initiation visit or at an investigator’s meeting, an Albireo 
representative or designee will review the protocol and eCRF with the investigators and the 
investigative staff.  During the study, the clinical monitor (clinical research associate [CRA]) 
will visit the site regularly to check the completeness of patient records, the accuracy of 
entries on the eCRFs, the adherence to the protocol and to GCP, the progress of enrollment, 
and to ensure that study drug is being stored, dispensed, and accounted for according to 
specifications. 
The investigator must ensure that eCRFs are completed within a timely period of patient 
visits, as per individual site agreements, and must allow the CRA and Albireo representative 
or designee periodic access to patient records and all study-related materials, including 
relevant hospital or clinical records, to confirm their consistency with the eCRF entries.  No 
information in these records about the identity of the patients will leave the study center.  
Albireo monitoring standards require full verification for the presence of the signed ICF, 
adherence to the inclusion/exclusion criteria, documentation of SAEs, and recording of 
primary efficacy and safety variables.  The CRA will review source data compared with the 
eCRFs and will verify source data according to the study-specific monitoring plan.  The 
design of the study, the frequency of patient visits, and the site enrollment rate will determine 
the frequency of monitoring visits.  Upon study completion, the CRA will visit the site to 
conduct a study termination visit, which will include collection of any outstanding 
documentation. 
It is recommended that the investigator/investigative staff log into the eCRF system every 
working day or at minimum twice weekly in order to provide a prompt response to queries.  
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The investigator/investigative staff should respond to queries and make any relevant changes 
to the study data within 3 working days. 
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14 
ETHICS 
14.1 
Independent Ethics Committee/Institutional Review Board 
Prior to the start of the study, the investigator is responsible for ensuring that the protocol and 
consent form have been reviewed and approved by a relevant IEC/IRB.  The IEC/IRB shall 
be appropriately constituted and perform its functions in accordance with FDA ICH GCP and 
local requirements as applicable. 
The IEC/IRB shall approve all protocol amendments (except for logistical or administrative 
changes), written informed consent documents and document updates, patient recruitment 
procedures (e.g., advertisements), written information to be provided to the patients and 
caregivers, IB, available safety information, information about payment and compensation 
available to patients, the investigator's curriculum vitae and/or other evidence of 
qualifications and any other documents requested by the IEC/IRB and regulatory authority 
(competent authority) as applicable. 
14.2 
Written Informed Consents and Assents 
The investigator (physician) or investigative staff, in accordance with local regulations, will 
explain to each patient (or legally authorized representative) the nature of the study, its 
purpose, the procedures involved, the expected duration, alternative treatment, the potential 
risks and benefits involved, and any discomfort that may occur.  Each caregiver/patient will 
be informed that participation in the study is voluntary and that he/she or their child may 
withdraw from the study at any time and that withdrawal of consent will not affect his/her 
subsequent medical treatment or relationship with the treating physician. 
Caregivers/patients will be informed that they/their children may experience side-effects or 
be at risk for symptoms, illnesses, or complications that cannot be foreseen by Albireo.  As 
with other medications, people treated with A4250 may be at risk of developing allergic 
reactions or anaphylaxis.  Caregivers/patients will be advised that study procedures include 
regular blood sampling for measurement of safety parameters and biological markers and that 
some minor risks are associated with these procedures. 
This informed consent should be given by means of a signed ICF, written in non-technical 
language in accordance with ICH GCP, the Declaration of Helsinki, and regulatory 
authorities.  The caregiver(s)/patient should read and consider the statements before signing 
and dating them and should be given a copy of each signed document.  If written consent is 
not possible, oral consent can be obtained if witnessed and followed by a signed statement 
from one or more persons not involved in the study, indicating why the patient was unable to 
sign the form.  No patient can enter the study before his/her or caregiver(s) informed consent 
has been obtained, as required by country regulations. 
The ICF is part of the protocol and must be submitted by the investigator/investigative staff 
with the protocol for IEC approval.  Albireo will supply an ICF which complies with 
regulatory requirements and country laws, and is considered appropriate for the study.  Any 
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changes to the ICF suggested by the investigator must be agreed to by Albireo before 
submission to the IEC and a copy of the approved version must be provided to the clinical 
monitor after IEC approval. 
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15 
DATA HANDLING AND RECORD KEEPING 
15.1 
Case Report Forms/Source Data Handling 
The investigator shall be provided with standardized eCRFs and shall ensure that all data 
from patient visits are promptly entered into the eCRFs in accordance with the specific 
instructions given.  The investigator must sign each eCRF to verify the integrity of the data 
recorded. 
A list of the normal ranges for all laboratory tests to be undertaken forms part of the 
documentation to be collated prior to study start.  As a central laboratory has been selected to 
conduct any or all tests, it is essential that all samples be analyzed at that laboratory. 
The investigator must maintain source documents such as laboratory reports, consultation 
reports, and complete medical history and physical examination reports. 
As no ethnicity data can be collected in the source document for study sites in France, the 
eCRF will be the Source Document for “origines ethniques”,  
15.2 
Retention of Essential Documents 
Essential documents, as defined by ICH GCP, include: the signed protocol and any 
amendment(s); copies of the completed eCRFs (for site archiving, compact discs of eCRF 
data for specific patients will be provided); signed ICFs; hospital records and other source 
documents; IEC approvals and all related correspondence including approved documents; 
drug accountability records; study correspondence; and a list of patients’ names and 
addresses. 
The investigator/investigative staff must retain copies of these essential documents for the 
period specified by ICH GCP and by applicable regulatory requirements.  The 
investigator/investigative staff will inform Albireo of the location where the essential 
documents are stored and must contact Albireo for approval before disposing of any essential 
documents.  The investigator/investigative staff should take measures to prevent accidental or 
premature destruction of these documents. 
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16 
FINANCING AND INSURANCE 
Financing and insurance are addressed in a separate agreement. 
 
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17 
PUBLICATION POLICY 
Albireo will retain the ownership of all data.  When the study is complete, Albireo shall 
arrange the analysis, tabulation of data and preparation of a clinical study report. Albireo may 
also use the data for publication, presentation at scientific meetings and submission to 
regulatory authorities.  All proposed publications based on this study must be subject to the 
sponsor's approval requirements. 
 
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18 
REFERENCE LIST 
1. A4250 Investigator’s Brochure. 
2. Alissa FT, Jaffe R, Shneider BL. Update on progressive familial intrahepatic cholestasis. 
J Pediatr Gastroenterol Nutr. 2008;46(3):241-52. 
3. Davit-Spraul A, Gonzales E, Baussan C, et al. Progressive familial intrahepatic 
cholestasis. Orphanet J Rare Disease 2009;4:1-12. 
4. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and 
functions. Front Biosci (Landmark Ed). 2009 Jan 1;14:2584-98. 
5. Hori T, Justin H, Nguyen JH and Uemoto S. Progressive familial intrahepatic cholestasis. 
Hepatobiliary pancreat Dis Int. 2010; 9(6):570-8. 
6. Jacquemin E. Progressive familial intrahepatic cholestasis. Genetic basis and treatment. 
Clin Liver Dis. 2000;4(4):753-63. 
7. Miethke AG, Zhang W, Simmons J, et al. Pharmacological inhibition of ASBT changes 
bile composition and blocks progression of sclerosing cholangitis in mdr2 knockout mice. 
Hepatology. 2016; 63(2):512–23. 
8. Murray CS, Rees JL. Are subjective accounts of itch to be relied on? The lack of relation 
between visual analogue itch scores and actigraphic measures of scratch. Acta Derm 
Venereol. 2011;91(1):18-23. 
9. Pawlikowska L, Strautnieks S, Jankowska I, et al. Differences in presentation and 
progression between severe FIC1 and BSEP deficiencies. J Hepatol. 2010;53(1):170-8. 
10. Shneider BL, Magee JC, Bezerra JA, et al. Efficacy of fat-soluble vitamin 
supplementation in infants with biliary atresia. Pediatrics, 2012;130(3):e607-14.  
11. Suchy FJ, Sokol RJ, Balistreri WF, eds. Liver Disease in Children. 3rd Edition. New 
York, NY: Cambridge University Press; 2007:310–4. 
12. Venkat VL, Shneider BL, Magee JC, et al. Total Serum Bilirubin Predicts Fat-Soluble 
Vitamin Deficiency Better Than Serum Bile Acids in Infants with Biliary Atresia. J 
Pediatr Gastroenterol Nutr. 2014;59(6):702-7.  
13. Whitington PF, Whitington GL. Partial external diversion of bile for the treatment of 
intractable 
pruritus 
associated 
with 
intrahepatic 
cholestasis. 
Gastroenterology. 
1988;95(1):130-6. 
 
 
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M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 02, 10 May 2018 
 
19 
APPENDICES 
Appendix 1 
Concomitant Medication Guidelines 
 
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M5.3.5.1 – Protocol A4250-005 
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Final Amendment 02, 10 May 2018 
Appendix 2 
Diary Questions including Albireo Patient-Reported 
Outcomes/Observer-Reported Outcome Instrument 
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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
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Final Amendment 02, 10 May 2018 
Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) 
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EudraCT 2017-002338-21 
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M5.3.5.1 – Protocol A4250-005 
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Final Amendment 02, 10 May 2018 
Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaire 
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EudraCT 2017-002338-21 
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M5.3.5.1 – Protocol A4250-005 
Confidential 
 
Final Amendment 02, 10 May 2018 
Appendix 5 
Guideline for Fat-Soluble Vitamin Supplementation 
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M5.3.5.1 – Protocol A4250-005 
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Final Amendment 02, 10 May 2018 
Appendix 6 
Contraceptive Requirements 
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Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
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Final Amendment 02, 10 May 2018 
Appendix 7 
Blood Volumes 
Page 109


 
 
 
CLINICAL STUDY PROTOCOL A4250-005 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
 
IND Number:   
130591 
EudraCT Number 
2017-002338-21 
Test Product: 
A4250 
Indication: 
Progressive Familial Intrahepatic Cholestasis (PFIC) 
Sponsor: 
Albireo AB 
Development Phase: 
Phase 3 
Sponsor Signatory: 
 
Sponsor Medical Monitor: 
 
Principal Investigator: 
 
Date of the Protocol: 
15 March 2018 
Version of the Protocol: 
Final Amendment 1.3 Canada 
 
 
 
 
Confidential 
The information in this document contains trade secrets and commercial information that are 
privileged or confidential and may not be used, divulged, published, or otherwise disclosed 
without the written consent of Albireo AB.  These restrictions on disclosure apply equally to 
all future information supplied to you or generated by you in connection with the study. 






 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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INVESTIGATOR SIGNATURE PAGE 
 
PROTOCOL TITLE:  A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children with Progressive 
Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
 
PROTOCOL NUMBER:  A4250-005 
I have read this protocol and agree that it contains all necessary details for performing this 
study.  I will conduct the study as outlined herein and will complete the study within the time 
designated, in accordance with all stipulations of the protocol and in accordance with Good 
Clinical Practice (GCP), local regulatory requirements, and the Declaration of Helsinki. 
I will provide copies of the protocol and all pertinent information to all individuals 
responsible to me who assist in the conduct of this study.  I will discuss this material with 
them to ensure that they are fully informed regarding the study drug and the conduct of the 
study. 
I will use only the informed consent approved by the Independent Ethics Committee (IEC) 
and will fulfil all responsibilities for submitting pertinent information to the IEC responsible 
for this study. 
I agree that the sponsor (Albireo AB) shall have access to any source documents from which 
case report form information may have been generated. 
I further agree not to originate or use the name of Albireo AB, A4250 in any publicity, news 
release, or other public announcement, written or oral, whether to the public, press, or 
otherwise, relating to this protocol, to any amendment to the protocol, or to the performance 
of this protocol without the prior written consent of Albireo AB. 
 
 
 
 
 
 
Name of Investigator 
 
 
 
 
 
 
Signature 
 
Date (day/month/year) 


 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
Confidential 
 
Page 5 
Final Amendment 1.3 Canada 15 March 2018 
 
1 
ADMINISTRATIVE INFORMATION 
A Double-blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
Protocol No.: 
A4250-005 
Date of the initial Protocol: 
20 September 2017 
Date and Number of Amendment(s): 
Amendment 01, 06 December 2017 
 
Amendment 1.3 Canada, 15 March 2018 
Sponsor: 
Albireo AB 
 
Arvid Wallgrens backe 20 
 
413 46 Gӧteborg 
Sweden 
Clinical Research Organization: 
 
 
 
 
 
 
 
Sponsor Signatory: 
 
 
  
 
  
 
  
 
 
  
Sponsor Medical Monitor: 
 
 
 
 
Principal Investigator: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 1.3 Canada 15 March 2018 
 
2 
STUDY SYNOPSIS 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Title of Study: 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate Efficacy and Safety of A4250 
in Children with Progressive Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
Principal Investigator: 
 
Study Centers: 
Up to 50 sites will be initiated for this study in the United States (US), Canada, European Union (EU), Middle 
East, and Australia. 
Publication(s): 
None. 
Planned Study Period: 
Q1 2018 to Q2 2019 
Development Phase: 
Phase 3 
Objectives: 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day A4250 in children with 
progressive familial intrahepatic cholestasis (PFIC) Types 1 and 2, as determined by the following: 
• 
Proportion of patients experiencing at least a 70% reduction in fasting serum bile acid (s-BA) 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L compared to placebo 
after 24 weeks of treatment 
• 
Change from baseline to Week 24 in pruritus compared to placebo  
Secondary Objectives 
• 
To evaluate the effect of A4250 on serum alanine aminotransferase (ALT) concentration 
• 
To evaluate the effect of A4250 on growth 
• 
To evaluate the effect of A4250 on sleep disturbance 
• 
To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or liver 
transplantation) 
• 
To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 
Methodology: 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the efficacy and safety of 
40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in children with PFIC (deficiencies of familial 
intrahepatic cholestasis-1 or bile salt export pump).  
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment Period and a 4-week 
Follow-up Period.  After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of A4250, or a 
matching placebo.  Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008) in which all patients will receive 
active treatment. 
There will be up to 10 clinic visits during the study and one scheduled telephone contact. 


 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 1.3 Canada 15 March 2018 
 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Number of Patients: 
Approximately 60 patients diagnosed with PFIC-1 or PFIC-2 will be randomized. 
Diagnosis and Main Criteria for Inclusion: 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages of ≥6 months 
and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have genetic confirmation of PFIC-1 or PFIC-2 through identification of biallelic 
pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, taken as the 
average of 2 samples 7 to 28 days apart (Visits 1 and 2) prior to randomization  
4. Patient must have history of significant pruritus and a caregiver-reported observed scratching in the 
electronic diary (eDiary) average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  Patients who 
turn 18 years of age (or legal age per country) during the study will be required to re-consent in order 
to remain in the study 
6. Patients are expected to have a consistent caregiver(s) for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use an eDiary 
device as required by the study 
Test Product, Dose and Mode of Administration: 
A4250, 40 µg/kg/day or 120 µg/kg/day, orally administered 
Reference Therapy, Dose and Duration of Administration: 
Matching placebo, orally administered 
Duration of Treatment: 
24 weeks 
Variables 
Efficacy:  
Primary Efficacy Endpoints 
The primary efficacy endpoints are: 
• 
EU and rest of world (RoW): Proportion of patients experiencing at least a 70% reduction in fasting 
s-BA concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L compared to 
placebo after 24 weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization and the previous 
visit (Clinic Visit 2).  The end value is the average of the values at Weeks 22 and 24 after the start of 
double-blind treatment. 
• 
US: Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline to 
Week 24 as measured by the worst scratch of the Albireo observer-reported outcome (ObsRO) 
instrument.  Scores for the 14 days baseline and post-treatment intervals will be calculated by 
averaging the worst scratching scores from each day in the interval. 
Secondary Efficacy Endpoints 
Secondary efficacy endpoints include the following: 
• 
EU and RoW: Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
to Week 24 as measured by the worst scratch of the Albireo ObsRO instrument.  Scores for the 14 days 
baseline and post-treatment intervals will be calculated by averaging the worst scratching scores from 
each day in the interval. 
• 
US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA concentration from 
baseline to the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 weeks of 
treatment. 
All Regions: 
All secondary endpoints will be compared to placebo. 


 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 1.3 Canada 15 March 2018 
 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
• 
Change from baseline to Week 24 in fasting s-BA 
• 
Change from baseline to Week 24 in serum ALT concentration 
• 
Change in growth from baseline to Week 24, defined as the linear deficit (weight for age and body 
mass index) compared to the national growth curves (Z-score, standard deviation from P50) 
• 
Proportion of patients achieving meaningful reduction in caregiver-reported observed scratching at 
Week 24 
• 
Change from baseline to Week 24 in sleep parameters measured with the Albireo patient-reported 
outcome (PRO) and ObsRO instruments 
• 
Change from baseline to Week 24 in patient-reported itch severity as measured by the average daily 
score of the Albireo PRO instrument; only patients ≥8 years of age will complete the Albireo PRO 
instrument 
• 
Number of patients undergoing biliary diversion surgery or being listed for liver transplantation 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• 
Proportion of patients achieving meaningful reduction in patient-reported itch severity at Week 24 
• 
Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, caregiver, and 
clinician via the patient global impression of change (PGIC) items 
• 
Change from baseline in fasting s-BA at Weeks 4 and 12 
• 
Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline to Weeks 4, 
12, and 24 as measured by the worst scratch.  Scores for the 7 worst scratching days of the last 14 days 
at baseline and post-treatment intervals will be selected 
• 
Change from baseline in patient-reported itch severity and observer-reported scratching at Weeks 4 and 
12 
• 
Change from baseline to Week 24 in patient-reported and observer-reported night time itching and 
scratching severity scores, respectively 
• 
Change from baseline to Week 24 in pooled pruritus score including observer-reported scratching for 
patients <8 years of age and patient-reported itch severity for patients ≥8 years of age 
• 
Change from baseline to Week 24 in additional patient-reported and observer-reported sleep 
parameters (e.g., tiredness and number of awakenings) 
• 
Change from baseline to Week 24 in Pediatric Quality of Life Inventory (PedsQL) 
• 
Change from baseline in serum ALT concentration at Weeks 4 and 12 
• 
Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• 
Change from baseline in AST at Weeks 4, 12, and 24 
• 
Change from baseline for other biochemical markers and measures of bile acid synthesis (autotaxin, 
plasma 7α-hydroxy-4-cholesten-3-one concentration [p-C4]) at Week 24 
• 
Change in pediatric end-stage liver disease/model for end-stage liver disease (PELD/MELD) score, 
AST to platelet ratio index (APRI) score, and Fib-4 score from baseline to Week 24 
• 
Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® (where available) 
• 
Liver-related mortality and liver decompensation events. 
• 
All-cause mortality 
Safety:   
Safety criteria are as follows: 
• 
Occurrence of treatment-emergent adverse events including severity and relatedness to study drug at all 
visits 
• 
The incidence of treatment-emergent serious adverse events (SAEs), based upon information from 
patient reports, including the description, incidence, and severity of an SAE 
• 
Trends evaluated for the following assessments: physical examinations, concomitant medications, vital 
signs, laboratory test results (including clinical chemistry, hematology, and urinalysis), and imaging 


 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 1.3 Canada 15 March 2018 
 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
studies 
Statistical Methods: 
The primary objective of this study is to show superiority in efficacy of A4250 (120 µg/kg/day and 
40 µg/kg/day) compared to placebo in patients with PFIC Types 1 and 2. 
In the EU and RoW, for the primary efficacy variable fasting bile acid responder endpoint (at least a 70% 
reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the end of treatment), the null 
hypothesis will be tested against the alternative hypothesis (α=0.0125, 1-sided).  Generalized linear mixed 
modelling will be used with a logistic link, to allow for repeated assessments over time.  The model will include 
baseline value, treatment group, PFIC class, age class, region, visit and visit*treatment group interaction.  The 
contrasts of primary interest will be the treatment difference at Week 24 but also the difference at Week 4, 
Week 8, Week 12, and Week 18 will be estimated and analyzed. 
In the US, for the primary efficacy variable pruritus score endpoint (change from baseline to Week 24 in 
observer-reported scratching), the null hypothesis will be tested against the alternative hypothesis (α=0.0125, 
1-sided).  The primary analysis of the change from baseline in pruritus score (indexed by observer-reported 
scratching) will be analyzed using a mixed model repeated measures analysis.  The contrasts of primary interest 
will be the treatment difference at Week 24 but also the difference at the other weeks will be estimated and 
analyzed.  
To strictly control overall Type I error rate across the dose groups and also across the primary and the main 
secondary endpoint to a 1-sided 2.5% level, a Bonferroni-based parallel gate-keeping procedure will be used. 
Other secondary and exploratory outcome variables shall provide supportive efficacy and safety information 
regarding the differences between the treatment groups.  No adjustments will be performed for multiple-dose 
comparisons when testing the other secondary and exploratory efficacy variables. 
Date of the Protocol:  15 March 2018 


 
A4250 – IND 130591 
EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 1.3 Canada 15 March 2018 
 
3 
TABLE OF CONTENTS 
1 
ADMINISTRATIVE INFORMATION ..................................................................... 5 
2 
STUDY SYNOPSIS .................................................................................................. 6 
3 
TABLE OF CONTENTS ........................................................................................ 10 
3.1 
List of Tables .............................................................................................. 14 
3.2 
List of Figures ............................................................................................ 14 
3.3 
List of Appendices ...................................................................................... 14 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS .............................. 15 
5 
INTRODUCTION ................................................................................................... 17 
5.1 
Investigational Medicinal Product ............................................................... 17 
5.2 
Background ................................................................................................ 17 
5.2.1 
Progressive Familial Intrahepatic Cholestasis ......................................... 17 
5.2.2 
Summary of Clinical and Nonclinical Studies ......................................... 19 
5.3 
Rationale .................................................................................................... 20 
5.3.1 
Risk/Benefit ........................................................................................... 21 
6 
STUDY OBJECTIVES ........................................................................................... 22 
6.1 
Primary Objectives ..................................................................................... 22 
6.2 
Secondary Objectives ................................................................................. 22 
7 
INVESTIGATIONAL PLAN .................................................................................. 23 
7.1 
Overall Study Design and Plan ................................................................... 23 
7.1.1 
Description ............................................................................................ 23 
7.1.2 
Schedule of Assessments ........................................................................ 25 
7.1.3 
Study Procedures and Assessments ........................................................ 29 
7.1.3.1 
Screening Period (Days -56 to 0) ............................................. 29 
7.1.3.2 
Treatment Period (Day 0 to Day 168) ...................................... 30 
7.1.3.3 
Follow-Up Period .................................................................... 33 
7.2 
Study Population ........................................................................................ 33 
7.2.1 
Inclusion Criteria ................................................................................... 33 
7.2.2 
Exclusion Criteria .................................................................................. 34 
7.2.3 
Withdrawal of Patients ........................................................................... 36 
7.2.4 
Study Termination by Sponsor ............................................................... 36 
8 
TREATMENT OF PATIENTS ............................................................................... 38 
8.1 
Identity of Study Drug ................................................................................ 38 
8.2 
Administration of Study Drug ..................................................................... 38 
8.3 
Study Treatment Packaging and Labeling ................................................... 39 
8.3.1 
Packaging and Labeling ......................................................................... 39 


 
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M5.3.5.1 – Protocol A4250-005 
 
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8.3.2 
Storage................................................................................................... 39 
8.3.3 
Blinding and Randomization of Study Drug ........................................... 39 
8.4 
Procedure for Breaking the Randomization Code ........................................ 40 
8.5 
Patient Compliance ..................................................................................... 41 
8.6 
Study Drug Accountability ......................................................................... 41 
8.7 
Concomitant Therapy and Prohibited Medications ...................................... 41 
9 
ASSESSMENT OF EFFICACY .............................................................................. 42 
9.1 
Efficacy Endpoints ..................................................................................... 42 
9.1.1 
Primary Efficacy Endpoint ..................................................................... 42 
9.1.2 
Secondary Efficacy Endpoint ................................................................. 42 
9.1.3 
Exploratory Efficacy Endpoints .............................................................. 43 
9.2 
Efficacy Assessments ................................................................................. 44 
9.2.1 
Serum Bile Acids ................................................................................... 44 
9.2.2 
Itching, Scratching and Sleep Score ........................................................ 44 
9.2.3 
Growth................................................................................................... 45 
9.2.4 
Quality of Life Questionnaire (PedsQL) ................................................. 45 
9.2.5 
Biomarkers and Other Laboratory Samples ............................................ 45 
9.2.6 
PELD/MELD Score ............................................................................... 45 
9.2.7 
Fibroscan® ............................................................................................. 45 
9.2.8 
Markers of Fibrosis ................................................................................ 46 
9.2.9 
Global Impression of Change and Global Impression of Symptom 
Measures................................................................................................ 46 
10 
ASSESSMENT OF SAFETY .................................................................................. 47 
10.1 
Adverse Events ........................................................................................... 47 
10.1.1 
Definitions and Investigator Assessments ............................................... 47 
10.1.1.1 Clinical Significance ............................................................... 47 
10.1.1.2 Serious Adverse Events ........................................................... 47 
10.1.1.3 Severity Assessment ................................................................ 48 
10.1.1.4 Causality Assessment .............................................................. 48 
10.1.2 
Recording of Adverse Events ................................................................. 49 
10.1.3 
Recording and Reporting of Serious Adverse Events .............................. 50 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions ............. 51 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments ............................................................................................... 51 
10.2.1 
Laboratory Assessments ......................................................................... 51 
10.2.2 
Individual Patient Safety Monitoring ...................................................... 52 
10.2.2.1 Liver Monitoring ..................................................................... 52 


 
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10.2.2.2 Close Observation ................................................................... 54 
10.2.2.3 De-challenge/Re-challenge for Liver and Clinical Hepatitis 
Monitoring .............................................................................. 54 
10.2.2.4 Diarrhea .................................................................................. 54 
10.2.2.5 Monitoring after Permanent Discontinuation of Study Drug 
Due to Safety .......................................................................... 55 
10.2.3 
Demographics/Medical and Surgical History .......................................... 55 
10.2.4 
Clinical Genetic Testing ......................................................................... 55 
10.2.5 
Physical Examination ............................................................................. 55 
10.2.6 
Vital Signs ............................................................................................. 56 
10.2.7 
Abdominal Ultrasound ........................................................................... 56 
10.2.8 
Overdose................................................................................................ 56 
10.2.9 
Pregnancy .............................................................................................. 56 
11 
STATISTICAL EVALUATION ............................................................................. 58 
11.1 
Sample Size and Power ............................................................................... 58 
11.2 
Statistical Methods ..................................................................................... 58 
11.2.1 
Statistical Analysis Sets.......................................................................... 58 
11.2.2 
Methods of Statistical Analyses .............................................................. 59 
11.2.2.1 General Principles ................................................................... 59 
11.2.2.2 Missing Data ........................................................................... 60 
11.2.2.3 Demographic and Baseline Characteristics .............................. 60 
11.2.2.4 Subject Disposition ................................................................. 61 
11.2.2.5 Evaluation of Primary Efficacy Variables ................................ 61 
11.2.2.6 Evaluation 
of 
Secondary 
and 
Exploratory 
Efficacy 
Variables ................................................................................. 62 
11.2.2.7 Evaluation of Safety Variables ................................................ 63 
11.2.2.8 Compliance and Exposure ....................................................... 63 
11.2.3 
Blinded Analyses to Confirm Responder Definitions .............................. 64 
11.2.4 
Data Safety Monitoring Board ................................................................ 64 
12 
DIRECT ACCESS TO SOURCE DATA/NOTES ................................................... 65 
13 
QUALITY CONTROL AND QUALITY ASSURANCE ........................................ 66 
13.1 
Conduct of the Study .................................................................................. 66 
13.2 
Study Monitoring........................................................................................ 66 
14 
ETHICS .................................................................................................................. 68 
14.1 
Independent Ethics Committee/Institutional Review Board ......................... 68 
14.2 
Written Informed Consents and Assents ..................................................... 68 


 
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M5.3.5.1 – Protocol A4250-005 
 
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15 
DATA HANDLING AND RECORD KEEPING..................................................... 70 
15.1 
Case Report Forms/Source Data Handling .................................................. 70 
15.2 
Retention of Essential Documents............................................................... 70 
16 
FINANCING AND INSURANCE .......................................................................... 71 
17 
PUBLICATION POLICY ....................................................................................... 72 
18 
REFERENCE LIST ................................................................................................. 73 
19 
APPENDICES ........................................................................................................ 74 
 


 
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3.1 
List of Tables 
Table 1 
Schedule of Assessments ................................................................................ 26 
Table 2 
Dosing and Capsule Strength .......................................................................... 39 
Table 3 
Routine Laboratory Parameters ...................................................................... 52 
 
3.2 
List of Figures 
Figure 1 
Study Design .................................................................................................. 25 
Figure 2 
Bonferroni Holm Multiple Comparison Procedure.......................................... 59 
 
3.3 
List of Appendices 
Appendix 1 Concomitant Medications Prohibited During the Study .................................. 74 
Appendix 2 Diary Questions including Albireo Patient-Reported Outcomes/Observer-
Reported Outcome Instrument ........................................................................ 75 
Appendix 3 Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) .................................................................... 76 
Appendix 4 Pediatric Quality of Life Inventory (PedQL) Questionnaire ............................ 77 
Appendix 5 Blood Volumes………………………………………………………….……..78 
 


 
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4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
ADME 
absorption, distribution, metabolism, and excretion 
AE(s) 
adverse event(s) 
AFP 
alfa-fetoprotein 
ALP 
alkaline phosphatase 
ALT 
alanine aminotransferase 
APRI 
AST to platelet ratio index 
ASBT 
apical sodium-dependent bile transporter (also known as IBAT) 
AST 
aspartate aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BMI 
body mass index 
BSEP 
bile salt export pump 
CPK 
creatine phosphokinase 
CRA 
clinical research associate 
CYP 
cytochrome P450 
DSMB 
Data and Safety Monitoring Board 
eCRF 
electronic case report form 
eDiary 
electronic diary 
EOT 
End of Treatment 
EU 
European Union 
EudraCT 
European Union drug regulatory agency clinical trial 
FAS 
full analysis set 
FDA 
United States Food and Drug Administration 
FIC1 
familial intrahepatic cholestasis-1 
GCP 
Good Clinical Practice 
GI 
gastrointestinal 
GLMM 
generalized linear mixed modelling 
IB 
Investigator’s Brochure 
IBAT 
ileal bile acid transporter (also known as ASBT) 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
 
 
IEC 
Independent Ethics Committee 
INR 
international normalized ratio 
IRB 
Institutional Review Board 
IWRS 
Interactive Web Response System 


 
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LDH 
lactate dehydrogenase 
LFT 
liver function test 
MDR3 
multidrug-resistance protein 3 
MedDRA 
Medical Dictionary for Regulatory Activities 
MELD 
model for end-stage liver disease 
MMRM 
mixed model repeated measures 
ObsRO 
observer-reported outcome 
PBC 
primary biliary cholangitis 
p-C4 
plasma 7α-hydroxy-4-cholesten-3-one concentration 
PCS 
potentially clinically significant 
PedsQL 
Pediatric Quality of Life Inventory 
PELD 
pediatric end-stage liver disease 
PFIC 
progressive familial intrahepatic cholestasis 
PGIC 
patient global impression of change 
PGIS 
patient global impression of symptoms 
PP 
per protocol 
PRO 
patient-reported outcome 
PT 
prothrombin time 
PVM 
pharmacovigilance manager 
QoL 
quality of life 
RoW 
rest of world 
RR 
respiratory rate 
SAE(s) 
serious adverse event(s) 
SAP 
statistical analysis plan 
s-BA 
serum bile acid 
SD 
standard deviation 
s-FGF19 
serum fibroblast growth factor 19 concentration 
SOC 
system organ class 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE(s) 
treatment-emergent adverse event(s) 
ULN 
upper limit of normal 
US 
United States 
WHO 
World Health Organization 
 


 
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5 
INTRODUCTION 
5.1 
Investigational Medicinal Product 
A4250 is a small molecule and a potent selective inhibitor of the ileal bile acid transporter 
(IBAT), also known as the apical sodium-dependent bile acid transporter (ASBT).  IBAT is 
an integral brush border membrane glycoprotein that co-transports sodium and bile acids and 
appears to be a critical component in the regulation of the bile acid pool size in animals and 
man.  This transporter, expressed in the distal ileum, is a key element in the enterohepatic 
circulation of bile acids since it facilitates the high-affinity, high-capacity reabsorption of bile 
acids.  Indeed, 95% of secreted bile acids are reabsorbed via IBAT [Hofmann 2009; 
Miethke 2016].  A4250 is orally administered and acts locally in the gut where it binds 
reversibly to IBAT to decrease the reuptake of bile acids.  A4250 has minimal systemic 
exposure at the predicted therapeutic dose ranges. 
No safety signals were found with A4250 in preclinical safety and toxicity studies.  In the 
first clinical study of A4250 in healthy volunteers, A4250 was given as a single oral dose up 
to 10 mg or daily oral doses up to 3 mg/day for 7 days.  A4250 was well tolerated and the 
only findings were gastrointestinal (GI) related effects, such as diarrhea, at higher doses. 
In an exploratory Phase 2 study in children with cholestatic pruritus, A4250 was given as oral 
doses of 10 to 200 µg/kg/day for 4 weeks.  A4250 reduced mean levels of serum bile acids 
(s-BA) in all cohorts.  In 11 of 13 progressive familial intrahepatic cholestasis 
(PFIC) patients, substantial reductions (ranging from 43% to 98%) were observed.  
Patient-reported diary data documented improvement of pruritus during treatment.  A4250 
was well tolerated.  No serious adverse events (SAEs) were deemed to be related to study 
drug, and most adverse events (AEs), including some increased transaminases, were 
considered unrelated to study drug and transient in nature. 
5.2 
Background 
5.2.1 
Progressive Familial Intrahepatic Cholestasis 
PFIC is a rare autosomal recessive cholestatic liver disease estimated to affect between 1 in 
every 50,000 to 100,000 children born worldwide.  PFIC represents 10% to 15% of causes of 
cholestasis in children and 10% to 15% of liver transplantation indications in children.  All 
types of PFIC exist worldwide and both sexes appear to be equally affected. 
The common underlying pathogenesis of PFIC is disruption of bile formation and bile 
transport through the liver [Jacquemin 2000].  The classification of PFIC has evolved over 
the years.  The most commonly used sub-classifications are PFIC Types 1, 2, and 3, which 
are based on the associated affected gene and described in more detail below. 
• PFIC, Type 1:  also referred to as “Byler disease” or “familial intrahepatic 
cholestasis-1 (FIC1) protein deficiency.” FIC1 protein is located on the canalicular 
membrane of hepatocytes and facilitates movement of aminophospholipids from the outer 


 
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to inner leaflet of the plasma membrane of the hepatocyte.  The ATP8B1 gene encodes 
FIC1 protein.  Biallelic pathologic variants in the ATP8B1 gene are associated with FIC1 
dysfunction and classified clinically as PFIC Type 1 disease. 
• PFIC, Type 2:  also referred to as “Byler syndrome” or “bile salt export pump (BSEP) 
deficiency.”  BSEP is a transporter protein that is expressed at the canalicular membrane 
of hepatocytes, and is the primary exporter of bile acids.  The ABCB11 gene encodes the 
BSEP protein.  Biallelic pathologic variations in the ABCB11 gene are associated with 
BSEP dysfunction and classified clinically as PFIC Type 2 disease. 
• PFIC, Type 3:  is caused by a deficiency of the multidrug-resistance protein 3 (MDR3) 
due to mutations in the ABCB4 gene.  MDR3 is a phospholipid translocase critical for 
phospholipid secretion. 
Severe pruritus is common in children diagnosed with PFIC.  Itching (and subsequent 
scratching) is a significant morbidity for these patients and their families [Suchy 2007].  A 
more severe degree of pruritus is experienced compared to patients with other forms of liver 
disease and to other pruritic conditions such as atopic dermatitis [Murray 2011].  In patients 
with PFIC, liver biopsy reveals canalicular cholestasis and, later, the appearance of portal 
fibrosis.  Serum biochemistry indicates cholestasis with hyperbilirubinemia, elevated alanine 
aminotransferase (ALT) and aspartate aminotransferase (AST).  The concentrations of bile 
acids in serum are very high, while serum gamma-glutamyl transferase activity (exception 
being MDR3 variants) and cholesterol are normal [Hori 2010].  Symptoms of portal 
hypertension and liver failure will develop during the course of the disease [Davit-Spraul 
2009; Alissa 2008].  Symptoms develop early; median age at onset of symptoms is 2 months, 
and 78% of PFIC patients present with jaundice [Pawlikowska 2010].  The life-threatening 
and debilitating nature of PFIC is reflected by the fact that survival in those not resorting to 
surgery is 50% at 10 years of age and almost zero at 20 years of age.  Approximately half of 
PFIC patients undergo liver transplantation [Davit-Spraul 2009] and treatment-resistant 
pruritus is the leading indication for the surgical procedure partial external biliary diversion, 
mostly in PFIC Type 1 and 2 patients. 
PFIC is life-threatening and debilitating.  Currently, the treatment for PFIC is palliative and 
there is no pharmaceutical treatment approved for use in PFIC.  The therapeutic choices are 
restricted to non-specific therapy of the symptoms and signs of the disease such as nutritional 
support, preventing vitamin deficiencies, and treatment of extrahepatic features.  Medical 
treatment options include off-label use of ursodeoxycholic acid, rifampin, antihistamines, and 
naltrexone.  A minority of patients respond nominally and transiently to these interventions.  
Biliary diversion is used to decrease systemic bile acids through interruption of the 
enterohepatic circulation and so avoid transplantation.  Liver transplantation is typically only 
viewed as an option when patients have failed medical treatment and/or biliary diversion and 
have liver failure or persistent uncontrolled pruritus. 


 
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5.2.2 
Summary of Clinical and Nonclinical Studies 
This is a summary of nonclinical and clinical studies.  More detail is provided in the 
Investigator’s Brochure ([IB] see A4250 IB).  
A4250 is a potent, selective IBAT inhibitor with no safety signals seen in the safety 
pharmacological studies.  In addition, no safety signals were seen in repeat-dose oral toxicity 
studies in rats with up to 26 weeks of daily dosing and in dogs with up to 39 weeks of daily 
dosing.  No safety signals were identified when A4250 was administered daily to juvenile 
rats from age 14 days to 63 days. 
A4250 is minimally absorbed with very low systemic exposure in both humans and other 
species.  In blood, protein binding is high (>99.7%).  Systemically absorbed A4250 is slowly 
metabolized in humans and in preclinical studies by hydroxylation only, a metabolite that 
retains IBAT activity.  A4250 did not show any significant inhibition of cytochrome 
P450 (CYP) 
enzymes 
in 
the 
rat. 
 
A4250 
inhibited 
human 
CYP3A4 
and 
CYP2D6 (IC50: 16 µmol/L for both enzymes), and CYP2C9 (IC50:1.2 µmol/L).  
To date, A4250 has been studied in 3 Albireo-sponsored clinical studies: a study in healthy 
volunteers evaluating single and multiple administration of A4250; a single-dose absorption, 
distribution, metabolism, and excretion (ADME) study; and a Phase 2 study in children with 
cholestatic pruritus.  In addition, an investigator-sponsored study has been conducted in 
patients with primary biliary cholangitis (PBC).  In total, 98 subjects have been exposed to 
A4250. 
In the first clinical study (A4250-001), A4250 was given to healthy volunteers as a single oral 
dose up to 10 mg or daily oral doses up to 3 mg/day for 7 days.  Following a single oral dose 
of 10 mg, the median maximum drug concentration was 0.2 nmol/L and the area under the 
curve was 0.8 nmol × h/L (n=6).  Pharmacodynamic evaluation of bile acid synthesis 
biomarkers 
(serum 
fibroblast 
growth 
factor 
19 [s-FGF19] 
and 
plasma 
7α Hydroxy 4-cholesten-3-one [p-C4]) identified the expected decrease in s-FGF19 and an 
increase in p-C4.  In addition, total plasma bile acids decreased and total fecal bile acids 
increased in A4250-treated patients compared to placebo. A4250 was generally well 
tolerated.  The only findings were GI-related effects, such as diarrhea at higher doses. 
In the ADME study (A4250-007), healthy subjects were administered a single oral dose of 
3 mg [14C]-A4250 capsule.  The main excretion route was determined to be fecal.  There 
were no quantifiable concentrations of A4250 in plasma or total radioactivity in plasma or 
whole blood.  A4250 was well tolerated.  AEs were reported and most were mild and not 
related to A4250. The 3 AEs experienced by 2 subjects reported as related to study drug were 
GI in nature, including diarrhea (2 AEs; 2 subjects) and abdominal pain (1 AE; 1 subject). 
In an exploratory Phase 2 study in children with cholestatic pruritus (A4250-003), A4250 was 
administered as oral daily doses of 0.01 to 0.2 mg/kg for 4 weeks.  Twenty 
patients (8 females and 12 males) aged 1 to 17 years were enrolled in this open-label 
dose-escalation study.  Four patients were re-enrolled into a second dose level.  No 


 
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treatment-related SAEs were reported, and most AEs, including some increased 
transaminases, were transient.  Four-week, once-daily treatment with A4250 reduced mean 
levels of s-BA in all cohorts.  Substantial s-BA reductions in 11 of 13 PFIC patients (ranging 
from 43% to 98%) were observed.  A4250 also reduced s-BA in the 11 non-PFIC patients, 
but to a lesser extent than in PFIC patients.  Patient-reported diaries documented 
improvement of the visual analogue scale itch score in 17 of 24 patients during treatment.  
The dose with the greatest improvement showed a mean decrease (± standard error of the 
mean) of 2.9 ±1.1 from baseline.  The results from this study form the basis of the current 
(A4250-005) study design. 
5.3 
Rationale 
In this investigational study, A4250 is under development for treatment of PFIC and is also 
being considered for other orphan cholestatic liver diseases. 
There is currently no medical treatment approved for PFIC.  In PFIC patients with severe 
pruritus, biliary diversion surgery is used to decrease systemic bile acids through interruption 
of the enterohepatic circulation and thereby reducing pruritus [Whitington 1988].  Liver 
transplantation is typically used as an option when patients have failed medical treatment 
and/or biliary diversion and have liver failure or persistent uncontrolled pruritus. 
By inhibiting IBAT with high selectivity and potency, A4250 has the potential to relieve 
cholestasis and improve liver function without surgical intervention in patients with PFIC.  
Since A4250 is minimally absorbed and achieves a substantial reduction of s-BA, it has the 
potential to be a safer and less invasive alternative to surgical biliary drainage.  The rationale 
for using A4250 is to decrease s-BA levels, and to reduce the major morbidity of pruritus, 
improving the health and wellbeing of patients affected with PFIC.  Ultimately, this 
noninvasive therapeutic approach has the potential to not only improve quality of life (QoL), 
but also reduce the need for biliary diversion and subsequent liver failure and liver 
transplantation. 
The doses selected, 40 and 120 µg/kg/day, were based on the efficacy and safety data 
generated from patients with PFIC (n=10 + 3 re-entered) and supporting data from all 
patients (n=20 + 4 re-entered) in the five dose groups dosed in the A4250-003 study.  
Improvements in s-BA and pruritus were observed with doses from 30 µg/kg to 100 µg/kg, 
with a dose-related trend.  The best dose response in all patients was at the 60 µg/kg/day for 
s-BA and 100 µg/kg/day for pruritus.  The best dose response in the PFIC subgroup was at 
30 µg/kg/day for both s-BA and pruritus.  Patients with PFIC responded well to the doses 
given from 30 µg/kg to 200 µg/kg; however, a clear dose relationship could not be 
established in order to select a single dose for this Phase 3 study.  As efficacy was observed 
at both low and high doses, 40 µg/kg and 120 µg/kg/day are considered to be the most 
optimal for this study.  
This study will be conducted in compliance with the protocol and with the International 
Council for Harmonisation (ICH) guidelines on Good Clinical Practice (GCP). 


 
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Patients completing 24 weeks of double-blind treatment will be offered to enter Study 
A4250-008 and receive open-label treatment with A4250. 
5.3.1 
Risk/Benefit 
A4250 has been evaluated in three Albireo-sponsored clinical studies: a double-blind 
placebo-controlled study in healthy volunteers, a single-dose ADME study, and a Phase 2 
study in children with cholestatic pruritus.  In addition, an investigator-sponsored study has 
been conducted in patients with PBC. 
A total of 98 subjects/patients have been exposed to A4250.  Healthy subjects have been 
exposed to up to 10 mg as a single dose and up to 3 mg daily as part of a multiple-dose 
evaluation.  Children with cholestatic liver disease have been treated with up to 
0.2 mg/kg/day (200 µg/kg/day) for 4 weeks.  In total, 2 SAEs have been reported; both were 
judged by the physician to be not related to the study drug. 
Patients with cholestatic liver diseases suffer from excess bile acids in the liver resulting in 
tissue damage.  A commonly used treatment in these patients is bile diversion surgery, 
whereby approximately 50% to 100% of the enterohepatic circulation of bile acids is 
interrupted.  Inhibition of IBAT with A4250, thereby interrupting the enterohepatic 
circulation of bile acids, is therefore a potential medical alternative to surgery which could be 
of benefit to these patients if shown to be effective and safe.  Data from the Phase 2 study 
showed efficacy of A4250 in reducing s-BA concentrations and pruritus in such patients. 
Based on the mode of action of an IBAT inhibitor, loose stools or diarrhea could be expected.  
However, in the pediatric Phase 2 study with 4 weeks daily treatment, only one patient had 
mild transient diarrhea after a single dose that did not recur on multiple dosing. 


 
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6 
STUDY OBJECTIVES 
6.1 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day 
A4250 in children with PFIC Types 1 and 2, as determined by the following: 
• The proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment 
• Change from baseline to Week 24 in pruritus compared to placebo  
6.2 
Secondary Objectives 
• To evaluate the effect of A4250 on serum ALT concentration 
• To evaluate the effect of A4250 on growth 
• To evaluate the effect of A4250 on sleep disturbance 
• To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or 
liver transplantation)  
• To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 


 
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7 
INVESTIGATIONAL PLAN 
7.1 
Overall Study Design and Plan 
7.1.1 
Description 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the 
efficacy and safety of 40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in 
children with PFIC (deficiencies of FIC1 or BSEP). 
Study data will be periodically reviewed by an unblinded Data Safety and Monitoring Board 
(DSMB). 
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment 
Period and a 4-week Follow-Up Period. 
After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of 
A4250, or a matching placebo. 
Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008) in which all 
patients will receive active treatment.  A patient who prematurely withdraws due to no 
improvement/intolerable symptoms, and has completed at least 12 weeks of the Treatment 
Period and End of Treatment (EOT) Visit, will also be offered the opportunity to enter 
Study A4250-008. 
Visit windows applicable to each visit are presented in the schedule of assessments (Table 1). 
There will be up to 10 clinic visits during the study (Figure 1) and one scheduled telephone 
contact, as follows: 
• Visit 1: Screening Period start. Patient is considered enrolled after signing the informed 
consent form (ICF) (Days -56 to -35) 
• Visit 2: second Screening Visit (Days -28 to -7) 
• Visit 3: Randomization Visit (Day 0) 
• Telephone Contact 1: (Day 14) 
• Visit 4: Clinic Visit (Day 28) 
• Visit 5: Clinic Visit (Day 56) 
• Visit 6: Clinic Visit (Day 84) 
• Visit 7: Clinic Visit (Day 126) 
• Visit 8: Clinic Visit (Day 154) 
• Visit 9: Clinic Visit, EOT (Day 168) 


 
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• Visit 10: Follow-Up Visit, 28 days after last dose of study drug (Day 196).  All patients 
continuing in Study A4250-008 will not have this Follow-up Visit. 
Additional clinic visits may be required for patients who need direct site assistance, e.g., due 
to AE monitoring, to fulfil screening requirements, and/or for safety maintenance. 
Screening Period 
Informed consent must be obtained at Visit 1 prior to performing any study procedures.  After 
signing the ICF, patients will be considered enrolled and evaluated for study eligibility. 
Daily recording of pruritus using an electronic diary (eDiary) will be started to determine 
confirmation of the magnitude and the baseline pruritus symptoms before the study.  After 
consultation with the Medical Monitor, patients failing eligibility criteria may be re-screened 
after 2 months.  Patients not fulfilling inclusion/exclusion criteria after 3 attempts are not 
allowed to re-screen. 
Caregivers/patients will be instructed on the daily use of the eDiary.  The eDiary will include 
patient-reported outcome (PRO) and observer-reported outcome (ObsRO) items for 
evaluation of itching (PRO), scratching (ObsRO), and sleep disturbance (PRO and ObsRO) 
during the Screening Period (study Days -56 to 0) and throughout the 24-week Treatment 
Period (study Days 0 to 168). 
Only the 14 most recent days before randomization will be used for eligibility calculation of 
the diary entries.  Observer-reported outcomes in patients of all ages will be recorded by a 
caregiver (or caregiver’s designee).  If possible, the same caregiver will complete the Albireo 
ObsRO items throughout the study. 
At a second Screening Visit (Visit 2) additional laboratory samples will be taken for 
eligibility assessments. 
Treatment Period 
Eligibility for randomization will be determined at Visit 3 using the pruritus (scratching 
reports from the Albireo ObsRO) data in the 2 weeks before Visit 3, genetic confirmation of 
diagnosis, and the liver biochemistry evaluations from the previous screening visits.  Two 
weeks after Visit 3, there will be a telephone contact with the caregiver/patient to evaluate 
any AEs. 
The patient will return to the clinic at Visit 4, Visit 5, Visit 6, Visit 7, Visit 8, and Visit 9 for 
assessments.  If a new caregiver will begin to complete the Albireo ObsRO during the course 
of the trial, they will be provided with training during a clinic visit before they begin to use 
the eDiary.  Additionally, caregivers/patients will be requested to use the diary to report the 
date and time each dose of study drug is administered during the Treatment Period. 
The last dose of study drug will be administered the day before Visit 9.  At study Visit 9, the 
patient has the option to enroll in an open-label extension study (A4250-008) should they 
meet the required entry criteria.  If the patient is continuing into Study A4250-008 this will be 


 
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the last visit in this study and the first visit in Study A4250-008.  Visit 9/EOT should also be 
performed at the time patients are prematurely withdrawn from the study. 
Clinic Visit 10 will take place 28 days after Visit 9 for all patients not continuing into Study 
A4250-008. 
Figure 1 
Study Design 
 
T: telephone contact; V: study visit 
7.1.2 
Schedule of Assessments 
The schedule of assessments is presented in Table 1. For information on blood volumes 
please see Appendix 5. 
 


 
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Table 1 
Schedule of Assessments 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Informed consent c 
X 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
Concomitant medicationd 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical and surgical history 
X 
 
X 
 
 
 
 
 
 
 
 
Blood sampling for genetic 
testinge 
X 
 
 
 
 
 
 
 
 
 
 
Physical examination including 
voluntary photography 
X 
 
X 
 
 
 
 
 
 
X 
 
Skin examination 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
Vital signsf 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
eDiary: itching, scratching, and 
sleep scoresg 
Daily diary entry 
Clinical chemistryh 
X 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Hematologyh 
 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Urinalysish 
 
X 
 
 
 
 
X 
 
 
X 
 


 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Serum bile acidsi 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
INR 
 
X 
 
 
X 
 
X 
 
X 
 
X 
Autotaxinj 
 
 
X 
 
X 
 
 
 
 
X 
 
p-C4j 
 
 
X 
 
X 
 
 
 
 
X 
 
AFP 
 
 
X 
 
 
 
 
 
 
X 
 
Fat-soluble vitamins A & E 
 
X 
 
 
 
 
 
 
 
X 
 
Fat-soluble vitamin D 
 
 
X 
 
 
 
 
 
 
X 
 
Abdominal Ultrasound 
 
 
X 
 
 
 
 
 
 
X 
 
Fibroscan® (where available) 
 
 
X 
 
 
 
 
 
 
X 
 
QoL questionnaire (PedsQL) 
 
 
X 
 
 
 
 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of Change 
 
 
 
 
X 
 
X 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of 
Symptoms 
 
 
X 
 
X 
 
X 
 
 
X 
 
Pregnancy testk 
Xk 
X 
X 
 
X 
X 
X 
X 
X 
Xk 
 
Study drug dispensedl 
 
 
X 
 
X 
X 
X 
X 
X 
 
 


 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Adverse eventsm 
Continuous collection 
Study drug compliance 
 
 
 
X 
X 
X 
X 
X 
X 
X 
 
AFP: alfa-fetoprotein; BMI: body mass index; eCRF: electronic case report form; eDiary: electronic diary; EOT: End of Treatment; ICF: informed consent form; 
INR: international normalized ratio; p-C4: plasma 7α Hydroxy 4-cholesten-3-one; PedsQL: Pediatric Quality of Life Inventory; QoL: quality of life; RR: respiratory rate; 
s-BA: serum bile acid. 
 
a 
Must also be performed at the time a patient is prematurely withdrawn from the study.  This visit is the same as Visit 1 in the extension protocol (Study A4250-008). 
b 
Assessments must be performed 28 days following the final dose of study drug.  This visit is omitted if patients are entering Study A4250-008. 
c 
The ICF (and assent) must be signed before any study procedures are performed. 
d 
Past (within 1 month prior to Visit 1) and current medication will be documented in the eCRF. 
e 
Blood sample for genetic testing will be drawn for all patients.  Genetic analysis will only be performed on a patient’s extracted DNA if previous the clinical genetic 
result is equivocal, unavailable, or unobtainable. 
f 
Includes blood pressure, pulse, RR, temperature, height (or length depending on age), and body weight (using a certified weight scale).  BMI will be calculated. 
g 
Includes training on use of the device at Screening (Visit 1) and review of patient daily entries during 14 days prior to Clinic Visit 3 to determine eligibility.  
h 
See Table 3 for detailed parameters. 
i 
Patients will fast (water intake only is permissible) for at least 4 hours prior to the collection of samples for s-BA.  Exceptions can be made for infants, less than 
12 months of age, if unable to fast for the full 4 hours.  All s-BA results during the treatment period and follow-up will be blinded. 
j 
Samples will not be collected for patients ≤10 kg. 
k 
For girls who have reached menarche.  Serum test at Visit 1; urine test at other visits.  A serum pregnancy test will also be conducted at Visit 9 for patients entering 
Study A4250-008.  If a urine pregnancy test is positive, a serum pregnancy test should be performed to confirm the pregnancy. 
l 
Study drug to be taken once daily on Day 1 through Day 167 as described in Section 8.2. 
m 
Adverse event information will be collected from the time of signing of the ICF to study discontinuation. 


 
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7.1.3 
Study Procedures and Assessments 
7.1.3.1 
Screening Period (Days -56 to 0) 
Day -56 to Day -35/Clinic Visit 1 
Patients will undergo a Screening Visit up to 56 days prior to the planned first day of study 
treatment.  Screening procedures and assessments are as follows: 
• Obtain written informed consent 
• Assess inclusion/exclusion criteria (Section 7.2) 
• Record demographics (age, full date of birth, gender, race, and ethnicity) 
• Document concomitant medications 
• Medical and surgical history (date of diagnosis of PFIC Types 1 or 2, prior investigational 
medications for PFIC, historical liver function test [LFT] values, any surgery performed, 
any other diagnosis, and historical liver biopsy data) 
• Blood sample for genetic testing 
• Collect, if available, past confirmatory clinical genetic laboratory report for PFIC1/PFIC2 
and send to central reader for review 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• eDiary training and compliance requirements; patients begin daily recording of pruritus 
using the eDiary (Appendix 2) 
• Clinical chemistry (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Serum pregnancy test for girls of who have reached menarche 
• AE monitoring 
Day -28 to Day -7/Clinic Visit 2 
A second Screening Visit will be performed.  Screening procedures and assessments are as 
follows: 
• Review concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for itching, scratching, and sleep scores and 
compliance 
• Urinalysis (Section 10.2.1) 


 
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• s-BA (Section 9.2.1) 
• International normalized ratio (INR) 
• Fat-soluble vitamins A and E 
• Urine pregnancy test for girls of who have reached menarche 
• AE monitoring 
7.1.3.2 
Treatment Period (Day 0 to Day 168) 
Study Day 0/Clinic Visit 3 (Randomization) 
At the randomization visit, the following assessments will be conducted: 
• Review inclusion/exclusion criteria (Section 7.2) 
• Review concomitant medications, medical and surgical history 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance and entrance criteria requirement (see 
Inclusion Criteria 4, Section 7.2.1) 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1)  
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• Fat-soluble vitamin D 
• Alfa-fetoprotein (AFP) 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer QoL (Pediatric Quality of Life Inventory [PedsQL]) questionnaire 
• Patient/caregiver/clinician complete the patient global impression of symptoms (PGIS) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Study drug is dispensed and patients are instructed to administer daily from Day 1 
Study Day 14 (Telephone Contact) 
A study nurse will contact the patient/caregiver by telephone for monitoring of the following:  
• Review concomitant medications 


 
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• Albireo PRO/ObsRO eDiary review for compliance 
• AEs 
• Evaluation of study drug compliance 
Study Day 28 and Day 84/Clinic Visits 4 and 6  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• Urinalysis (at Clinic Visit 6 only; Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Autotaxin and p-C4 (at Clinic Visit 4 only; samples will not be taken for patients ≤10 kg) 
• Patient/caregiver/clinician complete the patient global impression of change (PGIC) and 
PGIS 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 56 and Day 126/Clinic Visits 5 and 7 
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 


 
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• Study drug is dispensed 
Study Day 154/Clinic Visit 8  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 168/Clinic Visit 9/EOT 
The last dose of study drug is taken in the morning the day before Clinic Visit 9, and the 
following procedures and assessments will be performed (also performed at the time a patient 
prematurely withdraws):  
• Review of concomitant medications 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry, hematology, and urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• AFP  
• Fat-soluble vitamins A, E, and D 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer PedsQL questionnaire 
• Patient/caregiver/clinician complete the PGIC and PGIS 


 
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• Urine pregnancy test for girls who have reached menarche, or serum pregnancy test for 
girls continuing to study A4250-008 
• AE monitoring 
• Evaluation of study drug compliance 
7.1.3.3 
Follow-Up Period 
Study Day 196/Clinic Visit 10 
All patients not continuing into Study A4250-008 will return to the study site 28 days after 
Clinic Visit 9 or the last dose of study drug for the following assessments: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• AE monitoring 
7.2 
Study Population 
A total of 60 patients with clinical diagnosis of PFIC Type 1 or 2 that have been genetically 
confirmed will be randomized.  
An attempt will be made to randomize a minimum of 10 patients from each age category 
(6 months to 5 years, 6 to 12 years, and 13 to ≤18 years).  
A maximum of 5 patients that were treated in Study A4250-003 will be allowed to participate 
in this study. 
7.2.1 
Inclusion Criteria 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages 
of ≥6 months and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have genetic confirmation of PFIC-1 or PFIC-2 through identification of 
biallelic pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, 
taken as the average of 2 samples 7 to 28 days apart (Visits 1 and 2) prior to 
randomization 


 
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4. Patient must have history of significant pruritus and a caregiver-reported observed 
scratching in the eDiary average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to 
randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  
Patients who turn 18 years of age (or legal age per country) during the study will be 
required to re-consent in order to remain in the study 
6. Patients will be expected to have a consistent caregiver(s) for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use 
an eDiary device as required by the study 
7.2.2 
Exclusion Criteria 
1. Patient with pathologic variations of the ABCB11 gene that predict complete absence of 
the BSEP function 
2. Patient with past medical history or ongoing presence of other types of liver disease 
including, but not limited to, the following: 
a) Biliary atresia of any kind 
b) Benign recurrent intrahepatic cholestasis, indicated by any history of normal s-BAs 
c) Suspected or proven liver cancer or metastasis to the liver on imaging studies 
d) Histopathology on liver biopsy is suggestive of alternate non-PFIC related etiology of 
cholestasis 
3. Patient with a past medical history or ongoing presence of any other disease or condition 
known to interfere with the absorption, distribution, metabolism (specifically bile acid 
metabolism), or excretion of drugs in the intestine, including but not limited to, 
inflammatory bowel disease 
4. Patient with past medical history or ongoing chronic (i.e., >3 months) diarrhea requiring 
intravenous fluid or nutritional intervention for treatment of the diarrhea and/or its 
sequelae 
5. Patient has a confirmed past diagnosis of infection with human immunodeficiency virus 
or other present and active, clinically significant, acute, or chronic infection, or past 
medical history of any major episode of infection requiring hospitalization or treatment 
with parenteral anti-infective treatment within 4 weeks of treatment start (study Day 1) or 
completion of oral anti-infective treatment within 2 weeks prior to start of Screening 
Period 
6. Any patient with suspected or confirmed cancers except for basal cell carcinoma, and 
non-liver cancers treated at least 5 years prior to Screening with no evidence of 
recurrence 


 
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7. Patient with a past medical history of chronic kidney disease with an impaired renal 
function and a glomerular filtration rate <70 mL/min/1.73 m2  
8. Patient with surgical history of disruption of the enterohepatic circulation excluding those 
who have undergone a successful reversal procedure that has permanently restored flow 
of bile acids from the liver to the duodenum 
9. Patient has had a liver transplant or listed for liver transplant 
10. Decompensated liver disease, coagulopathy, history or presence of clinically significant 
ascites, variceal hemorrhage, and/or encephalopathy 
11. INR >1.4 (the patient may be treated with Vitamin K intravenously, and if INR is <1.4 at 
resampling the patient may be randomized) 
12. Serum ALT >10 × upper limit of normal (ULN) at Screening 
13. Serum ALT >15 × ULN at any time point during the last 6 months unless an alternate 
etiology was confirmed for the elevation 
14. Total bilirubin >5 × ULN at Screening 
15. Patient suffers from uncontrolled, recalcitrant pruritic condition other than PFIC.  
Examples include, but not limited to, refractory atopic dermatitis or other primary pruritic 
skin diseases 
16. Any patient who is pregnant or lactating or who is planning to become pregnant within 
24 weeks of randomization 
17. Sexually active males and females who are not using a reliable contraceptive method with 
≤1% failure rate (such as barrier protection, hormonal contraception, intrauterine device, 
or abstinence) throughout the duration of the study 
18. Patient with a past medical history of alcohol or substance abuse will be excluded.  
Patient must agree to refrain from illicit drug and alcohol use during the study 
19. Administration of bile acid or lipid binding resins and medications that slow GI motility 
(Refer to Appendix 1 – Concomitant Medications Prohibited during the study) 
20. Patient has had investigational exposure to a drug, biologic agent, or medical device 
within 30 days prior to Screening, or 5 half-lives of the study agent, whichever is longer 
21. Patient who has been previously treated with an IBAT inhibitor whose pruritus has not 
responded to treatment 
22. Any other conditions or abnormalities which, in the opinion of the investigator or Medical 
Monitor, may compromise the safety of the patient, or interfere with the patient 
participating in or completing the study 


 
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7.2.3 
Withdrawal of Patients 
Patients/caregivers will be informed that they have the right to withdraw from the study at 
any time, without prejudice to their medical care, and that they are not obliged to state their 
reasons. 
Any withdrawal, and reasons for withdrawal, must be fully documented in the electronic case 
report form (eCRF) and source documents and the patient followed by the 
investigator/investigative staff.  Even after the study is completed at the Follow-up Visit, the 
investigator/investigative staff will, regardless of reason for withdrawal, record any patient 
data they receive concerning SAEs, and all drug-related non-serious AEs, and subsequently 
report these to Albireo.  Withdrawn patients will not be replaced. 
Patients will be withdrawn in the following circumstances: 
• A patient’s/caregiver’s desire for withdrawal for any reason 
• Lost to follow-up (every effort must be made to contact the patient/caregiver; a certified 
letter must be sent) 
• An AE that, in the opinion of the investigator, necessitates withdrawal 
• A worsening of the symptoms related to diarrhea occurs 
• Death 
• Patient/caregiver estimation of lack of improvement/intolerable symptoms 
• A patient’s/caregiver’s substantial noncompliance (eDiary and study drug compliance) or 
protocol violation 
• An investigator’s opinion that continuing the patient in the study is not appropriate.  The 
investigator may withdraw a patient at any time, if it is considered to be in the patient’s 
best interest 
A patient who prematurely withdraws due to no improvement/intolerable symptoms, and has 
completed at least 12 weeks treatment in the double-blind study, will be offered the 
opportunity to enter open-label Study A4250-008.  A patient who withdraws from treatment 
prematurely and will not enter Study A4250-008 will be requested to complete all visits and 
safety assessments per the current protocol (A4250-005).  As a minimum, patients will have 
EOT (Visit 9) assessments at the time of withdrawal and a follow-up assessment (Visit 10) 
28 days following the last dose of study drug, and the patient will be recorded as withdrawn. 
7.2.4 
Study Termination by Sponsor 
This study may be terminated at any time by Albireo if serious side effects should occur, if 
the investigator does not adhere to the protocol; or if, in the sponsor's judgment, there are no 
further benefits to be achieved from the study.  In this event, Albireo/designee will inform the 
study investigators, institutions, and all regulatory authorities. 


 
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Albireo may temporarily or permanently discontinue the study at an investigative site at any 
time for safety, ethical, compliance, or other reasons.  If this is necessary, Albireo will 
endeavor to provide advance notification to the site.  If a site or the study is suspended or 
discontinued, the investigator/investigative staff will be responsible for promptly informing 
the Independent Ethics Committee (IEC).  If required by local regulations, Albireo/designee 
will be responsible for informing the IEC and the regulatory authority of study or site 
discontinuation.  In such an event, all study data and unused study drug must be returned to 
Albireo. 


 
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8 
TREATMENT OF PATIENTS 
8.1 
Identity of Study Drug 
A4250 and placebo will be supplied as capsules for oral administration.  White opaque 
capsules filled with pellets containing A4250 or placebo will be provided.  Two different 
capsule sizes will be available, as follows: 
• Capsule size 0 that can be opened 
• Capsule size 3 that can be opened or swallowed intact 
The content of the A4250 and placebo capsules will be identical in appearance.  Capsule 
filling weight will also be identical for high dose, low dose, and placebo.  Bottles with 
34 capsules will be given to the patient at each visit.  A patient who requires 2 or more 
capsules per day will be given multiple bottles.  Refer to the investigational product manual. 
8.2 
Administration of Study Drug 
Patients will be dosed with A4250 at a dose of 40 µg/kg/day, 120 µg/kg/day, or placebo for 
24 weeks.  Study drug will be dispensed to the patient at Visits 3, 4, 5, 6, 7, and 8, together 
with instructions on how to store and take the drug.  Study drug administration data, 
including whether each patient took each dose or partial doses of study drug, whether there 
were any delayed or missed doses, doses, and whether the capsule was opened or swallowed 
whole will be documented through the eDiary and transferred to the study database. 
A4250 should be taken in the morning, together with food.  On clinic visits days when 
laboratory assessments are conducted, study drug should be taken after the visit and after 
laboratory samples are taken.  Patients should not crush or chew the capsule(s).  When 
swallowing the capsule intact, the patient should administer the dose with a glass of water. 
For a patient unable to swallow capsule intact, the capsule can be opened and the contents 
sprinkled and mixed in a small amount of soft room-temperature yogurt, apple sauce, or fruit 
purée.  The amount of food should be less than what the patient/infant is expected to eat and 
should be followed by water.  
1. Hold the capsule over the prepared food, gently twist open and allow the content to fall 
into food 
2. Mix the entire capsule content with 1 or 2 tablespoons (15 to 30 mL) of soft food (apple 
sauce, fruit purée, or yogurt) 
3. Administer the mixture immediately after mixing. 
The number of capsules provided to the patient will be based on body weight at Visit 3.  At 
Visit 6, if a patient’s weight has shifted to another weight category (identified in Table 2), the 
number of capsules per day may be adjusted.   
 


 
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Table 2 
Dosing and Capsule Strength 
Body Weight 
(kg) 
Capsule 
Size 
Number of 
Capsules 
per Day 
Capsule 
Strength, 
Low Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Strength, 
High Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Size, 
Placebo 
(µg) 
5.0 to <7.5 
0 
1 
200 
200 
600 
600 
0 
7.5 to <12.5 
0 
2 
200 
400 
600 
1200 
0 
12.5 to <17.5 
0 
3 
200 
600 
600 
1800 
0 
17.5 to <19.5 
0 
4 
200 
800 
600 
2400 
0 
19.5 to <25.5 
3 
2 
400 
800 
1200 
2400 
3 
25.5 to <35.5 
3 
3 
400 
1200 
1200 
3600 
3 
35.5 to <45.5 
3 
4 
400 
1600 
1200 
4800 
3 
45.5 to 55.5 
3 
5 
400 
2000 
1200 
6000 
3 
>55.5 
3 
6 
400 
2400 
1200 
7200 
3 
8.3 
Study Treatment Packaging and Labeling 
8.3.1 
Packaging and Labeling 
The capsules will be packed in high-density polyethylene containers, with child-proof 
polypropylene caps.  Study drug capsules containing A4250 in the strength specified in 
Table 2 above will be manufactured. 
Packaging and labeling will be prepared to comply with applicable regulatory requirements. 
8.3.2 
Storage 
Treatment packs containing A4250 capsules should be stored and dispensed in accordance 
with regulations in their original containers.  The storage facility at the investigative site 
should be locked and the storage temperature should be between 15°C and 25°C. 
Patients/caregivers should be informed of appropriate storage conditions (i.e., room 
temperature, between 15°C and 25°C). 
Any deviations from the recommended storage conditions should be immediately reported to 
Albireo and the study drug should not be used until authorization has been given by Albireo. 
8.3.3 
Blinding and Randomization of Study Drug 
A double-blind design is employed so that both the investigators and the patients will be 
unaware of the treatment assignment during the study.  
After written informed consent is obtained from an eligible patient, an 8-digit patient 
identification number will be assigned.  The first 2 digits will denote the country, followed by 
a 3-digit site number and a 3-digit patient sequence number.  This number will be created and 
allocated by the Interactive Web Response System (IWRS), when the patient first enters the 
system at Screening.  The randomization codes will be computer-generated by 
 


 
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Biostatistics, and kept by a statistician independent from the project team.  The 
randomization will be done in blocks and stratified according to PFIC class (Type 1 and 2) 
and age class (6 months to 5 years, 6 to 12 years, and 13 to ≤18 years) to ensure approximate 
balance between dose schemes (1:1:1).  A separate randomization list will be prepared for the 
maximum 5 patients from Study A4250-003 regardless of stratification.  Randomization 
codes will be assigned sequentially as patients become eligible for randomization. 
Patients who are eligible for randomization will be assigned a unique 4-digit randomization 
number by IWRS.  This randomization number identifies which treatment will be allocated to 
the patient.  Patients who withdraw from the study after randomization visit are not to be 
replaced and their randomization number will not be reused.   
To ensure blinding, the study drug and the matching placebo have the same shape and size.  
Patients will receive capsule(s) of A4250 according to their dose group or capsule(s) of 
matching placebo once a day during the double-blind Treatment Period.  Labels on the study 
drug containers will not identify the treatment that a patient is randomized to.  Traceability of 
the treatment is ensured by the study drug number. 
The 5-digit study drug number will identify study drug packs and will be detailed on the 
study drug label.  Dispensing of study drug will be coordinated by IWRS.  
The system will assign a study drug number corresponding to the randomization arm.  The 
randomization number will be used in the background only to ensure no unblinding and will 
not be displayed to end-users in the IWRS. 
8.4 
Procedure for Breaking the Randomization Code 
Should a situation arise where unblinding is urgently required, i.e., only where knowledge of 
the study drug is required to adequately manage a life-threatening situation, the investigator 
at that study site may perform immediate unblinding through IWRS.  The investigator should 
make every effort to discuss the rationale for emergency unblinding with the sponsor Medical 
Monitor or the 
 Medical Monitor prior to unblinding the individual patient. 
The study site investigator and appropriate 
 project team members will be authorized to 
access the emergency unblinding functionality within the IWRS.  Emergency unblinding 
takes place in the IWRS via a dedicated emergency unblinding module.  The system will 
require the user to enter their emergency unblinding, which is typically provided at the same 
time login credentials are distributed.  The exact description of the treatment assigned to the 
individual patient then will be accessible to the user.  Once the randomization code is broken 
for a patient, he/she must be withdrawn from the study and all assessments and procedures at 
EOT should be performed.  As a follow-up, Visit 10 will be performed according to the time 
schedule.  Once a randomization code has been broken, the investigator must inform the 
 and sponsor Medical Monitor in writing within 24 hours. 


 
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8.5 
Patient Compliance 
The study nurse will monitor eDiary compliance by routine review of the CRF health 
website.  If both diary entries on a day are missing during, the study nurse will call the 
patient/caregiver to remind them to complete all scheduled entries.  Any noncompliance will 
be documented and explained in the source documents and in the eCRF. 
Study drug compliance will also be checked through diary responses.  Additionally, patients 
will return all unused study drug at Visits 4, 5, 6, 7, 8, and 9.  The study site staff will count 
all returned drug, assess compliance, and record details in the eCRF. 
Treatment Compliance = 100 × ((Number of study drug dispensed – number of study drug 
returned)/number of study drug that should be taken). 
Treatment compliance between 80% and 120% will be acceptable. 
8.6 
Study Drug Accountability 
Records shall be maintained of the delivery of study treatment(s) to the study site(s), of the 
inventory at the study site(s), of each use of the study treatment(s) for each patient, and of the 
return of unused study treatment(s) to Albireo. 
These records shall include dates, quantities, batch numbers, expiry dates, and the unique 
code numbers assigned to the study drug and to the study patients. 
The investigator will be responsible for ensuring that the records adequately document that 
the patients were provided the quantities specified in the protocol and that all study drug 
received from Albireo is reconciled.  
8.7 
Concomitant Therapy and Prohibited Medications 
The investigator will note all ongoing medication and any medication recently stopped 
(within 1 month prior to Visit 1) in the eCRF.  At Visits 2 through 10, all changes in 
medication (stopping or starting new medication or changes in dose) will be recorded in the 
eCRF.  All medications (prescribed or over the counter) for pruritus and sleep will be 
recorded. 
All medications taken by a patient within 3 months prior to the first intake of study drug are 
regarded as prior medication. 
All medications taken by a patient on or after the first intake of study drug, and which 
continue to be taken during the study, are regarded as concomitant medication.  
Prohibited medications are listed in Appendix 1. 
 


 
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9 
ASSESSMENT OF EFFICACY 
9.1 
Efficacy Endpoints 
9.1.1 
Primary Efficacy Endpoint 
The primary efficacy endpoints are as follows: 
• European Union (EU) and rest of the world (RoW): Proportion of patients 
experiencing at least a 70% reduction in fasting s-BA concentration from baseline to 
the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 
weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization 
and the previous visit (Clinic Visit 2).  The end value is the average of the values at 
Weeks 22 and 24 after the start of double-blind treatment. 
• United States (US): Change in pruritus, as indexed by caregiver-reported observed 
scratching, from baseline to Week 24 as measured by the worst scratch of the Albireo 
ObsRO instrument (Appendix 2).  Scores for the 14 days baseline and post-treatment 
intervals will be calculated by averaging the worst scratching scores from each day in 
the interval. 
9.1.2 
Secondary Efficacy Endpoint 
Secondary efficacy endpoints include the following:  
• EU and RoW: Change in pruritus, as indexed by caregiver-reported observed 
scratching, from baseline to Week 24 as measured by the worst scratch of the Albireo 
ObsRO instrument (Appendix 2).  Scores for the 14 days baseline and post-treatment 
intervals will be calculated by averaging the worst scratching scores from each day in 
the interval. 
• US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment. 
• All Regions: 
• All secondary endpoints are compared to placebo. 
o Change from baseline to Week 24 in fasting s-BA 
o Change from baseline to Week 24 in serum ALT concentration 
o Change in growth from baseline to Week 24, defined as the linear deficit 
(weight for age, and body mass index [BMI]) compared to the national growth 
curves (Z-score, standard deviation [SD] from P50)  
o Proportion of patients achieving meaningful reduction in caregiver-reported 
observed scratching at Week 24 


 
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o Change from baseline to Week 24 in sleep parameters measured with the 
Albireo PRO and ObsRO instruments 
o Change from baseline to Week 24 in patient-reported itch severity as 
measured by the average daily score of the Albireo PRO instrument 
(Appendix 2); only patients ≥8 years of age will complete the Albireo PRO 
instrument  
o Number of patients undergoing biliary diversion surgery or being listed for 
liver transplantation 
9.1.3 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• Proportion of patients achieving meaningful reduction in patient-reported itch severity at 
Week 24 
• Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, 
caregiver, and clinician via the PGIC items (Appendix 3) 
• Change from baseline in fasting s-BA at Weeks 4 and 12 
• Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
to Weeks 4, 12, and 24 as measured by the worst scratch.  Scores for the 7 worst 
scratching days of the last 14 days at baseline and post-treatment intervals will be selected 
• Change from baseline in patient-reported itch severity and observer-reported scratching at 
Weeks 4 and 12  
• Change from baseline to Week 24 in patient-reported and observer-reported night time 
itching and scratching severity scores, respectively 
• Change from baseline to Week 24 in pooled pruritus score including observer-reported 
scratching for patients <8 years of age and patient-reported itch severity for patients 
≥8 years of age 
• Change from baseline to Week 24 in additional patient-reported and observer-reported 
sleep parameters (e.g., tiredness and number of awakenings) 
• Change from baseline to Week 24 in PedsQL (Appendix 4) 
• Change from baseline in serum ALT concentration at Weeks 4 and 12 
• Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• Change from baseline in AST at Weeks 4, 12, and 24 
• Change from baseline for other biochemical markers and measures of bile acid synthesis 
(autotaxin, p-C4) at Week 24 


 
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• Change in pediatric end-stage liver disease/model for end-stage liver disease 
(PELD/MELD) score, AST to platelet ratio index (APRI) score, and Fib-4 score from 
baseline to Week 24 
• Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® 
(where available) 
• Liver-related mortality and liver decompensation events 
• All-cause mortality 
9.2 
Efficacy Assessments 
9.2.1 
Serum Bile Acids 
Blood samples for analysis of fasting total s-BA will be drawn at all visits.  Patients will fast 
(water intake only is permissible) for at least 4 hours prior to the collection of samples for 
s-BA.  Exceptions can be made for infants, <12 months of age, if unable to fast for the full 
4 hours.  Any visit at which a bile acid sample result is unreportable, an additional 
unscheduled visit for a repeat sample collection may be scheduled.  All s-BA results during 
the treatment period and follow-up will be blinded.  Samples will be processed and 
transported to a central laboratory per instructions in the laboratory manual. 
9.2.2 
Itching, Scratching and Sleep Score 
Itching, observed scratching, and sleep disturbance will be recorded twice each day via 
eDiary.  Patients and/or caregivers will be instructed to complete the eDiary every day in the 
morning after the patient wakes and in the evening just before the patient goes to sleep 
throughout the study.  Patients and caregivers will receive a formal training session during 
Visit 1. 
The eDiary includes Albireo ObsRO and PRO items.  Patients <8 years of age will not be 
asked to complete the Albireo PRO items; only the Albireo ObsRO will be completed by 
caregivers of patients in this age group.  Older patients, 8 to 17 years of age, will complete 
the Albireo PRO items and the caregiver will complete the Albireo ObsRO items.  The 
Albireo PRO items assess severity of itch, aspects of sleep disturbance (morning diary only), 
and tiredness.  For patients 8 to 12 years of age, the caregiver will read the Albireo PRO 
items along with the child and record the child’s response.  A guide will be provided to the 
caregivers that provides standardized explanations of the Albireo PRO items, in case the 
patient is confused or requires clarification about the meaning of a question.  The Albireo 
ObsRO items assess severity of observed scratching, aspects of observed sleep disturbance 
(morning diary only), and observed signs of tiredness (evening diary only).  The Albireo 
ObsRO and PRO scratching and itch severity items use 0 to 4 response scales, where each 
response is distinguished by a unique facial expression, verbal anchor, number, and color 
code (Appendix 2).  


 
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A daily score for the Albireo ObsRO scratching item will be calculated by selecting the 
maximum scratching severity score from the 2 ratings for each day.  A daily score will be 
considered missing if both of the daily assessments are missing.  A 14-days score will be 
calculated by averaging the daily scratching scores.  A 14-days score will be considered 
missing if >4 out of 7 days a week of data are missing.  The same approach will be used to 
calculate a patient-reported itch severity score. 
9.2.3 
Growth 
Growth will be measured as height (velocity) and weight (Z-score) using a certified weight 
scale.  BMI will be calculated by weight (kg) / height (m)2.  Change will be defined as linear 
growth deficit (weight and BMI for age) compared to national growth curves (Z-score, SD 
from P50). 
9.2.4 
Quality of Life Questionnaire (PedsQL) 
Patients and caregivers will be asked to complete a QoL questionnaire (PedsQL) at Visit 3 
and Visit 9.  Details of the questions included on the questionnaire are provided in 
Appendix 4. 
9.2.5 
Biomarkers and Other Laboratory Samples 
Blood samples for autotaxin and p-C4 will be drawn at Visits 3, 4, and 9 only for children 
with body weight ≥10 kg.  Samples will be processed and transported to a laboratory per 
instructions in the laboratory manual. 
9.2.6 
PELD/MELD Score 
At randomization and end of treatment, PELD score will be calculated for children up to 
12 years of age.  For children 13 years or older, the MELD score will be calculated. 
PELD Score = 4.80 * ln(total bilirubin in mg/dL) + 18.57 * ln(INR) - 6.87 * ln(albumin in 
g/dL) + 4.36 (if patient <1 year: scores for patients listed for liver transplantation before the 
patient's first birthday continue to include the value assigned for age (<1 year) until the 
patient reaches the age of 24 months) + 6.67 (if the patient has growth failure [≤2 SD]) 
Laboratory values less than 1.0 will be set to 1.0 for the calculation of the PELD score. 
MELD Score = 9.57 * ln(creatinine in mg/dL) + 3.78 * ln(total bilirubin in mg/dL) + 
11.2 * ln(INR) + 6.43 
Laboratory values less than 1.0 will be set to 1.0 and serum creatinine values greater than 
4.0 mg/dL will be set to 4.0 for calculation of the MELD score. 
9.2.7 
Fibroscan® 
Where available, Fibroscan® will be performed as per institution standard practice at Visits 3 
and 9. 


 
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9.2.8 
Markers of Fibrosis 
APRI score and Fib-4 score will be calculated at randomization and at end of treatment. 
APRI = [(AST in U/L) / (AST ULN in U/L)] / (Platelets in 109/L) 
Fibrosis 4 Score = (Age*AST in U/L) / (Platelets in 109/L * √(ALT in U/L)). 
9.2.9 
Global Impression of Change and Global Impression of Symptom Measures 
Patients (≥8 years of age), caregivers, and clinicians will complete the PGIC and the PGIS 
measures (Appendix 3) at randomization (Visit 3; PGIS only), Visits 4 and 6, and at EOT 
(Visit 9). 
The PGIC items assess change in itch (patient version), scratching (caregiver and clinician 
versions), and sleep (all versions) since starting the study drug.  The PGIS items assess itch 
(patient version), scratching (caregiver and clinician versions), and sleep (all versions) in the 
past week.  Caregivers and clinicians will complete the PGIC and PGIS for all patients; those 
patients ≥8 years of age will complete the patient version. 


 
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10 
ASSESSMENT OF SAFETY 
The timing and frequency of safety assessments are described in Section 7.1.3 and in Table 1 
above. 
The primary safety analysis for this study will include the incidence of total 
treatment-emergent adverse events (TEAE) and TEAEs categorized by causality, severity, 
and seriousness assessments made by the investigator by comparing study drug exposure to 
placebo. 
Trends in safety will also be evaluated for the following assessments: 
• Physical examinations 
• Concomitant medications 
• Vital signs 
• Laboratory test results (including clinical chemistry, hematology, urinalysis, AFP, and 
fat-soluble vitamins A, D, and E) 
• Abdominal ultrasound 
• Discontinuations due to AEs 
10.1 
Adverse Events 
10.1.1 
Definitions and Investigator Assessments 
An AE is defined as any untoward medical occurrence in an enrolled patient regardless of 
causal relationship with study drug.  An AE can therefore be any clinically significant 
unfavorable and unintended sign, symptom, or disease that occurs once a patient is enrolled 
(ICF is signed) in the study until the patient is discharged from the study, whether or not 
related to the study drug. 
10.1.1.1 
Clinical Significance 
Clinical significance is defined as any abnormal finding that results in further clinical 
investigation(s), treatment(s), or the diagnosis of new condition.  Patient-reported events and 
protocol-mandated laboratory values, vital signs, and physical examination findings can be 
considered clinically significant (i.e., an AE) if there is a deterioration as compared to 
baseline.  Examples of clinically significant worsening from baseline could include, but is not 
limited to, events causing withdrawal from the study and events requiring medical 
intervention outside of the study causing apparent clinical manifestations, or judged relevant 
by the investigator.   
10.1.1.2 
Serious Adverse Events 
Serious criteria are applied by the investigator to each AE as specifically defined below.  
These criteria are used to determine whether an AE is serious or non-serious.  The assessment 
is made independently of severity assessment (Section 10.1.1.3).  For example, the 


 
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development of a severe rash that occurs after signing of informed consent may not meet 
serious criteria as defined below and therefore would be considered a severe, non-serious AE. 
Any AE that meets any 1 of the following 6 criteria is considered an SAE: 
• The outcome of the AE is death 
• The AE is immediately life-threatening.  Life-threatening means that the patient is, in the 
opinion of the investigator, at immediate risk of death from the reaction as it occurred.  
This does not include an AE that, if more severe, might have caused death 
• The AE results in persistent or significant disability/incapacity.  Disability means a 
substantial disruption of a person's ability to conduct normal life functions 
• The AE requires or prolongs hospitalization  
• The AE results in a congenital anomaly/birth defect 
• The AE is an important medical event.  Important medical events may meet serious 
criteria should the investigator assess that they may significantly jeopardize the patient, 
represent a significant hazard, or requires medical/surgical intervention to prevent one of 
the outcomes listed above.  Examples of potential SAEs based on this criteria include, but 
are not limited to, allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions even if they do not result in inpatient 
hospitalization, or the development of drug dependency and drug abuse. 
10.1.1.3 
Severity Assessment 
Severity assessments are based on the intensity of the event in relation to expectation.  The 
investigator will assess the intensity of AEs based on the following definitions: 
• Mild (awareness of sign or symptom, but easily tolerated) 
• Moderate (discomfort sufficient to cause interference with normal activities) 
• Severe (incapacitating, with inability to perform normal activities) 
Severity is a measure of intensity where seriousness is defined by the criteria outlined in 
Section 10.1.1.2.  An AE of severe intensity need not necessarily be considered serious.  For 
example, nausea that persists for several hours may be considered severe nausea, but not meet 
serious criteria, and therefore would be assessed as a severe AE but not an SAE. 
10.1.1.4 
Causality Assessment 
The investigator determines the causality of all AEs to the study drug using medical judgment 
and considering all relevant factors such as (but not limited to) the underlying study 
indication, coexisting disease, concomitant medication, relevant history, pattern of the AE, 
temporal relationship to the study drug, and de-challenge or re-challenge.  The causality 
assessment of the AE/SAE is to be made as follows. 


 
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Related to study drug (possibly, probably, or definitely related) 
Based on medical judgment, there is at least a reasonable possibility that the study drug 
caused the event; one or more of the following criteria apply: 
• The event follows a reasonable temporal sequence from administration of study drug 
• The event could not be reasonably attributed to the known characteristics of the patient's 
clinical state, environmental or toxic factors, or other modes of therapy administered to 
the patient 
• The event follows a known pattern of response to study drug 
• The event disappears or decreases on cessation or reduction in dose of the study drug.  (It 
should be noted that in some situations an AE will not disappear or decrease in intensity 
upon discontinuation of study drug despite other clear indications of relatedness) 
• The event reappears or worsens when the study drug is re-administered 
Unrelated to study drug (unlikely or unrelated) 
Based on medical judgment there is no reasonable possibility that the study drug caused the 
event; one or more of the following criteria apply: 
• The event does not follow a reasonable temporal sequence from administration of study 
drug 
• The event could be reasonably attributed to the known characteristics of the patient's 
clinical state, concurrent illness, environment or toxic factors, or other modes of therapy 
administered to the patient 
• The event does not follow a known pattern of response to study drug 
• The event does not disappear or decrease on cessation or reduction in dose of the study 
drug, and it does not reappear or worsen when the study drug is re-administered 
10.1.2 
Recording of Adverse Events 
It is the investigator’s responsibility to assess whether each untoward event is a clinically 
significant worsening from baseline, thereby considered an AE.  For all AEs, the severity, 
seriousness, and causality to study drug for each AE as outlined in Section 10.1.1 will be 
assessed and recorded in the eCRF. 
All serious and non-serious AEs are collected once the caregiver/patient has signed the ICF 
and until the post-treatment follow-up (Visit 10) or 28 days after the last dose of study drug. 
Any AEs or SAEs that are unresolved at the patient’s last AE assessment in the study are 
followed up by the investigator until resolution or stabilization up to the database lock and 
recorded in the eCRF.  Albireo retains the right to request additional information for any 
patient with ongoing AE(s) or SAE(s) at the end of the study. 


 
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TEAEs are defined as any AE that occurs after randomization (Day 0).  All AEs that occur in 
the Screening Period, i.e., after enrollment and prior to randomization, will be collected on 
the eCRF as non-treatment-emergent AEs. 
If there is a clinically significant deterioration of a laboratory value/vital sign or other routine 
study assessment that is associated with a diagnosis, the clinical diagnosis will be reported as 
an AE and the associated signs and symptoms will be considered additional information 
unless the sign or symptom is more severe than expected given the diagnosis.  For example, if 
an investigator diagnoses a study patient with hepatitis during the study period, hepatitis 
would be considered the AE and the concomitant signs and symptoms of abdominal pain, 
vomiting, and elevated ALT and AST would not be reported separately unless, in the opinion 
of the investigator, one of these signs or symptoms is more severe than expected and 
therefore a separate AE assessment is indicated. 
10.1.3 
Recording and Reporting of Serious Adverse Events 
Every SAE (regardless of severity and causality) that occurs once the caregiver/patient has 
signed the ICF and until the post-treatment follow-up (Visit 10), and 28 days after the final 
dose of study drug, should immediately and not later than within 24 hours of knowledge of 
the event, be reported by the investigator or delegate in the SAE report form.    
Report of a SAE must include at least the following information: 
• Patient identification information (study number, site number, initials, and date of birth 
[as per local country requirements for data protection]) 
• The last study drug administration date 
• The diagnosis of the event with the description (or a brief description of 
signs/symptoms/clinical course if the diagnosis is not available) and the date of onset 
• The action used to treat the event (i.e., treatment medications, temporary discontinuation) 
• The reason(s) for considering the event serious 
• The relationship of the event to the study drug or to the study procedure (e.g., the 
investigator's assessment of causality) 
• A brief narrative of the SAE 
Follow-up reports including all new information obtained of the subsequent course of the 
SAE must be prepared and the information collected in the SAE submitted to 
 
 (Pharmacovigilance and Safety Services Department) by e-mail within 24 hours of 
knowledge of the event. 
The pharmacovigilance manager (PVM) may contact the investigator to obtain further 
information on a reported SAE.  The investigator/investigative staff must respond to any 
request for follow-up information or answers to questions regarding the SAE within the same 
timelines as for initial reports. 


 
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The PVM reports the occurrence of the SAE and follow-up to the Albireo medical monitor 
for medical assessment of the case. 
Should an outcome of death occur within the study period or within 28 days after the last 
administration of study drug, an SAE form should be completed including the main and 
contributory causes of death. 
All SAE reports must be e-mailed to the following e-mail address within 24 hours: 
 
 
If email is unavailable, SAEs may be transmitted via fax to the following number: 
 
In a study-related medical emergency situation, when the assigned medical monitors for the 
study cannot be reached by a caller, an on call physician can be reached 24 hours per day, 
7 days per week, via the 
 call center.  The following number is a chargeable telephone 
number allowing a global reach from both landlines and mobile phones and the internet page 
indicated below contains a list of country-specific toll-free telephone numbers that is 
provided.  It should be noted that not all countries globally have access to toll-free numbers 
as indicated on the “24/7 Medical Help desk” index.  Countries without toll-free numbers 
need to dial the chargeable number as indicated below.  Furthermore, toll-free numbers might 
not be available from mobile phones. 
 
 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions 
Suspected unexpected serious adverse reaction (SUSAR) is an SAE that occurs in a patient, 
the nature or severity of which is not expected per the applicable product information (e.g., 
the IB for an unauthorized investigational product or summary of product characteristics for 
an authorized product).  
Reporting and tracking of SUSARs will be in accordance to all applicable competent 
authority regulations.  The Ethics Committee and all investigators involved in this study will 
be informed according to national and international requirements. 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments 
10.2.1 
Laboratory Assessments 
Samples will be collected for clinical chemistry, hematology, and urinalysis testing at the 
time points specified in Table 1. The parameters assessed are presented in Table 3. 


 
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Blood for AFP will be drawn at Visits 3 and 9 and for fat-soluble vitamins at Visits 2, 3, 
and 9. 
A serum pregnancy test will be performed at Visit 1 and Visit 9 if the patient is entering the 
A4250-008 study. A urine pregnancy test will be collected at all other visits for girls who 
have reached menarche.  If a urine pregnancy test is positive, a serum pregnancy test should 
be performed to confirm the pregnancy.  Study drug administration will be held pending the 
confirmatory results.  If the serum pregnancy test is negative, the patient can resume dosing.  
If the serum pregnancy test is positive, the patient should be discontinued (see 
Section 10.2.9). 
All samples will be processed and transported to a laboratory per instructions in the 
laboratory manual. 
The observed values will be recorded and assessed as “normal” or “abnormal not clinically 
significant” or “abnormal clinically significant”. 
Additional safety blood samples may be needed due to follow-up of an abnormal value or 
analysis failure.  The blood samples collected for safety laboratory analysis will be destroyed 
after the analyses have been completed.  
Table 3 
Routine Laboratory Parameters 
Clinical Chemistry 
Hematology 
Urinalysis 
• 
Albumin 
• 
ALT 
• 
Alkaline phosphatase  
• 
AST 
• 
Bilirubin – total and direct 
• 
Calcium 
• 
Chloride 
• 
Creatinine 
• 
Creatine kinase 
• 
Gamma-glutamyl transferase 
• 
Potassium 
• 
Sodium 
• 
Hematocrit 
• 
Hemoglobin 
• 
Platelet count 
• 
Red blood cell count 
• 
White blood cell count 
• 
Blood 
• 
Glucose 
• 
Ketones 
• 
Leukocytes 
• 
Nitrites 
• 
pH 
• 
Protein 
 
10.2.2 
Individual Patient Safety Monitoring 
10.2.2.1 
Liver Monitoring 
Strategies to monitor markers of liver disease throughout the study are outlined below where 
the ULN will be based on central laboratory reference values for age and gender. 
If isolated transaminase elevations are observed, defined as: 
1. Normal bilirubin AND absence of clinical hepatitis symptoms AND 


 
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• ALT or AST ≥5 × ULN (if normal at baseline) or an absolute threshold of 800 U/L, 
whichever comes first  
• OR ALT or AST ≥3 × baseline (if abnormal at baseline) or an absolute threshold of 
800 U/L, whichever comes first 
Then: 
a) Repeat liver profile (AST, ALT, bilirubin, and prothrombin time [PT] or INR) within 
2 to 3 days 
b) Evaluate creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) 
c) As needed (for example, persistent ALT/total bilirubin elevation or worsening of liver 
function), consider evaluation for alternative etiologies 
Monitor the patient using close observation found in Section 10.2.2.2 
If any ONE of the following criteria are met: 
1. Transaminases (ALT or AST ≥3 × baseline or 800 U/L, whichever comes first) AND 
bilirubin (total bilirubin > 2 × ULN) elevations 
2. Transaminase elevations alone (ALT or AST >10 × ULN or 5 × baseline or absolute 
threshold of 800 U/L, whichever comes first) in presence of normal LDH and CPK 
3. Cholestatic marker elevations (alkaline phosphatase [ALP] or gamma-glutamyl 
transferase >2 × baseline) without alternative explanation 
4. Total bilirubin increased, unrelated to hemolysis (elevated reticulocyte count) or 
established genetic diseases, such as Gilbert’s syndrome 
a. Doubling if total bilirubin was <3 mg/dL at baseline  
b. OR Increase by >3 mg/dL if total bilirubin was ≥3 mg/dL at baseline 
5. INR increase refractory to vitamin K administration 
a. Increase by >1.5 if INR was normal at baseline  
b. OR Increase by >0.4 if INR was abnormal at baseline 
6. Any increase in total bilirubin and transaminases if accompanied by EITHER a symptom 
of clinical hepatitis (e.g., vomiting, nausea, right upper quadrant pain) OR immunological 
reaction (rash or >5% eosinophilia) 
Then: 
a) Interrupt study medication 
b) Initiate drug-induced liver injury work-up for alternative etiologies 
c) Repeat liver profile (AST, ALT, total bilirubin, direct bilirubin, ALP) and PT or INR 
within 48 to 72 hours 


 
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d) Monitor the patient using close observation found in Section 10.2.2.2 
e) If a patient lives in a remote area, they may be tested locally and the results 
communicated to the investigator site promptly 
10.2.2.2 
Close Observation 
• Repeat liver enzyme and serum bilirubin tests two or three times weekly.  Frequency 
of re-testing can decrease to once a week or less if abnormalities stabilize or the trial 
drug has been discontinued and the subject is asymptomatic  
• Obtain a more detailed history of symptoms and prior or concurrent diseases 
• Obtain a history of concomitant drug use (including non-prescription medications and 
herbal and dietary supplement preparations), alcohol use, recreational drug use, and 
special diets  
• Rule out alternate etiology including acute viral hepatitis types A, B, C, D, and E; 
autoimmune or alcoholic hepatitis; non-alcoholic steatohepatitis; hypoxic/ischemic 
hepatopathy; and biliary tract disease 
• Obtain a history of exposure to environmental chemical agents.  
• Obtain additional tests to evaluate liver function, as appropriate (e.g., INR, direct 
bilirubin) 
• Consider gastroenterology or hepatology consultations. 
10.2.2.3 
De-challenge/Re-challenge for Liver and Clinical Hepatitis Monitoring 
1. Re-challenge is not recommended: 
a. If a patient has had possible/probable drug-induced liver injury 
b. If a decompensation event has occurred (i.e., variceal hemorrhage, ascites, hepatic 
encephalopathy, etc.). 
2. If due to underlying cholestatic liver disease variability or another alternative etiology is 
identified AND liver tests returned to baseline, re-challenge may be considered after 
consultation with the sponsor Medical Monitor. 
3. If the ALT/total bilirubin elevations are observed after re-challenge, then repeat 
re-challenge is discouraged. 
If patient is permanently discontinued, monitoring should be continued as outlined in 
Section 10.2.2.5.  
10.2.2.4 
Diarrhea 
Study drug should be discontinued if a patient develops diarrhea with at least 1 of the 
following concomitant signs or symptoms: grossly bloody stools, vomiting, dehydration 


 
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requiring treatment with oral or intravenous rehydration and/or electrolyte imbalances, fever 
(≥38 C) and/or the diarrhea persists for 7 or more days. 
If there is gross blood in stool, INR and platelets should be measured.  
Study drug will be reintroduced (re-challenge) when the symptoms have resolved.  If the 
diarrhea re-occurs within 1 week after the re-challenge with no alternate etiology, the patient 
will be permanently discontinued and monitored as outlined in Section 10.2.2.5. 
10.2.2.5 
Monitoring after Permanent Discontinuation of Study Drug Due to Safety 
Once study drug is permanently discontinued for liver, hepatitis, diarrhea, or other severe AE 
related to study drug, the patient will be monitored weekly until the laboratory and clinical 
parameters have normalized/stabilized. 
10.2.3 
Demographics/Medical and Surgical History 
Demographic information per country regulations (age, full date of birth, gender, race, and 
ethnicity), along with medical and surgical history, will be obtained and recorded in the eCRF 
at Visit 1. 
Medical and surgical history will be entered in the eCRF at Visit 1.  This includes date of 
diagnosis of PFIC, prior investigational medications for PFIC, historical (3 months prior to 
Screening) LFT values (AST, ALT, and total bilirubin), ongoing medication, any surgery 
performed, any other diagnosis, and historical biopsy data.   
If a liver biopsy has been performed within 1 year prior to Screening, or during the study, the 
results will be recorded in the eCRF. 
10.2.4 
Clinical Genetic Testing 
If confirmatory clinical genetic testing for PFIC Type 1 or 2 has been performed prior to 
Screening Visit 1, the confirmatory clinical genetic testing laboratory report will be verified 
by a central reader to determine eligibility. 
Regardless of previous genetic testing, a blood sample will be drawn at Visit 1 for all patients 
for DNA extraction.  Genetic testing will only be performed on a patient’s extracted DNA if 
the previous clinical genetic result is equivocal, unavailable, or unobtainable.  In such cases, 
screening for pathologic biallelic variations in the ATP8B1 or ABCB11 genes will be 
performed if patient meets remaining inclusion and exclusion criteria and only after sponsor 
case review.  No other diagnostic genetic testing will be offered. 
10.2.5 
Physical Examination 
A physician or suitably trained qualified assistant will perform a complete physical 
examination, including voluntary photography, at Visits 1, 3, and 9.  A skin examination will 
be conducted at Visits 1, 3, 4, 6, 9, and 10 (see Table 1).  


 
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A complete physical examination will include assessment of general appearance, eyes, ears, 
nose, throat, head/neck/thyroid, lymph nodes, cardiovascular, lungs/chest, abdomen, 
genitourinary, extremities, skin, musculoskeletal, neurologic, and other.  
Skin will be examined thoroughly and excoriations/scratch marks recorded.  
10.2.6 
Vital Signs 
Evaluation of vital signs will be performed at all visits.  This includes blood pressure (systolic 
and diastolic), pulse, respiratory rate, temperature, height (or length depending on age), and 
body weight (using a certified weight scale) at clinic visits.  BMI will be calculated. 
10.2.7 
Abdominal Ultrasound 
Ultrasound of the liver and spleen will be performed at randomization (Visit 3) and at EOT 
(Visit 9).  Liver size, echogenicity, and presence of masses/nodules as well as spleen size will 
be recorded. 
10.2.8 
Overdose 
A4250 is minimally absorbed and has a very low systemic availability.  Based on toxicology 
data, for study purposes any dose exceeding a total of >3 mg/kg body weight of A4250 taken 
as a single dose or as a cumulative dose within 24 hours is defined as an overdose.   
The no-adverse-effect dose level in the most sensitive species (defined as 20 mg/kg/day) in 
the rat 1 month toxicity study gives a human equivalent dose of approximately 194 mg/day in 
a 60 kg person.  This dose is approximately 10-fold higher than the maximum possible dose 
predicted in human studies (20 mg).  
The investigator/investigative staff should immediately and not later than within 24 hours of 
knowledge of the overdose, be reported by the investigator or delegate on the SAE report 
form.  In the event of an A4250 overdose, the patient should be monitored closely. 
10.2.9 
Pregnancy 
If a pregnancy is discovered in a female patient enrolled in the study before the end of dosing, 
the patient will be immediately discontinued from the study and will attend the same visits as 
a prematurely withdrawn patient.  If the pregnancy is discovered in a female patient enrolled 
in the study after the end of dosing, the patient will continue in the study per protocol.  If a 
pregnancy occurs in a male patient’s partner at any time during the study, the pregnancy 
should also be reported and followed. 
Pregnancy is not considered to be an AE.  However, if the patient has been dosed with the 
study drug, the pregnancy must be reported on the Paper Pregnancy Notification Form 
immediately within 24 hours after recognition to Albireo/Safety Manager (please refer to 
Section 10.1.3).  Date of exposure and as far as possible, details of the period of gestation at 
the time of exposure must be given. 


 
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The pregnancy should be followed to determine outcome, including spontaneous termination, 
details of birth, and presence of any birth defects, congenital anomalies or newborn or 
maternal complication.  The infant who was exposed in-utero will be followed for up to 
2 years after delivery.  Individual cases with an abnormal outcome in association with the 
study drug should be reported on an expedited basis, i.e., reported rapidly to a competent 
authority. 


 
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11 
STATISTICAL EVALUATION 
All personnel involved with the analysis of the study will remain blinded until database lock 
for the double-blind Treatment Period and until major protocol violations have been 
identified. 
All statistical analyses will be performed using SAS version 9.3 or higher. 
11.1 
Sample Size and Power 
For the EU and RoW, the primary analysis will relate to the bile acid responder endpoint (at 
the 1-sided 1.25% level).  For the US, the primary analysis will relate to the pruritus endpoint 
(at the 1-sided 1.25% level).  The aim is to enroll 60 to 70 patients in order to get at least 
60 evaluable patients. 
Assuming 60% responders (patients experiencing at least 70% reduction in fasting s-BA 
concentration or reaching ≤70 µmol/L) in the A4250 dose group and 10% responders in the 
placebo group, approximately 20 patients per group are required to achieve a power of 84% 
with a 1.25% 1-sided Type-1 error. 
For pruritus, with approximately 20 patients per treatment group, the power is 83% given an 
assumed treatment effect difference of -1.0 in change from baseline (-1.6 for A4250 
and -0.6 for placebo) on the Albireo ObsRO scratching score with a SD of 0.95 and a Type I 
error of 1.25% (1-sided). 
11.2 
Statistical Methods 
11.2.1 
Statistical Analysis Sets 
Safety Analysis Set 
The safety analysis set will consist of all patients who received at least 1 dose of study drug.  
Patients will be analyzed according to the treatment they actually received.  The safety 
analysis set will be the primary analysis set for safety analyses.  
Full Analysis Set 
The full analysis set (FAS) will consist of all randomized patients who received at least 
1 dose of assigned treatment.  Patients will be analyzed in the treatment group they were 
randomized to even if they received incorrect study drug.  The FAS will be the primary 
analysis set for efficacy analyses. 
Per Protocol Analysis Set 
The per protocol (PP) analysis set is a subset of the FAS, and will consist of all randomized 
patients for whom no major protocol violation (no violation which may affect the study 
efficacy outcome) is documented.  The PP analysis set will provide supportive data for the 
efficacy analyses. 
Allocation of patients to the PP analysis set will be performed before unblinding of the study. 


 
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11.2.2 
Methods of Statistical Analyses 
11.2.2.1 
General Principles 
The primary objective of this study is to show superiority in efficacy of 
A4250 (120 µg/kg/day and 40 µg/kg/day) compared to placebo in patients with PFIC Types 1 
and 2. 
In the EU and RoW, for the primary efficacy variable fasting bile acid responder endpoint (at 
least a 70% reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the end 
of treatment), the null hypothesis will be tested against the alternative hypothesis (α = 0.0125, 
1-sided). 
The test will be performed at a Type I error rate of ≤0.0125 1-sided, as described above, 
based on the least squares means of the treatment difference in mean probability scale. 
To strictly control overall Type I error rate across the dose groups and also across the primary 
and the main secondary endpoint to a 1-sided 2.5% level, a Bonferroni-based parallel 
gate-keeping procedure will be used.  In this procedure the total significance level of 2.5% 
(1-sided) will be split equally between the dose groups.  Only a dose group that is significant 
for the primary endpoint will be tested, at the nominal significance level of 1.25% (1-sided), 
for the main secondary endpoint (here the pruritus endpoint).  If the previous step was not 
successful, the analysis of the following step will be considered descriptive.  If the procedure 
reaches the secondary endpoint for both dose groups, these will be evaluated using the 
Bonferroni Holm procedure.  The multiple comparison procedure is presented in Figure 2. 
Figure 2 
Bonferroni Holm Multiple Comparison Procedure 
 
In the US, for the primary efficacy variable pruritus score endpoint (change from baseline to 
Week 24 in observer-reported scratching), the null hypothesis will be tested against the 
alternative hypothesis (α=0.0125, 1-sided). 


 
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The test will be performed at a Type I error rate of ≤0.0125 1-sided, as described above, 
based on the least squares means of the treatment difference in mean change. 
To strictly control overall Type I error rate across the dose groups and also across the primary 
and the main secondary endpoint to a one-sided 2.5% level, a Bonferroni-based parallel 
gate-keeping procedure will be used.  In this procedure the total significance level of 2.5% 
(1-sided) will be split equally between the dose groups.  Only a dose group that is significant 
for the primary endpoint will be tested, at the nominal significance level of 1.25% (1-sided), 
for the main secondary endpoint (here the bile acid responder endpoint).  If the previous step 
was not successful, the analysis of the following step will be considered descriptive.  If the 
procedure reaches the secondary endpoint for both dose groups, these will be evaluated using 
the Bonferroni Holm procedure. 
Other secondary and exploratory outcome variables shall provide supportive efficacy and 
safety information regarding the differences between the treatment groups.  No adjustments 
will be performed for multiple dose comparisons when testing the other secondary and 
exploratory efficacy variables. 
A more detailed statistical analysis plan (SAP) will be prepared and finalized before study 
data are locked and unblinded.  
If not otherwise specified, the baseline value of a parameter is defined as the last assessment 
of that parameter on or prior to the first intake of study treatment date. 
11.2.2.2 
Missing Data 
For efficacy variables, with repeated measures, a mixed model will be employed with no 
additional imputation for missing values, i.e., based on “observed cases”, except where 
otherwise specified. 
Follow-up and continued collection of efficacy data for patients who discontinue treatment 
will be made.  These data will be included in the sensitivity analysis for primary endpoints 
estimating a treatment effect taking loss of effect after discontinuation into account.  Because 
complete follow-up of all patients discontinuing treatment may not be possible, a method to 
replace missing data for patients who discontinue the study will be prespecified (in the SAP) 
taking into account loss of effect after discontinuation (for example multiple imputation). 
No other replacement of missing values will be planned. 
11.2.2.3 
Demographic and Baseline Characteristics 
Descriptive summaries of demographics and other baseline characteristics (including medical 
and surgical history) will be presented by treatment group and overall using the FAS.  
Prior medication will be summarized by treatment group and overall using the safety analysis 
set. 


 
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11.2.2.4 
Subject Disposition 
The following will be summarized (by treatment group and overall where applicable): 
• Patients enrolled (who signed the informed consent) 
• Patients randomized 
• Patients treated 
• Patients completing the study/withdrawing early (including withdrawal reason) 
Additionally, patients enrolled, included in the safety, in the FAS, and in the PP analysis set 
will be summarized by region. 
11.2.2.5 
Evaluation of Primary Efficacy Variables 
The responder endpoint based on change in fasting bile acids will be analyzed in terms of the 
proportion of patients achieving the event of interest.  Patients in need of rescue therapy, i.e., 
biliary diversion will be classified as non-responders after the biliary diversion.  
Generalized linear mixed modelling (GLMM) will be used with a logistic link, to allow for 
repeated assessments over time.  The model will include baseline value, treatment group, 
PFIC class, age class, region, visit, and visit*treatment group interaction.  The treatment by 
visit interaction will remain in the model regardless of significance.  Region will be included 
in the model as a random effect.  The contrasts of primary interest will be the treatment 
difference at Week 24; also, the difference at Week 4, Week 8, Week 12, and Week 18 will 
be estimated and analyzed.  An unstructured covariance matrix will be used to model the 
within-subject error.  Another covariance structure may be chosen if the fit of this structure 
fails to converge.  Model diagnostics checks will be made to ensure appropriate fit. 
The primary analysis of the change from baseline in pruritus score (indexed by 
observer-reported scratching) will be analyzed using a mixed model repeated measures 
(MMRM) analysis.  The contrast of primary interest will be treatment difference at Week 24 
but also the differences at Week 4, Week 8, Week 12, and Week 18 will be estimated and 
analyzed.  Baseline pruritus score will be included as covariate.  The model will include 
baseline value, treatment group, PFIC class, age class, region, visit, and visit*treatment group 
interaction.  The treatment by visit interaction will remain in the model regardless of 
significance.  Region will be included in the model as a random effect and will be defined on 
country level.  An unstructured covariance matrix will be used to model the within-subject 
error.  Another covariance structure may be chosen if the fit of this structure fails to 
converge. 
Non-parametric approach may be considered if more appropriate. 
The primary analyses will be based on the FAS. 
To assess the robustness of the primary efficacy analysis, the following analyses will be 
performed: 


 
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• Primary efficacy analysis using the PP set  
• Analyses to investigate the influence of missing values, for example, by using pattern 
mixture models and tipping point analysis 
Subgroup analyses will also be performed for each of the 3 age groups (6 months to 5 years, 
6 to 12 years, and 13 to ≤18 years) and for patients who were not included in the previous 
Study A4250-003. 
Several sensitivity analyses will be performed, such as the following: 
• Cochran Mantel Haenzsel test stratified on PFIC class to compare the proportion in 
fasting bile acid responder endpoint at the end of treatment in the two A4250 dose groups 
to placebo group.  Dropouts will be treated as non-responders 
• Based on the primary analysis including the interaction age class*treatment 
• Same as primary analysis in which the random factor region is considered as fixed factor.  
The region*treatment interaction will also be explored 
• Same as primary analysis including the interaction PFIC*treatment  
Other sensitivity analyses may be performed and will be detailed in the SAP. 
11.2.2.6 
Evaluation of Secondary and Exploratory Efficacy Variables 
The change in the secondary and explorative endpoints such as s-BA, total bilirubin, ALT, 
growth and patient-reported itch severity will also be analyzed using the MMRM model as 
specified for the primary analysis.  Nonparametric methods may be applied as an alternative 
approach for some endpoints, if appropriate. Change in growth (weight for age and BMI for 
age) will also be displayed using graphical presentations. 
Proportions of responders (e.g., meaningful reduction in itch severity and scratching at 
Week 24) will be analyzed using the GLMM model as specified for the primary analysis. 
Comparisons of the change from baseline at Week 24 in PedsQL between the treatment 
groups will be conducted using analysis of covariance.  The model will include terms for 
baseline, PFIC class, age class, region, and treatment.  
Undergoing biliary diversion surgery and/or listed for liver transplant, all-cause mortality, 
and liver decompensation events will mainly be analyzed by using descriptive statistics.  
Kaplan-Meier curves will be used when appropriate for time to event data. 
Other exploratory variables (Global Symptom Relief, biochemical markers and measures of 
bile acid synthesis, Albireo PRO and ObsRO sleep parameters, PELD/MELD score, APRI 
score, and Fib-4 score) will be analyzed descriptively for categorical and continuous data, as 
applicable.  For continuous data, the change from baseline will be analyzed in addition to the 
actual visit values.  For categorical data, shift tables or frequency and percentages of patients 
will be presented as appropriate. 


 
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In addition, subgroup analyses will be performed for each of the 3 age classes (6 months to 
5 years, 6 to 12 years, and 13 to ≤18 years). 
11.2.2.7 
Evaluation of Safety Variables 
Safety data will be analyzed using descriptive statistics and summaries by treatment group of 
SAEs, AEs, vital signs, clinical laboratory tests (hematology, clinical chemistry and 
urinalysis), and concomitant medication.  Analyses will be performed using the safety 
analysis set.  
Summaries of AEs (coded according to the Medical Dictionary for Regulatory Activities 
[MedDRA] system organ class [SOC] and MedDRA preferred term) will include the 
following:  
• Overview of the incidence of TEAEs (TEAEs, drug-related TEAEs, TEAEs leading to 
study discontinuation, and treatment-emergent SAEs) 
• TEAEs by SOC and preferred term 
• Intensity of TEAEs by SOC and preferred term 
• Drug-related TEAEs by SOC and preferred term 
• TEAEs leading to study discontinuation by SOC and preferred term 
• Treatment-emergent SAEs by SOC and preferred term 
Concomitant medication use during the Treatment Period will be summarized by Anatomical 
Therapeutic Chemical (ATC) class and World Health Organization (WHO) preferred name.  
Summaries of vital signs will be presented.  For each visit, the actual results and the change 
from baseline, and the number and percentage of patients with potentially clinically 
significant (PCS) values observed post-baseline will be presented. 
Summaries of clinical safety laboratory data will be presented.  For each visit, the actual 
result and the change from baseline, and the number and percentage of patients with PCS 
values observed post-baseline will be presented. 
Data listings will be provided for each patient for all safety parameters. 
11.2.2.8 
Compliance and Exposure 
Exposure will be analyzed by calculating the number of days with exposure to study drug.  
Results will be presented by treatment group using the safety analysis set.  
The percentage compliance will be described by treatment group and the number of patients 
with a compliance <80%, between 80% and 120%, and >120% will be presented based on the 
safety analysis set. 


 
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11.2.3 
Blinded Analyses to Confirm Responder Definitions 
A blinded analysis of Albireo ObsRO and PRO eDiary data will be performed after 50% or 
more of the planned sample has completed the Week 24 Follow-Up Period.  The blinded 
analysis will be used to estimate a threshold of clinically meaningful change (i.e., responder 
definition) in Albireo ObsRO and PRO pruritus scores.  The analysis will be performed by a 
group independent from both the study team and Albireo.  Blinded data will be used for this 
analysis; data will be collapsed across treatment groups. 
The analyses will include distribution- and anchor-based analyses to examine a responder 
definition for the Albireo PRO/ObsRO pruritus scores.  Distribution-based analyses will 
include calculation of the SD and standard error of measurement for the baseline Albireo 
PRO and ObsRO scores.  Anchor-based analyses will involve examining the degree of 
change on the Albireo PRO and ObsRO scores from baseline to Week 24 for patients who 
experience change in pruritus according to PGIC and symptom items.  Details of this analysis 
will be included in a stand-alone SAP for assessing the measurement characteristics 
(reliability, validity, sensitivity to change, responder definition) of the Albireo PRO and 
ObsRO items. 
11.2.4 
Data Safety Monitoring Board 
A DSMB consisting of sponsor-independent clinical and statistical experts will be 
established.  The DSMB will periodically meet for the review of accumulating study data, 
including safety (AE and laboratory data) and liver values.  
The DSMB will have access to unblinded data.  The DSMB will make recommendations for 
the remaining part of the study (further details will be provided in the DSMB charter).  The 
DSMB may recommend continuing with the study as planned or stopping the study early for 
safety reasons.  The DSMB will submit its recommendations in writing to Albireo 
Medical/
 who are responsible for responding to the recommendations of the DSMB and 
taking appropriate action.  The investigators will only be informed by Albireo Medical/
 
if the study requires a protocol amendment or is stopped.  The DSMB may choose to make 
additional evaluations at any time if they feel this is warranted from a safety point of view.  
The DSMB will act according to its own written standard operating procedure described in a 
charter and will prepare written minutes of its meetings.  The charter of the DSMB will be 
stored in the trial master file.  The DSMB will maintain records of its meetings and these will 
become part of the study file when the study is complete.  In order not to disseminate 
unblinded data and to ensure that all staff involved in the conduct and final analysis of the 
study remain blind to the data, only the members of the DSMB and the unblinded DSMB 
statistician will have access to these data. 
 


 
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12 
DIRECT ACCESS TO SOURCE DATA/NOTES 
The investigator/institution shall provide direct access to source data/documents for 
study-related monitoring, audits, IEC/Institutional Review Board (IRB) review and 
regulatory inspection. 


 
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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
13.1 
Conduct of the Study 
Albireo/
 shall implement and maintain quality control and quality assurance procedures 
with written standard operating procedures to ensure that the study is conducted and data are 
generated, documented, and reported in compliance with the protocol, ICH GCP and 
applicable regulatory requirements. 
This study shall be conducted in accordance with the provisions of the Declaration of 
Helsinki (October 1996) and all revisions thereof, and in accordance with United States Food 
and Drug Administration (FDA) regulations (Code of Federal Regulations, Sections 312.50 
and 312.56) and with ICH GCP (CPMP 135/95). 
The investigator may not deviate from the protocol without a formal protocol amendment 
having been established and approved by an appropriate IEC/IRB, except when necessary to 
eliminate immediate hazards to the patient or when the change(s) involve(s) only logistical or 
administrative aspects of the study.  Any deviations may result in the patient having to be 
withdrawn from the study and render that patient non-evaluable. 
13.2 
Study Monitoring 
Before study initiation, at a site initiation visit or at an investigator’s meeting, an Albireo 
representative or designee will review the protocol and eCRF with the investigators and the 
investigative staff.  During the study, the clinical monitor (clinical research associate [CRA]) 
will visit the site regularly to check the completeness of patient records, the accuracy of 
entries on the eCRFs, the adherence to the protocol and to GCP, the progress of enrollment, 
and to ensure that study drug is being stored, dispensed, and accounted for according to 
specifications. 
The investigator must ensure that eCRFs are completed within a timely period of patient 
visits, as per individual site agreements, and must allow the CRA and Albireo representative 
or designee periodic access to patient records and all study-related materials, including 
relevant hospital or clinical records, to confirm their consistency with the eCRF entries.  No 
information in these records about the identity of the patients will leave the study center.  
Albireo monitoring standards require full verification for the presence of the signed ICF, 
adherence to the inclusion/exclusion criteria, documentation of SAEs, and recording of 
primary efficacy and safety variables.  The CRA will review source data compared with the 
eCRFs and will verify source data according to the study-specific monitoring plan.  The 
design of the study, the frequency of patient visits, and the site enrollment rate will determine 
the frequency of monitoring visits.  Upon study completion, the CRA will visit the site to 
conduct a study termination visit, which will include collection of any outstanding 
documentation. 
It is recommended that the investigator/investigative staff log into the eCRF system every 
working day or at minimum twice weekly in order to provide a prompt response to queries.  


 
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The investigator/investigative staff should respond to queries and make any relevant changes 
to the study data within 3 working days. 


 
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14 
ETHICS 
14.1 
Independent Ethics Committee/Institutional Review Board 
Prior to the start of the study, the investigator is responsible for ensuring that the protocol and 
consent form have been reviewed and approved by a relevant IEC/IRB.  The IEC/IRB shall 
be appropriately constituted and perform its functions in accordance with FDA ICH GCP and 
local requirements as applicable. 
The IEC/IRB shall approve all protocol amendments (except for logistical or administrative 
changes), written informed consent documents and document updates, patient recruitment 
procedures (e.g., advertisements), written information to be provided to the patients and 
caregivers, IB, available safety information, information about payment and compensation 
available to patients, the investigator's curriculum vitae and/or other evidence of 
qualifications and any other documents requested by the IEC/IRB and regulatory authority 
(competent authority) as applicable. 
14.2 
Written Informed Consents and Assents 
The investigator/investigative staff will explain to each patient (or legally authorized 
representative) the nature of the study, its purpose, the procedures involved, the expected 
duration, alternative treatment, the potential risks and benefits involved, and any discomfort 
that may occur.  Each caregiver/patient will be informed that participation in the study is 
voluntary and that he/she or their child may withdraw from the study at any time and that 
withdrawal of consent will not affect his/her subsequent medical treatment or relationship 
with the treating physician. 
Caregivers/patients will be informed that they/their children may experience side-effects or 
be at risk for symptoms, illnesses, or complications that cannot be foreseen by Albireo.  As 
with other medications, people treated with A4250 may be at risk of developing allergic 
reactions or anaphylaxis.  Caregivers/patients will be advised that study procedures include 
regular blood sampling for measurement of safety parameters and biological markers and that 
some minor risks are associated with these procedures. 
This informed consent should be given by means of a signed ICF, written in non-technical 
language in accordance with ICH GCP, the Declaration of Helsinki, and regulatory 
authorities.  The caregiver/patient should read and consider the statements before signing and 
dating them and should be given a copy of each signed document.  If written consent is not 
possible, oral consent can be obtained if witnessed and followed by a signed statement from 
one or more persons not involved in the study, indicating why the patient was unable to sign 
the form.  No patient can enter the study before his/her or caregiver informed consent has 
been obtained. 
The ICF is part of the protocol and must be submitted by the investigator/investigative staff 
with the protocol for IEC approval.  Albireo will supply an ICF which complies with 
regulatory requirements and is considered appropriate for the study.  Any changes to the ICF 


 
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suggested by the investigator must be agreed to by Albireo before submission to the IEC and 
a copy of the approved version must be provided to the clinical monitor after IEC approval. 


 
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15 
DATA HANDLING AND RECORD KEEPING 
15.1 
Case Report Forms/Source Data Handling 
The investigator shall be provided with standardized eCRFs and shall ensure that all data 
from patient visits are promptly entered into the eCRFs in accordance with the specific 
instructions given.  The investigator must sign each eCRF to verify the integrity of the data 
recorded. 
A list of the normal ranges for all laboratory tests to be undertaken forms part of the 
documentation to be collated prior to study start.  As a central laboratory has been selected to 
conduct any or all tests, it is essential that all samples be analyzed at that laboratory. 
The investigator must maintain source documents such as laboratory reports, consultation 
reports, and complete medical history and physical examination reports. 
As no ethnicity data can be collected in the source document for study sites in France, the 
eCRF will be the Source Document for “origines ethniques”,  
15.2 
Retention of Essential Documents 
Essential documents, as defined by ICH GCP, include: the signed protocol and any 
amendment(s); copies of the completed eCRFs (for site archiving, compact discs of eCRF 
data for specific patients will be provided); signed ICFs; hospital records and other source 
documents; IEC approvals and all related correspondence including approved documents; 
drug accountability records; study correspondence; and a list of patients’ names and 
addresses. 
The investigator/investigative staff must retain copies of these essential documents for the 
period specified by ICH GCP and by applicable regulatory requirements.  The 
investigator/investigative staff will inform Albireo of the location where the essential 
documents are stored and must contact Albireo for approval before disposing of any essential 
documents.  The investigator/investigative staff should take measures to prevent accidental or 
premature destruction of these documents. 


 
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16 
FINANCING AND INSURANCE 
Financing and insurance are addressed in a separate agreement. 
 


 
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17 
PUBLICATION POLICY 
Albireo will retain the ownership of all data.  When the study is complete, Albireo shall 
arrange the analysis, tabulation of data and preparation of a clinical study report. Albireo may 
also use the data for publication, presentation at scientific meetings and submission to 
regulatory authorities.  All proposed publications based on this study must be subject to the 
sponsor's approval requirements. 
 


 
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18 
REFERENCE LIST 
1. A4250 Investigator’s Brochure. 
2. Alissa FT, Jaffe R, Shneider BL. Update on progressive familial intrahepatic cholestasis. 
J Pediatr Gastroenterol Nutr. 2008;46(3):241-52. 
3. Davit-Spraul A, Gonzales E, Baussan C, et al. Progressive familial intrahepatic 
cholestasis. Orphanet J Rare Disease 2009;4:1-12. 
4. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and 
functions. Front Biosci (Landmark Ed). 2009 Jan 1;14:2584-98. 
5. Hori T, Justin H, Nguyen JH and Uemoto S. Progressive familial intrahepatic cholestasis. 
Hepatobiliary pancreat Dis Int. 2010; 9(6):570-8. 
6. Jacquemin E. Progressive familial intrahepatic cholestasis. Genetic basis and treatment. 
Clin Liver Dis. 2000;4(4):753-63. 
7. Miethke AG, Zhang W, Simmons J, et al. Pharmacological inhibition of ASBT changes 
bile composition and blocks progression of sclerosing cholangitis in mdr2 knockout mice. 
Hepatology. 2016; 63(2):512–23. 
8. Murray CS, Rees JL. Are subjective accounts of itch to be relied on? The lack of relation 
between visual analogue itch scores and actigraphic measures of scratch. Acta Derm 
Venereol. 2011;91(1):18-23. 
9. Pawlikowska L, Strautnieks S, Jankowska I, et al. Differences in presentation and 
progression between severe FIC1 and BSEP deficiencies. J Hepatol. 2010;53(1):170-8. 
10. Suchy FJ, Sokol RJ, Balistreri WF, eds. Liver Disease in Children. 3rd Edition. New 
York, NY: Cambridge University Press; 2007:310–4. 
11. Whitington PF, Whitington GL. Partial external diversion of bile for the treatment of 
intractable 
pruritus 
associated 
with 
intrahepatic 
cholestasis. 
Gastroenterology. 
1988;95(1):130-6. 
 
 


 
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19 
APPENDICES 
Appendix 1 
Concomitant Medications Prohibited During the Study 
 


 
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Appendix 2 
Diary Questions including Albireo Patient-Reported Outcomes/Observer-
Reported Outcome Instrument 
 


 
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Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) 
 


 
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Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaire 


 
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Appendix 5 
Blood Volumes 
 


CLINICAL STUDY PROTOCOL A4250-005 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
IND Number:   
130591 
EudraCT Number 
2017-002338-21 
Test Product: 
A4250 
Indication: 
Progressive Familial Intrahepatic Cholestasis (PFIC) 
Sponsor: 
Albireo AB 
Development Phase: 
Phase 3 
Sponsor Signatory: 
 
Sponsor Medical Monitor: 
 
Principal Investigator: 
 
Date of the Protocol: 
15 March 2018 
Version of the Protocol: 
Final Amendment 1.2 France and UK 
Confidential 
The information in this document contains trade secrets and commercial information that are 
privileged or confidential and may not be used, divulged, published, or otherwise disclosed 
without the written consent of Albireo AB.  These restrictions on disclosure apply equally to 
all future information supplied to you or generated by you in connection with the study. 






 
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INVESTIGATOR SIGNATURE PAGE 
 
PROTOCOL TITLE:  A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children with Progressive 
Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
 
PROTOCOL NUMBER:  A4250-005 
I have read this protocol and agree that it contains all necessary details for performing this 
study.  I will conduct the study as outlined herein and will complete the study within the time 
designated, in accordance with all stipulations of the protocol and in accordance with Good 
Clinical Practice (GCP), local regulatory requirements, and the Declaration of Helsinki. 
I will provide copies of the protocol and all pertinent information to all individuals 
responsible to me who assist in the conduct of this study.  I will discuss this material with 
them to ensure that they are fully informed regarding the study drug and the conduct of the 
study. 
I will use only the informed consent approved by the Independent Ethics Committee (IEC) 
and will fulfil all responsibilities for submitting pertinent information to the IEC responsible 
for this study. 
I agree that the sponsor (Albireo AB) shall have access to any source documents from which 
case report form information may have been generated. 
I further agree not to originate or use the name of Albireo AB, A4250 in any publicity, news 
release, or other public announcement, written or oral, whether to the public, press, or 
otherwise, relating to this protocol, to any amendment to the protocol, or to the performance 
of this protocol without the prior written consent of Albireo AB. 
 
 
 
 
 
 
Name of Investigator 
 
 
 
 
 
 
Signature 
 
Date (day/month/year) 


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1 
ADMINISTRATIVE INFORMATION 
A Double-blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
Protocol No.: 
Date of the initial Protocol: 
Date and Number of Amendment(s): 
Sponsor: 
Clinical Research Organization: 
Sponsor Signatory: 
Sponsor Medical Monitor: 
Principal Investigator: 
A4250-005 
20 September 2017 
Amendment 01,06 December 2017
Amendment 1.2 Fr, UK, 15 March 2018 
Albireo AB 
Arvid Wallgrens backe 20 
413 46 Gӧteborg 
Sweden 
 
 
 
 
 
  
 
  
  
 
  
 
 
 
 
 
 
 
 


 
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2 
STUDY SYNOPSIS 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Title of Study: 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate Efficacy and Safety of A4250 
in Children with Progressive Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
Principal Investigator: 
 
Study Centers: 
Up to 50 sites will be initiated for this study in the United States (US), Canada, European Union (EU), Middle 
East, and Australia. 
Publication(s): 
None. 
Planned Study Period: 
Q1 2018 to Q2 2019 
Development Phase: 
Phase 3 
Objectives: 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day A4250 in children with 
progressive familial intrahepatic cholestasis (PFIC) Types 1 and 2, as determined by the following: 
• 
Proportion of patients experiencing at least a 70% reduction in fasting serum bile acid (s-BA) 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L compared to placebo 
after 24 weeks of treatment 
• 
Change from baseline to Week 24 in pruritus compared to placebo  
Secondary Objectives 
• 
To evaluate the effect of A4250 on serum alanine aminotransferase (ALT) concentration 
• 
To evaluate the effect of A4250 on growth 
• 
To evaluate the effect of A4250 on sleep disturbance 
• 
To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or liver 
transplantation) 
• 
To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 
Methodology: 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the efficacy and safety of 
40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in children with PFIC (deficiencies of familial 
intrahepatic cholestasis-1 or bile salt export pump).  
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment Period and a 4-week 
Follow-up Period.  After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of A4250, or a 
matching placebo.  Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008) in which all patients will receive 
active treatment. 
There will be up to 10 clinic visits during the study and one scheduled telephone contact. 


 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Number of Patients: 
Approximately 60 patients diagnosed with PFIC-1 or PFIC-2 will be randomized. 
Diagnosis and Main Criteria for Inclusion: 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages of ≥6 months 
and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have genetic confirmation of PFIC-1 or PFIC-2 through identification of biallelic 
pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, taken as the 
average of 2 samples 7 to 28 days apart (Visits 1 and 2) prior to randomization  
4. Patient must have history of significant pruritus and a caregiver-reported observed scratching in the 
electronic diary (eDiary) average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  Patients who 
turn 18 years of age (or legal age per country) during the study will be required to re-consent in order 
to remain in the study 
6. Patients are expected to have a consistent caregiver(s) for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use an eDiary 
device as required by the study 
Test Product, Dose and Mode of Administration: 
A4250, 40 µg/kg/day or 120 µg/kg/day, orally administered 
Reference Therapy, Dose and Duration of Administration: 
Matching placebo, orally administered 
Duration of Treatment: 
24 weeks 
Variables 
Efficacy:  
Primary Efficacy Endpoints 
The primary efficacy endpoints are: 
• 
EU and rest of world (RoW): Proportion of patients experiencing at least a 70% reduction in fasting 
s-BA concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L compared to 
placebo after 24 weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization and the previous 
visit (Clinic Visit 2).  The end value is the average of the values at Weeks 22 and 24 after the start of 
double-blind treatment. 
• 
US: Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline to 
Week 24 as measured by the worst scratch of the Albireo observer-reported outcome (ObsRO) 
instrument.  Scores for the 14 days baseline and post-treatment intervals will be calculated by 
averaging the worst scratching scores from each day in the interval. 
Secondary Efficacy Endpoints 
Secondary efficacy endpoints include the following: 
• 
EU and RoW: Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
to Week 24 as measured by the worst scratch of the Albireo ObsRO instrument.  Scores for the 14 days 
baseline and post-treatment intervals will be calculated by averaging the worst scratching scores from 
each day in the interval. 
• 
US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA concentration from 
baseline to the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 weeks of 
treatment. 
All Regions: 
All secondary endpoints will be compared to placebo. 


 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
• 
Change from baseline to Week 24 in fasting s-BA 
• 
Change from baseline to Week 24 in serum ALT concentration 
• 
Change in growth from baseline to Week 24, defined as the linear deficit (weight for age and body 
mass index) compared to the national growth curves (Z-score, standard deviation from P50) 
• 
Proportion of patients achieving meaningful reduction in caregiver-reported observed scratching at 
Week 24 
• 
Change from baseline to Week 24 in sleep parameters measured with the Albireo patient-reported 
outcome (PRO) and ObsRO instruments 
• 
Change from baseline to Week 24 in patient-reported itch severity as measured by the average daily 
score of the Albireo PRO instrument; only patients ≥8 years of age will complete the Albireo PRO 
instrument 
• 
Number of patients undergoing biliary diversion surgery or being listed for liver transplantation 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• 
Proportion of patients achieving meaningful reduction in patient-reported itch severity at Week 24 
• 
Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, caregiver, and 
clinician via the patient global impression of change (PGIC) items 
• 
Change from baseline in fasting s-BA at Weeks 4 and 12 
• 
Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline to Weeks 4, 
12, and 24 as measured by the worst scratch.  Scores for the 7 worst scratching days of the last 14 days 
at baseline and post-treatment intervals will be selected 
• 
Change from baseline in patient-reported itch severity and observer-reported scratching at Weeks 4 and 
12 
• 
Change from baseline to Week 24 in patient-reported and observer-reported night time itching and 
scratching severity scores, respectively 
• 
Change from baseline to Week 24 in pooled pruritus score including observer-reported scratching for 
patients <8 years of age and patient-reported itch severity for patients ≥8 years of age 
• 
Change from baseline to Week 24 in additional patient-reported and observer-reported sleep 
parameters (e.g., tiredness and number of awakenings) 
• 
Change from baseline to Week 24 in Pediatric Quality of Life Inventory (PedsQL) 
• 
Change from baseline in serum ALT concentration at Weeks 4 and 12 
• 
Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• 
Change from baseline in AST at Weeks 4, 12, and 24 
• 
Change from baseline for other biochemical markers and measures of bile acid synthesis (autotaxin, 
plasma 7α-hydroxy-4-cholesten-3-one concentration [p-C4]) at Week 24 
• 
Change in pediatric end-stage liver disease/model for end-stage liver disease (PELD/MELD) score, 
AST to platelet ratio index (APRI) score, and Fib-4 score from baseline to Week 24 
• 
Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® (where available) 
• 
Liver-related mortality and liver decompensation events. 
• 
All-cause mortality 
Safety:   
Safety criteria are as follows: 
• 
Occurrence of treatment-emergent adverse events including severity and relatedness to study drug at all 
visits 
• 
The incidence of treatment-emergent serious adverse events (SAEs), based upon information from 
patient reports, including the description, incidence, and severity of an SAE 
• 
Trends evaluated for the following assessments: physical examinations, concomitant medications, vital 
signs, laboratory test results (including clinical chemistry, hematology, and urinalysis), and imaging 


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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
studies 
Statistical Methods: 
The primary objective of this study is to show superiority in efficacy of A4250 (120 µg/kg/day and 
40 µg/kg/day) compared to placebo in patients with PFIC Types 1 and 2. 
In the EU and RoW, for the primary efficacy variable fasting bile acid responder endpoint (at least a 70% 
reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the end of treatment), the null 
hypothesis will be tested against the alternative hypothesis (α=0.0125, 1-sided).  Generalized linear mixed 
modelling will be used with a logistic link, to allow for repeated assessments over time.  The model will include 
baseline value, treatment group, PFIC class, age class, region, visit and visit*treatment group interaction.  The 
contrasts of primary interest will be the treatment difference at Week 24 but also the difference at Week 4, 
Week 8, Week 12, and Week 18 will be estimated and analyzed. 
In the US, for the primary efficacy variable pruritus score endpoint (change from baseline to Week 24 in 
observer-reported scratching), the null hypothesis will be tested against the alternative hypothesis (α=0.0125, 
1-sided).  The primary analysis of the change from baseline in pruritus score (indexed by observer-reported 
scratching) will be analyzed using a mixed model repeated measures analysis.  The contrasts of primary interest 
will be the treatment difference at Week 24 but also the difference at the other weeks will be estimated and 
analyzed.  
To strictly control overall Type I error rate across the dose groups and also across the primary and the main 
secondary endpoint to a 1-sided 2.5% level, a Bonferroni-based parallel gate-keeping procedure will be used. 
Other secondary and exploratory outcome variables shall provide supportive efficacy and safety information 
regarding the differences between the treatment groups.  No adjustments will be performed for multiple-dose 
comparisons when testing the other secondary and exploratory efficacy variables. 
Date of the Protocol:  15 March 2018 


 
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3 
TABLE OF CONTENTS 
1 
ADMINISTRATIVE INFORMATION ........................................................................ 5 
2 
STUDY SYNOPSIS ...................................................................................................... 6 
3 
TABLE OF CONTENTS ............................................................................................ 10 
3.1 
List of Tables .................................................................................................. 14 
3.2 
List of Figures ................................................................................................ 14 
3.3 
List of Appendices .......................................................................................... 14 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ................................ 15 
5 
INTRODUCTION ....................................................................................................... 17 
5.1 
Investigational Medicinal Product .................................................................. 17 
5.2 
Background .................................................................................................... 17 
5.2.1 
Progressive Familial Intrahepatic Cholestasis ........................................... 17 
5.2.2 
Summary of Clinical and Nonclinical Studies ........................................... 19 
5.3 
Rationale ......................................................................................................... 20 
5.3.1 
Risk/Benefit ............................................................................................... 21 
6 
STUDY OBJECTIVES ............................................................................................... 22 
6.1 
Primary Objectives ......................................................................................... 22 
6.2 
Secondary Objectives ..................................................................................... 22 
7 
INVESTIGATIONAL PLAN ...................................................................................... 23 
7.1 
Overall Study Design and Plan....................................................................... 23 
7.1.1 
Description ................................................................................................. 23 
7.1.2 
Schedule of Assessments ........................................................................... 25 
7.1.3 
Study Procedures and Assessments ........................................................... 29 
7.1.3.1 
Screening Period (Days -56 to 0) ............................................... 29 
7.1.3.2 
Treatment Period (Day 0 to Day 168) ........................................ 30 
7.1.3.3 
Follow-Up Period ....................................................................... 33 
7.2 
Study Population ............................................................................................ 33 
7.2.1 
Inclusion Criteria ....................................................................................... 33 
7.2.2 
Exclusion Criteria ...................................................................................... 34 
7.2.3 
Withdrawal of Patients............................................................................... 36 
7.2.4 
Study Termination by Sponsor .................................................................. 36 
8 
TREATMENT OF PATIENTS ................................................................................... 38 
8.1 
Identity of Study Drug .................................................................................... 38 
8.2 
Administration of Study Drug ........................................................................ 38 
8.3 
Study Treatment Packaging and Labeling ...................................................... 39 
8.3.1 
Packaging and Labeling ............................................................................. 39 


 
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8.3.2 
Storage ....................................................................................................... 39 
8.3.3 
Blinding and Randomization of Study Drug ............................................. 39 
8.4 
Procedure for Breaking the Randomization Code .......................................... 40 
8.5 
Patient Compliance ......................................................................................... 41 
8.6 
Study Drug Accountability ............................................................................. 41 
8.7 
Concomitant Therapy and Prohibited Medications ........................................ 41 
9 
ASSESSMENT OF EFFICACY ................................................................................. 42 
9.1 
Efficacy Endpoints ......................................................................................... 42 
9.1.1 
Primary Efficacy Endpoint ........................................................................ 42 
9.1.2 
Secondary Efficacy Endpoint .................................................................... 42 
9.1.3 
Exploratory Efficacy Endpoints ................................................................. 43 
9.2 
Efficacy Assessments ..................................................................................... 44 
9.2.1 
Serum Bile Acids ....................................................................................... 44 
9.2.2 
Itching, Scratching and Sleep Score .......................................................... 44 
9.2.3 
Growth ....................................................................................................... 45 
9.2.4 
Quality of Life Questionnaire (PedsQL).................................................... 45 
9.2.5 
Biomarkers and Other Laboratory Samples ............................................... 45 
9.2.6 
PELD/MELD Score ................................................................................... 45 
9.2.7 
Fibroscan® .................................................................................................. 45 
9.2.8 
Markers of Fibrosis .................................................................................... 46 
9.2.9 
Global Impression of Change and Global Impression of Symptom 
Measures .................................................................................................... 46 
10 
ASSESSMENT OF SAFETY ..................................................................................... 47 
10.1 
Adverse Events ............................................................................................... 47 
10.1.1 
Definitions and Investigator Assessments ................................................. 47 
10.1.1.1 Clinical Significance .................................................................. 47 
10.1.1.2 Serious Adverse Events .............................................................. 47 
10.1.1.3 Severity Assessment ................................................................... 48 
10.1.1.4 Causality Assessment ................................................................. 48 
10.1.2 
Recording of Adverse Events .................................................................... 49 
10.1.3 
Recording and Reporting of Serious Adverse Events ................................ 50 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions .............. 51 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments .................................................................................................... 51 
10.2.1 
Laboratory Assessments ............................................................................ 51 
10.2.2 
Individual Patient Safety Monitoring ........................................................ 52 
10.2.2.1 Liver Monitoring ........................................................................ 52 


 
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10.2.2.2 Close Observation ...................................................................... 54 
10.2.2.3 De-challenge/Re-challenge for Liver and Clinical Hepatitis 
Monitoring .................................................................................. 54 
10.2.2.4 Diarrhea ...................................................................................... 54 
10.2.2.5 Monitoring after Permanent Discontinuation of Study Drug 
Due to Safety .............................................................................. 55 
10.2.3 
Demographics/Medical and Surgical History ............................................ 55 
10.2.4 
Clinical Genetic Testing ............................................................................ 55 
10.2.5 
Physical Examination ................................................................................ 55 
10.2.6 
Vital Signs ................................................................................................. 56 
10.2.7 
Abdominal Ultrasound ............................................................................... 56 
10.2.8 
Overdose .................................................................................................... 56 
10.2.9 
Pregnancy .................................................................................................. 56 
11 
STATISTICAL EVALUATION ................................................................................. 58 
11.1 
Sample Size and Power .................................................................................. 58 
11.2 
Statistical Methods ......................................................................................... 58 
11.2.1 
Statistical Analysis Sets ............................................................................. 58 
11.2.2 
Methods of Statistical Analyses ................................................................. 59 
11.2.2.1 General Principles ...................................................................... 59 
11.2.2.2 Missing Data .............................................................................. 60 
11.2.2.3 Demographic and Baseline Characteristics ................................ 60 
11.2.2.4 Subject Disposition..................................................................... 61 
11.2.2.5 Evaluation of Primary Efficacy Variables .................................. 61 
11.2.2.6 Evaluation 
of 
Secondary 
and 
Exploratory 
Efficacy 
Variables ..................................................................................... 62 
11.2.2.7 Evaluation of Safety Variables ................................................... 63 
11.2.2.8 Compliance and Exposure .......................................................... 63 
11.2.3 
Blinded Analyses to Confirm Responder Definitions ............................... 64 
11.2.4 
Data Safety Monitoring Board ................................................................... 64 
12 
DIRECT ACCESS TO SOURCE DATA/NOTES ..................................................... 65 
13 
QUALITY CONTROL AND QUALITY ASSURANCE .......................................... 66 
13.1 
Conduct of the Study ...................................................................................... 66 
13.2 
Study Monitoring ........................................................................................... 66 
14 
ETHICS ....................................................................................................................... 68 
14.1 
Independent Ethics Committee/Institutional Review Board .......................... 68 
14.2 
Written Informed Consents and Assents ........................................................ 68 


 
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15 
DATA HANDLING AND RECORD KEEPING ....................................................... 70 
15.1 
Case Report Forms/Source Data Handling..................................................... 70 
15.2 
Retention of Essential Documents .................................................................. 70 
16 
FINANCING AND INSURANCE .............................................................................. 71 
17 
PUBLICATION POLICY ........................................................................................... 72 
18 
REFERENCE LIST ..................................................................................................... 73 
19 
APPENDICES ............................................................................................................. 74 
 


 
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3.1 
List of Tables 
Table 1 
Schedule of Assessments ................................................................................... 26 
Table 2 
Dosing and Capsule Strength ............................................................................. 39 
Table 3 
Routine Laboratory Parameters .......................................................................... 52 
 
3.2 
List of Figures 
Figure 1 
Study Design ...................................................................................................... 25 
Figure 2 
Bonferroni Holm Multiple Comparison Procedure ............................................ 59 
 
3.3 
List of Appendices 
Appendix 1 Concomitant Medications Prohibited During the Study .................................... 74 
Appendix 2 Diary Questions including Albireo Patient-Reported Outcomes/Observer-
Reported Outcome Instrument ........................................................................... 75 
Appendix 3 Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) ....................................................................... 76 
Appendix 4 Pediatric Quality of Life Inventory (PedQL) Questionnaires ............................ 77 
Appendix 5 Contraceptive advice .......................................................................................... 78 
Appendix 6 Blood volumes.................................................................................................... 79 
 


 
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4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
ADME 
absorption, distribution, metabolism, and excretion 
AE(s) 
adverse event(s) 
AFP 
alfa-fetoprotein 
ALP 
alkaline phosphatase 
ALT 
alanine aminotransferase 
APRI 
AST to platelet ratio index 
ASBT 
apical sodium-dependent bile transporter (also known as IBAT) 
AST 
aspartate aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BMI 
body mass index 
BSEP 
bile salt export pump 
CPK 
creatine phosphokinase 
CRA 
clinical research associate 
CYP 
cytochrome P450 
DSMB 
Data and Safety Monitoring Board 
eCRF 
electronic case report form 
eDiary 
electronic diary 
EOT 
End of Treatment 
EU 
European Union 
EudraCT 
European Union drug regulatory agency clinical trial 
FAS 
full analysis set 
FDA 
United States Food and Drug Administration 
FIC1 
familial intrahepatic cholestasis-1 
GCP 
Good Clinical Practice 
GI 
gastrointestinal 
GLMM 
generalized linear mixed modelling 
IB 
Investigator’s Brochure 
IBAT 
ileal bile acid transporter (also known as ASBT) 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
 
 
IEC 
Independent Ethics Committee 
INR 
international normalized ratio 
IRB 
Institutional Review Board 
IWRS 
Interactive Web Response System 


 
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LDH 
lactate dehydrogenase 
LFT 
liver function test 
MDR3 
multidrug-resistance protein 3 
MedDRA 
Medical Dictionary for Regulatory Activities 
MELD 
model for end-stage liver disease 
MMRM 
mixed model repeated measures 
ObsRO 
observer-reported outcome 
PBC 
primary biliary cholangitis 
p-C4 
plasma 7α-hydroxy-4-cholesten-3-one concentration 
PCS 
potentially clinically significant 
PedsQL 
Pediatric Quality of Life Inventory 
PELD 
pediatric end-stage liver disease 
PFIC 
progressive familial intrahepatic cholestasis 
PGIC 
patient global impression of change 
PGIS 
patient global impression of symptoms 
PP 
per protocol 
PRO 
patient-reported outcome 
PT 
prothrombin time 
PVM 
pharmacovigilance manager 
QoL 
quality of life 
RoW 
rest of world 
RR 
respiratory rate 
SAE(s) 
serious adverse event(s) 
SAP 
statistical analysis plan 
s-BA 
serum bile acid 
SD 
standard deviation 
s-FGF19 
serum fibroblast growth factor 19 concentration 
SOC 
system organ class 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE(s) 
treatment-emergent adverse event(s) 
ULN 
upper limit of normal 
US 
United States 
WHO 
World Health Organization 
 


 
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5 
INTRODUCTION 
5.1 
Investigational Medicinal Product 
A4250 is a small molecule and a potent selective inhibitor of the ileal bile acid transporter 
(IBAT), also known as the apical sodium-dependent bile acid transporter (ASBT).  IBAT is 
an integral brush border membrane glycoprotein that co-transports sodium and bile acids and 
appears to be a critical component in the regulation of the bile acid pool size in animals and 
man.  This transporter, expressed in the distal ileum, is a key element in the enterohepatic 
circulation of bile acids since it facilitates the high-affinity, high-capacity reabsorption of bile 
acids.  Indeed, 95% of secreted bile acids are reabsorbed via IBAT [Hofmann 2009; 
Miethke 2016].  A4250 is orally administered and acts locally in the gut where it binds 
reversibly to IBAT to decrease the reuptake of bile acids.  A4250 has minimal systemic 
exposure at the predicted therapeutic dose ranges. 
No safety signals were found with A4250 in preclinical safety and toxicity studies.  In the 
first clinical study of A4250 in healthy volunteers, A4250 was given as a single oral dose up 
to 10 mg or daily oral doses up to 3 mg/day for 7 days.  A4250 was well tolerated and the 
only findings were gastrointestinal (GI) related effects, such as diarrhea, at higher doses. 
In an exploratory Phase 2 study in children with cholestatic pruritus, A4250 was given as oral 
doses of 10 to 200 µg/kg/day for 4 weeks.  A4250 reduced mean levels of serum bile acids 
(s-BA) in all cohorts.  In 11 of 13 progressive familial intrahepatic cholestasis 
(PFIC) patients, substantial reductions (ranging from 43% to 98%) were observed.  
Patient-reported diary data documented improvement of pruritus during treatment.  A4250 
was well tolerated.  No serious adverse events (SAEs) were deemed to be related to study 
drug, and most adverse events (AEs), including some increased transaminases, were 
considered unrelated to study drug and transient in nature. 
5.2 
Background 
5.2.1 
Progressive Familial Intrahepatic Cholestasis 
PFIC is a rare autosomal recessive cholestatic liver disease estimated to affect between 1 in 
every 50,000 to 100,000 children born worldwide.  PFIC represents 10% to 15% of causes of 
cholestasis in children and 10% to 15% of liver transplantation indications in children.  All 
types of PFIC exist worldwide and both sexes appear to be equally affected. 
The common underlying pathogenesis of PFIC is disruption of bile formation and bile 
transport through the liver [Jacquemin 2000].  The classification of PFIC has evolved over 
the years.  The most commonly used sub-classifications are PFIC Types 1, 2, and 3, which 
are based on the associated affected gene and described in more detail below. 
• PFIC, Type 1:  also referred to as “Byler disease” or “familial intrahepatic 
cholestasis-1 (FIC1) protein deficiency.” FIC1 protein is located on the canalicular 
membrane of hepatocytes and facilitates movement of aminophospholipids from the outer 


 
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to inner leaflet of the plasma membrane of the hepatocyte.  The ATP8B1 gene encodes 
FIC1 protein.  Biallelic pathologic variants in the ATP8B1 gene are associated with FIC1 
dysfunction and classified clinically as PFIC Type 1 disease. 
• PFIC, Type 2:  also referred to as “Byler syndrome” or “bile salt export pump (BSEP) 
deficiency.”  BSEP is a transporter protein that is expressed at the canalicular membrane 
of hepatocytes, and is the primary exporter of bile acids.  The ABCB11 gene encodes the 
BSEP protein.  Biallelic pathologic variations in the ABCB11 gene are associated with 
BSEP dysfunction and classified clinically as PFIC Type 2 disease. 
• PFIC, Type 3:  is caused by a deficiency of the multidrug-resistance protein 3 (MDR3) 
due to mutations in the ABCB4 gene.  MDR3 is a phospholipid translocase critical for 
phospholipid secretion. 
Severe pruritus is common in children diagnosed with PFIC.  Itching (and subsequent 
scratching) is a significant morbidity for these patients and their families [Suchy 2007].  A 
more severe degree of pruritus is experienced compared to patients with other forms of liver 
disease and to other pruritic conditions such as atopic dermatitis [Murray 2011].  In patients 
with PFIC, liver biopsy reveals canalicular cholestasis and, later, the appearance of portal 
fibrosis.  Serum biochemistry indicates cholestasis with hyperbilirubinemia, elevated alanine 
aminotransferase (ALT) and aspartate aminotransferase (AST).  The concentrations of bile 
acids in serum are very high, while serum gamma-glutamyl transferase activity (exception 
being MDR3 variants) and cholesterol are normal [Hori 2010].  Symptoms of portal 
hypertension and liver failure will develop during the course of the disease [Davit-Spraul 
2009; Alissa 2008].  Symptoms develop early; median age at onset of symptoms is 2 months, 
and 78% of PFIC patients present with jaundice [Pawlikowska 2010].  The life-threatening 
and debilitating nature of PFIC is reflected by the fact that survival in those not resorting to 
surgery is 50% at 10 years of age and almost zero at 20 years of age.  Approximately half of 
PFIC patients undergo liver transplantation [Davit-Spraul 2009] and treatment-resistant 
pruritus is the leading indication for the surgical procedure partial external biliary diversion, 
mostly in PFIC Type 1 and 2 patients. 
PFIC is life-threatening and debilitating.  Currently, the treatment for PFIC is palliative and 
there is no pharmaceutical treatment approved for use in PFIC.  The therapeutic choices are 
restricted to non-specific therapy of the symptoms and signs of the disease such as nutritional 
support, preventing vitamin deficiencies, and treatment of extrahepatic features.  Medical 
treatment options include off-label use of ursodeoxycholic acid, rifampin, antihistamines, and 
naltrexone.  A minority of patients respond nominally and transiently to these interventions.  
Biliary diversion is used to decrease systemic bile acids through interruption of the 
enterohepatic circulation and so avoid transplantation.  Liver transplantation is typically only 
viewed as an option when patients have failed medical treatment and/or biliary diversion and 
have liver failure or persistent uncontrolled pruritus. 


 
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5.2.2 
Summary of Clinical and Nonclinical Studies 
This is a summary of nonclinical and clinical studies.  More detail is provided in the 
Investigator’s Brochure ([IB] see A4250 IB).  
A4250 is a potent, selective IBAT inhibitor with no safety signals seen in the safety 
pharmacological studies.  In addition, no safety signals were seen in repeat-dose oral toxicity 
studies in rats with up to 26 weeks of daily dosing and in dogs with up to 39 weeks of daily 
dosing.  No safety signals were identified when A4250 was administered daily to juvenile 
rats from age 14 days to 63 days. 
A4250 is minimally absorbed with very low systemic exposure in both humans and other 
species.  In blood, protein binding is high (>99.7%).  Systemically absorbed A4250 is slowly 
metabolized in humans and in preclinical studies by hydroxylation only, a metabolite that 
retains IBAT activity.  A4250 did not show any significant inhibition of cytochrome 
P450 (CYP) 
enzymes 
in 
the 
rat. 
 
A4250 
inhibited 
human 
CYP3A4 
and 
CYP2D6 (IC50: 16 µmol/L for both enzymes), and CYP2C9 (IC50:1.2 µmol/L).  
To date, A4250 has been studied in 3 Albireo-sponsored clinical studies: a study in healthy 
volunteers evaluating single and multiple administration of A4250; a single-dose absorption, 
distribution, metabolism, and excretion (ADME) study; and a Phase 2 study in children with 
cholestatic pruritus.  In addition, an investigator-sponsored study has been conducted in 
patients with primary biliary cholangitis (PBC).  In total, 98 subjects have been exposed to 
A4250. 
In the first clinical study (A4250-001), A4250 was given to healthy volunteers as a single oral 
dose up to 10 mg or daily oral doses up to 3 mg/day for 7 days.  Following a single oral dose 
of 10 mg, the median maximum drug concentration was 0.2 nmol/L and the area under the 
curve was 0.8 nmol × h/L (n=6).  Pharmacodynamic evaluation of bile acid synthesis 
biomarkers 
(serum 
fibroblast 
growth 
factor 
19 [s-FGF19] 
and 
plasma 
7α Hydroxy 4-cholesten-3-one [p-C4]) identified the expected decrease in s-FGF19 and an 
increase in p-C4.  In addition, total plasma bile acids decreased and total fecal bile acids 
increased in A4250-treated patients compared to placebo. A4250 was generally well 
tolerated.  The only findings were GI-related effects, such as diarrhea at higher doses. 
In the ADME study (A4250-007), healthy subjects were administered a single oral dose of 
3 mg [14C]-A4250 capsule.  The main excretion route was determined to be fecal.  There 
were no quantifiable concentrations of A4250 in plasma or total radioactivity in plasma or 
whole blood.  A4250 was well tolerated.  AEs were reported and most were mild and not 
related to A4250. The 3 AEs experienced by 2 subjects reported as related to study drug were 
GI in nature, including diarrhea (2 AEs; 2 subjects) and abdominal pain (1 AE; 1 subject). 
In an exploratory Phase 2 study in children with cholestatic pruritus (A4250-003), A4250 was 
administered as oral daily doses of 0.01 to 0.2 mg/kg for 4 weeks.  Twenty 
patients (8 females and 12 males) aged 1 to 17 years were enrolled in this open-label 
dose-escalation study.  Four patients were re-enrolled into a second dose level.  No 


 
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treatment-related SAEs were reported, and most AEs, including some increased 
transaminases, were transient.  Four-week, once-daily treatment with A4250 reduced mean 
levels of s-BA in all cohorts.  Substantial s-BA reductions in 11 of 13 PFIC patients (ranging 
from 43% to 98%) were observed.  A4250 also reduced s-BA in the 11 non-PFIC patients, 
but to a lesser extent than in PFIC patients.  Patient-reported diaries documented 
improvement of the visual analogue scale itch score in 17 of 24 patients during treatment.  
The dose with the greatest improvement showed a mean decrease (± standard error of the 
mean) of 2.9 ±1.1 from baseline.  The results from this study form the basis of the current 
(A4250-005) study design. 
5.3 
Rationale 
In this investigational study, A4250 is under development for treatment of PFIC and is also 
being considered for other orphan cholestatic liver diseases. 
There is currently no medical treatment approved for PFIC.  In PFIC patients with severe 
pruritus, biliary diversion surgery is used to decrease systemic bile acids through interruption 
of the enterohepatic circulation and thereby reducing pruritus [Whitington 1988].  Liver 
transplantation is typically used as an option when patients have failed medical treatment 
and/or biliary diversion and have liver failure or persistent uncontrolled pruritus. 
By inhibiting IBAT with high selectivity and potency, A4250 has the potential to relieve 
cholestasis and improve liver function without surgical intervention in patients with PFIC.  
Since A4250 is minimally absorbed and achieves a substantial reduction of s-BA, it has the 
potential to be a safer and less invasive alternative to surgical biliary drainage.  The rationale 
for using A4250 is to decrease s-BA levels, and to reduce the major morbidity of pruritus, 
improving the health and wellbeing of patients affected with PFIC.  Ultimately, this 
noninvasive therapeutic approach has the potential to not only improve quality of life (QoL), 
but also reduce the need for biliary diversion and subsequent liver failure and liver 
transplantation. 
The doses selected, 40 and 120 µg/kg/day, were based on the efficacy and safety data 
generated from patients with PFIC (n=10 + 3 re-entered) and supporting data from all 
patients (n=20 + 4 re-entered) in the five dose groups dosed in the A4250-003 study.  
Improvements in s-BA and pruritus were observed with doses from 30 µg/kg to 100 µg/kg, 
with a dose-related trend.  The best dose response in all patients was at the 60 µg/kg/day for 
s-BA and 100 µg/kg/day for pruritus.  The best dose response in the PFIC subgroup was at 
30 µg/kg/day for both s-BA and pruritus.  Patients with PFIC responded well to the doses 
given from 30 µg/kg to 200 µg/kg; however, a clear dose relationship could not be 
established in order to select a single dose for this Phase 3 study.  As efficacy was observed 
at both low and high doses, 40 µg/kg and 120 µg/kg/day are considered to be the most 
optimal for this study.  
This study will be conducted in compliance with the protocol and with the International 
Council for Harmonisation (ICH) guidelines on Good Clinical Practice (GCP). 


 
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Patients completing 24 weeks of double-blind treatment will be offered to enter Study 
A4250-008 and receive open-label treatment with A4250. 
5.3.1 
Risk/Benefit 
A4250 has been evaluated in three Albireo-sponsored clinical studies: a double-blind 
placebo-controlled study in healthy volunteers, a single-dose ADME study, and a Phase 2 
study in children with cholestatic pruritus.  In addition, an investigator-sponsored study has 
been conducted in patients with PBC. 
A total of 98 subjects/patients have been exposed to A4250.  Healthy subjects have been 
exposed to up to 10 mg as a single dose and up to 3 mg daily as part of a multiple-dose 
evaluation.  Children with cholestatic liver disease have been treated with up to 
0.2 mg/kg/day (200 µg/kg/day) for 4 weeks.  In total, 2 SAEs have been reported; both were 
judged by the physician to be not related to the study drug. 
Patients with cholestatic liver diseases suffer from excess bile acids in the liver resulting in 
tissue damage.  A commonly used treatment in these patients is bile diversion surgery, 
whereby approximately 50% to 100% of the enterohepatic circulation of bile acids is 
interrupted.  Inhibition of IBAT with A4250, thereby interrupting the enterohepatic 
circulation of bile acids, is therefore a potential medical alternative to surgery which could be 
of benefit to these patients if shown to be effective and safe.  Data from the Phase 2 study 
showed efficacy of A4250 in reducing s-BA concentrations and pruritus in such patients. 
Based on the mode of action of an IBAT inhibitor, loose stools or diarrhea could be expected.  
However, in the pediatric Phase 2 study with 4 weeks daily treatment, only one patient had 
mild transient diarrhea after a single dose that did not recur on multiple dosing. 


 
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6 
STUDY OBJECTIVES 
6.1 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day 
A4250 in children with PFIC Types 1 and 2, as determined by the following: 
• The proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment 
• Change from baseline to Week 24 in pruritus compared to placebo  
6.2 
Secondary Objectives 
• To evaluate the effect of A4250 on serum ALT concentration 
• To evaluate the effect of A4250 on growth 
• To evaluate the effect of A4250 on sleep disturbance 
• To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or 
liver transplantation)  
• To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 


 
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7 
INVESTIGATIONAL PLAN 
7.1 
Overall Study Design and Plan 
7.1.1 
Description 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the 
efficacy and safety of 40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in 
children with PFIC (deficiencies of FIC1 or BSEP). 
Study data will be periodically reviewed by an unblinded Data Safety and Monitoring Board 
(DSMB). 
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment 
Period and a 4-week Follow-Up Period. 
After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of 
A4250, or a matching placebo. 
Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008) in which all 
patients will receive active treatment.  A patient who prematurely withdraws due to no 
improvement/intolerable symptoms, and has completed at least 12 weeks of the Treatment 
Period and End of Treatment (EOT) Visit, will also be offered the opportunity to enter 
Study A4250-008. 
Visit windows applicable to each visit are presented in the schedule of assessments (Table 1). 
There will be up to 10 clinic visits during the study (Figure 1) and one scheduled telephone 
contact, as follows: 
• Visit 1: Screening Period start. Patient is considered enrolled after signing the informed 
consent form (ICF) (Days -56 to -35) 
• Visit 2: second Screening Visit (Days -28 to -7) 
• Visit 3: Randomization Visit (Day 0) 
• Telephone Contact 1: (Day 14) 
• Visit 4: Clinic Visit (Day 28) 
• Visit 5: Clinic Visit (Day 56) 
• Visit 6: Clinic Visit (Day 84) 
• Visit 7: Clinic Visit (Day 126) 
• Visit 8: Clinic Visit (Day 154) 
• Visit 9: Clinic Visit, EOT (Day 168) 


 
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• Visit 10: Follow-Up Visit, 28 days after last dose of study drug (Day 196).  All patients 
continuing in Study A4250-008 will not have this Follow-up Visit. 
Additional clinic visits may be required for patients who need direct site assistance, e.g., due 
to AE monitoring, to fulfil screening requirements, and/or for safety maintenance. 
Screening Period 
Informed consent must be obtained at Visit 1 prior to performing any study procedures.  After 
signing the ICF, patients will be considered enrolled and evaluated for study eligibility. 
Daily recording of pruritus using an electronic diary (eDiary) will be started to determine 
confirmation of the magnitude and the baseline pruritus symptoms before the study.  After 
consultation with the Medical Monitor, patients failing eligibility criteria may be re-screened 
after 2 months.  Patients not fulfilling inclusion/exclusion criteria after 3 attempts are not 
allowed to re-screen. 
Caregivers/patients will be instructed on the daily use of the eDiary.  The eDiary will include 
patient-reported outcome (PRO) and observer-reported outcome (ObsRO) items for 
evaluation of itching (PRO), scratching (ObsRO), and sleep disturbance (PRO and ObsRO) 
during the Screening Period (study Days -56 to 0) and throughout the 24-week Treatment 
Period (study Days 0 to 168). 
Only the 14 most recent days before randomization will be used for eligibility calculation of 
the diary entries.  Observer-reported outcomes in patients of all ages will be recorded by a 
caregiver (or caregiver’s designee).  If possible, the same caregiver will complete the Albireo 
ObsRO items throughout the study. 
At a second Screening Visit (Visit 2) additional laboratory samples will be taken for 
eligibility assessments. 
Treatment Period 
Eligibility for randomization will be determined at Visit 3 using the pruritus (scratching 
reports from the Albireo ObsRO) data in the 2 weeks before Visit 3, genetic confirmation of 
diagnosis, and the liver biochemistry evaluations from the previous screening visits.  Two 
weeks after Visit 3, there will be a telephone contact with the caregiver/patient to evaluate 
any AEs. 
The patient will return to the clinic at Visit 4, Visit 5, Visit 6, Visit 7, Visit 8, and Visit 9 for 
assessments.  If a new caregiver will begin to complete the Albireo ObsRO during the course 
of the trial, they will be provided with training during a clinic visit before they begin to use 
the eDiary.  Additionally, caregivers/patients will be requested to use the diary to report the 
date and time each dose of study drug is administered during the Treatment Period. 
The last dose of study drug will be administered the day before Visit 9.  At study Visit 9, the 
patient has the option to enroll in an open-label extension study (A4250-008) should they 
meet the required entry criteria.  If the patient is continuing into Study A4250-008 this will be 


 
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the last visit in this study and the first visit in Study A4250-008.  Visit 9/EOT should also be 
performed at the time patients are prematurely withdrawn from the study. 
Clinic Visit 10 will take place 28 days after Visit 9 for all patients not continuing into Study 
A4250-008. 
Figure 1 
Study Design 
 
T: telephone contact; V: study visit 
7.1.2 
Schedule of Assessments 
The schedule of assessments is presented in Table 1. For information on blood volumes 
please see Appendix 6. 
 


 
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Table 1 
Schedule of Assessments 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Informed consent c 
X 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
Concomitant medicationd 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical and surgical history 
X 
 
X 
 
 
 
 
 
 
 
 
Blood sampling for genetic 
testinge 
X 
 
 
 
 
 
 
 
 
 
 
Physical examination including 
voluntary photography 
X 
 
X 
 
 
 
 
 
 
X 
 
Skin examination 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
Vital signsf 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
eDiary: itching, scratching, and 
sleep scoresg 
Daily diary entry 
Clinical chemistryh 
X 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Hematologyh 
 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Urinalysish 
 
X 
 
 
 
 
X 
 
 
X 
 


 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Serum bile acidsi 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
INR 
 
X 
 
 
X 
 
X 
 
X 
 
X 
Autotaxinj 
 
 
X 
 
X 
 
 
 
 
X 
 
p-C4j 
 
 
X 
 
X 
 
 
 
 
X 
 
AFP 
 
 
X 
 
 
 
 
 
 
X 
 
Fat-soluble vitamins A & E 
 
X 
 
 
 
 
 
 
 
X 
 
Fat-soluble vitamin D 
 
 
X 
 
 
 
 
 
 
X 
 
Abdominal Ultrasound 
 
 
X 
 
 
 
 
 
 
X 
 
Fibroscan® (where available) 
 
 
X 
 
 
 
 
 
 
X 
 
QoL questionnaire (PedsQL) 
 
 
X 
 
 
 
 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of Change 
 
 
 
 
X 
 
X 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of 
Symptoms 
 
 
X 
 
X 
 
X 
 
 
X 
 
Pregnancy testk 
Xk 
X 
X 
 
X 
X 
X 
X 
X 
Xk 
X 
Study drug dispensedl 
 
 
X 
 
X 
X 
X 
X 
X 
 
 


 
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EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 1.2 Fr, UK 15 March 2018 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Adverse eventsm 
Continuous collection 
Study drug compliance 
 
 
 
X 
X 
X 
X 
X 
X 
X 
 
AFP: alfa-fetoprotein; BMI: body mass index; eCRF: electronic case report form; eDiary: electronic diary; EOT: End of Treatment; ICF: informed consent form; 
INR: international normalized ratio; p-C4: plasma 7α Hydroxy 4-cholesten-3-one; PedsQL: Pediatric Quality of Life Inventory; QoL: quality of life; RR: respiratory rate; 
s-BA: serum bile acid. 
 
a 
Must also be performed at the time a patient is prematurely withdrawn from the study.  This visit is the same as Visit 1 in the extension protocol (Study A4250-008). 
b 
Assessments must be performed 28 days following the final dose of study drug.  This visit is omitted if patients are entering Study A4250-008. 
c 
The ICF (and assent) must be signed before any study procedures are performed. 
d 
Past (within 1 month prior to Visit 1) and current medication will be documented in the eCRF. 
e 
Blood sample for genetic testing will be drawn for all patients.  Genetic analysis will only be performed on a patient’s extracted DNA if previous the clinical genetic 
result is equivocal, unavailable, or unobtainable. 
f 
Includes blood pressure, pulse, RR, temperature, height (or length depending on age), and body weight (using a certified weight scale).  BMI will be calculated. 
g 
Includes training on use of the device at Screening (Visit 1) and review of patient daily entries during 14 days prior to Clinic Visit 3 to determine eligibility.  
h 
See Table 3 for detailed parameters. 
i 
Patients will fast (water intake only is permissible) for at least 4 hours prior to the collection of samples for s-BA.  Exceptions can be made for infants, less than 
12 months of age, if unable to fast for the full 4 hours.  All s-BA results during the treatment period and follow-up will be blinded. 
j 
Samples will not be collected for patients ≤10 kg. 
k 
For girls who have reached menarche.  Serum test at Visit 1; urine test at other visits.  A serum pregnancy test will also be conducted at Visit 9 for patients entering 
Study A4250-008.  If a urine pregnancy test is positive, a serum pregnancy test should be performed to confirm the pregnancy. 
l 
Study drug to be taken once daily on Day 1 through Day 167 as described in Section 8.2. 
m 
Adverse event information will be collected from the time of signing of the ICF to study discontinuation. 


 
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M5.3.5.1 – Protocol A4250-005 
 
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7.1.3 
Study Procedures and Assessments 
7.1.3.1 
Screening Period (Days -56 to 0) 
Day -56 to Day -35/Clinic Visit 1 
Patients will undergo a Screening Visit up to 56 days prior to the planned first day of study 
treatment.  Screening procedures and assessments are as follows: 
• Obtain written informed consent 
• Assess inclusion/exclusion criteria (Section 7.2) 
• Record demographics (age, full date of birth, gender, race, and ethnicity) 
• Document concomitant medications 
• Medical and surgical history (date of diagnosis of PFIC Types 1 or 2, prior investigational 
medications for PFIC, historical liver function test [LFT] values, any surgery performed, 
any other diagnosis, and historical liver biopsy data) 
• Blood sample for genetic testing 
• Collect, if available, past confirmatory clinical genetic laboratory report for PFIC1/PFIC2 
and send to central reader for review 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• eDiary training and compliance requirements; patients begin daily recording of pruritus 
using the eDiary (Appendix 2) 
• Clinical chemistry (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Serum pregnancy test for girls of who have reached menarche. Please see Appendix 5 for 
contraceptive advice.  
• AE monitoring 
Day -28 to Day -7/Clinic Visit 2 
A second Screening Visit will be performed.  Screening procedures and assessments are as 
follows: 
• Review concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for itching, scratching, and sleep scores and 
compliance 


 
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EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 1.2 Fr, UK 15 March 2018 
 
• Urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• International normalized ratio (INR) 
• Fat-soluble vitamins A and E 
• Urine pregnancy test for girls of who have reached menarche 
• AE monitoring 
7.1.3.2 
Treatment Period (Day 0 to Day 168) 
Study Day 0/Clinic Visit 3 (Randomization) 
At the randomization visit, the following assessments will be conducted: 
• Review inclusion/exclusion criteria (Section 7.2) 
• Review concomitant medications, medical and surgical history 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance and entrance criteria requirement (see 
Inclusion Criteria 4, Section 7.2.1) 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1)  
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• Fat-soluble vitamin D 
• Alfa-fetoprotein (AFP) 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer QoL (Pediatric Quality of Life Inventory [PedsQL]) questionnaire 
• Patient/caregiver/clinician complete the patient global impression of symptoms (PGIS) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Study drug is dispensed and patients are instructed to administer daily from Day 1 
Study Day 14 (Telephone Contact) 
A study nurse will contact the patient/caregiver by telephone for monitoring of the following:  


 
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EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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• Review concomitant medications 
• Albireo PRO/ObsRO eDiary review for compliance 
• AEs 
• Evaluation of study drug compliance 
Study Day 28 and Day 84/Clinic Visits 4 and 6  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• Urinalysis (at Clinic Visit 6 only; Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Autotaxin and p-C4 (at Clinic Visit 4 only; samples will not be taken for patients ≤10 kg) 
• Patient/caregiver/clinician complete the patient global impression of change (PGIC) and 
PGIS 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 56 and Day 126/Clinic Visits 5 and 7 
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 


 
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EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 1.2 Fr, UK 15 March 2018 
 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 154/Clinic Visit 8  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 168/Clinic Visit 9/EOT 
The last dose of study drug is taken in the morning the day before Clinic Visit 9, and the 
following procedures and assessments will be performed (also performed at the time a patient 
prematurely withdraws):  
• Review of concomitant medications 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry, hematology, and urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• AFP  
• Fat-soluble vitamins A, E, and D 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer PedsQL questionnaire 
• Patient/caregiver/clinician complete the PGIC and PGIS 


 
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EudraCT 2017-002338-21 
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M5.3.5.1 – Protocol A4250-005 
 
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• Urine pregnancy test for girls who have reached menarche, or serum pregnancy test for 
girls continuing to study A4250-008 
• AE monitoring 
• Evaluation of study drug compliance 
7.1.3.3 
Follow-Up Period 
Study Day 196/Clinic Visit 10 
All patients not continuing into Study A4250-008 will return to the study site 28 days after 
Clinic Visit 9 or the last dose of study drug for the following assessments: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
 
• AE monitoring 
7.2 
Study Population 
A total of 60 patients with clinical diagnosis of PFIC Type 1 or 2 that have been genetically 
confirmed will be randomized.  
An attempt will be made to randomize a minimum of 10 patients from each age category 
(6 months to 5 years, 6 to 12 years, and 13 to ≤18 years).  
A maximum of 5 patients that were treated in Study A4250-003 will be allowed to participate 
in this study. 
7.2.1 
Inclusion Criteria 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages 
of ≥6 months and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have genetic confirmation of PFIC-1 or PFIC-2 through identification of 
biallelic pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, 
taken as the average of 2 samples 7 to 28 days apart (Visits 1 and 2) prior to 
randomization 


 
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EudraCT 2017-002338-21 
Module 5:  Clinical Study Reports 
M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 1.2 Fr, UK 15 March 2018 
 
4. Patient must have history of significant pruritus and a caregiver-reported observed 
scratching in the eDiary average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to 
randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  
Patients who turn 18 years of age (or legal age per country) during the study will be 
required to re-consent in order to remain in the study 
6. Patients will be expected to have a consistent caregiver(s) for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use 
an eDiary device as required by the study 
7.2.2 
Exclusion Criteria 
1. Patient with pathologic variations of the ABCB11 gene that predict complete absence of 
the BSEP function 
2. Patient with past medical history or ongoing presence of other types of liver disease 
including, but not limited to, the following: 
a) Biliary atresia of any kind 
b) Benign recurrent intrahepatic cholestasis, indicated by any history of normal s-BAs 
c) Suspected or proven liver cancer or metastasis to the liver on imaging studies 
d) Histopathology on liver biopsy is suggestive of alternate non-PFIC related etiology of 
cholestasis 
3. Patient with a past medical history or ongoing presence of any other disease or condition 
known to interfere with the absorption, distribution, metabolism (specifically bile acid 
metabolism), or excretion of drugs in the intestine, including but not limited to, 
inflammatory bowel disease 
4. Patient with past medical history or ongoing chronic (i.e., >3 months) diarrhea requiring 
intravenous fluid or nutritional intervention for treatment of the diarrhea and/or its 
sequelae 
5. Patient has a confirmed past diagnosis of infection with human immunodeficiency virus 
or other present and active, clinically significant, acute, or chronic infection, or past 
medical history of any major episode of infection requiring hospitalization or treatment 
with parenteral anti-infective treatment within 4 weeks of treatment start (study Day 1) or 
completion of oral anti-infective treatment within 2 weeks prior to start of Screening 
Period 
6. Any patient with suspected or confirmed cancers except for basal cell carcinoma, and 
non-liver cancers treated at least 5 years prior to Screening with no evidence of 
recurrence 


 
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EudraCT 2017-002338-21 
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M5.3.5.1 – Protocol A4250-005 
 
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7. Patient with a past medical history of chronic kidney disease with an impaired renal 
function and a glomerular filtration rate <70 mL/min/1.73 m2  
8. Patient with surgical history of disruption of the enterohepatic circulation excluding those 
who have undergone a successful reversal procedure that has permanently restored flow 
of bile acids from the liver to the duodenum 
9. Patient has had a liver transplant or listed for liver transplant 
10. Decompensated liver disease, coagulopathy, history or presence of clinically significant 
ascites, variceal hemorrhage, and/or encephalopathy 
11. INR >1.4 (the patient may be treated with Vitamin K intravenously, and if INR is <1.4 at 
resampling the patient may be randomized) 
12. Serum ALT >10 × upper limit of normal (ULN) at Screening 
13. Serum ALT >15 × ULN at any time point during the last 6 months unless an alternate 
etiology was confirmed for the elevation 
14. Total bilirubin >5 × ULN at Screening 
15. Patient suffers from uncontrolled, recalcitrant pruritic condition other than PFIC.  
Examples include, but not limited to, refractory atopic dermatitis or other primary pruritic 
skin diseases 
16. Any patient who is pregnant or lactating or who is planning to become pregnant within 
24 weeks of randomization 
17. Sexually active males and females who are not using a reliable contraceptive method with 
≤1% failure rate (such as barrier protection, hormonal contraception, intrauterine device, 
or complete abstinence) throughout the duration of the study and 90 days thereafter (from 
signed informed consent through 90 days after last day of treatment). See Appendix 5 for 
further details. 
18. Patient with a past medical history of alcohol or substance abuse will be excluded.  
Patient must agree to refrain from illicit drug and alcohol use during the study 
19. Administration of bile acid or lipid binding resins and medications that slow GI motility 
(Refer to Appendix 1 – Concomitant Medications Prohibited during the study) 
20. Patient has had investigational exposure to a drug, biologic agent, or medical device 
within 30 days prior to Screening, or 5 half-lives of the study agent, whichever is longer 
21. Patient who has been previously treated with an IBAT inhibitor whose pruritus has not 
responded to treatment 
22. Any other conditions or abnormalities which, in the opinion of the investigator or Medical 
Monitor, may compromise the safety of the patient, or interfere with the patient 
participating in or completing the study 


 
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M5.3.5.1 – Protocol A4250-005 
 
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7.2.3 
Withdrawal of Patients 
Patients/caregivers will be informed that they have the right to withdraw from the study at 
any time, without prejudice to their medical care, and that they are not obliged to state their 
reasons. 
Any withdrawal, and reasons for withdrawal, must be fully documented in the electronic case 
report form (eCRF) and source documents and the patient followed by the 
investigator/investigative staff.  Even after the study is completed at the Follow-up Visit, the 
investigator/investigative staff will, regardless of reason for withdrawal, record any patient 
data they receive concerning SAEs, and all drug-related non-serious AEs, and subsequently 
report these to Albireo.  Withdrawn patients will not be replaced. 
Patients will be withdrawn in the following circumstances: 
• A patient’s/caregiver’s desire for withdrawal for any reason 
• Lost to follow-up (every effort must be made to contact the patient/caregiver; a certified 
letter must be sent) 
• An AE that, in the opinion of the investigator, necessitates withdrawal 
• A worsening of the symptoms related to diarrhea occurs 
• Death 
• Patient/caregiver estimation of lack of improvement/intolerable symptoms 
• A patient’s/caregiver’s substantial noncompliance (eDiary and study drug compliance) or 
protocol violation 
• An investigator’s opinion that continuing the patient in the study is not appropriate.  The 
investigator may withdraw a patient at any time, if it is considered to be in the patient’s 
best interest 
A patient who prematurely withdraws due to no improvement/intolerable symptoms, and has 
completed at least 12 weeks treatment in the double-blind study, will be offered the 
opportunity to enter open-label Study A4250-008.  A patient who withdraws from treatment 
prematurely and will not enter Study A4250-008 will be requested to complete all visits and 
safety assessments per the current protocol (A4250-005).  As a minimum, patients will have 
EOT (Visit 9) assessments at the time of withdrawal and a follow-up assessment (Visit 10) 
28 days following the last dose of study drug, and the patient will be recorded as withdrawn. 
7.2.4 
Study Termination by Sponsor 
This study may be terminated at any time by Albireo if serious side effects should occur, if 
the investigator does not adhere to the protocol; or if, in the sponsor's judgment, there are no 
further benefits to be achieved from the study.  In this event, Albireo/designee will inform the 
study investigators, institutions, and all regulatory authorities. 


 
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Final Amendment 1.2 Fr, UK 15 March 2018 
 
Albireo may temporarily or permanently discontinue the study at an investigative site at any 
time for safety, ethical, compliance, or other reasons.  If this is necessary, Albireo will 
endeavor to provide advance notification to the site.  If a site or the study is suspended or 
discontinued, the investigator/investigative staff will be responsible for promptly informing 
the Independent Ethics Committee (IEC).  If required by local regulations, Albireo/designee 
will be responsible for informing the IEC and the regulatory authority of study or site 
discontinuation.  In such an event, all study data and unused study drug must be returned to 
Albireo. 


 
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EudraCT 2017-002338-21 
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M5.3.5.1 – Protocol A4250-005 
 
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Final Amendment 1.2 Fr, UK 15 March 2018 
 
8 
TREATMENT OF PATIENTS 
8.1 
Identity of Study Drug 
A4250 and placebo will be supplied as capsules for oral administration.  White opaque 
capsules filled with pellets containing A4250 or placebo will be provided.  Two different 
capsule sizes will be available, as follows: 
• Capsule size 0 that can be opened 
• Capsule size 3 that can be opened or swallowed intact 
The content of the A4250 and placebo capsules will be identical in appearance.  Capsule 
filling weight will also be identical for high dose, low dose, and placebo.  Bottles with 
34 capsules will be given to the patient at each visit.  A patient who requires 2 or more 
capsules per day will be given multiple bottles.  Refer to the investigational product manual. 
8.2 
Administration of Study Drug 
Patients will be dosed with A4250 at a dose of 40 µg/kg/day, 120 µg/kg/day, or placebo for 
24 weeks.  Study drug will be dispensed to the patient at Visits 3, 4, 5, 6, 7, and 8, together 
with instructions on how to store and take the drug.  Study drug administration data, 
including whether each patient took each dose or partial doses of study drug, whether there 
were any delayed or missed doses, doses, and whether the capsule was opened or swallowed 
whole will be documented through the eDiary and transferred to the study database. 
A4250 should be taken in the morning, together with food.  On clinic visits days when 
laboratory assessments are conducted, study drug should be taken after the visit and after 
laboratory samples are taken.  Patients should not crush or chew the capsule(s).  When 
swallowing the capsule intact, the patient should administer the dose with a glass of water. 
For a patient unable to swallow capsule intact, the capsule can be opened and the contents 
sprinkled and mixed in a small amount of soft room-temperature yogurt, apple sauce, or fruit 
purée.  The amount of food should be less than what the patient/infant is expected to eat and 
should be followed by water.  
1. Hold the capsule over the prepared food, gently twist open and allow the content to fall 
into food 
2. Mix the entire capsule content with 1 or 2 tablespoons (15 to 30 mL) of soft food (apple 
sauce, fruit purée, or yogurt) 
3. Administer the mixture immediately after mixing. 
The number of capsules provided to the patient will be based on body weight at Visit 3.  At 
Visit 6, if a patient’s weight has shifted to another weight category (identified in Table 2), the 
number of capsules per day may be adjusted.   
 


 
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Final Amendment 1.2 Fr, UK 15 March 2018 
 
Table 2 
Dosing and Capsule Strength 
Body Weight 
(kg) 
Capsule 
Size 
Number of 
Capsules 
per Day 
Capsule 
Strength, 
Low Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Strength, 
High Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Size, 
Placebo 
(µg) 
5.0 to <7.5 
0 
1 
200 
200 
600 
600 
0 
7.5 to <12.5 
0 
2 
200 
400 
600 
1200 
0 
12.5 to <17.5 
0 
3 
200 
600 
600 
1800 
0 
17.5 to <19.5 
0 
4 
200 
800 
600 
2400 
0 
19.5 to <25.5 
3 
2 
400 
800 
1200 
2400 
3 
25.5 to <35.5 
3 
3 
400 
1200 
1200 
3600 
3 
35.5 to <45.5 
3 
4 
400 
1600 
1200 
4800 
3 
45.5 to 55.5 
3 
5 
400 
2000 
1200 
6000 
3 
>55.5 
3 
6 
400 
2400 
1200 
7200 
3 
8.3 
Study Treatment Packaging and Labeling 
8.3.1 
Packaging and Labeling 
The capsules will be packed in high-density polyethylene containers, with child-proof 
polypropylene caps.  Study drug capsules containing A4250 in the strength specified in 
Table 2 above will be manufactured. 
Packaging and labeling will be prepared to comply with applicable regulatory requirements. 
8.3.2 
Storage 
Treatment packs containing A4250 capsules should be stored and dispensed in accordance 
with regulations in their original containers.  The storage facility at the investigative site 
should be locked and the storage temperature should be between 15°C and 25°C. 
Patients/caregivers should be informed of appropriate storage conditions (i.e., room 
temperature, between 15°C and 25°C). 
Any deviations from the recommended storage conditions should be immediately reported to 
Albireo and the study drug should not be used until authorization has been given by Albireo. 
8.3.3 
Blinding and Randomization of Study Drug 
A double-blind design is employed so that both the investigators and the patients will be 
unaware of the treatment assignment during the study.  
After written informed consent is obtained from an eligible patient, an 8-digit patient 
identification number will be assigned.  The first 2 digits will denote the country, followed by 
a 3-digit site number and a 3-digit patient sequence number.  This number will be created and 
allocated by the Interactive Web Response System (IWRS), when the patient first enters the 
system at Screening.  The randomization codes will be computer-generated by 
 


 
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Biostatistics, and kept by a statistician independent from the project team.  The 
randomization will be done in blocks and stratified according to PFIC class (Type 1 and 2) 
and age class (6 months to 5 years, 6 to 12 years, and 13 to ≤18 years) to ensure approximate 
balance between dose schemes (1:1:1).  A separate randomization list will be prepared for the 
maximum 5 patients from Study A4250-003 regardless of stratification.  Randomization 
codes will be assigned sequentially as patients become eligible for randomization. 
Patients who are eligible for randomization will be assigned a unique 4-digit randomization 
number by IWRS.  This randomization number identifies which treatment will be allocated to 
the patient.  Patients who withdraw from the study after randomization visit are not to be 
replaced and their randomization number will not be reused.   
To ensure blinding, the study drug and the matching placebo have the same shape and size.  
Patients will receive capsule(s) of A4250 according to their dose group or capsule(s) of 
matching placebo once a day during the double-blind Treatment Period.  Labels on the study 
drug containers will not identify the treatment that a patient is randomized to.  Traceability of 
the treatment is ensured by the study drug number. 
The 5-digit study drug number will identify study drug packs and will be detailed on the 
study drug label.  Dispensing of study drug will be coordinated by IWRS.  
The system will assign a study drug number corresponding to the randomization arm.  The 
randomization number will be used in the background only to ensure no unblinding and will 
not be displayed to end-users in the IWRS. 
8.4 
Procedure for Breaking the Randomization Code 
Should a situation arise where unblinding is urgently required, i.e., only where knowledge of 
the study drug is required to adequately manage a life-threatening situation, the investigator 
at that study site may perform immediate unblinding through IWRS. The responsibility to 
break the treatment code in emergency situations resides solely with the investigator. The 
investigator should make every effort to discuss the rationale (status and outcome) for 
emergency unblinding with the sponsor Medical Monitor or the 
 Medical Monitor as 
soon as possible to review the individual patient details. 
The study site investigator and appropriate 
 project team members will be authorized to 
access the emergency unblinding functionality within the IWRS.  Emergency unblinding 
takes place in the IWRS via a dedicated emergency unblinding module.  The system will 
require the user to enter their emergency unblinding, which is typically provided at the same 
time login credentials are distributed.  The exact description of the treatment assigned to the 
individual patient then will be accessible to the user.  Once the randomization code is broken 
for a patient, he/she must be withdrawn from the study and all assessments and procedures at 
EOT should be performed.  As a follow-up, Visit 10 will be performed according to the time 
schedule.  Once a randomization code has been broken, the investigator must inform the 
 and sponsor Medical Monitor in writing within 24 hours. 


 
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8.5 
Patient Compliance 
The study nurse will monitor eDiary compliance by routine review of the CRF health 
website.  If both diary entries on a day are missing during, the study nurse will call the 
patient/caregiver to remind them to complete all scheduled entries.  Any noncompliance will 
be documented and explained in the source documents and in the eCRF. 
Study drug compliance will also be checked through diary responses.  Additionally, patients 
will return all unused study drug at Visits 4, 5, 6, 7, 8, and 9.  The study site staff will count 
all returned drug, assess compliance, and record details in the eCRF. 
Treatment Compliance = 100 × ((Number of study drug dispensed – number of study drug 
returned)/number of study drug that should be taken). 
Treatment compliance between 80% and 120% will be acceptable. 
8.6 
Study Drug Accountability 
Records shall be maintained of the delivery of study treatment(s) to the study site(s), of the 
inventory at the study site(s), of each use of the study treatment(s) for each patient, and of the 
return of unused study treatment(s) to Albireo. 
These records shall include dates, quantities, batch numbers, expiry dates, and the unique 
code numbers assigned to the study drug and to the study patients. 
The investigator will be responsible for ensuring that the records adequately document that 
the patients were provided the quantities specified in the protocol and that all study drug 
received from Albireo is reconciled.  
8.7 
Concomitant Therapy and Prohibited Medications 
The investigator will note all ongoing medication and any medication recently stopped 
(within 1 month prior to Visit 1) in the eCRF.  At Visits 2 through 10, all changes in 
medication (stopping or starting new medication or changes in dose) will be recorded in the 
eCRF.  All medications (prescribed or over the counter) for pruritus and sleep will be 
recorded. 
All medications taken by a patient within 3 months prior to the first intake of study drug are 
regarded as prior medication. 
All medications taken by a patient on or after the first intake of study drug, and which 
continue to be taken during the study, are regarded as concomitant medication.  
Prohibited medications are listed in Appendix 1. 
 


 
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9 
ASSESSMENT OF EFFICACY 
9.1 
Efficacy Endpoints 
9.1.1 
Primary Efficacy Endpoint 
The primary efficacy endpoints are as follows: 
• European Union (EU) and rest of the world (RoW): Proportion of patients 
experiencing at least a 70% reduction in fasting s-BA concentration from baseline to 
the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 
weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization 
and the previous visit (Clinic Visit 2).  The end value is the average of the values at 
Weeks 22 and 24 after the start of double-blind treatment. 
• United States (US): Change in pruritus, as indexed by caregiver-reported observed 
scratching, from baseline to Week 24 as measured by the worst scratch of the Albireo 
ObsRO instrument (Appendix 2).  Scores for the 14 days baseline and post-treatment 
intervals will be calculated by averaging the worst scratching scores from each day in 
the interval. 
9.1.2 
Secondary Efficacy Endpoint 
Secondary efficacy endpoints include the following:  
• EU and RoW: Change in pruritus, as indexed by caregiver-reported observed 
scratching, from baseline to Week 24 as measured by the worst scratch of the Albireo 
ObsRO instrument (Appendix 2).  Scores for the 14 days baseline and post-treatment 
intervals will be calculated by averaging the worst scratching scores from each day in 
the interval. 
• US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment. 
• All Regions: 
• All secondary endpoints are compared to placebo. 
o Change from baseline to Week 24 in fasting s-BA 
o Change from baseline to Week 24 in serum ALT concentration 
o Change in growth from baseline to Week 24, defined as the linear deficit 
(weight for age, and body mass index [BMI]) compared to the national growth 
curves (Z-score, standard deviation [SD] from P50)  
o Proportion of patients achieving meaningful reduction in caregiver-reported 
observed scratching at Week 24 


 
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o Change from baseline to Week 24 in sleep parameters measured with the 
Albireo PRO and ObsRO instruments 
o Change from baseline to Week 24 in patient-reported itch severity as 
measured by the average daily score of the Albireo PRO instrument 
(Appendix 2); only patients ≥8 years of age will complete the Albireo PRO 
instrument  
o Number of patients undergoing biliary diversion surgery or being listed for 
liver transplantation 
9.1.3 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• Proportion of patients achieving meaningful reduction in patient-reported itch severity at 
Week 24 
• Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, 
caregiver, and clinician via the PGIC items (Appendix 3) 
• Change from baseline in fasting s-BA at Weeks 4 and 12 
• Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
to Weeks 4, 12, and 24 as measured by the worst scratch.  Scores for the 7 worst 
scratching days of the last 14 days at baseline and post-treatment intervals will be selected 
• Change from baseline in patient-reported itch severity and observer-reported scratching at 
Weeks 4 and 12  
• Change from baseline to Week 24 in patient-reported and observer-reported night time 
itching and scratching severity scores, respectively 
• Change from baseline to Week 24 in pooled pruritus score including observer-reported 
scratching for patients <8 years of age and patient-reported itch severity for patients 
≥8 years of age 
• Change from baseline to Week 24 in additional patient-reported and observer-reported 
sleep parameters (e.g., tiredness and number of awakenings) 
• Change from baseline to Week 24 in PedsQL (Appendix 4) 
• Change from baseline in serum ALT concentration at Weeks 4 and 12 
• Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• Change from baseline in AST at Weeks 4, 12, and 24 
• Change from baseline for other biochemical markers and measures of bile acid synthesis 
(autotaxin, p-C4) at Week 24 


 
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• Change in pediatric end-stage liver disease/model for end-stage liver disease 
(PELD/MELD) score, AST to platelet ratio index (APRI) score, and Fib-4 score from 
baseline to Week 24 
• Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® 
(where available) 
• Liver-related mortality and liver decompensation events 
• All-cause mortality 
9.2 
Efficacy Assessments 
9.2.1 
Serum Bile Acids 
Blood samples for analysis of fasting total s-BA will be drawn at all visits.  Patients will fast 
(water intake only is permissible) for at least 4 hours prior to the collection of samples for 
s-BA.  Exceptions can be made for infants, <12 months of age, if unable to fast for the full 
4 hours.  Any visit at which a bile acid sample result is unreportable, an additional 
unscheduled visit for a repeat sample collection may be scheduled.  All s-BA results during 
the treatment period and follow-up will be blinded.  Samples will be processed and 
transported to a central laboratory per instructions in the laboratory manual. 
9.2.2 
Itching, Scratching and Sleep Score 
Itching, observed scratching, and sleep disturbance will be recorded twice each day via 
eDiary.  Patients and/or caregivers will be instructed to complete the eDiary every day in the 
morning after the patient wakes and in the evening just before the patient goes to sleep 
throughout the study.  Patients and caregivers will receive a formal training session during 
Visit 1. 
The eDiary includes Albireo ObsRO and PRO items.  Patients <8 years of age will not be 
asked to complete the Albireo PRO items; only the Albireo ObsRO will be completed by 
caregivers of patients in this age group.  Older patients, 8 to 17 years of age, will complete 
the Albireo PRO items and the caregiver will complete the Albireo ObsRO items.  The 
Albireo PRO items assess severity of itch, aspects of sleep disturbance (morning diary only), 
and tiredness.  For patients 8 to 12 years of age, the caregiver will read the Albireo PRO 
items along with the child and record the child’s response.  A guide will be provided to the 
caregivers that provides standardized explanations of the Albireo PRO items, in case the 
patient is confused or requires clarification about the meaning of a question.  The Albireo 
ObsRO items assess severity of observed scratching, aspects of observed sleep disturbance 
(morning diary only), and observed signs of tiredness (evening diary only).  The Albireo 
ObsRO and PRO scratching and itch severity items use 0 to 4 response scales, where each 
response is distinguished by a unique facial expression, verbal anchor, number, and color 
code (Appendix 2).  


 
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A daily score for the Albireo ObsRO scratching item will be calculated by selecting the 
maximum scratching severity score from the 2 ratings for each day.  A daily score will be 
considered missing if both of the daily assessments are missing.  A 14-days score will be 
calculated by averaging the daily scratching scores.  A 14-days score will be considered 
missing if >4 out of 7 days a week of data are missing.  The same approach will be used to 
calculate a patient-reported itch severity score. 
9.2.3 
Growth 
Growth will be measured as height (velocity) and weight (Z-score) using a certified weight 
scale.  BMI will be calculated by weight (kg) / height (m)2.  Change will be defined as linear 
growth deficit (weight and BMI for age) compared to national growth curves (Z-score, SD 
from P50). 
9.2.4 
Quality of Life Questionnaire (PedsQL) 
Patients and caregivers will be asked to complete a QoL questionnaire (PedsQL) at Visit 3 
and Visit 9.  Details of the questions included on the questionnaire are provided in 
Appendix 4. 
9.2.5 
Biomarkers and Other Laboratory Samples 
Blood samples for autotaxin and p-C4 will be drawn at Visits 3, 4, and 9 only for children 
with body weight ≥10 kg.  Samples will be processed and transported to a laboratory per 
instructions in the laboratory manual. 
9.2.6 
PELD/MELD Score 
At randomization and end of treatment, PELD score will be calculated for children up to 
12 years of age.  For children 13 years or older, the MELD score will be calculated. 
PELD Score = 4.80 * ln(total bilirubin in mg/dL) + 18.57 * ln(INR) - 6.87 * ln(albumin in 
g/dL) + 4.36 (if patient <1 year: scores for patients listed for liver transplantation before the 
patient's first birthday continue to include the value assigned for age (<1 year) until the 
patient reaches the age of 24 months) + 6.67 (if the patient has growth failure [≤2 SD]) 
Laboratory values less than 1.0 will be set to 1.0 for the calculation of the PELD score. 
MELD Score = 9.57 * ln(creatinine in mg/dL) + 3.78 * ln(total bilirubin in mg/dL) + 
11.2 * ln(INR) + 6.43 
Laboratory values less than 1.0 will be set to 1.0 and serum creatinine values greater than 
4.0 mg/dL will be set to 4.0 for calculation of the MELD score. 
9.2.7 
Fibroscan® 
Where available, Fibroscan® will be performed as per institution standard practice at Visits 3 
and 9. 


 
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9.2.8 
Markers of Fibrosis 
APRI score and Fib-4 score will be calculated at randomization and at end of treatment. 
APRI = [(AST in U/L) / (AST ULN in U/L)] / (Platelets in 109/L) 
Fibrosis 4 Score = (Age*AST in U/L) / (Platelets in 109/L * √(ALT in U/L)). 
9.2.9 
Global Impression of Change and Global Impression of Symptom Measures 
Patients (≥8 years of age), caregivers, and clinicians will complete the PGIC and the PGIS 
measures (Appendix 3) at randomization (Visit 3; PGIS only), Visits 4 and 6, and at EOT 
(Visit 9). 
The PGIC items assess change in itch (patient version), scratching (caregiver and clinician 
versions), and sleep (all versions) since starting the study drug.  The PGIS items assess itch 
(patient version), scratching (caregiver and clinician versions), and sleep (all versions) in the 
past week.  Caregivers and clinicians will complete the PGIC and PGIS for all patients; those 
patients ≥8 years of age will complete the patient version. 


 
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10 
ASSESSMENT OF SAFETY 
The timing and frequency of safety assessments are described in Section 7.1.3 and in Table 1 
above. 
The primary safety analysis for this study will include the incidence of total 
treatment-emergent adverse events (TEAE) and TEAEs categorized by causality, severity, 
and seriousness assessments made by the investigator by comparing study drug exposure to 
placebo. 
Trends in safety will also be evaluated for the following assessments: 
• Physical examinations 
• Concomitant medications 
• Vital signs 
• Laboratory test results (including clinical chemistry, hematology, urinalysis, AFP, and 
fat-soluble vitamins A, D, and E) 
• Abdominal ultrasound 
• Discontinuations due to AEs 
10.1 
Adverse Events 
10.1.1 
Definitions and Investigator Assessments 
An AE is defined as any untoward medical occurrence in an enrolled patient regardless of 
causal relationship with study drug.  An AE can therefore be any clinically significant 
unfavorable and unintended sign, symptom, or disease that occurs once a patient is enrolled 
(ICF is signed) in the study until the patient is discharged from the study, whether or not 
related to the study drug. 
10.1.1.1 
Clinical Significance 
Clinical significance is defined as any abnormal finding that results in further clinical 
investigation(s), treatment(s), or the diagnosis of new condition.  Patient-reported events and 
protocol-mandated laboratory values, vital signs, and physical examination findings can be 
considered clinically significant (i.e., an AE) if there is a deterioration as compared to 
baseline.  Examples of clinically significant worsening from baseline could include, but is not 
limited to, events causing withdrawal from the study and events requiring medical 
intervention outside of the study causing apparent clinical manifestations, or judged relevant 
by the investigator.   
10.1.1.2 
Serious Adverse Events 
Serious criteria are applied by the investigator to each AE as specifically defined below.  
These criteria are used to determine whether an AE is serious or non-serious.  The assessment 
is made independently of severity assessment (Section 10.1.1.3).  For example, the 


 
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development of a severe rash that occurs after signing of informed consent may not meet 
serious criteria as defined below and therefore would be considered a severe, non-serious AE. 
Any AE that meets any 1 of the following 6 criteria is considered an SAE: 
• The outcome of the AE is death 
• The AE is immediately life-threatening.  Life-threatening means that the patient is, in the 
opinion of the investigator, at immediate risk of death from the reaction as it occurred.  
This does not include an AE that, if more severe, might have caused death 
• The AE results in persistent or significant disability/incapacity.  Disability means a 
substantial disruption of a person's ability to conduct normal life functions 
• The AE requires or prolongs hospitalization  
• The AE results in a congenital anomaly/birth defect 
• The AE is an important medical event.  Important medical events may meet serious 
criteria should the investigator assess that they may significantly jeopardize the patient, 
represent a significant hazard, or requires medical/surgical intervention to prevent one of 
the outcomes listed above.  Examples of potential SAEs based on this criteria include, but 
are not limited to, allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions even if they do not result in inpatient 
hospitalization, or the development of drug dependency and drug abuse. 
10.1.1.3 
Severity Assessment 
Severity assessments are based on the intensity of the event in relation to expectation.  The 
investigator will assess the intensity of AEs based on the following definitions: 
• Mild (awareness of sign or symptom, but easily tolerated) 
• Moderate (discomfort sufficient to cause interference with normal activities) 
• Severe (incapacitating, with inability to perform normal activities) 
Severity is a measure of intensity where seriousness is defined by the criteria outlined in 
Section 10.1.1.2.  An AE of severe intensity need not necessarily be considered serious.  For 
example, nausea that persists for several hours may be considered severe nausea, but not meet 
serious criteria, and therefore would be assessed as a severe AE but not an SAE. 
10.1.1.4 
Causality Assessment 
The investigator determines the causality of all AEs to the study drug using medical judgment 
and considering all relevant factors such as (but not limited to) the underlying study 
indication, coexisting disease, concomitant medication, relevant history, pattern of the AE, 
temporal relationship to the study drug, and de-challenge or re-challenge.  The causality 
assessment of the AE/SAE is to be made as follows. 


 
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Related to study drug (possibly, probably, or definitely related) 
Based on medical judgment, there is at least a reasonable possibility that the study drug 
caused the event; one or more of the following criteria apply: 
• The event follows a reasonable temporal sequence from administration of study drug 
• The event could not be reasonably attributed to the known characteristics of the patient's 
clinical state, environmental or toxic factors, or other modes of therapy administered to 
the patient 
• The event follows a known pattern of response to study drug 
• The event disappears or decreases on cessation or reduction in dose of the study drug.  (It 
should be noted that in some situations an AE will not disappear or decrease in intensity 
upon discontinuation of study drug despite other clear indications of relatedness) 
• The event reappears or worsens when the study drug is re-administered 
Unrelated to study drug (unlikely or unrelated) 
Based on medical judgment there is no reasonable possibility that the study drug caused the 
event; one or more of the following criteria apply: 
• The event does not follow a reasonable temporal sequence from administration of study 
drug 
• The event could be reasonably attributed to the known characteristics of the patient's 
clinical state, concurrent illness, environment or toxic factors, or other modes of therapy 
administered to the patient 
• The event does not follow a known pattern of response to study drug 
• The event does not disappear or decrease on cessation or reduction in dose of the study 
drug, and it does not reappear or worsen when the study drug is re-administered 
10.1.2 
Recording of Adverse Events 
It is the investigator’s responsibility to assess whether each untoward event is a clinically 
significant worsening from baseline, thereby considered an AE.  For all AEs, the severity, 
seriousness, and causality to study drug for each AE as outlined in Section 10.1.1 will be 
assessed and recorded in the eCRF. 
All serious and non-serious AEs are collected once the caregiver/patient has signed the ICF 
and until the post-treatment follow-up (Visit 10) or 28 days after the last dose of study drug. 
Any AEs or SAEs that are unresolved at the patient’s last AE assessment in the study are 
followed up by the investigator until resolution or stabilization up to the database lock and 
recorded in the eCRF.  Albireo retains the right to request additional information for any 
patient with ongoing AE(s) or SAE(s) at the end of the study. 


 
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TEAEs are defined as any AE that occurs after randomization (Day 0).  All AEs that occur in 
the Screening Period, i.e., after enrollment and prior to randomization, will be collected on 
the eCRF as non-treatment-emergent AEs. 
If there is a clinically significant deterioration of a laboratory value/vital sign or other routine 
study assessment that is associated with a diagnosis, the clinical diagnosis will be reported as 
an AE and the associated signs and symptoms will be considered additional information 
unless the sign or symptom is more severe than expected given the diagnosis.  For example, if 
an investigator diagnoses a study patient with hepatitis during the study period, hepatitis 
would be considered the AE and the concomitant signs and symptoms of abdominal pain, 
vomiting, and elevated ALT and AST would not be reported separately unless, in the opinion 
of the investigator, one of these signs or symptoms is more severe than expected and 
therefore a separate AE assessment is indicated. 
10.1.3 
Recording and Reporting of Serious Adverse Events 
Every SAE (regardless of severity and causality) that occurs once the caregiver/patient has 
signed the ICF and until the post-treatment follow-up (Visit 10), and 28 days after the final 
dose of study drug, should immediately and not later than within 24 hours of knowledge of 
the event, be reported by the investigator or delegate in the SAE report form.    
Report of a SAE must include at least the following information: 
• Patient identification information (study number, site number, initials, and date of birth 
[as per local country requirements for data protection]) 
• The last study drug administration date 
• The diagnosis of the event with the description (or a brief description of 
signs/symptoms/clinical course if the diagnosis is not available) and the date of onset 
• The action used to treat the event (i.e., treatment medications, temporary discontinuation) 
• The reason(s) for considering the event serious 
• The relationship of the event to the study drug or to the study procedure (e.g., the 
investigator's assessment of causality) 
• A brief narrative of the SAE 
Follow-up reports including all new information obtained of the subsequent course of the 
SAE must be prepared and the information collected in the SAE submitted to 
 
 (Pharmacovigilance and Safety Services Department) by e-mail within 24 hours of 
knowledge of the event. 
The pharmacovigilance manager (PVM) may contact the investigator to obtain further 
information on a reported SAE.  The investigator/investigative staff must respond to any 
request for follow-up information or answers to questions regarding the SAE within the same 
timelines as for initial reports. 


 
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The PVM reports the occurrence of the SAE and follow-up to the Albireo medical monitor 
for medical assessment of the case. 
Should an outcome of death occur within the study period or within 28 days after the last 
administration of study drug, an SAE form should be completed including the main and 
contributory causes of death. 
All SAE reports must be e-mailed to the following e-mail address within 24 hours: 
 
 
If email is unavailable, SAEs may be transmitted via fax to the following number: 
 
In a study-related medical emergency situation, when the assigned medical monitors for the 
study cannot be reached by a caller, an on call physician can be reached 24 hours per day, 
7 days per week, via the 
 call center.  The following number is a chargeable telephone 
number allowing a global reach from both landlines and mobile phones and the internet page 
indicated below contains a list of country-specific toll-free telephone numbers that is 
provided.  It should be noted that not all countries globally have access to toll-free numbers 
as indicated on the “24/7 Medical Help desk” index.  Countries without toll-free numbers 
need to dial the chargeable number as indicated below.  Furthermore, toll-free numbers might 
not be available from mobile phones. 
 
 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions 
Suspected unexpected serious adverse reaction (SUSAR) is an SAE that occurs in a patient, 
the nature or severity of which is not expected per the applicable product information (e.g., 
the IB for an unauthorized investigational product or summary of product characteristics for 
an authorized product).  
Reporting and tracking of SUSARs will be in accordance to all applicable competent 
authority regulations.  The Ethics Committee and all investigators involved in this study will 
be informed according to national and international requirements. 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments 
10.2.1 
Laboratory Assessments 
Samples will be collected for clinical chemistry, hematology, and urinalysis testing at the 
time points specified in Table 1. The parameters assessed are presented in Table 3. 


 
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Blood for AFP will be drawn at Visits 3 and 9 and for fat-soluble vitamins at Visits 2, 3, 
and 9. 
A serum pregnancy test will be performed at Visit 1 and Visit 9 if the patient is entering the 
A4250-008 study. A urine pregnancy test will be collected at all other visits for girls who 
have reached menarche.  If a urine pregnancy test is positive, a serum pregnancy test should 
be performed to confirm the pregnancy.  Study drug administration will be held pending the 
confirmatory results.  If the serum pregnancy test is negative, the patient can resume dosing.  
If the serum pregnancy test is positive, the patient should be discontinued (see 
Section 10.2.9). 
All samples will be processed and transported to a laboratory per instructions in the 
laboratory manual. 
The observed values will be recorded and assessed as “normal” or “abnormal not clinically 
significant” or “abnormal clinically significant”. 
Additional safety blood samples may be needed due to follow-up of an abnormal value or 
analysis failure.  The blood samples collected for safety laboratory analysis will be destroyed 
after the analyses have been completed.  
Table 3 
Routine Laboratory Parameters 
Clinical Chemistry 
Hematology 
Urinalysis 
• 
Albumin 
• 
ALT 
• 
Alkaline phosphatase  
• 
AST 
• 
Bilirubin – total and direct 
• 
Calcium 
• 
Chloride 
• 
Creatinine 
• 
Creatine kinase 
• 
Gamma-glutamyl transferase 
• 
Potassium 
• 
Sodium 
• 
Hematocrit 
• 
Hemoglobin 
• 
Platelet count 
• 
Red blood cell count 
• 
White blood cell count 
• 
Blood 
• 
Glucose 
• 
Ketones 
• 
Leukocytes 
• 
Nitrites 
• 
pH 
• 
Protein 
 
10.2.2 
Individual Patient Safety Monitoring 
10.2.2.1 
Liver Monitoring 
Strategies to monitor markers of liver disease throughout the study are outlined below where 
the ULN will be based on central laboratory reference values for age and gender. 
If isolated transaminase elevations are observed, defined as: 
1. Normal bilirubin AND absence of clinical hepatitis symptoms AND 


 
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• ALT or AST ≥5 × ULN (if normal at baseline) or an absolute threshold of 800 U/L, 
whichever comes first  
• OR ALT or AST ≥3 × baseline (if abnormal at baseline) or an absolute threshold of 
800 U/L, whichever comes first 
Then: 
a) Repeat liver profile (AST, ALT, bilirubin, and prothrombin time [PT] or INR) within 
2 to 3 days 
b) Evaluate creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) 
c) As needed (for example, persistent ALT/total bilirubin elevation or worsening of liver 
function), consider evaluation for alternative etiologies 
Monitor the patient using close observation found in Section 10.2.2.2 
If any ONE of the following criteria are met: 
1. Transaminases (ALT or AST ≥3 × baseline or 800 U/L, whichever comes first) AND 
bilirubin (total bilirubin > 2 × ULN) elevations 
2. Transaminase elevations alone (ALT or AST >10 × ULN or 5 × baseline or absolute 
threshold of 800 U/L, whichever comes first) in presence of normal LDH and CPK 
3. Cholestatic marker elevations (alkaline phosphatase [ALP] or gamma-glutamyl 
transferase >2 × baseline) without alternative explanation 
4. Total bilirubin increased, unrelated to hemolysis (elevated reticulocyte count) or 
established genetic diseases, such as Gilbert’s syndrome 
a. Doubling if total bilirubin was <3 mg/dL at baseline  
b. OR Increase by >3 mg/dL if total bilirubin was ≥3 mg/dL at baseline 
5. INR increase refractory to vitamin K administration 
a. Increase by >1.5 if INR was normal at baseline  
b. OR Increase by >0.4 if INR was abnormal at baseline 
6. Any increase in total bilirubin and transaminases if accompanied by EITHER a symptom 
of clinical hepatitis (e.g., vomiting, nausea, right upper quadrant pain) OR immunological 
reaction (rash or >5% eosinophilia) 
Then: 
a) Interrupt study medication 
b) Initiate drug-induced liver injury work-up for alternative etiologies 
c) Repeat liver profile (AST, ALT, total bilirubin, direct bilirubin, ALP) and PT or INR 
within 48 to 72 hours 


 
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d) Monitor the patient using close observation found in Section 10.2.2.2 
e) If a patient lives in a remote area, they may be tested locally and the results 
communicated to the investigator site promptly 
10.2.2.2 
Close Observation 
• Repeat liver enzyme and serum bilirubin tests two or three times weekly.  Frequency 
of re-testing can decrease to once a week or less if abnormalities stabilize or the trial 
drug has been discontinued and the subject is asymptomatic  
• Obtain a more detailed history of symptoms and prior or concurrent diseases 
• Obtain a history of concomitant drug use (including non-prescription medications and 
herbal and dietary supplement preparations), alcohol use, recreational drug use, and 
special diets  
• Rule out alternate etiology including acute viral hepatitis types A, B, C, D, and E; 
autoimmune or alcoholic hepatitis; non-alcoholic steatohepatitis; hypoxic/ischemic 
hepatopathy; and biliary tract disease 
• Obtain a history of exposure to environmental chemical agents.  
• Obtain additional tests to evaluate liver function, as appropriate (e.g., INR, direct 
bilirubin) 
• Consider gastroenterology or hepatology consultations. 
10.2.2.3 
De-challenge/Re-challenge for Liver and Clinical Hepatitis Monitoring 
1. Re-challenge is not recommended: 
a. If a patient has had possible/probable drug-induced liver injury 
b. If a decompensation event has occurred (i.e., variceal hemorrhage, ascites, hepatic 
encephalopathy, etc.). 
2. If due to underlying cholestatic liver disease variability or another alternative etiology is 
identified AND liver tests returned to baseline, re-challenge may be considered after 
consultation with the sponsor Medical Monitor. 
3. If the ALT/total bilirubin elevations are observed after re-challenge, then repeat 
re-challenge is discouraged. 
If patient is permanently discontinued, monitoring should be continued as outlined in 
Section 10.2.2.5.  
10.2.2.4 
Diarrhea 
Study drug should be discontinued if a patient develops diarrhea with at least 1 of the 
following concomitant signs or symptoms: grossly bloody stools, vomiting, dehydration 


 
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requiring treatment with oral or intravenous rehydration and/or electrolyte imbalances, fever 
(≥38 C) and/or the diarrhea persists for 7 or more days. 
If there is gross blood in stool, INR and platelets should be measured.  
Study drug will be reintroduced (re-challenge) when the symptoms have resolved.  If the 
diarrhea re-occurs within 1 week after the re-challenge with no alternate etiology, the patient 
will be permanently discontinued and monitored as outlined in Section 10.2.2.5. 
10.2.2.5 
Monitoring after Permanent Discontinuation of Study Drug Due to Safety 
Once study drug is permanently discontinued for liver, hepatitis, diarrhea, or other severe AE 
related to study drug, the patient will be monitored weekly until the laboratory and clinical 
parameters have normalized/stabilized. 
10.2.3 
Demographics/Medical and Surgical History 
Demographic information per country regulations (age, full date of birth, gender, race, and 
ethnicity), along with medical and surgical history, will be obtained and recorded in the eCRF 
at Visit 1. 
Medical and surgical history will be entered in the eCRF at Visit 1.  This includes date of 
diagnosis of PFIC, prior investigational medications for PFIC, historical (3 months prior to 
Screening) LFT values (AST, ALT, and total bilirubin), ongoing medication, any surgery 
performed, any other diagnosis, and historical biopsy data.   
If a liver biopsy has been performed within 1 year prior to Screening, or during the study, the 
results will be recorded in the eCRF. 
10.2.4 
Clinical Genetic Testing 
If confirmatory clinical genetic testing for PFIC Type 1 or 2 has been performed prior to 
Screening Visit 1, the confirmatory clinical genetic testing laboratory report will be verified 
by a central reader to determine eligibility. 
Regardless of previous genetic testing, a blood sample will be drawn at Visit 1 for all patients 
for DNA extraction.  Genetic testing will only be performed on a patient’s extracted DNA if 
the previous clinical genetic result is equivocal, unavailable, or unobtainable.  In such cases, 
screening for pathologic biallelic variations in the ATP8B1 or ABCB11 genes will be 
performed if patient meets remaining inclusion and exclusion criteria and only after sponsor 
case review.  No other diagnostic genetic testing will be offered. 
10.2.5 
Physical Examination 
A physician or suitably trained qualified assistant will perform a complete physical 
examination, including voluntary photography, at Visits 1, 3, and 9.  A skin examination will 
be conducted at Visits 1, 3, 4, 6, 9, and 10 (see Table 1).  


 
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A complete physical examination will include assessment of general appearance, eyes, ears, 
nose, throat, head/neck/thyroid, lymph nodes, cardiovascular, lungs/chest, abdomen, 
genitourinary, extremities, skin, musculoskeletal, neurologic, and other.  
Skin will be examined thoroughly and excoriations/scratch marks recorded.  
10.2.6 
Vital Signs 
Evaluation of vital signs will be performed at all visits.  This includes blood pressure (systolic 
and diastolic), pulse, respiratory rate, temperature, height (or length depending on age), and 
body weight (using a certified weight scale) at clinic visits.  BMI will be calculated. 
10.2.7 
Abdominal Ultrasound 
Ultrasound of the liver and spleen will be performed at randomization (Visit 3) and at EOT 
(Visit 9).  Liver size, echogenicity, and presence of masses/nodules as well as spleen size will 
be recorded. 
10.2.8 
Overdose 
A4250 is minimally absorbed and has a very low systemic availability.  Based on toxicology 
data, for study purposes any dose exceeding a total of >3 mg/kg body weight of A4250 taken 
as a single dose or as a cumulative dose within 24 hours is defined as an overdose.   
The no-adverse-effect dose level in the most sensitive species (defined as 20 mg/kg/day) in 
the rat 1 month toxicity study gives a human equivalent dose of approximately 194 mg/day in 
a 60 kg person.  This dose is approximately 10-fold higher than the maximum possible dose 
predicted in human studies (20 mg).  
The investigator/investigative staff should immediately and not later than within 24 hours of 
knowledge of the overdose, be reported by the investigator or delegate on the SAE report 
form.  In the event of an A4250 overdose, the patient should be monitored closely. 
10.2.9 
Pregnancy 
If a pregnancy is discovered in a female patient enrolled in the study before the end of dosing, 
the patient will be immediately discontinued from the study and will attend the same visits as 
a prematurely withdrawn patient.  If the pregnancy is discovered in a female patient enrolled 
in the study after the end of dosing, the patient will continue in the study per protocol.  If a 
pregnancy occurs in a male patient’s partner at any time during the study, the pregnancy 
should also be reported and followed. 
Pregnancy is not considered to be an AE.  However, if the patient has been dosed with the 
study drug, the pregnancy must be reported on the Paper Pregnancy Notification Form 
immediately within 24 hours after recognition to Albireo/Safety Manager (please refer to 
Section 10.1.3).  Date of exposure and as far as possible, details of the period of gestation at 
the time of exposure must be given. 


 
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The pregnancy should be followed to determine outcome, including spontaneous termination, 
details of birth, and presence of any birth defects, congenital anomalies or newborn or 
maternal complication.  The infant who was exposed in-utero will be followed for up to 
2 years after delivery.  Individual cases with an abnormal outcome in association with the 
study drug should be reported on an expedited basis, i.e., reported rapidly to a competent 
authority. 


 
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11 
STATISTICAL EVALUATION 
All personnel involved with the analysis of the study will remain blinded until database lock 
for the double-blind Treatment Period and until major protocol violations have been 
identified. 
All statistical analyses will be performed using SAS version 9.3 or higher. 
11.1 
Sample Size and Power 
For the EU and RoW, the primary analysis will relate to the bile acid responder endpoint (at 
the 1-sided 1.25% level).  For the US, the primary analysis will relate to the pruritus endpoint 
(at the 1-sided 1.25% level).  The aim is to enroll 60 to 70 patients in order to get at least 
60 evaluable patients. 
Assuming 60% responders (patients experiencing at least 70% reduction in fasting s-BA 
concentration or reaching ≤70 µmol/L) in the A4250 dose group and 10% responders in the 
placebo group, approximately 20 patients per group are required to achieve a power of 84% 
with a 1.25% 1-sided Type-1 error. 
For pruritus, with approximately 20 patients per treatment group, the power is 83% given an 
assumed treatment effect difference of -1.0 in change from baseline (-1.6 for A4250 
and -0.6 for placebo) on the Albireo ObsRO scratching score with a SD of 0.95 and a Type I 
error of 1.25% (1-sided). 
11.2 
Statistical Methods 
11.2.1 
Statistical Analysis Sets 
Safety Analysis Set 
The safety analysis set will consist of all patients who received at least 1 dose of study drug.  
Patients will be analyzed according to the treatment they actually received.  The safety 
analysis set will be the primary analysis set for safety analyses.  
Full Analysis Set 
The full analysis set (FAS) will consist of all randomized patients who received at least 
1 dose of assigned treatment.  Patients will be analyzed in the treatment group they were 
randomized to even if they received incorrect study drug.  The FAS will be the primary 
analysis set for efficacy analyses. 
Per Protocol Analysis Set 
The per protocol (PP) analysis set is a subset of the FAS, and will consist of all randomized 
patients for whom no major protocol violation (no violation which may affect the study 
efficacy outcome) is documented.  The PP analysis set will provide supportive data for the 
efficacy analyses. 
Allocation of patients to the PP analysis set will be performed before unblinding of the study. 


 
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11.2.2 
Methods of Statistical Analyses 
11.2.2.1 
General Principles 
The primary objective of this study is to show superiority in efficacy of 
A4250 (120 µg/kg/day and 40 µg/kg/day) compared to placebo in patients with PFIC Types 1 
and 2. 
In the EU and RoW, for the primary efficacy variable fasting bile acid responder endpoint (at 
least a 70% reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the end 
of treatment), the null hypothesis will be tested against the alternative hypothesis (α = 0.0125, 
1-sided). 
The test will be performed at a Type I error rate of ≤0.0125 1-sided, as described above, 
based on the least squares means of the treatment difference in mean probability scale. 
To strictly control overall Type I error rate across the dose groups and also across the primary 
and the main secondary endpoint to a 1-sided 2.5% level, a Bonferroni-based parallel 
gate-keeping procedure will be used.  In this procedure the total significance level of 2.5% 
(1-sided) will be split equally between the dose groups.  Only a dose group that is significant 
for the primary endpoint will be tested, at the nominal significance level of 1.25% (1-sided), 
for the main secondary endpoint (here the pruritus endpoint).  If the previous step was not 
successful, the analysis of the following step will be considered descriptive.  If the procedure 
reaches the secondary endpoint for both dose groups, these will be evaluated using the 
Bonferroni Holm procedure.  The multiple comparison procedure is presented in Figure 2. 
Figure 2 
Bonferroni Holm Multiple Comparison Procedure 
 
In the US, for the primary efficacy variable pruritus score endpoint (change from baseline to 
Week 24 in observer-reported scratching), the null hypothesis will be tested against the 
alternative hypothesis (α=0.0125, 1-sided). 


 
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The test will be performed at a Type I error rate of ≤0.0125 1-sided, as described above, 
based on the least squares means of the treatment difference in mean change. 
To strictly control overall Type I error rate across the dose groups and also across the primary 
and the main secondary endpoint to a one-sided 2.5% level, a Bonferroni-based parallel 
gate-keeping procedure will be used.  In this procedure the total significance level of 2.5% 
(1-sided) will be split equally between the dose groups.  Only a dose group that is significant 
for the primary endpoint will be tested, at the nominal significance level of 1.25% (1-sided), 
for the main secondary endpoint (here the bile acid responder endpoint).  If the previous step 
was not successful, the analysis of the following step will be considered descriptive.  If the 
procedure reaches the secondary endpoint for both dose groups, these will be evaluated using 
the Bonferroni Holm procedure. 
Other secondary and exploratory outcome variables shall provide supportive efficacy and 
safety information regarding the differences between the treatment groups.  No adjustments 
will be performed for multiple dose comparisons when testing the other secondary and 
exploratory efficacy variables. 
A more detailed statistical analysis plan (SAP) will be prepared and finalized before study 
data are locked and unblinded.  
If not otherwise specified, the baseline value of a parameter is defined as the last assessment 
of that parameter on or prior to the first intake of study treatment date. 
11.2.2.2 
Missing Data 
For efficacy variables, with repeated measures, a mixed model will be employed with no 
additional imputation for missing values, i.e., based on “observed cases”, except where 
otherwise specified. 
Follow-up and continued collection of efficacy data for patients who discontinue treatment 
will be made.  These data will be included in the sensitivity analysis for primary endpoints 
estimating a treatment effect taking loss of effect after discontinuation into account.  Because 
complete follow-up of all patients discontinuing treatment may not be possible, a method to 
replace missing data for patients who discontinue the study will be prespecified (in the SAP) 
taking into account loss of effect after discontinuation (for example multiple imputation). 
No other replacement of missing values will be planned. 
11.2.2.3 
Demographic and Baseline Characteristics 
Descriptive summaries of demographics and other baseline characteristics (including medical 
and surgical history) will be presented by treatment group and overall using the FAS.  
Prior medication will be summarized by treatment group and overall using the safety analysis 
set. 


 
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11.2.2.4 
Subject Disposition 
The following will be summarized (by treatment group and overall where applicable): 
• Patients enrolled (who signed the informed consent) 
• Patients randomized 
• Patients treated 
• Patients completing the study/withdrawing early (including withdrawal reason) 
Additionally, patients enrolled, included in the safety, in the FAS, and in the PP analysis set 
will be summarized by region. 
11.2.2.5 
Evaluation of Primary Efficacy Variables 
The responder endpoint based on change in fasting bile acids will be analyzed in terms of the 
proportion of patients achieving the event of interest.  Patients in need of rescue therapy, i.e., 
biliary diversion will be classified as non-responders after the biliary diversion.  
Generalized linear mixed modelling (GLMM) will be used with a logistic link, to allow for 
repeated assessments over time.  The model will include baseline value, treatment group, 
PFIC class, age class, region, visit, and visit*treatment group interaction.  The treatment by 
visit interaction will remain in the model regardless of significance.  Region will be included 
in the model as a random effect.  The contrasts of primary interest will be the treatment 
difference at Week 24; also, the difference at Week 4, Week 8, Week 12, and Week 18 will 
be estimated and analyzed.  An unstructured covariance matrix will be used to model the 
within-subject error.  Another covariance structure may be chosen if the fit of this structure 
fails to converge.  Model diagnostics checks will be made to ensure appropriate fit. 
The primary analysis of the change from baseline in pruritus score (indexed by 
observer-reported scratching) will be analyzed using a mixed model repeated measures 
(MMRM) analysis.  The contrast of primary interest will be treatment difference at Week 24 
but also the differences at Week 4, Week 8, Week 12, and Week 18 will be estimated and 
analyzed.  Baseline pruritus score will be included as covariate.  The model will include 
baseline value, treatment group, PFIC class, age class, region, visit, and visit*treatment group 
interaction.  The treatment by visit interaction will remain in the model regardless of 
significance.  Region will be included in the model as a random effect and will be defined on 
country level.  An unstructured covariance matrix will be used to model the within-subject 
error.  Another covariance structure may be chosen if the fit of this structure fails to 
converge. 
Non-parametric approach may be considered if more appropriate. 
The primary analyses will be based on the FAS. 
To assess the robustness of the primary efficacy analysis, the following analyses will be 
performed: 


 
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• Primary efficacy analysis using the PP set  
• Analyses to investigate the influence of missing values, for example, by using pattern 
mixture models and tipping point analysis 
Subgroup analyses will also be performed for each of the 3 age groups (6 months to 5 years, 
6 to 12 years, and 13 to ≤18 years) and for patients who were not included in the previous 
Study A4250-003. 
Several sensitivity analyses will be performed, such as the following: 
• Cochran Mantel Haenzsel test stratified on PFIC class to compare the proportion in 
fasting bile acid responder endpoint at the end of treatment in the two A4250 dose groups 
to placebo group.  Dropouts will be treated as non-responders 
• Based on the primary analysis including the interaction age class*treatment 
• Same as primary analysis in which the random factor region is considered as fixed factor.  
The region*treatment interaction will also be explored 
• Same as primary analysis including the interaction PFIC*treatment  
Other sensitivity analyses may be performed and will be detailed in the SAP. 
11.2.2.6 
Evaluation of Secondary and Exploratory Efficacy Variables 
The change in the secondary and explorative endpoints such as s-BA, total bilirubin, ALT, 
growth and patient-reported itch severity will also be analyzed using the MMRM model as 
specified for the primary analysis.  Nonparametric methods may be applied as an alternative 
approach for some endpoints, if appropriate. Change in growth (weight for age and BMI for 
age) will also be displayed using graphical presentations. 
Proportions of responders (e.g., meaningful reduction in itch severity and scratching at 
Week 24) will be analyzed using the GLMM model as specified for the primary analysis. 
Comparisons of the change from baseline at Week 24 in PedsQL between the treatment 
groups will be conducted using analysis of covariance.  The model will include terms for 
baseline, PFIC class, age class, region, and treatment.  
Undergoing biliary diversion surgery and/or listed for liver transplant, all-cause mortality, 
and liver decompensation events will mainly be analyzed by using descriptive statistics.  
Kaplan-Meier curves will be used when appropriate for time to event data. 
Other exploratory variables (Global Symptom Relief, biochemical markers and measures of 
bile acid synthesis, Albireo PRO and ObsRO sleep parameters, PELD/MELD score, APRI 
score, and Fib-4 score) will be analyzed descriptively for categorical and continuous data, as 
applicable.  For continuous data, the change from baseline will be analyzed in addition to the 
actual visit values.  For categorical data, shift tables or frequency and percentages of patients 
will be presented as appropriate. 


 
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In addition, subgroup analyses will be performed for each of the 3 age classes (6 months to 
5 years, 6 to 12 years, and 13 to ≤18 years). 
11.2.2.7 
Evaluation of Safety Variables 
Safety data will be analyzed using descriptive statistics and summaries by treatment group of 
SAEs, AEs, vital signs, clinical laboratory tests (hematology, clinical chemistry and 
urinalysis), and concomitant medication.  Analyses will be performed using the safety 
analysis set.  
Summaries of AEs (coded according to the Medical Dictionary for Regulatory Activities 
[MedDRA] system organ class [SOC] and MedDRA preferred term) will include the 
following:  
• Overview of the incidence of TEAEs (TEAEs, drug-related TEAEs, TEAEs leading to 
study discontinuation, and treatment-emergent SAEs) 
• TEAEs by SOC and preferred term 
• Intensity of TEAEs by SOC and preferred term 
• Drug-related TEAEs by SOC and preferred term 
• TEAEs leading to study discontinuation by SOC and preferred term 
• Treatment-emergent SAEs by SOC and preferred term 
Concomitant medication use during the Treatment Period will be summarized by Anatomical 
Therapeutic Chemical (ATC) class and World Health Organization (WHO) preferred name.  
Summaries of vital signs will be presented.  For each visit, the actual results and the change 
from baseline, and the number and percentage of patients with potentially clinically 
significant (PCS) values observed post-baseline will be presented. 
Summaries of clinical safety laboratory data will be presented.  For each visit, the actual 
result and the change from baseline, and the number and percentage of patients with PCS 
values observed post-baseline will be presented. 
Data listings will be provided for each patient for all safety parameters. 
11.2.2.8 
Compliance and Exposure 
Exposure will be analyzed by calculating the number of days with exposure to study drug.  
Results will be presented by treatment group using the safety analysis set.  
The percentage compliance will be described by treatment group and the number of patients 
with a compliance <80%, between 80% and 120%, and >120% will be presented based on the 
safety analysis set. 


 
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11.2.3 
Blinded Analyses to Confirm Responder Definitions 
A blinded analysis of Albireo ObsRO and PRO eDiary data will be performed after 50% or 
more of the planned sample has completed the Week 24 Follow-Up Period.  The blinded 
analysis will be used to estimate a threshold of clinically meaningful change (i.e., responder 
definition) in Albireo ObsRO and PRO pruritus scores.  The analysis will be performed by a 
group independent from both the study team and Albireo.  Blinded data will be used for this 
analysis; data will be collapsed across treatment groups. 
The analyses will include distribution- and anchor-based analyses to examine a responder 
definition for the Albireo PRO/ObsRO pruritus scores.  Distribution-based analyses will 
include calculation of the SD and standard error of measurement for the baseline Albireo 
PRO and ObsRO scores.  Anchor-based analyses will involve examining the degree of 
change on the Albireo PRO and ObsRO scores from baseline to Week 24 for patients who 
experience change in pruritus according to PGIC and symptom items.  Details of this analysis 
will be included in a stand-alone SAP for assessing the measurement characteristics 
(reliability, validity, sensitivity to change, responder definition) of the Albireo PRO and 
ObsRO items. 
11.2.4 
Data Safety Monitoring Board 
A DSMB consisting of sponsor-independent clinical and statistical experts will be 
established.  The DSMB will periodically meet for the review of accumulating study data, 
including safety (AE and laboratory data) and liver values.  
The DSMB will have access to unblinded data.  The DSMB will make recommendations for 
the remaining part of the study (further details will be provided in the DSMB charter).  The 
DSMB may recommend continuing with the study as planned or stopping the study early for 
safety reasons.  The DSMB will submit its recommendations in writing to Albireo 
Medical/
 who are responsible for responding to the recommendations of the DSMB and 
taking appropriate action.  The investigators will only be informed by Albireo Medical/
 
if the study requires a protocol amendment or is stopped.  The DSMB may choose to make 
additional evaluations at any time if they feel this is warranted from a safety point of view.  
The DSMB will act according to its own written standard operating procedure described in a 
charter and will prepare written minutes of its meetings.  The charter of the DSMB will be 
stored in the trial master file.  The DSMB will maintain records of its meetings and these will 
become part of the study file when the study is complete.  In order not to disseminate 
unblinded data and to ensure that all staff involved in the conduct and final analysis of the 
study remain blind to the data, only the members of the DSMB and the unblinded DSMB 
statistician will have access to these data. 
 


 
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12 
DIRECT ACCESS TO SOURCE DATA/NOTES 
The investigator/institution shall provide direct access to source data/documents for 
study-related monitoring, audits, IEC/Institutional Review Board (IRB) review and 
regulatory inspection. 


 
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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
13.1 
Conduct of the Study 
Albireo/
 shall implement and maintain quality control and quality assurance procedures 
with written standard operating procedures to ensure that the study is conducted and data are 
generated, documented, and reported in compliance with the protocol, ICH GCP and 
applicable regulatory requirements. 
This study shall be conducted in accordance with the provisions of the Declaration of 
Helsinki (October 1996) and all revisions thereof, and in accordance with United States Food 
and Drug Administration (FDA) regulations (Code of Federal Regulations, Sections 312.50 
and 312.56) and with ICH GCP (CPMP 135/95). 
The investigator may not deviate from the protocol without a formal protocol amendment 
having been established and approved by an appropriate IEC/IRB, except when necessary to 
eliminate immediate hazards to the patient or when the change(s) involve(s) only logistical or 
administrative aspects of the study.  Any deviations may result in the patient having to be 
withdrawn from the study and render that patient non-evaluable. 
13.2 
Study Monitoring 
Before study initiation, at a site initiation visit or at an investigator’s meeting, an Albireo 
representative or designee will review the protocol and eCRF with the investigators and the 
investigative staff.  During the study, the clinical monitor (clinical research associate [CRA]) 
will visit the site regularly to check the completeness of patient records, the accuracy of 
entries on the eCRFs, the adherence to the protocol and to GCP, the progress of enrollment, 
and to ensure that study drug is being stored, dispensed, and accounted for according to 
specifications. 
The investigator must ensure that eCRFs are completed within a timely period of patient 
visits, as per individual site agreements, and must allow the CRA and Albireo representative 
or designee periodic access to patient records and all study-related materials, including 
relevant hospital or clinical records, to confirm their consistency with the eCRF entries.  No 
information in these records about the identity of the patients will leave the study center.  
Albireo monitoring standards require full verification for the presence of the signed ICF, 
adherence to the inclusion/exclusion criteria, documentation of SAEs, and recording of 
primary efficacy and safety variables.  The CRA will review source data compared with the 
eCRFs and will verify source data according to the study-specific monitoring plan.  The 
design of the study, the frequency of patient visits, and the site enrollment rate will determine 
the frequency of monitoring visits.  Upon study completion, the CRA will visit the site to 
conduct a study termination visit, which will include collection of any outstanding 
documentation. 
It is recommended that the investigator/investigative staff log into the eCRF system every 
working day or at minimum twice weekly in order to provide a prompt response to queries.  


 
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The investigator/investigative staff should respond to queries and make any relevant changes 
to the study data within 3 working days. 


 
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14 
ETHICS 
14.1 
Independent Ethics Committee/Institutional Review Board 
Prior to the start of the study, the investigator is responsible for ensuring that the protocol and 
consent form have been reviewed and approved by a relevant IEC/IRB.  The IEC/IRB shall 
be appropriately constituted and perform its functions in accordance with FDA ICH GCP and 
local requirements as applicable. 
The IEC/IRB shall approve all protocol amendments (except for logistical or administrative 
changes), written informed consent documents and document updates, patient recruitment 
procedures (e.g., advertisements), written information to be provided to the patients and 
caregivers, IB, available safety information, information about payment and compensation 
available to patients, the investigator's curriculum vitae and/or other evidence of 
qualifications and any other documents requested by the IEC/IRB and regulatory authority 
(competent authority) as applicable. 
14.2 
Written Informed Consents and Assents 
The investigator/investigative staff will explain to each patient (or legally authorized 
representative) the nature of the study, its purpose, the procedures involved, the expected 
duration, alternative treatment, the potential risks and benefits involved, and any discomfort 
that may occur.  Each caregiver/patient will be informed that participation in the study is 
voluntary and that he/she or their child may withdraw from the study at any time and that 
withdrawal of consent will not affect his/her subsequent medical treatment or relationship 
with the treating physician. 
Caregivers/patients will be informed that they/their children may experience side-effects or 
be at risk for symptoms, illnesses, or complications that cannot be foreseen by Albireo.  As 
with other medications, people treated with A4250 may be at risk of developing allergic 
reactions or anaphylaxis.  Caregivers/patients will be advised that study procedures include 
regular blood sampling for measurement of safety parameters and biological markers and that 
some minor risks are associated with these procedures. 
This informed consent should be given by means of a signed ICF, written in non-technical 
language in accordance with ICH GCP, the Declaration of Helsinki, and regulatory 
authorities.  The caregiver/patient should read and consider the statements before signing and 
dating them and should be given a copy of each signed document.  If written consent is not 
possible, oral consent can be obtained if witnessed and followed by a signed statement from 
one or more persons not involved in the study, indicating why the patient was unable to sign 
the form.  No patient can enter the study before his/her or caregiver informed consent has 
been obtained. 
The ICF is part of the protocol and must be submitted by the investigator/investigative staff 
with the protocol for IEC approval.  Albireo will supply an ICF which complies with 
regulatory requirements and is considered appropriate for the study.  Any changes to the ICF 


 
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suggested by the investigator must be agreed to by Albireo before submission to the IEC and 
a copy of the approved version must be provided to the clinical monitor after IEC approval. 


 
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15 
DATA HANDLING AND RECORD KEEPING 
15.1 
Case Report Forms/Source Data Handling 
The investigator shall be provided with standardized eCRFs and shall ensure that all data 
from patient visits are promptly entered into the eCRFs in accordance with the specific 
instructions given.  The investigator must sign each eCRF to verify the integrity of the data 
recorded. 
A list of the normal ranges for all laboratory tests to be undertaken forms part of the 
documentation to be collated prior to study start.  As a central laboratory has been selected to 
conduct any or all tests, it is essential that all samples be analyzed at that laboratory. 
The investigator must maintain source documents such as laboratory reports, consultation 
reports, and complete medical history and physical examination reports. 
As no ethnicity data can be collected in the source document for study sites in France, the 
eCRF will be the Source Document for “origines ethniques”,  
15.2 
Retention of Essential Documents 
Essential documents, as defined by ICH GCP, include: the signed protocol and any 
amendment(s); copies of the completed eCRFs (for site archiving, compact discs of eCRF 
data for specific patients will be provided); signed ICFs; hospital records and other source 
documents; IEC approvals and all related correspondence including approved documents; 
drug accountability records; study correspondence; and a list of patients’ names and 
addresses. 
The investigator/investigative staff must retain copies of these essential documents for the 
period specified by ICH GCP and by applicable regulatory requirements.  The 
investigator/investigative staff will inform Albireo of the location where the essential 
documents are stored and must contact Albireo for approval before disposing of any essential 
documents.  The investigator/investigative staff should take measures to prevent accidental or 
premature destruction of these documents. 


 
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16 
FINANCING AND INSURANCE 
Financing and insurance are addressed in a separate agreement. 
 


 
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17 
PUBLICATION POLICY 
Albireo will retain the ownership of all data.  When the study is complete, Albireo shall 
arrange the analysis, tabulation of data and preparation of a clinical study report. Albireo may 
also use the data for publication, presentation at scientific meetings and submission to 
regulatory authorities.  All proposed publications based on this study must be subject to the 
sponsor's approval requirements. 
 


 
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18 
REFERENCE LIST 
1. A4250 Investigator’s Brochure. 
2. Alissa FT, Jaffe R, Shneider BL. Update on progressive familial intrahepatic cholestasis. 
J Pediatr Gastroenterol Nutr. 2008;46(3):241-52. 
3. Davit-Spraul A, Gonzales E, Baussan C, et al. Progressive familial intrahepatic 
cholestasis. Orphanet J Rare Disease 2009;4:1-12. 
4. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and 
functions. Front Biosci (Landmark Ed). 2009 Jan 1;14:2584-98. 
5. Hori T, Justin H, Nguyen JH and Uemoto S. Progressive familial intrahepatic cholestasis. 
Hepatobiliary pancreat Dis Int. 2010; 9(6):570-8. 
6. Jacquemin E. Progressive familial intrahepatic cholestasis. Genetic basis and treatment. 
Clin Liver Dis. 2000;4(4):753-63. 
7. Miethke AG, Zhang W, Simmons J, et al. Pharmacological inhibition of ASBT changes 
bile composition and blocks progression of sclerosing cholangitis in mdr2 knockout mice. 
Hepatology. 2016; 63(2):512–23. 
8. Murray CS, Rees JL. Are subjective accounts of itch to be relied on? The lack of relation 
between visual analogue itch scores and actigraphic measures of scratch. Acta Derm 
Venereol. 2011;91(1):18-23. 
9. Pawlikowska L, Strautnieks S, Jankowska I, et al. Differences in presentation and 
progression between severe FIC1 and BSEP deficiencies. J Hepatol. 2010;53(1):170-8. 
10. Suchy FJ, Sokol RJ, Balistreri WF, eds. Liver Disease in Children. 3rd Edition. New 
York, NY: Cambridge University Press; 2007:310–4. 
11. Whitington PF, Whitington GL. Partial external diversion of bile for the treatment of 
intractable 
pruritus 
associated 
with 
intrahepatic 
cholestasis. 
Gastroenterology. 
1988;95(1):130-6. 
 
 


 
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19 
APPENDICES 
Appendix 1 
Concomitant Medications Prohibited During the Study 
 


 
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Appendix 2 
Diary Questions including Albireo Patient-Reported Outcomes/Observer-
Reported Outcome Instrument 
 


 
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Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) 
 


 
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Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaires 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
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Appendix 5 
Contraceptive advice 
 


 
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Appendix 6 
Blood volumes 
 
 


 
 
 
CLINICAL STUDY PROTOCOL A4250-005 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
 
IND Number:   
130591 
EudraCT Number 
2017-002338-21 
Test Product: 
A4250 
Indication: 
Progressive Familial Intrahepatic Cholestasis (PFIC) 
Sponsor: 
Albireo AB 
Development Phase: 
Phase 3 
Sponsor Signatory: 
 
Sponsor Medical Monitor: 
 
Principal Investigator: 
 
Date of the Protocol: 
01 March 2018 
Version of the Protocol: 
Final Amendment 1.1 Sweden 
 
 
 
 
Confidential 
The information in this document contains trade secrets and commercial information that are 
privileged or confidential and may not be used, divulged, published, or otherwise disclosed 
without the written consent of Albireo AB.  These restrictions on disclosure apply equally to 
all future information supplied to you or generated by you in connection with the study. 






 
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INVESTIGATOR SIGNATURE PAGE 
 
PROTOCOL TITLE:  A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children with Progressive 
Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
 
PROTOCOL NUMBER:  A4250-005 
I have read this protocol and agree that it contains all necessary details for performing this 
study.  I will conduct the study as outlined herein and will complete the study within the time 
designated, in accordance with all stipulations of the protocol and in accordance with Good 
Clinical Practice (GCP), local regulatory requirements, and the Declaration of Helsinki. 
I will provide copies of the protocol and all pertinent information to all individuals 
responsible to me who assist in the conduct of this study.  I will discuss this material with 
them to ensure that they are fully informed regarding the study drug and the conduct of the 
study. 
I will use only the informed consent approved by the Independent Ethics Committee (IEC) 
and will fulfil all responsibilities for submitting pertinent information to the IEC responsible 
for this study. 
I agree that the sponsor (Albireo AB) shall have access to any source documents from which 
case report form information may have been generated. 
I further agree not to originate or use the name of Albireo AB, A4250 in any publicity, news 
release, or other public announcement, written or oral, whether to the public, press, or 
otherwise, relating to this protocol, to any amendment to the protocol, or to the performance 
of this protocol without the prior written consent of Albireo AB. 
 
 
 
 
 
 
Name of Investigator 
 
 
 
 
 
 
Signature 
 
Date (day/month/year) 


 
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1 
ADMINISTRATIVE INFORMATION 
A Double-blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
Protocol No.: 
A4250-005 
Date of the initial Protocol: 
20 September 2017 
Date and Number of Amendment(s): 
Amendment 01, 06 December 2017 
Sponsor: 
Albireo AB 
 
Arvid Wallgrens backe 20 
 
413 46 Gӧteborg 
Sweden 
Clinical Research Organization: 
 
 
 
 
 
 
 
 
Sponsor Signatory: 
 
 
 
  
 
 
 
 
 
  
Sponsor Medical Monitor: 
 
 
 
 
Principal Investigator: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
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2 
STUDY SYNOPSIS 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Title of Study: 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate Efficacy and Safety of A4250 
in Children with Progressive Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
Principal Investigator: 
 
Study Centers: 
Up to 50 sites will be initiated for this study in the United States (US), Canada, European Union (EU), Middle 
East, and Australia. 
Publication(s): 
None. 
Planned Study Period: 
Q1 2018 to Q2 2019 
Development Phase: 
Phase 3 
Objectives: 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day A4250 in children with 
progressive familial intrahepatic cholestasis (PFIC) Types 1 and 2, as determined by the following: 
• 
Proportion of patients experiencing at least a 70% reduction in fasting serum bile acid (s-BA) 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L compared to placebo 
after 24 weeks of treatment 
• 
Change from baseline to Week 24 in pruritus compared to placebo  
Secondary Objectives 
• 
To evaluate the effect of A4250 on serum alanine aminotransferase (ALT) concentration 
• 
To evaluate the effect of A4250 on growth 
• 
To evaluate the effect of A4250 on sleep disturbance 
• 
To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or liver 
transplantation) 
• 
To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 
Methodology: 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the efficacy and safety of 
40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in children with PFIC (deficiencies of familial 
intrahepatic cholestasis-1 or bile salt export pump).  
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment Period and a 4-week 
Follow-up Period.  After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of A4250, or a 
matching placebo.  Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008) in which all patients will receive 
active treatment. 
There will be up to 10 clinic visits during the study and one scheduled telephone contact. 


 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Number of Patients: 
Approximately 60 patients diagnosed with PFIC-1 or PFIC-2 will be randomized. 
Diagnosis and Main Criteria for Inclusion: 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages of ≥6 months 
and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have genetic confirmation of PFIC-1 or PFIC-2 through identification of biallelic 
pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, taken as the 
average of 2 samples 7 to 28 days apart (Visits 1 and 2) prior to randomization  
4. Patient must have history of significant pruritus and a caregiver-reported observed scratching in the 
electronic diary (eDiary) average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  Patients who 
turn 18 years of age (or legal age per country) during the study will be required to re-consent in order 
to remain in the study 
6. Patients are expected to have a consistent caregiver(s) for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use an eDiary 
device as required by the study 
Test Product, Dose and Mode of Administration: 
A4250, 40 µg/kg/day or 120 µg/kg/day, orally administered 
Reference Therapy, Dose and Duration of Administration: 
Matching placebo, orally administered 
Duration of Treatment: 
24 weeks 
Variables 
Efficacy:  
Primary Efficacy Endpoints 
The primary efficacy endpoints are: 
• 
EU and rest of world (RoW): Proportion of patients experiencing at least a 70% reduction in fasting 
s-BA concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L compared to 
placebo after 24 weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization and the previous 
visit (Clinic Visit 2).  The end value is the average of the values at Weeks 22 and 24 after the start of 
double-blind treatment. 
• 
US: Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline to 
Week 24 as measured by the worst scratch of the Albireo observer-reported outcome (ObsRO) 
instrument.  Scores for the 14 days baseline and post-treatment intervals will be calculated by 
averaging the worst scratching scores from each day in the interval. 
Secondary Efficacy Endpoints 
Secondary efficacy endpoints include the following: 
• 
EU and RoW: Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
to Week 24 as measured by the worst scratch of the Albireo ObsRO instrument.  Scores for the 14 days 
baseline and post-treatment intervals will be calculated by averaging the worst scratching scores from 
each day in the interval. 
• 
US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA concentration from 
baseline to the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 weeks of 
treatment. 
All Regions: 
All secondary endpoints will be compared to placebo. 


 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
• 
Change from baseline to Week 24 in fasting s-BA 
• 
Change from baseline to Week 24 in serum ALT concentration 
• 
Change in growth from baseline to Week 24, defined as the linear deficit (weight for age and body 
mass index) compared to the national growth curves (Z-score, standard deviation from P50) 
• 
Proportion of patients achieving meaningful reduction in caregiver-reported observed scratching at 
Week 24 
• 
Change from baseline to Week 24 in sleep parameters measured with the Albireo patient-reported 
outcome (PRO) and ObsRO instruments 
• 
Change from baseline to Week 24 in patient-reported itch severity as measured by the average daily 
score of the Albireo PRO instrument; only patients ≥8 years of age will complete the Albireo PRO 
instrument 
• 
Number of patients undergoing biliary diversion surgery or being listed for liver transplantation 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• 
Proportion of patients achieving meaningful reduction in patient-reported itch severity at Week 24 
• 
Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, caregiver, and 
clinician via the patient global impression of change (PGIC) items 
• 
Change from baseline in fasting s-BA at Weeks 4 and 12 
• 
Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline to Weeks 4, 
12, and 24 as measured by the worst scratch.  Scores for the 7 worst scratching days of the last 14 days 
at baseline and post-treatment intervals will be selected 
• 
Change from baseline in patient-reported itch severity and observer-reported scratching at Weeks 4 and 
12 
• 
Change from baseline to Week 24 in patient-reported and observer-reported night time itching and 
scratching severity scores, respectively 
• 
Change from baseline to Week 24 in pooled pruritus score including observer-reported scratching for 
patients <8 years of age and patient-reported itch severity for patients ≥8 years of age 
• 
Change from baseline to Week 24 in additional patient-reported and observer-reported sleep 
parameters (e.g., tiredness and number of awakenings) 
• 
Change from baseline to Week 24 in Pediatric Quality of Life Inventory (PedsQL) 
• 
Change from baseline in serum ALT concentration at Weeks 4 and 12 
• 
Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• 
Change from baseline in AST at Weeks 4, 12, and 24 
• 
Change from baseline for other biochemical markers and measures of bile acid synthesis (autotaxin, 
plasma 7α-hydroxy-4-cholesten-3-one concentration [p-C4]) at Week 24 
• 
Change in pediatric end-stage liver disease/model for end-stage liver disease (PELD/MELD) score, 
AST to platelet ratio index (APRI) score, and Fib-4 score from baseline to Week 24 
• 
Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® (where available) 
• 
Liver-related mortality and liver decompensation events. 
• 
All-cause mortality 
Safety:   
Safety criteria are as follows: 
• 
Occurrence of treatment-emergent adverse events including severity and relatedness to study drug at all 
visits 
• 
The incidence of treatment-emergent serious adverse events (SAEs), based upon information from 
patient reports, including the description, incidence, and severity of an SAE 
• 
Trends evaluated for the following assessments: physical examinations, concomitant medications, vital 
signs, laboratory test results (including clinical chemistry, hematology, and urinalysis), and imaging 


 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
studies 
Statistical Methods: 
The primary objective of this study is to show superiority in efficacy of A4250 (120 µg/kg/day and 
40 µg/kg/day) compared to placebo in patients with PFIC Types 1 and 2. 
In the EU and RoW, for the primary efficacy variable fasting bile acid responder endpoint (at least a 70% 
reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the end of treatment), the null 
hypothesis will be tested against the alternative hypothesis (α=0.0125, 1-sided).  Generalized linear mixed 
modelling will be used with a logistic link, to allow for repeated assessments over time.  The model will include 
baseline value, treatment group, PFIC class, age class, region, visit and visit*treatment group interaction.  The 
contrasts of primary interest will be the treatment difference at Week 24 but also the difference at Week 4, 
Week 8, Week 12, and Week 18 will be estimated and analyzed. 
In the US, for the primary efficacy variable pruritus score endpoint (change from baseline to Week 24 in 
observer-reported scratching), the null hypothesis will be tested against the alternative hypothesis (α=0.0125, 
1-sided).  The primary analysis of the change from baseline in pruritus score (indexed by observer-reported 
scratching) will be analyzed using a mixed model repeated measures analysis.  The contrasts of primary interest 
will be the treatment difference at Week 24 but also the difference at the other weeks will be estimated and 
analyzed.  
To strictly control overall Type I error rate across the dose groups and also across the primary and the main 
secondary endpoint to a 1-sided 2.5% level, a Bonferroni-based parallel gate-keeping procedure will be used. 
Other secondary and exploratory outcome variables shall provide supportive efficacy and safety information 
regarding the differences between the treatment groups.  No adjustments will be performed for multiple-dose 
comparisons when testing the other secondary and exploratory efficacy variables. 
Date of the Protocol:  06 December 2017 


 
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3 
TABLE OF CONTENTS 
1 
ADMINISTRATIVE INFORMATION ........................................................................ 5 
2 
STUDY SYNOPSIS ...................................................................................................... 6 
3 
TABLE OF CONTENTS ............................................................................................ 10 
3.1 
List of Tables .................................................................................................. 14 
3.2 
List of Figures ................................................................................................ 14 
3.3 
List of Appendices .......................................................................................... 14 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ................................ 15 
5 
INTRODUCTION ....................................................................................................... 17 
5.1 
Investigational Medicinal Product .................................................................. 17 
5.2 
Background .................................................................................................... 17 
5.2.1 
Progressive Familial Intrahepatic Cholestasis ........................................... 17 
5.2.2 
Summary of Clinical and Nonclinical Studies ........................................... 19 
5.3 
Rationale ......................................................................................................... 20 
5.3.1 
Risk/Benefit ............................................................................................... 21 
6 
STUDY OBJECTIVES ............................................................................................... 22 
6.1 
Primary Objectives ......................................................................................... 22 
6.2 
Secondary Objectives ..................................................................................... 22 
7 
INVESTIGATIONAL PLAN ...................................................................................... 23 
7.1 
Overall Study Design and Plan....................................................................... 23 
7.1.1 
Description ................................................................................................. 23 
7.1.2 
Schedule of Assessments ........................................................................... 25 
7.1.3 
Study Procedures and Assessments ........................................................... 29 
7.1.3.1 
Screening Period (Days -56 to 0) ............................................... 29 
7.1.3.2 
Treatment Period (Day 0 to Day 168) ........................................ 30 
7.1.3.3 
Follow-Up Period ....................................................................... 33 
7.2 
Study Population ............................................................................................ 33 
7.2.1 
Inclusion Criteria ....................................................................................... 33 
7.2.2 
Exclusion Criteria ...................................................................................... 34 
7.2.3 
Withdrawal of Patients............................................................................... 36 
7.2.4 
Study Termination by Sponsor .................................................................. 36 
8 
TREATMENT OF PATIENTS ................................................................................... 38 
8.1 
Identity of Study Drug .................................................................................... 38 
8.2 
Administration of Study Drug ........................................................................ 38 
8.3 
Study Treatment Packaging and Labeling ...................................................... 39 
8.3.1 
Packaging and Labeling ............................................................................. 39 


 
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8.3.2 
Storage ....................................................................................................... 39 
8.3.3 
Blinding and Randomization of Study Drug ............................................. 39 
8.4 
Procedure for Breaking the Randomization Code .......................................... 40 
8.5 
Patient Compliance ......................................................................................... 41 
8.6 
Study Drug Accountability ............................................................................. 41 
8.7 
Concomitant Therapy and Prohibited Medications ........................................ 41 
9 
ASSESSMENT OF EFFICACY ................................................................................. 42 
9.1 
Efficacy Endpoints ......................................................................................... 42 
9.1.1 
Primary Efficacy Endpoint ........................................................................ 42 
9.1.2 
Secondary Efficacy Endpoint .................................................................... 42 
9.1.3 
Exploratory Efficacy Endpoints ................................................................. 43 
9.2 
Efficacy Assessments ..................................................................................... 44 
9.2.1 
Serum Bile Acids ....................................................................................... 44 
9.2.2 
Itching, Scratching and Sleep Score .......................................................... 44 
9.2.3 
Growth ....................................................................................................... 45 
9.2.4 
Quality of Life Questionnaire (PedsQL).................................................... 45 
9.2.5 
Biomarkers and Other Laboratory Samples ............................................... 45 
9.2.6 
PELD/MELD Score ................................................................................... 45 
9.2.7 
Fibroscan® .................................................................................................. 45 
9.2.8 
Markers of Fibrosis .................................................................................... 46 
9.2.9 
Global Impression of Change and Global Impression of Symptom 
Measures .................................................................................................... 46 
10 
ASSESSMENT OF SAFETY ..................................................................................... 47 
10.1 
Adverse Events ............................................................................................... 47 
10.1.1 
Definitions and Investigator Assessments ................................................. 47 
10.1.1.1 Clinical Significance .................................................................. 47 
10.1.1.2 Serious Adverse Events .............................................................. 47 
10.1.1.3 Severity Assessment ................................................................... 48 
10.1.1.4 Causality Assessment ................................................................. 48 
10.1.2 
Recording of Adverse Events .................................................................... 49 
10.1.3 
Recording and Reporting of Serious Adverse Events ................................ 50 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions .............. 51 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments .................................................................................................... 51 
10.2.1 
Laboratory Assessments ............................................................................ 51 
10.2.2 
Individual Patient Safety Monitoring ........................................................ 52 
10.2.2.1 Liver Monitoring ........................................................................ 52 


 
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10.2.2.2 Close Observation ...................................................................... 54 
10.2.2.3 De-challenge/Re-challenge for Liver and Clinical Hepatitis 
Monitoring .................................................................................. 54 
10.2.2.4 Diarrhea ...................................................................................... 54 
10.2.2.5 Monitoring after Permanent Discontinuation of Study Drug 
Due to Safety .............................................................................. 55 
10.2.3 
Demographics/Medical and Surgical History ............................................ 55 
10.2.4 
Clinical Genetic Testing ............................................................................ 55 
10.2.5 
Physical Examination ................................................................................ 55 
10.2.6 
Vital Signs ................................................................................................. 56 
10.2.7 
Abdominal Ultrasound ............................................................................... 56 
10.2.8 
Overdose .................................................................................................... 56 
10.2.9 
Pregnancy .................................................................................................. 56 
11 
STATISTICAL EVALUATION ................................................................................. 58 
11.1 
Sample Size and Power .................................................................................. 58 
11.2 
Statistical Methods ......................................................................................... 58 
11.2.1 
Statistical Analysis Sets ............................................................................. 58 
11.2.2 
Methods of Statistical Analyses ................................................................. 59 
11.2.2.1 General Principles ...................................................................... 59 
11.2.2.2 Missing Data .............................................................................. 60 
11.2.2.3 Demographic and Baseline Characteristics ................................ 60 
11.2.2.4 Subject Disposition..................................................................... 61 
11.2.2.5 Evaluation of Primary Efficacy Variables .................................. 61 
11.2.2.6 Evaluation 
of 
Secondary 
and 
Exploratory 
Efficacy 
Variables ..................................................................................... 62 
11.2.2.7 Evaluation of Safety Variables ................................................... 63 
11.2.2.8 Compliance and Exposure .......................................................... 63 
11.2.3 
Blinded Analyses to Confirm Responder Definitions ............................... 64 
11.2.4 
Data Safety Monitoring Board ................................................................... 64 
12 
DIRECT ACCESS TO SOURCE DATA/NOTES ..................................................... 65 
13 
QUALITY CONTROL AND QUALITY ASSURANCE .......................................... 66 
13.1 
Conduct of the Study ...................................................................................... 66 
13.2 
Study Monitoring ........................................................................................... 66 
14 
ETHICS ....................................................................................................................... 68 
14.1 
Independent Ethics Committee/Institutional Review Board .......................... 68 
14.2 
Written Informed Consents and Assents ........................................................ 68 


 
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15 
DATA HANDLING AND RECORD KEEPING ....................................................... 70 
15.1 
Case Report Forms/Source Data Handling..................................................... 70 
15.2 
Retention of Essential Documents .................................................................. 70 
16 
FINANCING AND INSURANCE .............................................................................. 71 
17 
PUBLICATION POLICY ........................................................................................... 72 
18 
REFERENCE LIST ..................................................................................................... 73 
19 
APPENDICES ............................................................................................................. 74 
 


 
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3.1 
List of Tables 
Table 1 
Schedule of Assessments ................................................................................... 26 
Table 2 
Dosing and Capsule Strength ............................................................................. 39 
Table 3 
Routine Laboratory Parameters .......................................................................... 52 
 
3.2 
List of Figures 
Figure 1 
Study Design ...................................................................................................... 25 
Figure 2 
Bonferroni Holm Multiple Comparison Procedure ............................................ 59 
 
3.3 
List of Appendices 
Appendix 1 Concomitant Medications Prohibited During the Study .................................... 74 
Appendix 2 Diary Questions including Albireo Patient-Reported Outcomes/Observer-
Reported Outcome Instrument ........................................................................... 75 
Appendix 3 Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) ....................................................................... 76 
Appendix 4 Pediatric Quality of Life Inventory (PedQL) Questionnaire .............................. 77 
 


 
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4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
ADME 
absorption, distribution, metabolism, and excretion 
AE(s) 
adverse event(s) 
AFP 
alfa-fetoprotein 
ALP 
alkaline phosphatase 
ALT 
alanine aminotransferase 
APRI 
AST to platelet ratio index 
ASBT 
apical sodium-dependent bile transporter (also known as IBAT) 
AST 
aspartate aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BMI 
body mass index 
BSEP 
bile salt export pump 
CPK 
creatine phosphokinase 
CRA 
clinical research associate 
CYP 
cytochrome P450 
DSMB 
Data and Safety Monitoring Board 
eCRF 
electronic case report form 
eDiary 
electronic diary 
EOT 
End of Treatment 
EU 
European Union 
EudraCT 
European Union drug regulatory agency clinical trial 
FAS 
full analysis set 
FDA 
United States Food and Drug Administration 
FIC1 
familial intrahepatic cholestasis-1 
GCP 
Good Clinical Practice 
GI 
gastrointestinal 
GLMM 
generalized linear mixed modelling 
IB 
Investigator’s Brochure 
IBAT 
ileal bile acid transporter (also known as ASBT) 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
 
 
IEC 
Independent Ethics Committee 
INR 
international normalized ratio 
IRB 
Institutional Review Board 
IWRS 
Interactive Web Response System 


 
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LDH 
lactate dehydrogenase 
LFT 
LPLV 
liver function test 
last patient last visit 
MDR3 
multidrug-resistance protein 3 
MedDRA 
Medical Dictionary for Regulatory Activities 
MELD 
model for end-stage liver disease 
MMRM 
mixed model repeated measures 
ObsRO 
observer-reported outcome 
PBC 
primary biliary cholangitis 
p-C4 
plasma 7α-hydroxy-4-cholesten-3-one concentration 
PCS 
potentially clinically significant 
PedsQL 
Pediatric Quality of Life Inventory 
PELD 
pediatric end-stage liver disease 
PFIC 
progressive familial intrahepatic cholestasis 
PGIC 
patient global impression of change 
PGIS 
patient global impression of symptoms 
PP 
per protocol 
PRO 
patient-reported outcome 
PT 
prothrombin time 
PVM 
pharmacovigilance manager 
QoL 
quality of life 
RoW 
rest of world 
RR 
respiratory rate 
SAE(s) 
serious adverse event(s) 
SAP 
statistical analysis plan 
s-BA 
serum bile acid 
SD 
standard deviation 
s-FGF19 
serum fibroblast growth factor 19 concentration 
SOC 
system organ class 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE(s) 
treatment-emergent adverse event(s) 
ULN 
upper limit of normal 
US 
United States 
WHO 
World Health Organization 
 


 
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5 
INTRODUCTION 
5.1 
Investigational Medicinal Product 
A4250 is a small molecule and a potent selective inhibitor of the ileal bile acid transporter 
(IBAT), also known as the apical sodium-dependent bile acid transporter (ASBT).  IBAT is 
an integral brush border membrane glycoprotein that co-transports sodium and bile acids and 
appears to be a critical component in the regulation of the bile acid pool size in animals and 
man.  This transporter, expressed in the distal ileum, is a key element in the enterohepatic 
circulation of bile acids since it facilitates the high-affinity, high-capacity reabsorption of bile 
acids.  Indeed, 95% of secreted bile acids are reabsorbed via IBAT [Hofmann 2009; 
Miethke 2016].  A4250 is orally administered and acts locally in the gut where it binds 
reversibly to IBAT to decrease the reuptake of bile acids.  A4250 has minimal systemic 
exposure at the predicted therapeutic dose ranges. 
No safety signals were found with A4250 in preclinical safety and toxicity studies.  In the 
first clinical study of A4250 in healthy volunteers, A4250 was given as a single oral dose up 
to 10 mg or daily oral doses up to 3 mg/day for 7 days.  A4250 was well tolerated and the 
only findings were gastrointestinal (GI) related effects, such as diarrhea, at higher doses. 
In an exploratory Phase 2 study in children with cholestatic pruritus, A4250 was given as oral 
doses of 10 to 200 µg/kg/day for 4 weeks.  A4250 reduced mean levels of serum bile acids 
(s-BA) in all cohorts.  In 11 of 13 progressive familial intrahepatic cholestasis 
(PFIC) patients, substantial reductions (ranging from 43% to 98%) were observed.  
Patient-reported diary data documented improvement of pruritus during treatment.  A4250 
was well tolerated.  No serious adverse events (SAEs) were deemed to be related to study 
drug, and most adverse events (AEs), including some increased transaminases, were 
considered unrelated to study drug and transient in nature. 
5.2 
Background 
5.2.1 
Progressive Familial Intrahepatic Cholestasis 
PFIC is a rare autosomal recessive cholestatic liver disease estimated to affect between 1 in 
every 50,000 to 100,000 children born worldwide.  PFIC represents 10% to 15% of causes of 
cholestasis in children and 10% to 15% of liver transplantation indications in children.  All 
types of PFIC exist worldwide and both sexes appear to be equally affected. 
The common underlying pathogenesis of PFIC is disruption of bile formation and bile 
transport through the liver [Jacquemin 2000].  The classification of PFIC has evolved over 
the years.  The most commonly used sub-classifications are PFIC Types 1, 2, and 3, which 
are based on the associated affected gene and described in more detail below. 
• PFIC, Type 1:  also referred to as “Byler disease” or “familial intrahepatic 
cholestasis-1 (FIC1) protein deficiency.” FIC1 protein is located on the canalicular 
membrane of hepatocytes and facilitates movement of aminophospholipids from the outer 


 
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to inner leaflet of the plasma membrane of the hepatocyte.  The ATP8B1 gene encodes 
FIC1 protein.  Biallelic pathologic variants in the ATP8B1 gene are associated with FIC1 
dysfunction and classified clinically as PFIC Type 1 disease. 
• PFIC, Type 2:  also referred to as “Byler syndrome” or “bile salt export pump (BSEP) 
deficiency.”  BSEP is a transporter protein that is expressed at the canalicular membrane 
of hepatocytes, and is the primary exporter of bile acids.  The ABCB11 gene encodes the 
BSEP protein.  Biallelic pathologic variations in the ABCB11 gene are associated with 
BSEP dysfunction and classified clinically as PFIC Type 2 disease. 
• PFIC, Type 3:  is caused by a deficiency of the multidrug-resistance protein 3 (MDR3) 
due to mutations in the ABCB4 gene.  MDR3 is a phospholipid translocase critical for 
phospholipid secretion. 
Severe pruritus is common in children diagnosed with PFIC.  Itching (and subsequent 
scratching) is a significant morbidity for these patients and their families [Suchy 2007].  A 
more severe degree of pruritus is experienced compared to patients with other forms of liver 
disease and to other pruritic conditions such as atopic dermatitis [Murray 2011].  In patients 
with PFIC, liver biopsy reveals canalicular cholestasis and, later, the appearance of portal 
fibrosis.  Serum biochemistry indicates cholestasis with hyperbilirubinemia, elevated alanine 
aminotransferase (ALT) and aspartate aminotransferase (AST).  The concentrations of bile 
acids in serum are very high, while serum gamma-glutamyl transferase activity (exception 
being MDR3 variants) and cholesterol are normal [Hori 2010].  Symptoms of portal 
hypertension and liver failure will develop during the course of the disease [Davit-Spraul 
2009; Alissa 2008].  Symptoms develop early; median age at onset of symptoms is 2 months, 
and 78% of PFIC patients present with jaundice [Pawlikowska 2010].  The life-threatening 
and debilitating nature of PFIC is reflected by the fact that survival in those not resorting to 
surgery is 50% at 10 years of age and almost zero at 20 years of age.  Approximately half of 
PFIC patients undergo liver transplantation [Davit-Spraul 2009] and treatment-resistant 
pruritus is the leading indication for the surgical procedure partial external biliary diversion, 
mostly in PFIC Type 1 and 2 patients. 
PFIC is life-threatening and debilitating.  Currently, the treatment for PFIC is palliative and 
there is no pharmaceutical treatment approved for use in PFIC.  The therapeutic choices are 
restricted to non-specific therapy of the symptoms and signs of the disease such as nutritional 
support, preventing vitamin deficiencies, and treatment of extrahepatic features.  Medical 
treatment options include off-label use of ursodeoxycholic acid, rifampin, antihistamines, and 
naltrexone.  A minority of patients respond nominally and transiently to these interventions.  
Biliary diversion is used to decrease systemic bile acids through interruption of the 
enterohepatic circulation and so avoid transplantation.  Liver transplantation is typically only 
viewed as an option when patients have failed medical treatment and/or biliary diversion and 
have liver failure or persistent uncontrolled pruritus. 


 
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5.2.2 
Summary of Clinical and Nonclinical Studies 
This is a summary of nonclinical and clinical studies.  More detail is provided in the 
Investigator’s Brochure ([IB] see A4250 IB).  
A4250 is a potent, selective IBAT inhibitor with no safety signals seen in the safety 
pharmacological studies.  In addition, no safety signals were seen in repeat-dose oral toxicity 
studies in rats with up to 26 weeks of daily dosing and in dogs with up to 39 weeks of daily 
dosing.  No safety signals were identified when A4250 was administered daily to juvenile 
rats from age 14 days to 63 days. 
A4250 is minimally absorbed with very low systemic exposure in both humans and other 
species.  In blood, protein binding is high (>99.7%).  Systemically absorbed A4250 is slowly 
metabolized in humans and in preclinical studies by hydroxylation only, a metabolite that 
retains IBAT activity.  A4250 did not show any significant inhibition of cytochrome 
P450 (CYP) 
enzymes 
in 
the 
rat. 
 
A4250 
inhibited 
human 
CYP3A4 
and 
CYP2D6 (IC50: 16 µmol/L for both enzymes), and CYP2C9 (IC50:1.2 µmol/L).  
To date, A4250 has been studied in 3 Albireo-sponsored clinical studies: a study in healthy 
volunteers evaluating single and multiple administration of A4250; a single-dose absorption, 
distribution, metabolism, and excretion (ADME) study; and a Phase 2 study in children with 
cholestatic pruritus.  In addition, an investigator-sponsored study has been conducted in 
patients with primary biliary cholangitis (PBC).  In total, 98 subjects have been exposed to 
A4250. 
In the first clinical study (A4250-001), A4250 was given to healthy volunteers as a single oral 
dose up to 10 mg or daily oral doses up to 3 mg/day for 7 days.  Following a single oral dose 
of 10 mg, the median maximum drug concentration was 0.2 nmol/L and the area under the 
curve was 0.8 nmol × h/L (n=6).  Pharmacodynamic evaluation of bile acid synthesis 
biomarkers 
(serum 
fibroblast 
growth 
factor 
19 [s-FGF19] 
and 
plasma 
7α Hydroxy 4-cholesten-3-one [p-C4]) identified the expected decrease in s-FGF19 and an 
increase in p-C4.  In addition, total plasma bile acids decreased and total fecal bile acids 
increased in A4250-treated patients compared to placebo. A4250 was generally well 
tolerated.  The only findings were GI-related effects, such as diarrhea at higher doses. 
In the ADME study (A4250-007), healthy subjects were administered a single oral dose of 
3 mg [14C]-A4250 capsule.  The main excretion route was determined to be fecal.  There 
were no quantifiable concentrations of A4250 in plasma or total radioactivity in plasma or 
whole blood.  A4250 was well tolerated.  AEs were reported and most were mild and not 
related to A4250. The 3 AEs experienced by 2 subjects reported as related to study drug were 
GI in nature, including diarrhea (2 AEs; 2 subjects) and abdominal pain (1 AE; 1 subject). 
In an exploratory Phase 2 study in children with cholestatic pruritus (A4250-003), A4250 was 
administered as oral daily doses of 0.01 to 0.2 mg/kg for 4 weeks.  Twenty 
patients (8 females and 12 males) aged 1 to 17 years were enrolled in this open-label 
dose-escalation study.  Four patients were re-enrolled into a second dose level.  No 


 
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treatment-related SAEs were reported, and most AEs, including some increased 
transaminases, were transient.  Four-week, once-daily treatment with A4250 reduced mean 
levels of s-BA in all cohorts.  Substantial s-BA reductions in 11 of 13 PFIC patients (ranging 
from 43% to 98%) were observed.  A4250 also reduced s-BA in the 11 non-PFIC patients, 
but to a lesser extent than in PFIC patients.  Patient-reported diaries documented 
improvement of the visual analogue scale itch score in 17 of 24 patients during treatment.  
The dose with the greatest improvement showed a mean decrease (± standard error of the 
mean) of 2.9 ±1.1 from baseline.  The results from this study form the basis of the current 
(A4250-005) study design. 
5.3 
Rationale 
In this investigational study, A4250 is under development for treatment of PFIC and is also 
being considered for other orphan cholestatic liver diseases. 
There is currently no medical treatment approved for PFIC.  In PFIC patients with severe 
pruritus, biliary diversion surgery is used to decrease systemic bile acids through interruption 
of the enterohepatic circulation and thereby reducing pruritus [Whitington 1988].  Liver 
transplantation is typically used as an option when patients have failed medical treatment 
and/or biliary diversion and have liver failure or persistent uncontrolled pruritus. 
By inhibiting IBAT with high selectivity and potency, A4250 has the potential to relieve 
cholestasis and improve liver function without surgical intervention in patients with PFIC.  
Since A4250 is minimally absorbed and achieves a substantial reduction of s-BA, it has the 
potential to be a safer and less invasive alternative to surgical biliary drainage.  The rationale 
for using A4250 is to decrease s-BA levels, and to reduce the major morbidity of pruritus, 
improving the health and wellbeing of patients affected with PFIC.  Ultimately, this 
noninvasive therapeutic approach has the potential to not only improve quality of life (QoL), 
but also reduce the need for biliary diversion and subsequent liver failure and liver 
transplantation. 
The doses selected, 40 and 120 µg/kg/day, were based on the efficacy and safety data 
generated from patients with PFIC (n=10 + 3 re-entered) and supporting data from all 
patients (n=20 + 4 re-entered) in the five dose groups dosed in the A4250-003 study.  
Improvements in s-BA and pruritus were observed with doses from 30 µg/kg to 100 µg/kg, 
with a dose-related trend.  The best dose response in all patients was at the 60 µg/kg/day for 
s-BA and 100 µg/kg/day for pruritus.  The best dose response in the PFIC subgroup was at 
30 µg/kg/day for both s-BA and pruritus.  Patients with PFIC responded well to the doses 
given from 30 µg/kg to 200 µg/kg; however, a clear dose relationship could not be 
established in order to select a single dose for this Phase 3 study.  As efficacy was observed 
at both low and high doses, 40 µg/kg and 120 µg/kg/day are considered to be the most 
optimal for this study.  
This study will be conducted in compliance with the protocol and with the International 
Council for Harmonisation (ICH) guidelines on Good Clinical Practice (GCP). 


 
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Patients completing 24 weeks of double-blind treatment will be offered to enter Study 
A4250-008 and receive open-label treatment with A4250. 
5.3.1 
Risk/Benefit 
A4250 has been evaluated in three Albireo-sponsored clinical studies: a double-blind 
placebo-controlled study in healthy volunteers, a single-dose ADME study, and a Phase 2 
study in children with cholestatic pruritus.  In addition, an investigator-sponsored study has 
been conducted in patients with PBC. 
A total of 98 subjects/patients have been exposed to A4250.  Healthy subjects have been 
exposed to up to 10 mg as a single dose and up to 3 mg daily as part of a multiple-dose 
evaluation.  Children with cholestatic liver disease have been treated with up to 
0.2 mg/kg/day (200 µg/kg/day) for 4 weeks.  In total, 2 SAEs have been reported; both were 
judged by the physician to be not related to the study drug. 
Patients with cholestatic liver diseases suffer from excess bile acids in the liver resulting in 
tissue damage.  A commonly used treatment in these patients is bile diversion surgery, 
whereby approximately 50% to 100% of the enterohepatic circulation of bile acids is 
interrupted.  Inhibition of IBAT with A4250, thereby interrupting the enterohepatic 
circulation of bile acids, is therefore a potential medical alternative to surgery which could be 
of benefit to these patients if shown to be effective and safe.  Data from the Phase 2 study 
showed efficacy of A4250 in reducing s-BA concentrations and pruritus in such patients. 
Based on the mode of action of an IBAT inhibitor, loose stools or diarrhea could be expected.  
However, in the pediatric Phase 2 study with 4 weeks daily treatment, only one patient had 
mild transient diarrhea after a single dose that did not recur on multiple dosing. 


 
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6 
STUDY OBJECTIVES 
6.1 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day 
A4250 in children with PFIC Types 1 and 2, as determined by the following: 
• The proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment 
• Change from baseline to Week 24 in pruritus compared to placebo  
6.2 
Secondary Objectives 
• To evaluate the effect of A4250 on serum ALT concentration 
• To evaluate the effect of A4250 on growth 
• To evaluate the effect of A4250 on sleep disturbance 
• To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or 
liver transplantation)  
• To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 


 
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7 
INVESTIGATIONAL PLAN 
7.1 
Overall Study Design and Plan 
7.1.1 
Description 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the 
efficacy and safety of 40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in 
children with PFIC (deficiencies of FIC1 or BSEP). 
Study data will be periodically reviewed by an unblinded Data Safety and Monitoring Board 
(DSMB). 
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment 
Period and a 4-week Follow-Up Period. 
After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of 
A4250, or a matching placebo. 
Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008, EudraCT Number 
017-002325-38, subject to a separate study approval) in which all patients will receive active 
treatment.  A patient who prematurely withdraws due to no improvement/intolerable 
symptoms, and has completed at least 12 weeks of the Treatment Period and End of 
Treatment (EOT) Visit, will also be offered the opportunity to enter Study A4250-008. 
Visit windows applicable to each visit are presented in the schedule of assessments (Table 1). 
There will be up to 10 clinic visits during the study (Figure 1) and one scheduled telephone 
contact, as follows: 
• Visit 1: Screening Period start. Patient is considered enrolled after signing the informed 
consent form (ICF) (Days -56 to -35) 
• Visit 2: second Screening Visit (Days -28 to -7) 
• Visit 3: Randomization Visit (Day 0) 
• Telephone Contact 1: (Day 14) 
• Visit 4: Clinic Visit (Day 28) 
• Visit 5: Clinic Visit (Day 56) 
• Visit 6: Clinic Visit (Day 84) 
• Visit 7: Clinic Visit (Day 126) 
• Visit 8: Clinic Visit (Day 154) 
• Visit 9: Clinic Visit, EOT (Day 168) 


 
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• Visit 10: Follow-Up Visit, 28 days after last dose of study drug (Day 196).  All patients 
continuing in Study A4250-008 will not have this Follow-up Visit. 
Additional clinic visits may be required for patients who need direct site assistance, e.g., due 
to AE monitoring, to fulfil screening requirements, and/or for safety maintenance. 
Screening Period 
Informed consent must be obtained at Visit 1 prior to performing any study procedures.  After 
signing the ICF, patients will be considered enrolled and evaluated for study eligibility. 
Daily recording of pruritus using an electronic diary (eDiary) will be started to determine 
confirmation of the magnitude and the baseline pruritus symptoms before the study.  After 
consultation with the Medical Monitor, patients failing eligibility criteria may be re-screened 
after 2 months.  Patients not fulfilling inclusion/exclusion criteria after 3 attempts are not 
allowed to re-screen. 
Caregivers/patients will be instructed on the daily use of the eDiary.  The eDiary will include 
patient-reported outcome (PRO) and observer-reported outcome (ObsRO) items for 
evaluation of itching (PRO), scratching (ObsRO), and sleep disturbance (PRO and ObsRO) 
during the Screening Period (study Days -56 to 0) and throughout the 24-week Treatment 
Period (study Days 0 to 168). 
Only the 14 most recent days before randomization will be used for eligibility calculation of 
the diary entries.  Observer-reported outcomes in patients of all ages will be recorded by a 
caregiver (or caregiver’s designee).  If possible, the same caregiver will complete the Albireo 
ObsRO items throughout the study. 
At a second Screening Visit (Visit 2) additional laboratory samples will be taken for 
eligibility assessments. 
Treatment Period 
Eligibility for randomization will be determined at Visit 3 using the pruritus (scratching 
reports from the Albireo ObsRO) data in the 2 weeks before Visit 3, genetic confirmation of 
diagnosis, and the liver biochemistry evaluations from the previous screening visits.  Two 
weeks after Visit 3, there will be a telephone contact with the caregiver/patient to evaluate 
any AEs. 
The patient will return to the clinic at Visit 4, Visit 5, Visit 6, Visit 7, Visit 8, and Visit 9 for 
assessments.  If a new caregiver will begin to complete the Albireo ObsRO during the course 
of the trial, they will be provided with training during a clinic visit before they begin to use 
the eDiary.  Additionally, caregivers/patients will be requested to use the diary to report the 
date and time each dose of study drug is administered during the Treatment Period. 
The last dose of study drug will be administered the day before Visit 9.  At study Visit 9, the 
patient has the option to enroll in an open-label extension study (A4250-008) should they 
meet the required entry criteria.  If the patient is continuing into Study A4250-008 this will be 


 
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the last visit in this study and the first visit in Study A4250-008.  Visit 9/EOT should also be 
performed at the time patients are prematurely withdrawn from the study. 
Clinic Visit 10 will take place 28 days after Visit 9 for all patients not continuing into Study 
A4250-008. 
Figure 1 
Study Design 
 
T: telephone contact; V: study visit 
 
End of Study 
The end of the study is defined as follows: 
a) End of study in one country: last patient last visit (LPLV) and sites are closed. 
b) End of study globally: LPLV globally and all sites closed. 
 
7.1.2 
Schedule of Assessments 
The schedule of assessments is presented in Table 1. 
 


 
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Table 1 
Schedule of Assessments 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Informed consent c 
X 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
Concomitant medicationd 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical and surgical history 
X 
 
X 
 
 
 
 
 
 
 
 
Blood sampling for genetic 
testinge 
X 
 
 
 
 
 
 
 
 
 
 
Physical examination including 
voluntary photography 
X 
 
X 
 
 
 
 
 
 
X 
 
Skin examination 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
Vital signsf 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
eDiary: itching, scratching, and 
sleep scoresg 
Daily diary entry 
Clinical chemistryh 
X 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Hematologyh 
 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Urinalysish 
 
X 
 
 
 
 
X 
 
 
X 
 


 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Serum bile acidsi 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
INR 
 
X 
 
 
X 
 
X 
 
X 
 
X 
Autotaxinj 
 
 
X 
 
X 
 
 
 
 
X 
 
p-C4j 
 
 
X 
 
X 
 
 
 
 
X 
 
AFP 
 
 
X 
 
 
 
 
 
 
X 
 
Fat-soluble vitamins A & E 
 
X 
 
 
 
 
 
 
 
X 
 
Fat-soluble vitamin D 
 
 
X 
 
 
 
 
 
 
X 
 
Abdominal Ultrasound 
 
 
X 
 
 
 
 
 
 
X 
 
Fibroscan® (where available) 
 
 
X 
 
 
 
 
 
 
X 
 
QoL questionnaire (PedsQL) 
 
 
X 
 
 
 
 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of Change 
 
 
 
 
X 
 
X 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of 
Symptoms 
 
 
X 
 
X 
 
X 
 
 
X 
 
Pregnancy testk 
Xk 
X 
X 
 
X 
X 
X 
X 
X 
Xk 
 
Study drug dispensedl 
 
 
X 
 
X 
X 
X 
X 
X 
 
 


 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Adverse eventsm 
Continuous collection 
Study drug compliance 
 
 
 
X 
X 
X 
X 
X 
X 
X 
 
AFP: alfa-fetoprotein; BMI: body mass index; eCRF: electronic case report form; eDiary: electronic diary; EOT: End of Treatment; ICF: informed consent form; 
INR: international normalized ratio; p-C4: plasma 7α Hydroxy 4-cholesten-3-one; PedsQL: Pediatric Quality of Life Inventory; QoL: quality of life; RR: respiratory rate; 
s-BA: serum bile acid. 
 
a 
Must also be performed at the time a patient is prematurely withdrawn from the study.  This visit is the same as Visit 1 in the extension protocol (Study A4250-008). 
b 
Assessments must be performed 28 days following the final dose of study drug.  This visit is omitted if patients are entering Study A4250-008. 
c 
The ICF (and assent) must be signed before any study procedures are performed. 
d 
Past (within 1 month prior to Visit 1) and current medication will be documented in the eCRF. 
e 
Blood sample for genetic testing will be drawn for all patients.  Genetic analysis will only be performed on a patient’s extracted DNA if previous the clinical genetic 
result is equivocal, unavailable, or unobtainable. 
f 
Includes blood pressure, pulse, RR, temperature, height (or length depending on age), and body weight (using a certified weight scale).  BMI will be calculated. 
g 
Includes training on use of the device at Screening (Visit 1) and review of patient daily entries during 14 days prior to Clinic Visit 3 to determine eligibility.  
h 
See Table 3 for detailed parameters. 
i 
Patients will fast (water intake only is permissible) for at least 4 hours prior to the collection of samples for s-BA.  Exceptions can be made for infants, less than 
12 months of age, if unable to fast for the full 4 hours.  All s-BA results during the treatment period and follow-up will be blinded. 
j 
Samples will not be collected for patients ≤10 kg. 
k 
For girls who have reached menarche.  Serum test at Visit 1; urine test at other visits.  A serum pregnancy test will also be conducted at Visit 9 for patients entering 
Study A4250-008.  If a urine pregnancy test is positive, a serum pregnancy test should be performed to confirm the pregnancy. 
l 
Study drug to be taken once daily on Day 1 through Day 167 as described in Section 8.2. 
m 
Adverse event information will be collected from the time of signing of the ICF to study discontinuation. 


 
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7.1.3 
Study Procedures and Assessments 
7.1.3.1 
Screening Period (Days -56 to 0) 
Day -56 to Day -35/Clinic Visit 1 
Patients will undergo a Screening Visit up to 56 days prior to the planned first day of study 
treatment.  Screening procedures and assessments are as follows: 
• Obtain written informed consent 
• Assess inclusion/exclusion criteria (Section 7.2) 
• Record demographics (age, full date of birth, gender, race, and ethnicity) 
• Document concomitant medications 
• Medical and surgical history (date of diagnosis of PFIC Types 1 or 2, prior investigational 
medications for PFIC, historical liver function test [LFT] values, any surgery performed, 
any other diagnosis, and historical liver biopsy data) 
• Blood sample for genetic testing 
• Collect, if available, past confirmatory clinical genetic laboratory report for PFIC1/PFIC2 
and send to central reader for review 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• eDiary training and compliance requirements; patients begin daily recording of pruritus 
using the eDiary (Appendix 2) 
• Clinical chemistry (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Serum pregnancy test for girls of who have reached menarche 
• AE monitoring 
Day -28 to Day -7/Clinic Visit 2 
A second Screening Visit will be performed.  Screening procedures and assessments are as 
follows: 
• Review concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for itching, scratching, and sleep scores and 
compliance 
• Urinalysis (Section 10.2.1) 


 
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• s-BA (Section 9.2.1) 
• International normalized ratio (INR) 
• Fat-soluble vitamins A and E 
• Urine pregnancy test for girls of who have reached menarche 
• AE monitoring 
7.1.3.2 
Treatment Period (Day 0 to Day 168) 
Study Day 0/Clinic Visit 3 (Randomization) 
At the randomization visit, the following assessments will be conducted: 
• Review inclusion/exclusion criteria (Section 7.2) 
• Review concomitant medications, medical and surgical history 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance and entrance criteria requirement (see 
Inclusion Criteria 4, Section 7.2.1) 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1)  
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• Fat-soluble vitamin D 
• Alfa-fetoprotein (AFP) 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer QoL (Pediatric Quality of Life Inventory [PedsQL]) questionnaire 
• Patient/caregiver/clinician complete the patient global impression of symptoms (PGIS) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Study drug is dispensed and patients are instructed to administer daily from Day 1 
Study Day 14 (Telephone Contact) 
A study nurse will contact the patient/caregiver by telephone for monitoring of the following:  
• Review concomitant medications 


 
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• Albireo PRO/ObsRO eDiary review for compliance 
• AEs 
• Evaluation of study drug compliance 
Study Day 28 and Day 84/Clinic Visits 4 and 6  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• Urinalysis (at Clinic Visit 6 only; Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Autotaxin and p-C4 (at Clinic Visit 4 only; samples will not be taken for patients ≤10 kg) 
• Patient/caregiver/clinician complete the patient global impression of change (PGIC) and 
PGIS 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 56 and Day 126/Clinic Visits 5 and 7 
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 


 
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• Study drug is dispensed 
Study Day 154/Clinic Visit 8  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 168/Clinic Visit 9/EOT 
The last dose of study drug is taken in the morning the day before Clinic Visit 9, and the 
following procedures and assessments will be performed (also performed at the time a patient 
prematurely withdraws):  
• Review of concomitant medications 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry, hematology, and urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• AFP  
• Fat-soluble vitamins A, E, and D 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer PedsQL questionnaire 
• Patient/caregiver/clinician complete the PGIC and PGIS 


 
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• Urine pregnancy test for girls who have reached menarche, or serum pregnancy test for 
girls continuing to study A4250-008 
• AE monitoring 
• Evaluation of study drug compliance 
7.1.3.3 
Follow-Up Period 
Study Day 196/Clinic Visit 10 
All patients not continuing into Study A4250-008 will return to the study site 28 days after 
Clinic Visit 9 or the last dose of study drug for the following assessments: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• AE monitoring 
7.2 
Study Population 
A total of 60 patients with clinical diagnosis of PFIC Type 1 or 2 that have been genetically 
confirmed will be randomized.  
An attempt will be made to randomize a minimum of 10 patients from each age category 
(6 months to 5 years, 6 to 12 years, and 13 to ≤18 years).  
A maximum of 5 patients that were treated in Study A4250-003 will be allowed to participate 
in this study. 
7.2.1 
Inclusion Criteria 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages 
of ≥6 months and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have genetic confirmation of PFIC-1 or PFIC-2 through identification of 
biallelic pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, 
taken as the average of 2 samples 7 to 28 days apart (Visits 1 and 2) prior to 
randomization 


 
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4. Patient must have history of significant pruritus and a caregiver-reported observed 
scratching in the eDiary average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to 
randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  
Patients who turn 18 years of age (or legal age per country) during the study will be 
required to re-consent in order to remain in the study 
6. Patients will be expected to have a consistent caregiver(s) for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use 
an eDiary device as required by the study 
7.2.2 
Exclusion Criteria 
1. Patient with pathologic variations of the ABCB11 gene that predict complete absence of 
the BSEP function 
2. Patient with past medical history or ongoing presence of other types of liver disease 
including, but not limited to, the following: 
a) Biliary atresia of any kind 
b) Benign recurrent intrahepatic cholestasis, indicated by any history of normal s-BAs 
c) Suspected or proven liver cancer or metastasis to the liver on imaging studies 
d) Histopathology on liver biopsy is suggestive of alternate non-PFIC related etiology of 
cholestasis 
3. Patient with a past medical history or ongoing presence of any other disease or condition 
known to interfere with the absorption, distribution, metabolism (specifically bile acid 
metabolism), or excretion of drugs in the intestine, including but not limited to, 
inflammatory bowel disease 
4. Patient with past medical history or ongoing chronic (i.e., >3 months) diarrhea requiring 
intravenous fluid or nutritional intervention for treatment of the diarrhea and/or its 
sequelae 
5. Patient has a confirmed past diagnosis of infection with human immunodeficiency virus 
or other present and active, clinically significant, acute, or chronic infection, or past 
medical history of any major episode of infection requiring hospitalization or treatment 
with parenteral anti-infective treatment within 4 weeks of treatment start (study Day 1) or 
completion of oral anti-infective treatment within 2 weeks prior to start of Screening 
Period 
6. Any patient with suspected or confirmed cancers except for basal cell carcinoma, and 
non-liver cancers treated at least 5 years prior to Screening with no evidence of 
recurrence 


 
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7. Patient with a past medical history of chronic kidney disease with an impaired renal 
function and a glomerular filtration rate <70 mL/min/1.73 m2  
8. Patient with surgical history of disruption of the enterohepatic circulation excluding those 
who have undergone a successful reversal procedure that has permanently restored flow 
of bile acids from the liver to the duodenum 
9. Patient has had a liver transplant or listed for liver transplant 
10. Decompensated liver disease, coagulopathy, history or presence of clinically significant 
ascites, variceal hemorrhage, and/or encephalopathy 
11. INR >1.4 (the patient may be treated with Vitamin K intravenously, and if INR is <1.4 at 
resampling the patient may be randomized) 
12. Serum ALT >10 × upper limit of normal (ULN) at Screening 
13. Serum ALT >15 × ULN at any time point during the last 6 months unless an alternate 
etiology was confirmed for the elevation 
14. Total bilirubin >5 × ULN at Screening 
15. Patient suffers from uncontrolled, recalcitrant pruritic condition other than PFIC.  
Examples include, but not limited to, refractory atopic dermatitis or other primary pruritic 
skin diseases 
16. Any patient who is pregnant or lactating or who is planning to become pregnant within 
24 weeks of randomization 
17. Sexually active males and females who are not using a reliable contraceptive method with 
≤1% failure rate (such as barrier protection, hormonal contraception, intrauterine device, 
or complete abstinence) throughout the duration of the study (from signed informed 
consent through Follow-up, Visit 10). 
18. Patient with a past medical history of alcohol or substance abuse will be excluded.  
Patient must agree to refrain from illicit drug and alcohol use during the study 
19. Administration of bile acid or lipid binding resins and medications that slow GI motility 
(Refer to Appendix 1 – Concomitant Medications Prohibited during the study) 
20. Patient has had investigational exposure to a drug, biologic agent, or medical device 
within 30 days prior to Screening, or 5 half-lives of the study agent, whichever is longer 
21. Patient who has been previously treated with an IBAT inhibitor whose pruritus has not 
responded to treatment 
22. Any other conditions or abnormalities which, in the opinion of the investigator or Medical 
Monitor, may compromise the safety of the patient, or interfere with the patient 
participating in or completing the study 


 
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7.2.3 
Withdrawal of Patients 
Patients/caregivers will be informed that they have the right to withdraw from the study at 
any time, without prejudice to their medical care, and that they are not obliged to state their 
reasons. 
Any withdrawal, and reasons for withdrawal, must be fully documented in the electronic case 
report form (eCRF) and source documents and the patient followed by the 
investigator/investigative staff.  Even after the study is completed at the Follow-up Visit, the 
investigator/investigative staff will, regardless of reason for withdrawal, record any patient 
data they receive concerning SAEs, and all drug-related non-serious AEs, and subsequently 
report these to Albireo.  Withdrawn patients will not be replaced. 
Patients will be withdrawn in the following circumstances: 
• A patient’s/caregiver’s desire for withdrawal for any reason 
• Lost to follow-up (every effort must be made to contact the patient/caregiver; a certified 
letter must be sent) 
• An AE that, in the opinion of the investigator, necessitates withdrawal 
• A worsening of the symptoms related to diarrhea occurs 
• Death 
• Patient/caregiver estimation of lack of improvement/intolerable symptoms 
• A patient’s/caregiver’s substantial noncompliance (eDiary and study drug compliance) or 
protocol violation 
• An investigator’s opinion that continuing the patient in the study is not appropriate.  The 
investigator may withdraw a patient at any time, if it is considered to be in the patient’s 
best interest 
A patient who prematurely withdraws due to no improvement/intolerable symptoms, and has 
completed at least 12 weeks treatment in the double-blind study, will be offered the 
opportunity to enter open-label Study A4250-008.  A patient who withdraws from treatment 
prematurely and will not enter Study A4250-008 will be requested to complete all visits and 
safety assessments per the current protocol (A4250-005).  As a minimum, patients will have 
EOT (Visit 9) assessments at the time of withdrawal and a follow-up assessment (Visit 10) 
28 days following the last dose of study drug, and the patient will be recorded as withdrawn. 
7.2.4 
Study Termination by Sponsor 
This study may be terminated at any time by Albireo if serious side effects should occur, if 
the investigator does not adhere to the protocol; or if, in the sponsor's judgment, there are no 
further benefits to be achieved from the study.  In this event, Albireo/designee will inform the 
study investigators, institutions, and all regulatory authorities. 


 
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Albireo may temporarily or permanently discontinue the study at an investigative site at any 
time for safety, ethical, compliance, or other reasons.  If this is necessary, Albireo will 
endeavor to provide advance notification to the site.  If a site or the study is suspended or 
discontinued, the investigator/investigative staff will be responsible for promptly informing 
the Independent Ethics Committee (IEC).  If required by local regulations, Albireo/designee 
will be responsible for informing the IEC and the regulatory authority of study or site 
discontinuation.  In such an event, all study data and unused study drug must be returned to 
Albireo. 


 
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8 
TREATMENT OF PATIENTS 
8.1 
Identity of Study Drug 
A4250 and placebo will be supplied as capsules for oral administration.  White opaque 
capsules filled with pellets containing A4250 or placebo will be provided.  Two different 
capsule sizes will be available, as follows: 
• Capsule size 0 that can be opened 
• Capsule size 3 that can be opened or swallowed intact 
The content of the A4250 and placebo capsules will be identical in appearance.  Capsule 
filling weight will also be identical for high dose, low dose, and placebo.  Bottles with 
34 capsules will be given to the patient at each visit.  A patient who requires 2 or more 
capsules per day will be given multiple bottles.  Refer to the investigational product manual. 
8.2 
Administration of Study Drug 
Patients will be dosed with A4250 at a dose of 40 µg/kg/day, 120 µg/kg/day, or placebo for 
24 weeks.  Study drug will be dispensed to the patient at Visits 3, 4, 5, 6, 7, and 8, together 
with instructions on how to store and take the drug.  Study drug administration data, 
including whether each patient took each dose or partial doses of study drug, whether there 
were any delayed or missed doses, doses, and whether the capsule was opened or swallowed 
whole will be documented through the eDiary and transferred to the study database. 
A4250 should be taken in the morning, together with food.  On clinic visits days when 
laboratory assessments are conducted, study drug should be taken after the visit and after 
laboratory samples are taken.  Patients should not crush or chew the capsule(s).  When 
swallowing the capsule intact, the patient should administer the dose with a glass of water. 
For a patient unable to swallow capsule intact, the capsule can be opened and the contents 
sprinkled and mixed in a small amount of soft room-temperature yogurt, apple sauce, or fruit 
purée.  The amount of food should be less than what the patient/infant is expected to eat and 
should be followed by water.  
1. Hold the capsule over the prepared food, gently twist open and allow the content to fall 
into food 
2. Mix the entire capsule content with 1 or 2 tablespoons (15 to 30 mL) of soft food (apple 
sauce, fruit purée, or yogurt) 
3. Administer the mixture immediately after mixing. 
The number of capsules provided to the patient will be based on body weight at Visit 3.  At 
Visit 6, if a patient’s weight has shifted to another weight category (identified in Table 2), the 
number of capsules per day may be adjusted.   
 


 
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Table 2 
Dosing and Capsule Strength 
Body Weight 
(kg) 
Capsule 
Size 
Number of 
Capsules 
per Day 
Capsule 
Strength, 
Low Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Strength, 
High Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Size, 
Placebo 
(µg) 
5.0 to <7.5 
0 
1 
200 
200 
600 
600 
0 
7.5 to <12.5 
0 
2 
200 
400 
600 
1200 
0 
12.5 to <17.5 
0 
3 
200 
600 
600 
1800 
0 
17.5 to <19.5 
0 
4 
200 
800 
600 
2400 
0 
19.5 to <25.5 
3 
2 
400 
800 
1200 
2400 
3 
25.5 to <35.5 
3 
3 
400 
1200 
1200 
3600 
3 
35.5 to <45.5 
3 
4 
400 
1600 
1200 
4800 
3 
45.5 to 55.5 
3 
5 
400 
2000 
1200 
6000 
3 
>55.5 
3 
6 
400 
2400 
1200 
7200 
3 
8.3 
Study Treatment Packaging and Labeling 
8.3.1 
Packaging and Labeling 
The capsules will be packed in high-density polyethylene containers, with child-proof 
polypropylene caps.  Study drug capsules containing A4250 in the strength specified in 
Table 2 above will be manufactured. 
Packaging and labeling will be prepared to comply with applicable regulatory requirements. 
8.3.2 
Storage 
Treatment packs containing A4250 capsules should be stored and dispensed in accordance 
with regulations in their original containers.  The storage facility at the investigative site 
should be locked and the storage temperature should be between 15°C and 25°C. 
Patients/caregivers should be informed of appropriate storage conditions (i.e., room 
temperature, between 15°C and 25°C). 
Any deviations from the recommended storage conditions should be immediately reported to 
Albireo and the study drug should not be used until authorization has been given by Albireo. 
8.3.3 
Blinding and Randomization of Study Drug 
A double-blind design is employed so that both the investigators and the patients will be 
unaware of the treatment assignment during the study.  
After written informed consent is obtained from an eligible patient, an 8-digit patient 
identification number will be assigned.  The first 2 digits will denote the country, followed by 
a 3-digit site number and a 3-digit patient sequence number.  This number will be created and 
allocated by the Interactive Web Response System (IWRS), when the patient first enters the 
system at Screening.  The randomization codes will be computer-generated by 
 


 
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, and kept by a statistician independent from the project team.  The 
randomization will be done in blocks and stratified according to PFIC class (Type 1 and 2) 
and age class (6 months to 5 years, 6 to 12 years, and 13 to ≤18 years) to ensure approximate 
balance between dose schemes (1:1:1).  A separate randomization list will be prepared for the 
maximum 5 patients from Study A4250-003 regardless of stratification.  Randomization 
codes will be assigned sequentially as patients become eligible for randomization. 
Patients who are eligible for randomization will be assigned a unique 4-digit randomization 
number by IWRS.  This randomization number identifies which treatment will be allocated to 
the patient.  Patients who withdraw from the study after randomization visit are not to be 
replaced and their randomization number will not be reused.   
To ensure blinding, the study drug and the matching placebo have the same shape and size.  
Patients will receive capsule(s) of A4250 according to their dose group or capsule(s) of 
matching placebo once a day during the double-blind Treatment Period.  Labels on the study 
drug containers will not identify the treatment that a patient is randomized to.  Traceability of 
the treatment is ensured by the study drug number. 
The 5-digit study drug number will identify study drug packs and will be detailed on the 
study drug label.  Dispensing of study drug will be coordinated by IWRS.  
The system will assign a study drug number corresponding to the randomization arm.  The 
randomization number will be used in the background only to ensure no unblinding and will 
not be displayed to end-users in the IWRS. 
8.4 
Procedure for Breaking the Randomization Code 
Should a situation arise where unblinding is urgently required, i.e., only where knowledge of 
the study drug is required to adequately manage a life-threatening situation, the investigator 
at that study site may perform immediate unblinding through IWRS.  The investigator should 
make every effort to discuss the rationale for emergency unblinding with the sponsor Medical 
Monitor or the 
 Medical Monitor prior to unblinding the individual patient. 
The study site investigator and appropriate 
 project team members will be authorized to 
access the emergency unblinding functionality within the IWRS.  Emergency unblinding 
takes place in the IWRS via a dedicated emergency unblinding module.  The system will 
require the user to enter their emergency unblinding, which is typically provided at the same 
time login credentials are distributed.  The exact description of the treatment assigned to the 
individual patient then will be accessible to the user.  Once the randomization code is broken 
for a patient, he/she must be withdrawn from the study and all assessments and procedures at 
EOT should be performed.  As a follow-up, Visit 10 will be performed according to the time 
schedule.  Once a randomization code has been broken, the investigator must inform the 
 and sponsor Medical Monitor in writing within 24 hours. In case of technical issues 
accessing the system online, please see the IWRS site user manual for country specific 
contact telephone numbers to the 
 HelpDesk 24/7 system support. 


 
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8.5 
Patient Compliance 
The study nurse will monitor eDiary compliance by routine review of the CRF health 
website.  If both diary entries on a day are missing during, the study nurse will call the 
patient/caregiver to remind them to complete all scheduled entries.  Any noncompliance will 
be documented and explained in the source documents and in the eCRF. 
Study drug compliance will also be checked through diary responses.  Additionally, patients 
will return all unused study drug at Visits 4, 5, 6, 7, 8, and 9.  The study site staff will count 
all returned drug, assess compliance, and record details in the eCRF. 
Treatment Compliance = 100 × ((Number of study drug dispensed – number of study drug 
returned)/number of study drug that should be taken). 
Treatment compliance between 80% and 120% will be acceptable. 
8.6 
Study Drug Accountability 
Records shall be maintained of the delivery of study treatment(s) to the study site(s), of the 
inventory at the study site(s), of each use of the study treatment(s) for each patient, and of the 
return of unused study treatment(s) to Albireo. 
These records shall include dates, quantities, batch numbers, expiry dates, and the unique 
code numbers assigned to the study drug and to the study patients. 
The investigator will be responsible for ensuring that the records adequately document that 
the patients were provided the quantities specified in the protocol and that all study drug 
received from Albireo is reconciled.  
8.7 
Concomitant Therapy and Prohibited Medications 
The investigator will note all ongoing medication and any medication recently stopped 
(within 1 month prior to Visit 1) in the eCRF.  At Visits 2 through 10, all changes in 
medication (stopping or starting new medication or changes in dose) will be recorded in the 
eCRF.  All medications (prescribed or over the counter) for pruritus and sleep will be 
recorded. 
All medications taken by a patient within 3 months prior to the first intake of study drug are 
regarded as prior medication. 
All medications taken by a patient on or after the first intake of study drug, and which 
continue to be taken during the study, are regarded as concomitant medication.  
Prohibited medications are listed in Appendix 1. 
 


 
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9 
ASSESSMENT OF EFFICACY 
9.1 
Efficacy Endpoints 
9.1.1 
Primary Efficacy Endpoint 
The primary efficacy endpoints are as follows: 
• European Union (EU) and rest of the world (RoW): Proportion of patients 
experiencing at least a 70% reduction in fasting s-BA concentration from baseline to 
the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 
weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization 
and the previous visit (Clinic Visit 2).  The end value is the average of the values at 
Weeks 22 and 24 after the start of double-blind treatment. 
• United States (US): Change in pruritus, as indexed by caregiver-reported observed 
scratching, from baseline to Week 24 as measured by the worst scratch of the Albireo 
ObsRO instrument (Appendix 2).  Scores for the 14 days baseline and post-treatment 
intervals will be calculated by averaging the worst scratching scores from each day in 
the interval. 
9.1.2 
Secondary Efficacy Endpoint 
Secondary efficacy endpoints include the following:  
• EU and RoW: Change in pruritus, as indexed by caregiver-reported observed 
scratching, from baseline to Week 24 as measured by the worst scratch of the Albireo 
ObsRO instrument (Appendix 2).  Scores for the 14 days baseline and post-treatment 
intervals will be calculated by averaging the worst scratching scores from each day in 
the interval. 
• US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment. 
• All Regions: 
• All secondary endpoints are compared to placebo. 
o Change from baseline to Week 24 in fasting s-BA 
o Change from baseline to Week 24 in serum ALT concentration 
o Change in growth from baseline to Week 24, defined as the linear deficit 
(weight for age, and body mass index [BMI]) compared to the national growth 
curves (Z-score, standard deviation [SD] from P50)  
o Proportion of patients achieving meaningful reduction in caregiver-reported 
observed scratching at Week 24 


 
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o Change from baseline to Week 24 in sleep parameters measured with the 
Albireo PRO and ObsRO instruments 
o Change from baseline to Week 24 in patient-reported itch severity as 
measured by the average daily score of the Albireo PRO instrument 
(Appendix 2); only patients ≥8 years of age will complete the Albireo PRO 
instrument  
o Number of patients undergoing biliary diversion surgery or being listed for 
liver transplantation 
9.1.3 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• Proportion of patients achieving meaningful reduction in patient-reported itch severity at 
Week 24 
• Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, 
caregiver, and clinician via the PGIC items (Appendix 3) 
• Change from baseline in fasting s-BA at Weeks 4 and 12 
• Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
to Weeks 4, 12, and 24 as measured by the worst scratch.  Scores for the 7 worst 
scratching days of the last 14 days at baseline and post-treatment intervals will be selected 
• Change from baseline in patient-reported itch severity and observer-reported scratching at 
Weeks 4 and 12  
• Change from baseline to Week 24 in patient-reported and observer-reported night time 
itching and scratching severity scores, respectively 
• Change from baseline to Week 24 in pooled pruritus score including observer-reported 
scratching for patients <8 years of age and patient-reported itch severity for patients 
≥8 years of age 
• Change from baseline to Week 24 in additional patient-reported and observer-reported 
sleep parameters (e.g., tiredness and number of awakenings) 
• Change from baseline to Week 24 in PedsQL (Appendix 4) 
• Change from baseline in serum ALT concentration at Weeks 4 and 12 
• Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• Change from baseline in AST at Weeks 4, 12, and 24 
• Change from baseline for other biochemical markers and measures of bile acid synthesis 
(autotaxin, p-C4) at Week 24 


 
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• Change in pediatric end-stage liver disease/model for end-stage liver disease 
(PELD/MELD) score, AST to platelet ratio index (APRI) score, and Fib-4 score from 
baseline to Week 24 
• Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® 
(where available) 
• Liver-related mortality and liver decompensation events 
• All-cause mortality 
9.2 
Efficacy Assessments 
9.2.1 
Serum Bile Acids 
Blood samples for analysis of fasting total s-BA will be drawn at all visits.  Patients will fast 
(water intake only is permissible) for at least 4 hours prior to the collection of samples for 
s-BA.  Exceptions can be made for infants, <12 months of age, if unable to fast for the full 
4 hours.  Any visit at which a bile acid sample result is unreportable, an additional 
unscheduled visit for a repeat sample collection may be scheduled.  All s-BA results during 
the treatment period and follow-up will be blinded.  Samples will be processed and 
transported to a central laboratory per instructions in the laboratory manual. 
9.2.2 
Itching, Scratching and Sleep Score 
Itching, observed scratching, and sleep disturbance will be recorded twice each day via 
eDiary.  Patients and/or caregivers will be instructed to complete the eDiary every day in the 
morning after the patient wakes and in the evening just before the patient goes to sleep 
throughout the study.  Patients and caregivers will receive a formal training session during 
Visit 1. 
The eDiary includes Albireo ObsRO and PRO items.  Patients <8 years of age will not be 
asked to complete the Albireo PRO items; only the Albireo ObsRO will be completed by 
caregivers of patients in this age group.  Older patients, 8 to 17 years of age, will complete 
the Albireo PRO items and the caregiver will complete the Albireo ObsRO items.  The 
Albireo PRO items assess severity of itch, aspects of sleep disturbance (morning diary only), 
and tiredness.  For patients 8 to 12 years of age, the caregiver will read the Albireo PRO 
items along with the child and record the child’s response.  A guide will be provided to the 
caregivers that provides standardized explanations of the Albireo PRO items, in case the 
patient is confused or requires clarification about the meaning of a question.  The Albireo 
ObsRO items assess severity of observed scratching, aspects of observed sleep disturbance 
(morning diary only), and observed signs of tiredness (evening diary only).  The Albireo 
ObsRO and PRO scratching and itch severity items use 0 to 4 response scales, where each 
response is distinguished by a unique facial expression, verbal anchor, number, and color 
code (Appendix 2).  


 
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A daily score for the Albireo ObsRO scratching item will be calculated by selecting the 
maximum scratching severity score from the 2 ratings for each day.  A daily score will be 
considered missing if both of the daily assessments are missing.  A 14-days score will be 
calculated by averaging the daily scratching scores.  A 14-days score will be considered 
missing if >4 out of 7 days a week of data are missing.  The same approach will be used to 
calculate a patient-reported itch severity score. 
9.2.3 
Growth 
Growth will be measured as height (velocity) and weight (Z-score) using a certified weight 
scale.  BMI will be calculated by weight (kg) / height (m)2.  Change will be defined as linear 
growth deficit (weight and BMI for age) compared to national growth curves (Z-score, SD 
from P50). 
9.2.4 
Quality of Life Questionnaire (PedsQL) 
Patients and caregivers will be asked to complete a QoL questionnaire (PedsQL) at Visit 3 
and Visit 9.  Details of the questions included on the questionnaire are provided in 
Appendix 4. 
9.2.5 
Biomarkers and Other Laboratory Samples 
Blood samples for autotaxin and p-C4 will be drawn at Visits 3, 4, and 9 only for children 
with body weight ≥10 kg.  Samples will be processed and transported to a laboratory per 
instructions in the laboratory manual. 
9.2.6 
PELD/MELD Score 
At randomization and end of treatment, PELD score will be calculated for children up to 
12 years of age.  For children 13 years or older, the MELD score will be calculated. 
PELD Score = 4.80 * ln(total bilirubin in mg/dL) + 18.57 * ln(INR) - 6.87 * ln(albumin in 
g/dL) + 4.36 (if patient <1 year: scores for patients listed for liver transplantation before the 
patient's first birthday continue to include the value assigned for age (<1 year) until the 
patient reaches the age of 24 months) + 6.67 (if the patient has growth failure [≤2 SD]) 
Laboratory values less than 1.0 will be set to 1.0 for the calculation of the PELD score. 
MELD Score = 9.57 * ln(creatinine in mg/dL) + 3.78 * ln(total bilirubin in mg/dL) + 
11.2 * ln(INR) + 6.43 
Laboratory values less than 1.0 will be set to 1.0 and serum creatinine values greater than 
4.0 mg/dL will be set to 4.0 for calculation of the MELD score. 
9.2.7 
Fibroscan® 
Where available, Fibroscan® will be performed as per institution standard practice at Visits 3 
and 9. 


 
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9.2.8 
Markers of Fibrosis 
APRI score and Fib-4 score will be calculated at randomization and at end of treatment. 
APRI = [(AST in U/L) / (AST ULN in U/L)] / (Platelets in 109/L) 
Fibrosis 4 Score = (Age*AST in U/L) / (Platelets in 109/L * √(ALT in U/L)). 
9.2.9 
Global Impression of Change and Global Impression of Symptom Measures 
Patients (≥8 years of age), caregivers, and clinicians will complete the PGIC and the PGIS 
measures (Appendix 3) at randomization (Visit 3; PGIS only), Visits 4 and 6, and at EOT 
(Visit 9). 
The PGIC items assess change in itch (patient version), scratching (caregiver and clinician 
versions), and sleep (all versions) since starting the study drug.  The PGIS items assess itch 
(patient version), scratching (caregiver and clinician versions), and sleep (all versions) in the 
past week.  Caregivers and clinicians will complete the PGIC and PGIS for all patients; those 
patients ≥8 years of age will complete the patient version. 


 
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10 
ASSESSMENT OF SAFETY 
The timing and frequency of safety assessments are described in Section 7.1.3 and in Table 1 
above. 
The primary safety analysis for this study will include the incidence of total 
treatment-emergent adverse events (TEAE) and TEAEs categorized by causality, severity, 
and seriousness assessments made by the investigator by comparing study drug exposure to 
placebo. 
Trends in safety will also be evaluated for the following assessments: 
• Physical examinations 
• Concomitant medications 
• Vital signs 
• Laboratory test results (including clinical chemistry, hematology, urinalysis, AFP, and 
fat-soluble vitamins A, D, and E) 
• Abdominal ultrasound 
• Discontinuations due to AEs 
10.1 
Adverse Events 
10.1.1 
Definitions and Investigator Assessments 
An AE is defined as any untoward medical occurrence in an enrolled patient regardless of 
causal relationship with study drug.  An AE can therefore be any clinically significant 
unfavorable and unintended sign, symptom, or disease that occurs once a patient is enrolled 
(ICF is signed) in the study until the patient is discharged from the study, whether or not 
related to the study drug. 
10.1.1.1 
Clinical Significance 
Clinical significance is defined as any abnormal finding that results in further clinical 
investigation(s), treatment(s), or the diagnosis of new condition.  Patient-reported events and 
protocol-mandated laboratory values, vital signs, and physical examination findings can be 
considered clinically significant (i.e., an AE) if there is a deterioration as compared to 
baseline.  Examples of clinically significant worsening from baseline could include, but is not 
limited to, events causing withdrawal from the study and events requiring medical 
intervention outside of the study causing apparent clinical manifestations, or judged relevant 
by the investigator.   
10.1.1.2 
Serious Adverse Events 
Serious criteria are applied by the investigator to each AE as specifically defined below.  
These criteria are used to determine whether an AE is serious or non-serious.  The assessment 
is made independently of severity assessment (Section 10.1.1.3).  For example, the 


 
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development of a severe rash that occurs after signing of informed consent may not meet 
serious criteria as defined below and therefore would be considered a severe, non-serious AE. 
Any AE that meets any 1 of the following 6 criteria is considered an SAE: 
• The outcome of the AE is death 
• The AE is immediately life-threatening.  Life-threatening means that the patient is, in the 
opinion of the investigator, at immediate risk of death from the reaction as it occurred.  
This does not include an AE that, if more severe, might have caused death 
• The AE results in persistent or significant disability/incapacity.  Disability means a 
substantial disruption of a person's ability to conduct normal life functions 
• The AE requires or prolongs hospitalization  
• The AE results in a congenital anomaly/birth defect 
• The AE is an important medical event.  Important medical events may meet serious 
criteria should the investigator assess that they may significantly jeopardize the patient, 
represent a significant hazard, or requires medical/surgical intervention to prevent one of 
the outcomes listed above.  Examples of potential SAEs based on this criteria include, but 
are not limited to, allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions even if they do not result in inpatient 
hospitalization, or the development of drug dependency and drug abuse. 
10.1.1.3 
Severity Assessment 
Severity assessments are based on the intensity of the event in relation to expectation.  The 
investigator will assess the intensity of AEs based on the following definitions: 
• Mild (awareness of sign or symptom, but easily tolerated) 
• Moderate (discomfort sufficient to cause interference with normal activities) 
• Severe (incapacitating, with inability to perform normal activities) 
Severity is a measure of intensity where seriousness is defined by the criteria outlined in 
Section 10.1.1.2.  An AE of severe intensity need not necessarily be considered serious.  For 
example, nausea that persists for several hours may be considered severe nausea, but not meet 
serious criteria, and therefore would be assessed as a severe AE but not an SAE. 
10.1.1.4 
Causality Assessment 
The investigator determines the causality of all AEs to the study drug using medical judgment 
and considering all relevant factors such as (but not limited to) the underlying study 
indication, coexisting disease, concomitant medication, relevant history, pattern of the AE, 
temporal relationship to the study drug, and de-challenge or re-challenge.  The causality 
assessment of the AE/SAE is to be made as follows. 


 
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Related to study drug (possibly, probably, or definitely related) 
Based on medical judgment, there is at least a reasonable possibility that the study drug 
caused the event; one or more of the following criteria apply: 
• The event follows a reasonable temporal sequence from administration of study drug 
• The event could not be reasonably attributed to the known characteristics of the patient's 
clinical state, environmental or toxic factors, or other modes of therapy administered to 
the patient 
• The event follows a known pattern of response to study drug 
• The event disappears or decreases on cessation or reduction in dose of the study drug.  (It 
should be noted that in some situations an AE will not disappear or decrease in intensity 
upon discontinuation of study drug despite other clear indications of relatedness) 
• The event reappears or worsens when the study drug is re-administered 
Unrelated to study drug (unlikely or unrelated) 
Based on medical judgment there is no reasonable possibility that the study drug caused the 
event; one or more of the following criteria apply: 
• The event does not follow a reasonable temporal sequence from administration of study 
drug 
• The event could be reasonably attributed to the known characteristics of the patient's 
clinical state, concurrent illness, environment or toxic factors, or other modes of therapy 
administered to the patient 
• The event does not follow a known pattern of response to study drug 
• The event does not disappear or decrease on cessation or reduction in dose of the study 
drug, and it does not reappear or worsen when the study drug is re-administered 
10.1.2 
Recording of Adverse Events 
It is the investigator’s responsibility to assess whether each untoward event is a clinically 
significant worsening from baseline, thereby considered an AE.  For all AEs, the severity, 
seriousness, and causality to study drug for each AE as outlined in Section 10.1.1 will be 
assessed and recorded in the eCRF. 
All serious and non-serious AEs are collected once the caregiver/patient has signed the ICF 
and until the post-treatment follow-up (Visit 10) or 28 days after the last dose of study drug. 
Any AEs or SAEs that are unresolved at the patient’s last AE assessment in the study are 
followed up by the investigator until resolution or stabilization up to the database lock and 
recorded in the eCRF.  Albireo retains the right to request additional information for any 
patient with ongoing AE(s) or SAE(s) at the end of the study. 


 
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TEAEs are defined as any AE that occurs after randomization (Day 0).  All AEs that occur in 
the Screening Period, i.e., after enrollment and prior to randomization, will be collected on 
the eCRF as non-treatment-emergent AEs. 
If there is a clinically significant deterioration of a laboratory value/vital sign or other routine 
study assessment that is associated with a diagnosis, the clinical diagnosis will be reported as 
an AE and the associated signs and symptoms will be considered additional information 
unless the sign or symptom is more severe than expected given the diagnosis.  For example, if 
an investigator diagnoses a study patient with hepatitis during the study period, hepatitis 
would be considered the AE and the concomitant signs and symptoms of abdominal pain, 
vomiting, and elevated ALT and AST would not be reported separately unless, in the opinion 
of the investigator, one of these signs or symptoms is more severe than expected and 
therefore a separate AE assessment is indicated. 
10.1.3 
Recording and Reporting of Serious Adverse Events 
Every SAE (regardless of severity and causality) that occurs once the caregiver/patient has 
signed the ICF and until the post-treatment follow-up (Visit 10), and 28 days after the final 
dose of study drug, should immediately and not later than within 24 hours of knowledge of 
the event, be reported by the investigator or delegate in the SAE report form.    
Report of a SAE must include at least the following information: 
• Patient identification information (study number, site number, initials, and date of birth 
[as per local country requirements for data protection]) 
• The last study drug administration date 
• The diagnosis of the event with the description (or a brief description of 
signs/symptoms/clinical course if the diagnosis is not available) and the date of onset 
• The action used to treat the event (i.e., treatment medications, temporary discontinuation) 
• The reason(s) for considering the event serious 
• The relationship of the event to the study drug or to the study procedure (e.g., the 
investigator's assessment of causality) 
• A brief narrative of the SAE 
Follow-up reports including all new information obtained of the subsequent course of the 
SAE must be prepared and the information collected in the SAE submitted to 
 
 (Pharmacovigilance and Safety Services Department) by e-mail within 24 hours of 
knowledge of the event. 
The pharmacovigilance manager (PVM) may contact the investigator to obtain further 
information on a reported SAE.  The investigator/investigative staff must respond to any 
request for follow-up information or answers to questions regarding the SAE within the same 
timelines as for initial reports. 




 
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Blood for AFP will be drawn at Visits 3 and 9 and for fat-soluble vitamins at Visits 2, 3, 
and 9. 
A serum pregnancy test will be performed at Visit 1 and Visit 9 if the patient is entering the 
A4250-008 study. A urine pregnancy test will be collected at all other visits for girls who 
have reached menarche.  If a urine pregnancy test is positive, a serum pregnancy test should 
be performed to confirm the pregnancy.  Study drug administration will be held pending the 
confirmatory results.  If the serum pregnancy test is negative, the patient can resume dosing.  
If the serum pregnancy test is positive, the patient should be discontinued (see 
Section 10.2.9). 
All samples will be processed and transported to a laboratory per instructions in the 
laboratory manual. 
The observed values will be recorded and assessed as “normal” or “abnormal not clinically 
significant” or “abnormal clinically significant”. 
Additional safety blood samples may be needed due to follow-up of an abnormal value or 
analysis failure.  The blood samples collected for safety laboratory analysis will be destroyed 
after the analyses have been completed.  
Table 3 
Routine Laboratory Parameters 
Clinical Chemistry 
Hematology 
Urinalysis 
• 
Albumin 
• 
ALT 
• 
Alkaline phosphatase  
• 
AST 
• 
Bilirubin – total and direct 
• 
Calcium 
• 
Chloride 
• 
Creatinine 
• 
Creatine kinase 
• 
Gamma-glutamyl transferase 
• 
Potassium 
• 
Sodium 
• 
Hematocrit 
• 
Hemoglobin 
• 
Platelet count 
• 
Red blood cell count 
• 
White blood cell count 
• 
Blood 
• 
Glucose 
• 
Ketones 
• 
Leukocytes 
• 
Nitrites 
• 
pH 
• 
Protein 
 
10.2.2 
Individual Patient Safety Monitoring 
10.2.2.1 
Liver Monitoring 
Strategies to monitor markers of liver disease throughout the study are outlined below where 
the ULN will be based on central laboratory reference values for age and gender. 
If isolated transaminase elevations are observed, defined as: 
1. Normal bilirubin AND absence of clinical hepatitis symptoms AND 


 
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• ALT or AST ≥5 × ULN (if normal at baseline) or an absolute threshold of 800 U/L, 
whichever comes first  
• OR ALT or AST ≥3 × baseline (if abnormal at baseline) or an absolute threshold of 
800 U/L, whichever comes first 
Then: 
a) Repeat liver profile (AST, ALT, bilirubin, and prothrombin time [PT] or INR) within 
2 to 3 days 
b) Evaluate creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) 
c) As needed (for example, persistent ALT/total bilirubin elevation or worsening of liver 
function), consider evaluation for alternative etiologies 
Monitor the patient using close observation found in Section 10.2.2.2 
If any ONE of the following criteria are met: 
1. Transaminases (ALT or AST ≥3 × baseline or 800 U/L, whichever comes first) AND 
bilirubin (total bilirubin > 2 × ULN) elevations 
2. Transaminase elevations alone (ALT or AST >10 × ULN or 5 × baseline or absolute 
threshold of 800 U/L, whichever comes first) in presence of normal LDH and CPK 
3. Cholestatic marker elevations (alkaline phosphatase [ALP] or gamma-glutamyl 
transferase >2 × baseline) without alternative explanation 
4. Total bilirubin increased, unrelated to hemolysis (elevated reticulocyte count) or 
established genetic diseases, such as Gilbert’s syndrome 
a. Doubling if total bilirubin was <3 mg/dL at baseline  
b. OR Increase by >3 mg/dL if total bilirubin was ≥3 mg/dL at baseline 
5. INR increase refractory to vitamin K administration 
a. Increase by >1.5 if INR was normal at baseline  
b. OR Increase by >0.4 if INR was abnormal at baseline 
6. Any increase in total bilirubin and transaminases if accompanied by EITHER a symptom 
of clinical hepatitis (e.g., vomiting, nausea, right upper quadrant pain) OR immunological 
reaction (rash or >5% eosinophilia) 
Then: 
a) Interrupt study medication 
b) Initiate drug-induced liver injury work-up for alternative etiologies 
c) Repeat liver profile (AST, ALT, total bilirubin, direct bilirubin, ALP) and PT or INR 
within 48 to 72 hours 


 
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d) Monitor the patient using close observation found in Section 10.2.2.2 
e) If a patient lives in a remote area, they may be tested locally and the results 
communicated to the investigator site promptly 
10.2.2.2 
Close Observation 
• Repeat liver enzyme and serum bilirubin tests two or three times weekly.  Frequency 
of re-testing can decrease to once a week or less if abnormalities stabilize or the trial 
drug has been discontinued and the subject is asymptomatic  
• Obtain a more detailed history of symptoms and prior or concurrent diseases 
• Obtain a history of concomitant drug use (including non-prescription medications and 
herbal and dietary supplement preparations), alcohol use, recreational drug use, and 
special diets  
• Rule out alternate etiology including acute viral hepatitis types A, B, C, D, and E; 
autoimmune or alcoholic hepatitis; non-alcoholic steatohepatitis; hypoxic/ischemic 
hepatopathy; and biliary tract disease 
• Obtain a history of exposure to environmental chemical agents.  
• Obtain additional tests to evaluate liver function, as appropriate (e.g., INR, direct 
bilirubin) 
• Consider gastroenterology or hepatology consultations. 
10.2.2.3 
De-challenge/Re-challenge for Liver and Clinical Hepatitis Monitoring 
1. Re-challenge is not recommended: 
a. If a patient has had possible/probable drug-induced liver injury 
b. If a decompensation event has occurred (i.e., variceal hemorrhage, ascites, hepatic 
encephalopathy, etc.). 
2. If due to underlying cholestatic liver disease variability or another alternative etiology is 
identified AND liver tests returned to baseline, re-challenge may be considered after 
consultation with the sponsor Medical Monitor. 
3. If the ALT/total bilirubin elevations are observed after re-challenge, then repeat 
re-challenge is discouraged. 
If patient is permanently discontinued, monitoring should be continued as outlined in 
Section 10.2.2.5.  
10.2.2.4 
Diarrhea 
Study drug should be discontinued if a patient develops diarrhea with at least 1 of the 
following concomitant signs or symptoms: grossly bloody stools, vomiting, dehydration 


 
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requiring treatment with oral or intravenous rehydration and/or electrolyte imbalances, fever 
(≥38 C) and/or the diarrhea persists for 7 or more days. 
If there is gross blood in stool, INR and platelets should be measured.  
Study drug will be reintroduced (re-challenge) when the symptoms have resolved.  If the 
diarrhea re-occurs within 1 week after the re-challenge with no alternate etiology, the patient 
will be permanently discontinued and monitored as outlined in Section 10.2.2.5. 
10.2.2.5 
Monitoring after Permanent Discontinuation of Study Drug Due to Safety 
Once study drug is permanently discontinued for liver, hepatitis, diarrhea, or other severe AE 
related to study drug, the patient will be monitored weekly until the laboratory and clinical 
parameters have normalized/stabilized. 
10.2.3 
Demographics/Medical and Surgical History 
Demographic information per country regulations (age, full date of birth, gender, race, and 
ethnicity), along with medical and surgical history, will be obtained and recorded in the eCRF 
at Visit 1. 
Medical and surgical history will be entered in the eCRF at Visit 1.  This includes date of 
diagnosis of PFIC, prior investigational medications for PFIC, historical (3 months prior to 
Screening) LFT values (AST, ALT, and total bilirubin), ongoing medication, any surgery 
performed, any other diagnosis, and historical biopsy data.   
If a liver biopsy has been performed within 1 year prior to Screening, or during the study, the 
results will be recorded in the eCRF. 
10.2.4 
Clinical Genetic Testing 
If confirmatory clinical genetic testing for PFIC Type 1 or 2 has been performed prior to 
Screening Visit 1, the confirmatory clinical genetic testing laboratory report will be verified 
by a central reader to determine eligibility. 
Regardless of previous genetic testing, a blood sample will be drawn at Visit 1 for all patients 
for DNA extraction.  Genetic testing will only be performed on a patient’s extracted DNA if 
the previous clinical genetic result is equivocal, unavailable, or unobtainable.  In such cases, 
screening for pathologic biallelic variations in the ATP8B1 or ABCB11 genes will be 
performed if patient meets remaining inclusion and exclusion criteria and only after sponsor 
case review.  No other diagnostic genetic testing will be offered. 
10.2.5 
Physical Examination 
A physician or suitably trained qualified assistant will perform a complete physical 
examination, including voluntary photography, at Visits 1, 3, and 9.  A skin examination will 
be conducted at Visits 1, 3, 4, 6, 9, and 10 (see Table 1).  


 
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A complete physical examination will include assessment of general appearance, eyes, ears, 
nose, throat, head/neck/thyroid, lymph nodes, cardiovascular, lungs/chest, abdomen, 
genitourinary, extremities, skin, musculoskeletal, neurologic, and other.  
Skin will be examined thoroughly and excoriations/scratch marks recorded.  
10.2.6 
Vital Signs 
Evaluation of vital signs will be performed at all visits.  This includes blood pressure (systolic 
and diastolic), pulse, respiratory rate, temperature, height (or length depending on age), and 
body weight (using a certified weight scale) at clinic visits.  BMI will be calculated. 
10.2.7 
Abdominal Ultrasound 
Ultrasound of the liver and spleen will be performed at randomization (Visit 3) and at EOT 
(Visit 9).  Liver size, echogenicity, and presence of masses/nodules as well as spleen size will 
be recorded. 
10.2.8 
Overdose 
A4250 is minimally absorbed and has a very low systemic availability.  Based on toxicology 
data, for study purposes any dose exceeding a total of >3 mg/kg body weight of A4250 taken 
as a single dose or as a cumulative dose within 24 hours is defined as an overdose.   
The no-adverse-effect dose level in the most sensitive species (defined as 20 mg/kg/day) in 
the rat 1 month toxicity study gives a human equivalent dose of approximately 194 mg/day in 
a 60 kg person.  This dose is approximately 10-fold higher than the maximum possible dose 
predicted in human studies (20 mg).  
The investigator/investigative staff should immediately and not later than within 24 hours of 
knowledge of the overdose, be reported by the investigator or delegate on the SAE report 
form.  In the event of an A4250 overdose, the patient should be monitored closely. 
10.2.9 
Pregnancy 
If a pregnancy is discovered in a female patient enrolled in the study before the end of dosing, 
the patient will be immediately discontinued from the study and will attend the same visits as 
a prematurely withdrawn patient.  If the pregnancy is discovered in a female patient enrolled 
in the study after the end of dosing, the patient will continue in the study per protocol.  If a 
pregnancy occurs in a male patient’s partner at any time during the study, the pregnancy 
should also be reported and followed. 
Pregnancy is not considered to be an AE.  However, if the patient has been dosed with the 
study drug, the pregnancy must be reported on the Paper Pregnancy Notification Form 
immediately within 24 hours after recognition to Albireo/Safety Manager (please refer to 
Section 10.1.3).  Date of exposure and as far as possible, details of the period of gestation at 
the time of exposure must be given. 


 
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The pregnancy should be followed to determine outcome, including spontaneous termination, 
details of birth, and presence of any birth defects, congenital anomalies or newborn or 
maternal complication.  The infant who was exposed in-utero will be followed for up to 
2 years after delivery.  Individual cases with an abnormal outcome in association with the 
study drug should be reported on an expedited basis, i.e., reported rapidly to a competent 
authority. 


 
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11 
STATISTICAL EVALUATION 
All personnel involved with the analysis of the study will remain blinded until database lock 
for the double-blind Treatment Period and until major protocol violations have been 
identified. 
All statistical analyses will be performed using SAS version 9.3 or higher. 
11.1 
Sample Size and Power 
For the EU and RoW, the primary analysis will relate to the bile acid responder endpoint (at 
the 1-sided 1.25% level).  For the US, the primary analysis will relate to the pruritus endpoint 
(at the 1-sided 1.25% level).  The aim is to enroll 60 to 70 patients in order to get at least 
60 evaluable patients. 
Assuming 60% responders (patients experiencing at least 70% reduction in fasting s-BA 
concentration or reaching ≤70 µmol/L) in the A4250 dose group and 10% responders in the 
placebo group, approximately 20 patients per group are required to achieve a power of 84% 
with a 1.25% 1-sided Type-1 error. 
For pruritus, with approximately 20 patients per treatment group, the power is 83% given an 
assumed treatment effect difference of -1.0 in change from baseline (-1.6 for A4250 
and -0.6 for placebo) on the Albireo ObsRO scratching score with a SD of 0.95 and a Type I 
error of 1.25% (1-sided). 
11.2 
Statistical Methods 
11.2.1 
Statistical Analysis Sets 
Safety Analysis Set 
The safety analysis set will consist of all patients who received at least 1 dose of study drug.  
Patients will be analyzed according to the treatment they actually received.  The safety 
analysis set will be the primary analysis set for safety analyses.  
Full Analysis Set 
The full analysis set (FAS) will consist of all randomized patients who received at least 
1 dose of assigned treatment.  Patients will be analyzed in the treatment group they were 
randomized to even if they received incorrect study drug.  The FAS will be the primary 
analysis set for efficacy analyses. 
Per Protocol Analysis Set 
The per protocol (PP) analysis set is a subset of the FAS, and will consist of all randomized 
patients for whom no major protocol violation (no violation which may affect the study 
efficacy outcome) is documented.  The PP analysis set will provide supportive data for the 
efficacy analyses. 
Allocation of patients to the PP analysis set will be performed before unblinding of the study. 


 
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11.2.2 
Methods of Statistical Analyses 
11.2.2.1 
General Principles 
The primary objective of this study is to show superiority in efficacy of 
A4250 (120 µg/kg/day and 40 µg/kg/day) compared to placebo in patients with PFIC Types 1 
and 2. 
In the EU and RoW, for the primary efficacy variable fasting bile acid responder endpoint (at 
least a 70% reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the end 
of treatment), the null hypothesis will be tested against the alternative hypothesis (α = 0.0125, 
1-sided). 
The test will be performed at a Type I error rate of ≤0.0125 1-sided, as described above, 
based on the least squares means of the treatment difference in mean probability scale. 
To strictly control overall Type I error rate across the dose groups and also across the primary 
and the main secondary endpoint to a 1-sided 2.5% level, a Bonferroni-based parallel 
gate-keeping procedure will be used.  In this procedure the total significance level of 2.5% 
(1-sided) will be split equally between the dose groups.  Only a dose group that is significant 
for the primary endpoint will be tested, at the nominal significance level of 1.25% (1-sided), 
for the main secondary endpoint (here the pruritus endpoint).  If the previous step was not 
successful, the analysis of the following step will be considered descriptive.  If the procedure 
reaches the secondary endpoint for both dose groups, these will be evaluated using the 
Bonferroni Holm procedure.  The multiple comparison procedure is presented in Figure 2. 
Figure 2 
Bonferroni Holm Multiple Comparison Procedure 
 
In the US, for the primary efficacy variable pruritus score endpoint (change from baseline to 
Week 24 in observer-reported scratching), the null hypothesis will be tested against the 
alternative hypothesis (α=0.0125, 1-sided). 


 
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The test will be performed at a Type I error rate of ≤0.0125 1-sided, as described above, 
based on the least squares means of the treatment difference in mean change. 
To strictly control overall Type I error rate across the dose groups and also across the primary 
and the main secondary endpoint to a one-sided 2.5% level, a Bonferroni-based parallel 
gate-keeping procedure will be used.  In this procedure the total significance level of 2.5% 
(1-sided) will be split equally between the dose groups.  Only a dose group that is significant 
for the primary endpoint will be tested, at the nominal significance level of 1.25% (1-sided), 
for the main secondary endpoint (here the bile acid responder endpoint).  If the previous step 
was not successful, the analysis of the following step will be considered descriptive.  If the 
procedure reaches the secondary endpoint for both dose groups, these will be evaluated using 
the Bonferroni Holm procedure. 
Other secondary and exploratory outcome variables shall provide supportive efficacy and 
safety information regarding the differences between the treatment groups.  No adjustments 
will be performed for multiple dose comparisons when testing the other secondary and 
exploratory efficacy variables. 
A more detailed statistical analysis plan (SAP) will be prepared and finalized before study 
data are locked and unblinded.  
If not otherwise specified, the baseline value of a parameter is defined as the last assessment 
of that parameter on or prior to the first intake of study treatment date. 
11.2.2.2 
Missing Data 
For efficacy variables, with repeated measures, a mixed model will be employed with no 
additional imputation for missing values, i.e., based on “observed cases”, except where 
otherwise specified. 
Follow-up and continued collection of efficacy data for patients who discontinue treatment 
will be made.  These data will be included in the sensitivity analysis for primary endpoints 
estimating a treatment effect taking loss of effect after discontinuation into account.  Because 
complete follow-up of all patients discontinuing treatment may not be possible, a method to 
replace missing data for patients who discontinue the study will be prespecified (in the SAP) 
taking into account loss of effect after discontinuation (for example multiple imputation). 
No other replacement of missing values will be planned. 
11.2.2.3 
Demographic and Baseline Characteristics 
Descriptive summaries of demographics and other baseline characteristics (including medical 
and surgical history) will be presented by treatment group and overall using the FAS.  
Prior medication will be summarized by treatment group and overall using the safety analysis 
set. 


 
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11.2.2.4 
Subject Disposition 
The following will be summarized (by treatment group and overall where applicable): 
• Patients enrolled (who signed the informed consent) 
• Patients randomized 
• Patients treated 
• Patients completing the study/withdrawing early (including withdrawal reason) 
Additionally, patients enrolled, included in the safety, in the FAS, and in the PP analysis set 
will be summarized by region. 
11.2.2.5 
Evaluation of Primary Efficacy Variables 
The responder endpoint based on change in fasting bile acids will be analyzed in terms of the 
proportion of patients achieving the event of interest.  Patients in need of rescue therapy, i.e., 
biliary diversion will be classified as non-responders after the biliary diversion.  
Generalized linear mixed modelling (GLMM) will be used with a logistic link, to allow for 
repeated assessments over time.  The model will include baseline value, treatment group, 
PFIC class, age class, region, visit, and visit*treatment group interaction.  The treatment by 
visit interaction will remain in the model regardless of significance.  Region will be included 
in the model as a random effect.  The contrasts of primary interest will be the treatment 
difference at Week 24; also, the difference at Week 4, Week 8, Week 12, and Week 18 will 
be estimated and analyzed.  An unstructured covariance matrix will be used to model the 
within-subject error.  Another covariance structure may be chosen if the fit of this structure 
fails to converge.  Model diagnostics checks will be made to ensure appropriate fit. 
The primary analysis of the change from baseline in pruritus score (indexed by 
observer-reported scratching) will be analyzed using a mixed model repeated measures 
(MMRM) analysis.  The contrast of primary interest will be treatment difference at Week 24 
but also the differences at Week 4, Week 8, Week 12, and Week 18 will be estimated and 
analyzed.  Baseline pruritus score will be included as covariate.  The model will include 
baseline value, treatment group, PFIC class, age class, region, visit, and visit*treatment group 
interaction.  The treatment by visit interaction will remain in the model regardless of 
significance.  Region will be included in the model as a random effect and will be defined on 
country level.  An unstructured covariance matrix will be used to model the within-subject 
error.  Another covariance structure may be chosen if the fit of this structure fails to 
converge. 
Non-parametric approach may be considered if more appropriate. 
The primary analyses will be based on the FAS. 
To assess the robustness of the primary efficacy analysis, the following analyses will be 
performed: 


 
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• Primary efficacy analysis using the PP set  
• Analyses to investigate the influence of missing values, for example, by using pattern 
mixture models and tipping point analysis 
Subgroup analyses will also be performed for each of the 3 age groups (6 months to 5 years, 
6 to 12 years, and 13 to ≤18 years) and for patients who were not included in the previous 
Study A4250-003. 
Several sensitivity analyses will be performed, such as the following: 
• Cochran Mantel Haenzsel test stratified on PFIC class to compare the proportion in 
fasting bile acid responder endpoint at the end of treatment in the two A4250 dose groups 
to placebo group.  Dropouts will be treated as non-responders 
• Based on the primary analysis including the interaction age class*treatment 
• Same as primary analysis in which the random factor region is considered as fixed factor.  
The region*treatment interaction will also be explored 
• Same as primary analysis including the interaction PFIC*treatment  
Other sensitivity analyses may be performed and will be detailed in the SAP. 
11.2.2.6 
Evaluation of Secondary and Exploratory Efficacy Variables 
The change in the secondary and explorative endpoints such as s-BA, total bilirubin, ALT, 
growth and patient-reported itch severity will also be analyzed using the MMRM model as 
specified for the primary analysis.  Nonparametric methods may be applied as an alternative 
approach for some endpoints, if appropriate. Change in growth (weight for age and BMI for 
age) will also be displayed using graphical presentations. 
Proportions of responders (e.g., meaningful reduction in itch severity and scratching at 
Week 24) will be analyzed using the GLMM model as specified for the primary analysis. 
Comparisons of the change from baseline at Week 24 in PedsQL between the treatment 
groups will be conducted using analysis of covariance.  The model will include terms for 
baseline, PFIC class, age class, region, and treatment.  
Undergoing biliary diversion surgery and/or listed for liver transplant, all-cause mortality, 
and liver decompensation events will mainly be analyzed by using descriptive statistics.  
Kaplan-Meier curves will be used when appropriate for time to event data. 
Other exploratory variables (Global Symptom Relief, biochemical markers and measures of 
bile acid synthesis, Albireo PRO and ObsRO sleep parameters, PELD/MELD score, APRI 
score, and Fib-4 score) will be analyzed descriptively for categorical and continuous data, as 
applicable.  For continuous data, the change from baseline will be analyzed in addition to the 
actual visit values.  For categorical data, shift tables or frequency and percentages of patients 
will be presented as appropriate. 


 
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In addition, subgroup analyses will be performed for each of the 3 age classes (6 months to 
5 years, 6 to 12 years, and 13 to ≤18 years). 
11.2.2.7 
Evaluation of Safety Variables 
Safety data will be analyzed using descriptive statistics and summaries by treatment group of 
SAEs, AEs, vital signs, clinical laboratory tests (hematology, clinical chemistry and 
urinalysis), and concomitant medication.  Analyses will be performed using the safety 
analysis set.  
Summaries of AEs (coded according to the Medical Dictionary for Regulatory Activities 
[MedDRA] system organ class [SOC] and MedDRA preferred term) will include the 
following:  
• Overview of the incidence of TEAEs (TEAEs, drug-related TEAEs, TEAEs leading to 
study discontinuation, and treatment-emergent SAEs) 
• TEAEs by SOC and preferred term 
• Intensity of TEAEs by SOC and preferred term 
• Drug-related TEAEs by SOC and preferred term 
• TEAEs leading to study discontinuation by SOC and preferred term 
• Treatment-emergent SAEs by SOC and preferred term 
Concomitant medication use during the Treatment Period will be summarized by Anatomical 
Therapeutic Chemical (ATC) class and World Health Organization (WHO) preferred name.  
Summaries of vital signs will be presented.  For each visit, the actual results and the change 
from baseline, and the number and percentage of patients with potentially clinically 
significant (PCS) values observed post-baseline will be presented. 
Summaries of clinical safety laboratory data will be presented.  For each visit, the actual 
result and the change from baseline, and the number and percentage of patients with PCS 
values observed post-baseline will be presented. 
Data listings will be provided for each patient for all safety parameters. 
11.2.2.8 
Compliance and Exposure 
Exposure will be analyzed by calculating the number of days with exposure to study drug.  
Results will be presented by treatment group using the safety analysis set.  
The percentage compliance will be described by treatment group and the number of patients 
with a compliance <80%, between 80% and 120%, and >120% will be presented based on the 
safety analysis set. 


 
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11.2.3 
Blinded Analyses to Confirm Responder Definitions 
A blinded analysis of Albireo ObsRO and PRO eDiary data will be performed after 50% or 
more of the planned sample has completed the Week 24 Follow-Up Period.  The blinded 
analysis will be used to estimate a threshold of clinically meaningful change (i.e., responder 
definition) in Albireo ObsRO and PRO pruritus scores.  The analysis will be performed by a 
group independent from both the study team and Albireo.  Blinded data will be used for this 
analysis; data will be collapsed across treatment groups. 
The analyses will include distribution- and anchor-based analyses to examine a responder 
definition for the Albireo PRO/ObsRO pruritus scores.  Distribution-based analyses will 
include calculation of the SD and standard error of measurement for the baseline Albireo 
PRO and ObsRO scores.  Anchor-based analyses will involve examining the degree of 
change on the Albireo PRO and ObsRO scores from baseline to Week 24 for patients who 
experience change in pruritus according to PGIC and symptom items.  Details of this analysis 
will be included in a stand-alone SAP for assessing the measurement characteristics 
(reliability, validity, sensitivity to change, responder definition) of the Albireo PRO and 
ObsRO items. 
11.2.4 
Data Safety Monitoring Board 
A DSMB consisting of sponsor-independent clinical and statistical experts will be 
established.  The DSMB will periodically meet for the review of accumulating study data, 
including safety (AE and laboratory data) and liver values.  
The DSMB will have access to unblinded data.  The DSMB will make recommendations for 
the remaining part of the study (further details will be provided in the DSMB charter).  The 
DSMB may recommend continuing with the study as planned or stopping the study early for 
safety reasons.  The DSMB will submit its recommendations in writing to Albireo 
Medical/
 who are responsible for responding to the recommendations of the DSMB and 
taking appropriate action.  The investigators will only be informed by Albireo Medical/
 
if the study requires a protocol amendment or is stopped.  The DSMB may choose to make 
additional evaluations at any time if they feel this is warranted from a safety point of view.  
The DSMB will act according to its own written standard operating procedure described in a 
charter and will prepare written minutes of its meetings.  The charter of the DSMB will be 
stored in the trial master file.  The DSMB will maintain records of its meetings and these will 
become part of the study file when the study is complete.  In order not to disseminate 
unblinded data and to ensure that all staff involved in the conduct and final analysis of the 
study remain blind to the data, only the members of the DSMB and the unblinded DSMB 
statistician will have access to these data. 
 


 
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12 
DIRECT ACCESS TO SOURCE DATA/NOTES 
The investigator/institution shall provide direct access to source data/documents for 
study-related monitoring, audits, IEC/Institutional Review Board (IRB) review and 
regulatory inspection. 


 
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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
13.1 
Conduct of the Study 
Albireo/
 shall implement and maintain quality control and quality assurance procedures 
with written standard operating procedures to ensure that the study is conducted and data are 
generated, documented, and reported in compliance with the protocol, ICH GCP and 
applicable regulatory requirements. 
This study shall be conducted in accordance with the provisions of the Declaration of 
Helsinki (October 1996) and all revisions thereof, and in accordance with United States Food 
and Drug Administration (FDA) regulations (Code of Federal Regulations, Sections 312.50 
and 312.56) and with ICH GCP (CPMP 135/95). 
The investigator may not deviate from the protocol without a formal protocol amendment 
having been established and approved by an appropriate competent authority and IEC/IRB, 
except when necessary to eliminate immediate hazards to the patient or when the change(s) 
involve(s) only logistical or administrative aspects of the study.  Any deviations may result in 
the patient having to be withdrawn from the study and render that patient non-evaluable. 
13.2 
Study Monitoring 
Before study initiation, at a site initiation visit or at an investigator’s meeting, an Albireo 
representative or designee will review the protocol and eCRF with the investigators and the 
investigative staff.  During the study, the clinical monitor (clinical research associate [CRA]) 
will visit the site regularly to check the completeness of patient records, the accuracy of 
entries on the eCRFs, the adherence to the protocol and to GCP, the progress of enrollment, 
and to ensure that study drug is being stored, dispensed, and accounted for according to 
specifications. 
The investigator must ensure that eCRFs are completed within a timely period of patient 
visits, as per individual site agreements, and must allow the CRA and Albireo representative 
or designee periodic access to patient records and all study-related materials, including 
relevant hospital or clinical records, to confirm their consistency with the eCRF entries.  No 
information in these records about the identity of the patients will leave the study center.  
Albireo monitoring standards require full verification for the presence of the signed ICF, 
adherence to the inclusion/exclusion criteria, documentation of SAEs, and recording of 
primary efficacy and safety variables.  The CRA will review source data compared with the 
eCRFs and will verify source data according to the study-specific monitoring plan.  The 
design of the study, the frequency of patient visits, and the site enrollment rate will determine 
the frequency of monitoring visits.  Upon study completion, the CRA will visit the site to 
conduct a study termination visit, which will include collection of any outstanding 
documentation. 
It is recommended that the investigator/investigative staff log into the eCRF system every 
working day or at minimum twice weekly in order to provide a prompt response to queries.  


 
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The investigator/investigative staff should respond to queries and make any relevant changes 
to the study data within 3 working days. 


 
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14 
ETHICS 
14.1 
Independent Ethics Committee/Institutional Review Board 
Prior to the start of the study, the investigator is responsible for ensuring that the protocol and 
consent form have been reviewed and approved by a relevant IEC/IRB.  The IEC/IRB shall 
be appropriately constituted and perform its functions in accordance with FDA ICH GCP and 
local requirements as applicable. 
The IEC/IRB shall approve all protocol amendments (except for logistical or administrative 
changes), written informed consent documents and document updates, patient recruitment 
procedures (e.g., advertisements), written information to be provided to the patients and 
caregivers, IB, available safety information, information about payment and compensation 
available to patients, the investigator's curriculum vitae and/or other evidence of 
qualifications and any other documents requested by the IEC/IRB and regulatory authority 
(competent authority) as applicable. 
14.2 
Written Informed Consents and Assents 
The investigator (physician) or investigative staff, in accordance with local regulation, will 
explain to each patient (or legally authorized representative) the nature of the study, its 
purpose, the procedures involved, the expected duration, alternative treatment, the potential 
risks and benefits involved, and any discomfort that may occur.  Each caregiver/patient will 
be informed that participation in the study is voluntary and that he/she or their child may 
withdraw from the study at any time and that withdrawal of consent will not affect his/her 
subsequent medical treatment or relationship with the treating physician. 
Caregivers/patients will be informed that they/their children may experience side-effects or 
be at risk for symptoms, illnesses, or complications that cannot be foreseen by Albireo.  As 
with other medications, people treated with A4250 may be at risk of developing allergic 
reactions or anaphylaxis.  Caregivers/patients will be advised that study procedures include 
regular blood sampling for measurement of safety parameters and biological markers and that 
some minor risks are associated with these procedures. 
This informed consent should be given by means of a signed ICF, written in non-technical 
language in accordance with ICH GCP, the Declaration of Helsinki, and regulatory 
authorities.  The caregiver(s)/patient should read and consider the statements before signing 
and dating them and should be given a copy of each signed document.  If written consent is 
not possible, oral consent can be obtained if witnessed and followed by a signed statement 
from one or more persons not involved in the study, indicating why the patient was unable to 
sign the form.  No patient can enter the study before his/her or caregiver(s), as required by 
country regulation, informed consent has been obtained. 
The ICF is part of the protocol and must be submitted by the investigator/investigative staff 
with the protocol for IEC approval.  Albireo will supply an ICF which complies with 
regulatory requirements and country laws and is considered appropriate for the study.  Any 


 
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changes to the ICF suggested by the investigator must be agreed to by Albireo before 
submission to the IEC and a copy of the approved version must be provided to the clinical 
monitor after IEC approval. 


 
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15 
DATA HANDLING AND RECORD KEEPING 
15.1 
Case Report Forms/Source Data Handling 
The investigator shall be provided with standardized eCRFs and shall ensure that all data 
from patient visits are promptly entered into the eCRFs in accordance with the specific 
instructions given.  The investigator must sign each eCRF to verify the integrity of the data 
recorded. 
A list of the normal ranges for all laboratory tests to be undertaken forms part of the 
documentation to be collated prior to study start.  As a central laboratory has been selected to 
conduct any or all tests, it is essential that all samples be analyzed at that laboratory. 
The investigator must maintain source documents such as laboratory reports, consultation 
reports, and complete medical history and physical examination reports. 
As no ethnicity data can be collected in the source document for study sites in France, the 
eCRF will be the Source Document for “origines ethniques”,  
15.2 
Retention of Essential Documents 
Essential documents, as defined by ICH GCP, include: the signed protocol and any 
amendment(s); copies of the completed eCRFs (for site archiving, compact discs of eCRF 
data for specific patients will be provided); signed ICFs; hospital records and other source 
documents; IEC approvals and all related correspondence including approved documents; 
drug accountability records; study correspondence; and a list of patients’ names and 
addresses. 
The investigator/investigative staff must retain copies of these essential documents for the 
period specified by ICH GCP and by applicable regulatory requirements.  The 
investigator/investigative staff will inform Albireo of the location where the essential 
documents are stored and must contact Albireo for approval before disposing of any essential 
documents.  The investigator/investigative staff should take measures to prevent accidental or 
premature destruction of these documents. 


 
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16 
FINANCING AND INSURANCE 
Financing and insurance are addressed in a separate agreement. 
 


 
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17 
PUBLICATION POLICY 
Albireo will retain the ownership of all data.  When the study is complete, Albireo shall 
arrange the analysis, tabulation of data and preparation of a clinical study report. Albireo may 
also use the data for publication, presentation at scientific meetings and submission to 
regulatory authorities.  All proposed publications based on this study must be subject to the 
sponsor's approval requirements. 
 


 
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18 
REFERENCE LIST 
1. A4250 Investigator’s Brochure. 
2. Alissa FT, Jaffe R, Shneider BL. Update on progressive familial intrahepatic cholestasis. 
J Pediatr Gastroenterol Nutr. 2008;46(3):241-52. 
3. Davit-Spraul A, Gonzales E, Baussan C, et al. Progressive familial intrahepatic 
cholestasis. Orphanet J Rare Disease 2009;4:1-12. 
4. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and 
functions. Front Biosci (Landmark Ed). 2009 Jan 1;14:2584-98. 
5. Hori T, Justin H, Nguyen JH and Uemoto S. Progressive familial intrahepatic cholestasis. 
Hepatobiliary pancreat Dis Int. 2010; 9(6):570-8. 
6. Jacquemin E. Progressive familial intrahepatic cholestasis. Genetic basis and treatment. 
Clin Liver Dis. 2000;4(4):753-63. 
7. Miethke AG, Zhang W, Simmons J, et al. Pharmacological inhibition of ASBT changes 
bile composition and blocks progression of sclerosing cholangitis in mdr2 knockout mice. 
Hepatology. 2016; 63(2):512–23. 
8. Murray CS, Rees JL. Are subjective accounts of itch to be relied on? The lack of relation 
between visual analogue itch scores and actigraphic measures of scratch. Acta Derm 
Venereol. 2011;91(1):18-23. 
9. Pawlikowska L, Strautnieks S, Jankowska I, et al. Differences in presentation and 
progression between severe FIC1 and BSEP deficiencies. J Hepatol. 2010;53(1):170-8. 
10. Suchy FJ, Sokol RJ, Balistreri WF, eds. Liver Disease in Children. 3rd Edition. New 
York, NY: Cambridge University Press; 2007:310–4. 
11. Whitington PF, Whitington GL. Partial external diversion of bile for the treatment of 
intractable 
pruritus 
associated 
with 
intrahepatic 
cholestasis. 
Gastroenterology. 
1988;95(1):130-6. 
 
 


 
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19 
APPENDICES 
Appendix 1 
Concomitant Medications Prohibited During the Study 
 


 
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Appendix 2 
Diary Questions including Albireo Patient-Reported Outcomes/Observer-
Reported Outcome Instrument 
 


 
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Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) 
 


 
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Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaire 
 


 
 
 
CLINICAL STUDY PROTOCOL A4250-005 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
 
IND Number:   
130591 
EudraCT Number 
2017-002338-21 
Test Product: 
A4250 
Indication: 
Progressive Familial Intrahepatic Cholestasis (PFIC) 
Sponsor: 
Albireo AB 
Development Phase: 
Phase 3 
Sponsor Signatory: 
 
Sponsor Medical Monitor: 
 
Principal Investigator: 
 
Date of the Protocol: 
06 December 2017 
Version of the Protocol: 
Final Amendment 01 
 
 
 
 
Confidential 
The information in this document contains trade secrets and commercial information that are 
privileged or confidential and may not be used, divulged, published, or otherwise disclosed 
without the written consent of Albireo AB.  These restrictions on disclosure apply equally to 
all future information supplied to you or generated by you in connection with the study. 






 
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INVESTIGATOR SIGNATURE PAGE 
 
PROTOCOL TITLE:  A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children with Progressive 
Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
 
PROTOCOL NUMBER:  A4250-005 
I have read this protocol and agree that it contains all necessary details for performing this 
study.  I will conduct the study as outlined herein and will complete the study within the time 
designated, in accordance with all stipulations of the protocol and in accordance with Good 
Clinical Practice (GCP), local regulatory requirements, and the Declaration of Helsinki. 
I will provide copies of the protocol and all pertinent information to all individuals 
responsible to me who assist in the conduct of this study.  I will discuss this material with 
them to ensure that they are fully informed regarding the study drug and the conduct of the 
study. 
I will use only the informed consent approved by the Independent Ethics Committee (IEC) 
and will fulfil all responsibilities for submitting pertinent information to the IEC responsible 
for this study. 
I agree that the sponsor (Albireo AB) shall have access to any source documents from which 
case report form information may have been generated. 
I further agree not to originate or use the name of Albireo AB, A4250 in any publicity, news 
release, or other public announcement, written or oral, whether to the public, press, or 
otherwise, relating to this protocol, to any amendment to the protocol, or to the performance 
of this protocol without the prior written consent of Albireo AB. 
 
 
 
 
 
 
Name of Investigator 
 
 
 
 
 
 
Signature 
 
Date (day/month/year) 
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1 
ADMINISTRATIVE INFORMATION 
A Double-blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
Protocol No.: 
A4250-005 
Date of the initial Protocol: 
20 September 2017 
Date and Number of Amendment(s): 
Amendment 01, 06 December 2017 
Sponsor: 
Albireo AB 
 
Arvid Wallgrens backe 20 
 
413 46 Gӧteborg 
Sweden 
Clinical Research Organization: 
 
 
 
 
 
 
 
 
Sponsor Signatory: 
 
 
  
 
  
 
  
 
 
 
  
Sponsor Medical Monitor: 
 
 
 
 
Principal Investigator: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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2 
STUDY SYNOPSIS 
Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Title of Study: 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate Efficacy and Safety of A4250 
in Children with Progressive Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
Principal Investigator: 
 
Study Centers: 
Up to 50 sites will be initiated for this study in the United States (US), Canada, European Union (EU), Middle 
East, and Australia. 
Publication(s): 
None. 
Planned Study Period: 
Q1 2018 to Q2 2019 
Development Phase: 
Phase 3 
Objectives: 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day A4250 in children with 
progressive familial intrahepatic cholestasis (PFIC) Types 1 and 2, as determined by the following: 
• 
Proportion of patients experiencing at least a 70% reduction in fasting serum bile acid (s-BA) 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L compared to placebo 
after 24 weeks of treatment 
• 
Change from baseline to Week 24 in pruritus compared to placebo  
Secondary Objectives 
• 
To evaluate the effect of A4250 on serum alanine aminotransferase (ALT) concentration 
• 
To evaluate the effect of A4250 on growth 
• 
To evaluate the effect of A4250 on sleep disturbance 
• 
To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or liver 
transplantation) 
• 
To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 
Methodology: 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the efficacy and safety of 
40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in children with PFIC (deficiencies of familial 
intrahepatic cholestasis-1 or bile salt export pump).  
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment Period and a 4-week 
Follow-up Period.  After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of A4250, or a 
matching placebo.  Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008) in which all patients will receive 
active treatment. 
There will be up to 10 clinic visits during the study and one scheduled telephone contact. 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
Number of Patients: 
Approximately 60 patients diagnosed with PFIC-1 or PFIC-2 will be randomized. 
Diagnosis and Main Criteria for Inclusion: 
1. 
A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages of ≥6 months 
and ≤18 years at Visit 1 with a body weight above 5 kg 
2. 
Patient must have genetic confirmation of PFIC-1 or PFIC-2 through identification of biallelic 
pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. 
Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, taken as the 
average of 2 samples 7 to 28 days apart (Visits 1 and 2) prior to randomization  
4. 
Patient must have history of significant pruritus and a caregiver-reported observed scratching in the 
electronic diary (eDiary) average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to randomization 
5. 
Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  Patients who 
turn 18 years of age (or legal age per country) during the study will be required to re-consent in order 
to remain in the study 
6. 
Patients are expected to have a consistent caregiver(s) for the duration of the study 
7. 
Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use an eDiary 
device as required by the study 
Test Product, Dose and Mode of Administration: 
A4250, 40 µg/kg/day or 120 µg/kg/day, orally administered 
Reference Therapy, Dose and Duration of Administration: 
Matching placebo, orally administered 
Duration of Treatment: 
24 weeks 
Variables 
Efficacy:  
Primary Efficacy Endpoints 
The primary efficacy endpoints are: 
• 
EU and rest of world (RoW): Proportion of patients experiencing at least a 70% reduction in fasting 
s-BA concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L compared to 
placebo after 24 weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization and the previous 
visit (Clinic Visit 2).  The end value is the average of the values at Weeks 22 and 24 after the start of 
double-blind treatment. 
• 
US: Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline to 
Week 24 as measured by the worst scratch of the Albireo observer-reported outcome (ObsRO) 
instrument.  Scores for the 14 days baseline and post-treatment intervals will be calculated by 
averaging the worst scratching scores from each day in the interval. 
Secondary Efficacy Endpoints 
Secondary efficacy endpoints include the following: 
• 
EU and RoW: Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
to Week 24 as measured by the worst scratch of the Albireo ObsRO instrument.  Scores for the 14 days 
baseline and post-treatment intervals will be calculated by averaging the worst scratching scores from 
each day in the interval. 
• 
US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA concentration from 
baseline to the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 weeks of 
treatment. 
All Regions: 
All secondary endpoints will be compared to placebo. 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
• 
Change from baseline to Week 24 in fasting s-BA 
• 
Change from baseline to Week 24 in serum ALT concentration 
• 
Change in growth from baseline to Week 24, defined as the linear deficit (weight for age and body 
mass index) compared to the national growth curves (Z-score, standard deviation from P50) 
• 
Proportion of patients achieving meaningful reduction in caregiver-reported observed scratching at 
Week 24 
• 
Change from baseline to Week 24 in sleep parameters measured with the Albireo patient-reported 
outcome (PRO) and ObsRO instruments 
• 
Change from baseline to Week 24 in patient-reported itch severity as measured by the average daily 
score of the Albireo PRO instrument; only patients ≥8 years of age will complete the Albireo PRO 
instrument 
• 
Number of patients undergoing biliary diversion surgery or being listed for liver transplantation 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• 
Proportion of patients achieving meaningful reduction in patient-reported itch severity at Week 24 
• 
Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, caregiver, and 
clinician via the patient global impression of change (PGIC) items 
• 
Change from baseline in fasting s-BA at Weeks 4 and 12 
• 
Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline to Weeks 4, 
12, and 24 as measured by the worst scratch.  Scores for the 7 worst scratching days of the last 14 days 
at baseline and post-treatment intervals will be selected 
• 
Change from baseline in patient-reported itch severity and observer-reported scratching at Weeks 4 and 
12 
• 
Change from baseline to Week 24 in patient-reported and observer-reported night time itching and 
scratching severity scores, respectively 
• 
Change from baseline to Week 24 in pooled pruritus score including observer-reported scratching for 
patients <8 years of age and patient-reported itch severity for patients ≥8 years of age 
• 
Change from baseline to Week 24 in additional patient-reported and observer-reported sleep 
parameters (e.g., tiredness and number of awakenings) 
• 
Change from baseline to Week 24 in Pediatric Quality of Life Inventory (PedsQL) 
• 
Change from baseline in serum ALT concentration at Weeks 4 and 12 
• 
Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• 
Change from baseline in AST at Weeks 4, 12, and 24 
• 
Change from baseline for other biochemical markers and measures of bile acid synthesis (autotaxin, 
plasma 7α-hydroxy-4-cholesten-3-one concentration [p-C4]) at Week 24 
• 
Change in pediatric end-stage liver disease/model for end-stage liver disease (PELD/MELD) score, 
AST to platelet ratio index (APRI) score, and Fib-4 score from baseline to Week 24 
• 
Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® (where available) 
• 
Liver-related mortality and liver decompensation events. 
• 
All-cause mortality 
Safety:   
Safety criteria are as follows: 
• 
Occurrence of treatment-emergent adverse events including severity and relatedness to study drug at all 
visits 
• 
The incidence of treatment-emergent serious adverse events (SAEs), based upon information from 
patient reports, including the description, incidence, and severity of an SAE 
• 
Trends evaluated for the following assessments: physical examinations, concomitant medications, vital 
signs, laboratory test results (including clinical chemistry, hematology, and urinalysis), and imaging 
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Name of Sponsor/Company: 
Albireo 
Name of Product: 
A4250 
Name of Active Ingredient: 
A4250 
studies 
Statistical Methods: 
The primary objective of this study is to show superiority in efficacy of A4250 (120 µg/kg/day and 
40 µg/kg/day) compared to placebo in patients with PFIC Types 1 and 2. 
In the EU and RoW, for the primary efficacy variable fasting bile acid responder endpoint (at least a 70% 
reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the end of treatment), the null 
hypothesis will be tested against the alternative hypothesis (α=0.0125, 1-sided).  Generalized linear mixed 
modelling will be used with a logistic link, to allow for repeated assessments over time.  The model will include 
baseline value, treatment group, PFIC class, age class, region, visit and visit*treatment group interaction.  The 
contrasts of primary interest will be the treatment difference at Week 24 but also the difference at Week 4, 
Week 8, Week 12, and Week 18 will be estimated and analyzed. 
In the US, for the primary efficacy variable pruritus score endpoint (change from baseline to Week 24 in 
observer-reported scratching), the null hypothesis will be tested against the alternative hypothesis (α=0.0125, 
1-sided).  The primary analysis of the change from baseline in pruritus score (indexed by observer-reported 
scratching) will be analyzed using a mixed model repeated measures analysis.  The contrasts of primary interest 
will be the treatment difference at Week 24 but also the difference at the other weeks will be estimated and 
analyzed.  
To strictly control overall Type I error rate across the dose groups and also across the primary and the main 
secondary endpoint to a 1-sided 2.5% level, a Bonferroni-based parallel gate-keeping procedure will be used. 
Other secondary and exploratory outcome variables shall provide supportive efficacy and safety information 
regarding the differences between the treatment groups.  No adjustments will be performed for multiple-dose 
comparisons when testing the other secondary and exploratory efficacy variables. 
Date of the Protocol:  06 December 2017 
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3 
TABLE OF CONTENTS 
1 
ADMINISTRATIVE INFORMATION ........................................................................ 5 
2 
STUDY SYNOPSIS ...................................................................................................... 6 
3 
TABLE OF CONTENTS ............................................................................................ 10 
3.1 
List of Tables .................................................................................................. 14 
3.2 
List of Figures................................................................................................. 14 
3.3 
List of Appendices .......................................................................................... 14 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ................................ 15 
5 
INTRODUCTION ....................................................................................................... 17 
5.1 
Investigational Medicinal Product .................................................................. 17 
5.2 
Background..................................................................................................... 17 
5.2.1 
Progressive Familial Intrahepatic Cholestasis ........................................... 17 
5.2.2 
Summary of Clinical and Nonclinical Studies ........................................... 19 
5.3 
Rationale ......................................................................................................... 20 
5.3.1 
Risk/Benefit ............................................................................................... 21 
6 
STUDY OBJECTIVES ............................................................................................... 22 
6.1 
Primary Objectives ......................................................................................... 22 
6.2 
Secondary Objectives ..................................................................................... 22 
7 
INVESTIGATIONAL PLAN ...................................................................................... 23 
7.1 
Overall Study Design and Plan....................................................................... 23 
7.1.1 
Description ................................................................................................. 23 
7.1.2 
Schedule of Assessments ........................................................................... 25 
7.1.3 
Study Procedures and Assessments ........................................................... 29 
7.1.3.1 
Screening Period (Days -56 to 0) ............................................... 29 
7.1.3.2 
Treatment Period (Day 0 to Day 168) ........................................ 30 
7.1.3.3 
Follow-Up Period ....................................................................... 33 
7.2 
Study Population ............................................................................................ 33 
7.2.1 
Inclusion Criteria ....................................................................................... 33 
7.2.2 
Exclusion Criteria ...................................................................................... 34 
7.2.3 
Withdrawal of Patients............................................................................... 36 
7.2.4 
Study Termination by Sponsor .................................................................. 36 
8 
TREATMENT OF PATIENTS ................................................................................... 38 
8.1 
Identity of Study Drug .................................................................................... 38 
8.2 
Administration of Study Drug ........................................................................ 38 
8.3 
Study Treatment Packaging and Labeling ...................................................... 39 
8.3.1 
Packaging and Labeling ............................................................................. 39 
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8.3.2 
Storage ....................................................................................................... 39 
8.3.3 
Blinding and Randomization of Study Drug ............................................. 39 
8.4 
Procedure for Breaking the Randomization Code .......................................... 40 
8.5 
Patient Compliance ......................................................................................... 41 
8.6 
Study Drug Accountability ............................................................................. 41 
8.7 
Concomitant Therapy and Prohibited Medications ........................................ 41 
9 
ASSESSMENT OF EFFICACY ................................................................................. 42 
9.1 
Efficacy Endpoints ......................................................................................... 42 
9.1.1 
Primary Efficacy Endpoint ........................................................................ 42 
9.1.2 
Secondary Efficacy Endpoint .................................................................... 42 
9.1.3 
Exploratory Efficacy Endpoints ................................................................. 43 
9.2 
Efficacy Assessments ..................................................................................... 44 
9.2.1 
Serum Bile Acids ....................................................................................... 44 
9.2.2 
Itching, Scratching and Sleep Score .......................................................... 44 
9.2.3 
Growth ....................................................................................................... 45 
9.2.4 
Quality of Life Questionnaire (PedsQL) ................................................... 45 
9.2.5 
Biomarkers and Other Laboratory Samples ............................................... 45 
9.2.6 
PELD/MELD Score ................................................................................... 45 
9.2.7 
Fibroscan® .................................................................................................. 45 
9.2.8 
Markers of Fibrosis .................................................................................... 46 
9.2.9 
Global Impression of Change and Global Impression of Symptom 
Measures .................................................................................................... 46 
10 
ASSESSMENT OF SAFETY...................................................................................... 47 
10.1 
Adverse Events ............................................................................................... 47 
10.1.1 
Definitions and Investigator Assessments ................................................. 47 
10.1.1.1 Clinical Significance .................................................................. 47 
10.1.1.2 Serious Adverse Events .............................................................. 47 
10.1.1.3 Severity Assessment ................................................................... 48 
10.1.1.4 Causality Assessment ................................................................. 48 
10.1.2 
Recording of Adverse Events .................................................................... 49 
10.1.3 
Recording and Reporting of Serious Adverse Events ................................ 50 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions .............. 51 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments .................................................................................................... 51 
10.2.1 
Laboratory Assessments ............................................................................ 51 
10.2.2 
Individual Patient Safety Monitoring ........................................................ 52 
10.2.2.1 Liver Monitoring ........................................................................ 52 
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10.2.2.2 Close Observation ...................................................................... 54 
10.2.2.3 De-challenge/Re-challenge for Liver and Clinical Hepatitis 
Monitoring .................................................................................. 54 
10.2.2.4 Diarrhea ...................................................................................... 54 
10.2.2.5 Monitoring after Permanent Discontinuation of Study Drug 
Due to Safety .............................................................................. 55 
10.2.3 
Demographics/Medical and Surgical History ............................................ 55 
10.2.4 
Clinical Genetic Testing ............................................................................ 55 
10.2.5 
Physical Examination ................................................................................ 55 
10.2.6 
Vital Signs ................................................................................................. 56 
10.2.7 
Abdominal Ultrasound ............................................................................... 56 
10.2.8 
Overdose .................................................................................................... 56 
10.2.9 
Pregnancy .................................................................................................. 56 
11 
STATISTICAL EVALUATION ................................................................................. 58 
11.1 
Sample Size and Power .................................................................................. 58 
11.2 
Statistical Methods ......................................................................................... 58 
11.2.1 
Statistical Analysis Sets ............................................................................. 58 
11.2.2 
Methods of Statistical Analyses ................................................................. 59 
11.2.2.1 General Principles ...................................................................... 59 
11.2.2.2 Missing Data............................................................................... 60 
11.2.2.3 Demographic and Baseline Characteristics ................................ 60 
11.2.2.4 Subject Disposition..................................................................... 61 
11.2.2.5 Evaluation of Primary Efficacy Variables .................................. 61 
11.2.2.6 Evaluation 
of 
Secondary 
and 
Exploratory 
Efficacy 
Variables ..................................................................................... 62 
11.2.2.7 Evaluation of Safety Variables ................................................... 63 
11.2.2.8 Compliance and Exposure .......................................................... 63 
11.2.3 
Blinded Analyses to Confirm Responder Definitions ............................... 64 
11.2.4 
Data Safety Monitoring Board ................................................................... 64 
12 
DIRECT ACCESS TO SOURCE DATA/NOTES ..................................................... 65 
13 
QUALITY CONTROL AND QUALITY ASSURANCE .......................................... 66 
13.1 
Conduct of the Study ...................................................................................... 66 
13.2 
Study Monitoring............................................................................................ 66 
14 
ETHICS ....................................................................................................................... 68 
14.1 
Independent Ethics Committee/Institutional Review Board .......................... 68 
14.2 
Written Informed Consents and Assents ........................................................ 68 
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15 
DATA HANDLING AND RECORD KEEPING ....................................................... 70 
15.1 
Case Report Forms/Source Data Handling..................................................... 70 
15.2 
Retention of Essential Documents .................................................................. 70 
16 
FINANCING AND INSURANCE .............................................................................. 71 
17 
PUBLICATION POLICY ........................................................................................... 72 
18 
REFERENCE LIST ..................................................................................................... 73 
19 
APPENDICES ............................................................................................................. 74 
 
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3.1 
List of Tables 
Table 1 
Schedule of Assessments ................................................................................... 26 
Table 2 
Dosing and Capsule Strength ............................................................................. 39 
Table 3 
Routine Laboratory Parameters .......................................................................... 52 
 
3.2 
List of Figures 
Figure 1 
Study Design ...................................................................................................... 25 
Figure 2 
Bonferroni Holm Multiple Comparison Procedure ............................................ 59 
 
3.3 
List of Appendices 
Appendix 1 
Concomitant Medications Prohibited During the Study .................................... 74 
Appendix 2 
Diary Questions including Albireo Patient-Reported Outcomes/Observer-
Reported Outcome Instrument ........................................................................... 75 
Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) ....................................................................... 76 
Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaire .............................. 77 
 
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4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
ADME 
absorption, distribution, metabolism, and excretion 
AE(s) 
adverse event(s) 
AFP 
alfa-fetoprotein 
ALP 
alkaline phosphatase 
ALT 
alanine aminotransferase 
APRI 
AST to platelet ratio index 
ASBT 
apical sodium-dependent bile transporter (also known as IBAT) 
AST 
aspartate aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BMI 
body mass index 
BSEP 
bile salt export pump 
CPK 
creatine phosphokinase 
CRA 
clinical research associate 
CYP 
cytochrome P450 
DSMB 
Data and Safety Monitoring Board 
eCRF 
electronic case report form 
eDiary 
electronic diary 
EOT 
End of Treatment 
EU 
European Union 
EudraCT 
European Union drug regulatory agency clinical trial 
FAS 
full analysis set 
FDA 
United States Food and Drug Administration 
FIC1 
familial intrahepatic cholestasis-1 
GCP 
Good Clinical Practice 
GI 
gastrointestinal 
GLMM 
generalized linear mixed modelling 
IB 
Investigator’s Brochure 
IBAT 
ileal bile acid transporter (also known as ASBT) 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
 
 
IEC 
Independent Ethics Committee 
INR 
international normalized ratio 
IRB 
Institutional Review Board 
IWRS 
Interactive Web Response System 
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LDH 
lactate dehydrogenase 
LFT 
liver function test 
MDR3 
multidrug-resistance protein 3 
MedDRA 
Medical Dictionary for Regulatory Activities 
MELD 
model for end-stage liver disease 
MMRM 
mixed model repeated measures 
ObsRO 
observer-reported outcome 
PBC 
primary biliary cholangitis 
p-C4 
plasma 7α-hydroxy-4-cholesten-3-one concentration 
PCS 
potentially clinically significant 
PedsQL 
Pediatric Quality of Life Inventory 
PELD 
pediatric end-stage liver disease 
PFIC 
progressive familial intrahepatic cholestasis 
PGIC 
patient global impression of change 
PGIS 
patient global impression of symptoms 
PP 
per protocol 
PRO 
patient-reported outcome 
PT 
prothrombin time 
PVM 
pharmacovigilance manager 
QoL 
quality of life 
RoW 
rest of world 
RR 
respiratory rate 
SAE(s) 
serious adverse event(s) 
SAP 
statistical analysis plan 
s-BA 
serum bile acid 
SD 
standard deviation 
s-FGF19 
serum fibroblast growth factor 19 concentration 
SOC 
system organ class 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE(s) 
treatment-emergent adverse event(s) 
ULN 
upper limit of normal 
US 
United States 
WHO 
World Health Organization 
 
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5 
INTRODUCTION 
5.1 
Investigational Medicinal Product 
A4250 is a small molecule and a potent selective inhibitor of the ileal bile acid transporter 
(IBAT), also known as the apical sodium-dependent bile acid transporter (ASBT).  IBAT is 
an integral brush border membrane glycoprotein that co-transports sodium and bile acids and 
appears to be a critical component in the regulation of the bile acid pool size in animals and 
man.  This transporter, expressed in the distal ileum, is a key element in the enterohepatic 
circulation of bile acids since it facilitates the high-affinity, high-capacity reabsorption of bile 
acids.  Indeed, 95% of secreted bile acids are reabsorbed via IBAT [Hofmann 2009; 
Miethke 2016].  A4250 is orally administered and acts locally in the gut where it binds 
reversibly to IBAT to decrease the reuptake of bile acids.  A4250 has minimal systemic 
exposure at the predicted therapeutic dose ranges. 
No safety signals were found with A4250 in preclinical safety and toxicity studies.  In the 
first clinical study of A4250 in healthy volunteers, A4250 was given as a single oral dose up 
to 10 mg or daily oral doses up to 3 mg/day for 7 days.  A4250 was well tolerated and the 
only findings were gastrointestinal (GI) related effects, such as diarrhea, at higher doses. 
In an exploratory Phase 2 study in children with cholestatic pruritus, A4250 was given as oral 
doses of 10 to 200 µg/kg/day for 4 weeks.  A4250 reduced mean levels of serum bile acids 
(s-BA) in all cohorts.  In 11 of 13 progressive familial intrahepatic cholestasis 
(PFIC) patients, substantial reductions (ranging from 43% to 98%) were observed.  
Patient-reported diary data documented improvement of pruritus during treatment.  A4250 
was well tolerated.  No serious adverse events (SAEs) were deemed to be related to study 
drug, and most adverse events (AEs), including some increased transaminases, were 
considered unrelated to study drug and transient in nature. 
5.2 
Background 
5.2.1 
Progressive Familial Intrahepatic Cholestasis 
PFIC is a rare autosomal recessive cholestatic liver disease estimated to affect between 1 in 
every 50,000 to 100,000 children born worldwide.  PFIC represents 10% to 15% of causes of 
cholestasis in children and 10% to 15% of liver transplantation indications in children.  All 
types of PFIC exist worldwide and both sexes appear to be equally affected. 
The common underlying pathogenesis of PFIC is disruption of bile formation and bile 
transport through the liver [Jacquemin 2000].  The classification of PFIC has evolved over 
the years.  The most commonly used sub-classifications are PFIC Types 1, 2, and 3, which 
are based on the associated affected gene and described in more detail below. 
• PFIC, Type 1:  also referred to as “Byler disease” or “familial intrahepatic 
cholestasis-1 (FIC1) protein deficiency.” FIC1 protein is located on the canalicular 
membrane of hepatocytes and facilitates movement of aminophospholipids from the outer 
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to inner leaflet of the plasma membrane of the hepatocyte.  The ATP8B1 gene encodes 
FIC1 protein.  Biallelic pathologic variants in the ATP8B1 gene are associated with FIC1 
dysfunction and classified clinically as PFIC Type 1 disease. 
• PFIC, Type 2:  also referred to as “Byler syndrome” or “bile salt export pump (BSEP) 
deficiency.”  BSEP is a transporter protein that is expressed at the canalicular membrane 
of hepatocytes, and is the primary exporter of bile acids.  The ABCB11 gene encodes the 
BSEP protein.  Biallelic pathologic variations in the ABCB11 gene are associated with 
BSEP dysfunction and classified clinically as PFIC Type 2 disease. 
• PFIC, Type 3:  is caused by a deficiency of the multidrug-resistance protein 3 (MDR3) 
due to mutations in the ABCB4 gene.  MDR3 is a phospholipid translocase critical for 
phospholipid secretion. 
Severe pruritus is common in children diagnosed with PFIC.  Itching (and subsequent 
scratching) is a significant morbidity for these patients and their families [Suchy 2007].  A 
more severe degree of pruritus is experienced compared to patients with other forms of liver 
disease and to other pruritic conditions such as atopic dermatitis [Murray 2011].  In patients 
with PFIC, liver biopsy reveals canalicular cholestasis and, later, the appearance of portal 
fibrosis.  Serum biochemistry indicates cholestasis with hyperbilirubinemia, elevated alanine 
aminotransferase (ALT) and aspartate aminotransferase (AST).  The concentrations of bile 
acids in serum are very high, while serum gamma-glutamyl transferase activity (exception 
being MDR3 variants) and cholesterol are normal [Hori 2010].  Symptoms of portal 
hypertension and liver failure will develop during the course of the disease [Davit-Spraul 
2009; Alissa 2008].  Symptoms develop early; median age at onset of symptoms is 2 months, 
and 78% of PFIC patients present with jaundice [Pawlikowska 2010].  The life-threatening 
and debilitating nature of PFIC is reflected by the fact that survival in those not resorting to 
surgery is 50% at 10 years of age and almost zero at 20 years of age.  Approximately half of 
PFIC patients undergo liver transplantation [Davit-Spraul 2009] and treatment-resistant 
pruritus is the leading indication for the surgical procedure partial external biliary diversion, 
mostly in PFIC Type 1 and 2 patients. 
PFIC is life-threatening and debilitating.  Currently, the treatment for PFIC is palliative and 
there is no pharmaceutical treatment approved for use in PFIC.  The therapeutic choices are 
restricted to non-specific therapy of the symptoms and signs of the disease such as nutritional 
support, preventing vitamin deficiencies, and treatment of extrahepatic features.  Medical 
treatment options include off-label use of ursodeoxycholic acid, rifampin, antihistamines, and 
naltrexone.  A minority of patients respond nominally and transiently to these interventions.  
Biliary diversion is used to decrease systemic bile acids through interruption of the 
enterohepatic circulation and so avoid transplantation.  Liver transplantation is typically only 
viewed as an option when patients have failed medical treatment and/or biliary diversion and 
have liver failure or persistent uncontrolled pruritus. 
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5.2.2 
Summary of Clinical and Nonclinical Studies 
This is a summary of nonclinical and clinical studies.  More detail is provided in the 
Investigator’s Brochure ([IB] see A4250 IB).  
A4250 is a potent, selective IBAT inhibitor with no safety signals seen in the safety 
pharmacological studies.  In addition, no safety signals were seen in repeat-dose oral toxicity 
studies in rats with up to 26 weeks of daily dosing and in dogs with up to 39 weeks of daily 
dosing.  No safety signals were identified when A4250 was administered daily to juvenile 
rats from age 14 days to 63 days. 
A4250 is minimally absorbed with very low systemic exposure in both humans and other 
species.  In blood, protein binding is high (>99.7%).  Systemically absorbed A4250 is slowly 
metabolized in humans and in preclinical studies by hydroxylation only, a metabolite that 
retains IBAT activity.  A4250 did not show any significant inhibition of cytochrome 
P450 (CYP) 
enzymes 
in 
the 
rat. 
 
A4250 
inhibited 
human 
CYP3A4 
and 
CYP2D6 (IC50: 16 µmol/L for both enzymes), and CYP2C9 (IC50:1.2 µmol/L).  
To date, A4250 has been studied in 3 Albireo-sponsored clinical studies: a study in healthy 
volunteers evaluating single and multiple administration of A4250; a single-dose absorption, 
distribution, metabolism, and excretion (ADME) study; and a Phase 2 study in children with 
cholestatic pruritus.  In addition, an investigator-sponsored study has been conducted in 
patients with primary biliary cholangitis (PBC).  In total, 98 subjects have been exposed to 
A4250. 
In the first clinical study (A4250-001), A4250 was given to healthy volunteers as a single oral 
dose up to 10 mg or daily oral doses up to 3 mg/day for 7 days.  Following a single oral dose 
of 10 mg, the median maximum drug concentration was 0.2 nmol/L and the area under the 
curve was 0.8 nmol × h/L (n=6).  Pharmacodynamic evaluation of bile acid synthesis 
biomarkers 
(serum 
fibroblast 
growth 
factor 
19 [s-FGF19] 
and 
plasma 
7α Hydroxy 4-cholesten-3-one [p-C4]) identified the expected decrease in s-FGF19 and an 
increase in p-C4.  In addition, total plasma bile acids decreased and total fecal bile acids 
increased in A4250-treated patients compared to placebo. A4250 was generally well 
tolerated.  The only findings were GI-related effects, such as diarrhea at higher doses. 
In the ADME study (A4250-007), healthy subjects were administered a single oral dose of 
3 mg [14C]-A4250 capsule.  The main excretion route was determined to be fecal.  There 
were no quantifiable concentrations of A4250 in plasma or total radioactivity in plasma or 
whole blood.  A4250 was well tolerated.  AEs were reported and most were mild and not 
related to A4250. The 3 AEs experienced by 2 subjects reported as related to study drug were 
GI in nature, including diarrhea (2 AEs; 2 subjects) and abdominal pain (1 AE; 1 subject). 
In an exploratory Phase 2 study in children with cholestatic pruritus (A4250-003), A4250 was 
administered as oral daily doses of 0.01 to 0.2 mg/kg for 4 weeks.  Twenty 
patients (8 females and 12 males) aged 1 to 17 years were enrolled in this open-label 
dose-escalation study.  Four patients were re-enrolled into a second dose level.  No 
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treatment-related SAEs were reported, and most AEs, including some increased 
transaminases, were transient.  Four-week, once-daily treatment with A4250 reduced mean 
levels of s-BA in all cohorts.  Substantial s-BA reductions in 11 of 13 PFIC patients (ranging 
from 43% to 98%) were observed.  A4250 also reduced s-BA in the 11 non-PFIC patients, 
but to a lesser extent than in PFIC patients.  Patient-reported diaries documented 
improvement of the visual analogue scale itch score in 17 of 24 patients during treatment.  
The dose with the greatest improvement showed a mean decrease (± standard error of the 
mean) of 2.9 ±1.1 from baseline.  The results from this study form the basis of the current 
(A4250-005) study design. 
5.3 
Rationale 
In this investigational study, A4250 is under development for treatment of PFIC and is also 
being considered for other orphan cholestatic liver diseases. 
There is currently no medical treatment approved for PFIC.  In PFIC patients with severe 
pruritus, biliary diversion surgery is used to decrease systemic bile acids through interruption 
of the enterohepatic circulation and thereby reducing pruritus [Whitington 1988].  Liver 
transplantation is typically used as an option when patients have failed medical treatment 
and/or biliary diversion and have liver failure or persistent uncontrolled pruritus. 
By inhibiting IBAT with high selectivity and potency, A4250 has the potential to relieve 
cholestasis and improve liver function without surgical intervention in patients with PFIC.  
Since A4250 is minimally absorbed and achieves a substantial reduction of s-BA, it has the 
potential to be a safer and less invasive alternative to surgical biliary drainage.  The rationale 
for using A4250 is to decrease s-BA levels, and to reduce the major morbidity of pruritus, 
improving the health and wellbeing of patients affected with PFIC.  Ultimately, this 
noninvasive therapeutic approach has the potential to not only improve quality of life (QoL), 
but also reduce the need for biliary diversion and subsequent liver failure and liver 
transplantation. 
The doses selected, 40 and 120 µg/kg/day, were based on the efficacy and safety data 
generated from patients with PFIC (n=10 + 3 re-entered) and supporting data from all 
patients (n=20 + 4 re-entered) in the five dose groups dosed in the A4250-003 study.  
Improvements in s-BA and pruritus were observed with doses from 30 µg/kg to 100 µg/kg, 
with a dose-related trend.  The best dose response in all patients was at the 60 µg/kg/day for 
s-BA and 100 µg/kg/day for pruritus.  The best dose response in the PFIC subgroup was at 
30 µg/kg/day for both s-BA and pruritus.  Patients with PFIC responded well to the doses 
given from 30 µg/kg to 200 µg/kg; however, a clear dose relationship could not be 
established in order to select a single dose for this Phase 3 study.  As efficacy was observed 
at both low and high doses, 40 µg/kg and 120 µg/kg/day are considered to be the most 
optimal for this study.  
This study will be conducted in compliance with the protocol and with the International 
Council for Harmonisation (ICH) guidelines on Good Clinical Practice (GCP). 
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Patients completing 24 weeks of double-blind treatment will be offered to enter Study 
A4250-008 and receive open-label treatment with A4250. 
5.3.1 
Risk/Benefit 
A4250 has been evaluated in three Albireo-sponsored clinical studies: a double-blind 
placebo-controlled study in healthy volunteers, a single-dose ADME study, and a Phase 2 
study in children with cholestatic pruritus.  In addition, an investigator-sponsored study has 
been conducted in patients with PBC. 
A total of 98 subjects/patients have been exposed to A4250.  Healthy subjects have been 
exposed to up to 10 mg as a single dose and up to 3 mg daily as part of a multiple-dose 
evaluation.  Children with cholestatic liver disease have been treated with up to 
0.2 mg/kg/day (200 µg/kg/day) for 4 weeks.  In total, 2 SAEs have been reported; both were 
judged by the physician to be not related to the study drug. 
Patients with cholestatic liver diseases suffer from excess bile acids in the liver resulting in 
tissue damage.  A commonly used treatment in these patients is bile diversion surgery, 
whereby approximately 50% to 100% of the enterohepatic circulation of bile acids is 
interrupted.  Inhibition of IBAT with A4250, thereby interrupting the enterohepatic 
circulation of bile acids, is therefore a potential medical alternative to surgery which could be 
of benefit to these patients if shown to be effective and safe.  Data from the Phase 2 study 
showed efficacy of A4250 in reducing s-BA concentrations and pruritus in such patients. 
Based on the mode of action of an IBAT inhibitor, loose stools or diarrhea could be expected.  
However, in the pediatric Phase 2 study with 4 weeks daily treatment, only one patient had 
mild transient diarrhea after a single dose that did not recur on multiple dosing. 
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6 
STUDY OBJECTIVES 
6.1 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day 
A4250 in children with PFIC Types 1 and 2, as determined by the following: 
• The proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment 
• Change from baseline to Week 24 in pruritus compared to placebo  
6.2 
Secondary Objectives 
• To evaluate the effect of A4250 on serum ALT concentration 
• To evaluate the effect of A4250 on growth 
• To evaluate the effect of A4250 on sleep disturbance 
• To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion or 
liver transplantation)  
• To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 
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7 
INVESTIGATIONAL PLAN 
7.1 
Overall Study Design and Plan 
7.1.1 
Description 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the 
efficacy and safety of 40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in 
children with PFIC (deficiencies of FIC1 or BSEP). 
Study data will be periodically reviewed by an unblinded Data Safety and Monitoring Board 
(DSMB). 
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment 
Period and a 4-week Follow-Up Period. 
After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of 
A4250, or a matching placebo. 
Patients who complete the Treatment Period and Visit 9 (Day 168) in this study will be 
invited to participate in a 72-week open-label extension study (A4250-008) in which all 
patients will receive active treatment.  A patient who prematurely withdraws due to no 
improvement/intolerable symptoms, and has completed at least 12 weeks of the Treatment 
Period and End of Treatment (EOT) Visit, will also be offered the opportunity to enter 
Study A4250-008. 
Visit windows applicable to each visit are presented in the schedule of assessments (Table 1). 
There will be up to 10 clinic visits during the study (Figure 1) and one scheduled telephone 
contact, as follows: 
• Visit 1: Screening Period start. Patient is considered enrolled after signing the informed 
consent form (ICF) (Days -56 to -35) 
• Visit 2: second Screening Visit (Days -28 to -7) 
• Visit 3: Randomization Visit (Day 0) 
• Telephone Contact 1: (Day 14) 
• Visit 4: Clinic Visit (Day 28) 
• Visit 5: Clinic Visit (Day 56) 
• Visit 6: Clinic Visit (Day 84) 
• Visit 7: Clinic Visit (Day 126) 
• Visit 8: Clinic Visit (Day 154) 
• Visit 9: Clinic Visit, EOT (Day 168) 
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• Visit 10: Follow-Up Visit, 28 days after last dose of study drug (Day 196).  All patients 
continuing in Study A4250-008 will not have this Follow-up Visit. 
Additional clinic visits may be required for patients who need direct site assistance, e.g., due 
to AE monitoring, to fulfil screening requirements, and/or for safety maintenance. 
Screening Period 
Informed consent must be obtained at Visit 1 prior to performing any study procedures.  After 
signing the ICF, patients will be considered enrolled and evaluated for study eligibility. 
Daily recording of pruritus using an electronic diary (eDiary) will be started to determine 
confirmation of the magnitude and the baseline pruritus symptoms before the study.  After 
consultation with the Medical Monitor, patients failing eligibility criteria may be re-screened 
after 2 months.  Patients not fulfilling inclusion/exclusion criteria after 3 attempts are not 
allowed to re-screen. 
Caregivers/patients will be instructed on the daily use of the eDiary.  The eDiary will include 
patient-reported outcome (PRO) and observer-reported outcome (ObsRO) items for 
evaluation of itching (PRO), scratching (ObsRO), and sleep disturbance (PRO and ObsRO) 
during the Screening Period (study Days -56 to 0) and throughout the 24-week Treatment 
Period (study Days 0 to 168). 
Only the 14 most recent days before randomization will be used for eligibility calculation of 
the diary entries.  Observer-reported outcomes in patients of all ages will be recorded by a 
caregiver (or caregiver’s designee).  If possible, the same caregiver will complete the Albireo 
ObsRO items throughout the study. 
At a second Screening Visit (Visit 2) additional laboratory samples will be taken for 
eligibility assessments. 
Treatment Period 
Eligibility for randomization will be determined at Visit 3 using the pruritus (scratching 
reports from the Albireo ObsRO) data in the 2 weeks before Visit 3, genetic confirmation of 
diagnosis, and the liver biochemistry evaluations from the previous screening visits.  Two 
weeks after Visit 3, there will be a telephone contact with the caregiver/patient to evaluate 
any AEs. 
The patient will return to the clinic at Visit 4, Visit 5, Visit 6, Visit 7, Visit 8, and Visit 9 for 
assessments.  If a new caregiver will begin to complete the Albireo ObsRO during the course 
of the trial, they will be provided with training during a clinic visit before they begin to use 
the eDiary.  Additionally, caregivers/patients will be requested to use the diary to report the 
date and time each dose of study drug is administered during the Treatment Period. 
The last dose of study drug will be administered the day before Visit 9.  At study Visit 9, the 
patient has the option to enroll in an open-label extension study (A4250-008) should they 
meet the required entry criteria.  If the patient is continuing into Study A4250-008 this will be 
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the last visit in this study and the first visit in Study A4250-008.  Visit 9/EOT should also be 
performed at the time patients are prematurely withdrawn from the study. 
Clinic Visit 10 will take place 28 days after Visit 9 for all patients not continuing into Study 
A4250-008. 
Figure 1 
Study Design 
 
T: telephone contact; V: study visit 
7.1.2 
Schedule of Assessments 
The schedule of assessments is presented in Table 1. 
 
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Table 1 
Schedule of Assessments 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Informed consent c 
X 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
Concomitant medicationd 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical and surgical history 
X 
 
X 
 
 
 
 
 
 
 
 
Blood sampling for genetic 
testinge 
X 
 
 
 
 
 
 
 
 
 
 
Physical examination including 
voluntary photography 
X 
 
X 
 
 
 
 
 
 
X 
 
Skin examination 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
Vital signsf 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
eDiary: itching, scratching, and 
sleep scoresg 
Daily diary entry 
Clinical chemistryh 
X 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Hematologyh 
 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Urinalysish 
 
X 
 
 
 
 
X 
 
 
X 
 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Serum bile acidsi 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
INR 
 
X 
 
 
X 
 
X 
 
X 
 
X 
Autotaxinj 
 
 
X 
 
X 
 
 
 
 
X 
 
p-C4j 
 
 
X 
 
X 
 
 
 
 
X 
 
AFP 
 
 
X 
 
 
 
 
 
 
X 
 
Fat-soluble vitamins A & E 
 
X 
 
 
 
 
 
 
 
X 
 
Fat-soluble vitamin D 
 
 
X 
 
 
 
 
 
 
X 
 
Abdominal Ultrasound 
 
 
X 
 
 
 
 
 
 
X 
 
Fibroscan® (where available) 
 
 
X 
 
 
 
 
 
 
X 
 
QoL questionnaire (PedsQL) 
 
 
X 
 
 
 
 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of Change 
 
 
 
 
X 
 
X 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of 
Symptoms 
 
 
X 
 
X 
 
X 
 
 
X 
 
Pregnancy testk 
Xk 
X 
X 
 
X 
X 
X 
X 
X 
Xk 
 
Study drug dispensedl 
 
 
X 
 
X 
X 
X 
X 
X 
 
 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Adverse eventsm 
Continuous collection 
Study drug compliance 
 
 
 
X 
X 
X 
X 
X 
X 
X 
 
AFP: alfa-fetoprotein; BMI: body mass index; eCRF: electronic case report form; eDiary: electronic diary; EOT: End of Treatment; ICF: informed consent form; 
INR: international normalized ratio; p-C4: plasma 7α Hydroxy 4-cholesten-3-one; PedsQL: Pediatric Quality of Life Inventory; QoL: quality of life; RR: respiratory rate; 
s-BA: serum bile acid. 
 
a 
Must also be performed at the time a patient is prematurely withdrawn from the study.  This visit is the same as Visit 1 in the extension protocol (Study A4250-008). 
b 
Assessments must be performed 28 days following the final dose of study drug.  This visit is omitted if patients are entering Study A4250-008. 
c 
The ICF (and assent) must be signed before any study procedures are performed. 
d 
Past (within 1 month prior to Visit 1) and current medication will be documented in the eCRF. 
e 
Blood sample for genetic testing will be drawn for all patients.  Genetic analysis will only be performed on a patient’s extracted DNA if previous the clinical genetic 
result is equivocal, unavailable, or unobtainable. 
f 
Includes blood pressure, pulse, RR, temperature, height (or length depending on age), and body weight (using a certified weight scale).  BMI will be calculated. 
g 
Includes training on use of the device at Screening (Visit 1) and review of patient daily entries during 14 days prior to Clinic Visit 3 to determine eligibility.  
h 
See Table 3 for detailed parameters. 
i 
Patients will fast (water intake only is permissible) for at least 4 hours prior to the collection of samples for s-BA.  Exceptions can be made for infants, less than 
12 months of age, if unable to fast for the full 4 hours.  All s-BA results during the treatment period and follow-up will be blinded. 
j 
Samples will not be collected for patients ≤10 kg. 
k 
For girls who have reached menarche.  Serum test at Visit 1; urine test at other visits.  A serum pregnancy test will also be conducted at Visit 9 for patients entering 
Study A4250-008.  If a urine pregnancy test is positive, a serum pregnancy test should be performed to confirm the pregnancy. 
l 
Study drug to be taken once daily on Day 1 through Day 167 as described in Section 8.2. 
m 
Adverse event information will be collected from the time of signing of the ICF to study discontinuation. 
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7.1.3 
Study Procedures and Assessments 
7.1.3.1 
Screening Period (Days -56 to 0) 
Day -56 to Day -35/Clinic Visit 1 
Patients will undergo a Screening Visit up to 56 days prior to the planned first day of study 
treatment.  Screening procedures and assessments are as follows: 
• Obtain written informed consent 
• Assess inclusion/exclusion criteria (Section 7.2) 
• Record demographics (age, full date of birth, gender, race, and ethnicity) 
• Document concomitant medications 
• Medical and surgical history (date of diagnosis of PFIC Types 1 or 2, prior investigational 
medications for PFIC, historical liver function test [LFT] values, any surgery performed, 
any other diagnosis, and historical liver biopsy data) 
• Blood sample for genetic testing 
• Collect, if available, past confirmatory clinical genetic laboratory report for PFIC1/PFIC2 
and send to central reader for review 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• eDiary training and compliance requirements; patients begin daily recording of pruritus 
using the eDiary (Appendix 2) 
• Clinical chemistry (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Serum pregnancy test for girls of who have reached menarche 
• AE monitoring 
Day -28 to Day -7/Clinic Visit 2 
A second Screening Visit will be performed.  Screening procedures and assessments are as 
follows: 
• Review concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for itching, scratching, and sleep scores and 
compliance 
• Urinalysis (Section 10.2.1) 
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• s-BA (Section 9.2.1) 
• International normalized ratio (INR) 
• Fat-soluble vitamins A and E 
• Urine pregnancy test for girls of who have reached menarche 
• AE monitoring 
7.1.3.2 
Treatment Period (Day 0 to Day 168) 
Study Day 0/Clinic Visit 3 (Randomization) 
At the randomization visit, the following assessments will be conducted: 
• Review inclusion/exclusion criteria (Section 7.2) 
• Review concomitant medications, medical and surgical history 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance and entrance criteria requirement (see 
Inclusion Criteria 4, Section 7.2.1) 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1)  
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• Fat-soluble vitamin D 
• Alfa-fetoprotein (AFP) 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer QoL (Pediatric Quality of Life Inventory [PedsQL]) questionnaire 
• Patient/caregiver/clinician complete the patient global impression of symptoms (PGIS) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Study drug is dispensed and patients are instructed to administer daily from Day 1 
Study Day 14 (Telephone Contact) 
A study nurse will contact the patient/caregiver by telephone for monitoring of the following:  
• Review concomitant medications 
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• Albireo PRO/ObsRO eDiary review for compliance 
• AEs 
• Evaluation of study drug compliance 
Study Day 28 and Day 84/Clinic Visits 4 and 6  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• Urinalysis (at Clinic Visit 6 only; Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• Autotaxin and p-C4 (at Clinic Visit 4 only; samples will not be taken for patients ≤10 kg) 
• Patient/caregiver/clinician complete the patient global impression of change (PGIC) and 
PGIS 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 56 and Day 126/Clinic Visits 5 and 7 
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance  
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
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• Study drug is dispensed 
Study Day 154/Clinic Visit 8  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 168/Clinic Visit 9/EOT 
The last dose of study drug is taken in the morning the day before Clinic Visit 9, and the 
following procedures and assessments will be performed (also performed at the time a patient 
prematurely withdraws):  
• Review of concomitant medications 
• Physical examination, including voluntary photography (Section 10.2.5) 
• Skin examination (Section 10.2.5) 
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry, hematology, and urinalysis (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• Autotaxin and p-C4 (samples will not be taken for patients ≤10 kg) 
• AFP  
• Fat-soluble vitamins A, E, and D 
• Abdominal ultrasound (Section 10.2.7) 
• Fibroscan®, where available (Section 9.2.7) 
• Administer PedsQL questionnaire 
• Patient/caregiver/clinician complete the PGIC and PGIS 
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• Urine pregnancy test for girls who have reached menarche, or serum pregnancy test for 
girls continuing to study A4250-008 
• AE monitoring 
• Evaluation of study drug compliance 
7.1.3.3 
Follow-Up Period 
Study Day 196/Clinic Visit 10 
All patients not continuing into Study A4250-008 will return to the study site 28 days after 
Clinic Visit 9 or the last dose of study drug for the following assessments: 
• Review of concomitant medications 
• Skin examination (Section 10.2.5)  
• Vital signs (Section 10.2.6) 
• Albireo PRO/ObsRO eDiary review for compliance 
• Clinical chemistry and hematology (Section 10.2.1) 
• s-BA (Section 9.2.1) 
• INR 
• AE monitoring 
7.2 
Study Population 
A total of 60 patients with clinical diagnosis of PFIC Type 1 or 2 that have been genetically 
confirmed will be randomized.  
An attempt will be made to randomize a minimum of 10 patients from each age category 
(6 months to 5 years, 6 to 12 years, and 13 to ≤18 years).  
A maximum of 5 patients that were treated in Study A4250-003 will be allowed to participate 
in this study. 
7.2.1 
Inclusion Criteria 
1. A male or female patient, with clinical diagnosis of PFIC Type 1 or 2, between the ages 
of ≥6 months and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have genetic confirmation of PFIC-1 or PFIC-2 through identification of 
biallelic pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA concentration, specifically measured to be ≥100 µmol/L, 
taken as the average of 2 samples 7 to 28 days apart (Visits 1 and 2) prior to 
randomization 
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4. Patient must have history of significant pruritus and a caregiver-reported observed 
scratching in the eDiary average of ≥2 (on 0 to 4 scale) in the 2 weeks prior to 
randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  
Patients who turn 18 years of age (or legal age per country) during the study will be 
required to re-consent in order to remain in the study 
6. Patients will be expected to have a consistent caregiver(s) for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use 
an eDiary device as required by the study 
7.2.2 
Exclusion Criteria 
1. Patient with pathologic variations of the ABCB11 gene that predict complete absence of 
the BSEP function 
2. Patient with past medical history or ongoing presence of other types of liver disease 
including, but not limited to, the following: 
a) Biliary atresia of any kind 
b) Benign recurrent intrahepatic cholestasis, indicated by any history of normal s-BAs 
c) Suspected or proven liver cancer or metastasis to the liver on imaging studies 
d) Histopathology on liver biopsy is suggestive of alternate non-PFIC related etiology of 
cholestasis 
3. Patient with a past medical history or ongoing presence of any other disease or condition 
known to interfere with the absorption, distribution, metabolism (specifically bile acid 
metabolism), or excretion of drugs in the intestine, including but not limited to, 
inflammatory bowel disease 
4. Patient with past medical history or ongoing chronic (i.e., >3 months) diarrhea requiring 
intravenous fluid or nutritional intervention for treatment of the diarrhea and/or its 
sequelae 
5. Patient has a confirmed past diagnosis of infection with human immunodeficiency virus 
or other present and active, clinically significant, acute, or chronic infection, or past 
medical history of any major episode of infection requiring hospitalization or treatment 
with parenteral anti-infective treatment within 4 weeks of treatment start (study Day 1) or 
completion of oral anti-infective treatment within 2 weeks prior to start of Screening 
Period 
6. Any patient with suspected or confirmed cancers except for basal cell carcinoma, and 
non-liver cancers treated at least 5 years prior to Screening with no evidence of 
recurrence 
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7. Patient with a past medical history of chronic kidney disease with an impaired renal 
function and a glomerular filtration rate <70 mL/min/1.73 m2  
8. Patient with surgical history of disruption of the enterohepatic circulation excluding those 
who have undergone a successful reversal procedure that has permanently restored flow 
of bile acids from the liver to the duodenum 
9. Patient has had a liver transplant or listed for liver transplant 
10. Decompensated liver disease, coagulopathy, history or presence of clinically significant 
ascites, variceal hemorrhage, and/or encephalopathy 
11. INR >1.4 (the patient may be treated with Vitamin K intravenously, and if INR is <1.4 at 
resampling the patient may be randomized) 
12. Serum ALT >10 × upper limit of normal (ULN) at Screening 
13. Serum ALT >15 × ULN at any time point during the last 6 months unless an alternate 
etiology was confirmed for the elevation 
14. Total bilirubin >5 × ULN at Screening 
15. Patient suffers from uncontrolled, recalcitrant pruritic condition other than PFIC.  
Examples include, but not limited to, refractory atopic dermatitis or other primary pruritic 
skin diseases 
16. Any patient who is pregnant or lactating or who is planning to become pregnant within 
24 weeks of randomization 
17. Sexually active males and females who are not using a reliable contraceptive method with 
≤1% failure rate (such as barrier protection, hormonal contraception, intrauterine device, 
or abstinence) throughout the duration of the study 
18. Patient with a past medical history of alcohol or substance abuse will be excluded.  
Patient must agree to refrain from illicit drug and alcohol use during the study 
19. Administration of bile acid or lipid binding resins and medications that slow GI motility 
(Refer to Appendix 1 – Concomitant Medications Prohibited during the study) 
20. Patient has had investigational exposure to a drug, biologic agent, or medical device 
within 30 days prior to Screening, or 5 half-lives of the study agent, whichever is longer 
21. Patient who has been previously treated with an IBAT inhibitor whose pruritus has not 
responded to treatment 
22. Any other conditions or abnormalities which, in the opinion of the investigator or Medical 
Monitor, may compromise the safety of the patient, or interfere with the patient 
participating in or completing the study 
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7.2.3 
Withdrawal of Patients 
Patients/caregivers will be informed that they have the right to withdraw from the study at 
any time, without prejudice to their medical care, and that they are not obliged to state their 
reasons. 
Any withdrawal, and reasons for withdrawal, must be fully documented in the electronic case 
report form (eCRF) and source documents and the patient followed by the 
investigator/investigative staff.  Even after the study is completed at the Follow-up Visit, the 
investigator/investigative staff will, regardless of reason for withdrawal, record any patient 
data they receive concerning SAEs, and all drug-related non-serious AEs, and subsequently 
report these to Albireo.  Withdrawn patients will not be replaced. 
Patients will be withdrawn in the following circumstances: 
• A patient’s/caregiver’s desire for withdrawal for any reason 
• Lost to follow-up (every effort must be made to contact the patient/caregiver; a certified 
letter must be sent) 
• An AE that, in the opinion of the investigator, necessitates withdrawal 
• A worsening of the symptoms related to diarrhea occurs 
• Death 
• Patient/caregiver estimation of lack of improvement/intolerable symptoms 
• A patient’s/caregiver’s substantial noncompliance (eDiary and study drug compliance) or 
protocol violation 
• An investigator’s opinion that continuing the patient in the study is not appropriate.  The 
investigator may withdraw a patient at any time, if it is considered to be in the patient’s 
best interest 
A patient who prematurely withdraws due to no improvement/intolerable symptoms, and has 
completed at least 12 weeks treatment in the double-blind study, will be offered the 
opportunity to enter open-label Study A4250-008.  A patient who withdraws from treatment 
prematurely and will not enter Study A4250-008 will be requested to complete all visits and 
safety assessments per the current protocol (A4250-005).  As a minimum, patients will have 
EOT (Visit 9) assessments at the time of withdrawal and a follow-up assessment (Visit 10) 
28 days following the last dose of study drug, and the patient will be recorded as withdrawn. 
7.2.4 
Study Termination by Sponsor 
This study may be terminated at any time by Albireo if serious side effects should occur, if 
the investigator does not adhere to the protocol; or if, in the sponsor's judgment, there are no 
further benefits to be achieved from the study.  In this event, Albireo/designee will inform the 
study investigators, institutions, and all regulatory authorities. 
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Albireo may temporarily or permanently discontinue the study at an investigative site at any 
time for safety, ethical, compliance, or other reasons.  If this is necessary, Albireo will 
endeavor to provide advance notification to the site.  If a site or the study is suspended or 
discontinued, the investigator/investigative staff will be responsible for promptly informing 
the Independent Ethics Committee (IEC).  If required by local regulations, Albireo/designee 
will be responsible for informing the IEC and the regulatory authority of study or site 
discontinuation.  In such an event, all study data and unused study drug must be returned to 
Albireo. 
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8 
TREATMENT OF PATIENTS 
8.1 
Identity of Study Drug 
A4250 and placebo will be supplied as capsules for oral administration.  White opaque 
capsules filled with pellets containing A4250 or placebo will be provided.  Two different 
capsule sizes will be available, as follows: 
• Capsule size 0 that can be opened 
• Capsule size 3 that can be opened or swallowed intact 
The content of the A4250 and placebo capsules will be identical in appearance.  Capsule 
filling weight will also be identical for high dose, low dose, and placebo.  Bottles with 
34 capsules will be given to the patient at each visit.  A patient who requires 2 or more 
capsules per day will be given multiple bottles.  Refer to the investigational product manual. 
8.2 
Administration of Study Drug 
Patients will be dosed with A4250 at a dose of 40 µg/kg/day, 120 µg/kg/day, or placebo for 
24 weeks.  Study drug will be dispensed to the patient at Visits 3, 4, 5, 6, 7, and 8, together 
with instructions on how to store and take the drug.  Study drug administration data, 
including whether each patient took each dose or partial doses of study drug, whether there 
were any delayed or missed doses, doses, and whether the capsule was opened or swallowed 
whole will be documented through the eDiary and transferred to the study database. 
A4250 should be taken in the morning, together with food.  On clinic visits days when 
laboratory assessments are conducted, study drug should be taken after the visit and after 
laboratory samples are taken.  Patients should not crush or chew the capsule(s).  When 
swallowing the capsule intact, the patient should administer the dose with a glass of water. 
For a patient unable to swallow capsule intact, the capsule can be opened and the contents 
sprinkled and mixed in a small amount of soft room-temperature yogurt, apple sauce, or fruit 
purée.  The amount of food should be less than what the patient/infant is expected to eat and 
should be followed by water.  
1. Hold the capsule over the prepared food, gently twist open and allow the content to fall 
into food 
2. Mix the entire capsule content with 1 or 2 tablespoons (15 to 30 mL) of soft food (apple 
sauce, fruit purée, or yogurt) 
3. Administer the mixture immediately after mixing. 
The number of capsules provided to the patient will be based on body weight at Visit 3.  At 
Visit 6, if a patient’s weight has shifted to another weight category (identified in Table 2), the 
number of capsules per day may be adjusted.   
 
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8.5 
Patient Compliance 
The study nurse will monitor eDiary compliance by routine review of the CRF health 
website.  If both diary entries on a day are missing during, the study nurse will call the 
patient/caregiver to remind them to complete all scheduled entries.  Any noncompliance will 
be documented and explained in the source documents and in the eCRF. 
Study drug compliance will also be checked through diary responses.  Additionally, patients 
will return all unused study drug at Visits 4, 5, 6, 7, 8, and 9.  The study site staff will count 
all returned drug, assess compliance, and record details in the eCRF. 
Treatment Compliance = 100 × ((Number of study drug dispensed – number of study drug 
returned)/number of study drug that should be taken). 
Treatment compliance between 80% and 120% will be acceptable. 
8.6 
Study Drug Accountability 
Records shall be maintained of the delivery of study treatment(s) to the study site(s), of the 
inventory at the study site(s), of each use of the study treatment(s) for each patient, and of the 
return of unused study treatment(s) to Albireo. 
These records shall include dates, quantities, batch numbers, expiry dates, and the unique 
code numbers assigned to the study drug and to the study patients. 
The investigator will be responsible for ensuring that the records adequately document that 
the patients were provided the quantities specified in the protocol and that all study drug 
received from Albireo is reconciled.  
8.7 
Concomitant Therapy and Prohibited Medications 
The investigator will note all ongoing medication and any medication recently stopped 
(within 1 month prior to Visit 1) in the eCRF.  At Visits 2 through 10, all changes in 
medication (stopping or starting new medication or changes in dose) will be recorded in the 
eCRF.  All medications (prescribed or over the counter) for pruritus and sleep will be 
recorded. 
All medications taken by a patient within 3 months prior to the first intake of study drug are 
regarded as prior medication. 
All medications taken by a patient on or after the first intake of study drug, and which 
continue to be taken during the study, are regarded as concomitant medication.  
Prohibited medications are listed in Appendix 1. 
 
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9 
ASSESSMENT OF EFFICACY 
9.1 
Efficacy Endpoints 
9.1.1 
Primary Efficacy Endpoint 
The primary efficacy endpoints are as follows: 
• European Union (EU) and rest of the world (RoW): Proportion of patients 
experiencing at least a 70% reduction in fasting s-BA concentration from baseline to 
the end of treatment or reaching a level ≤70 µmol/L compared to placebo after 24 
weeks of treatment. 
Fasting s-BA baseline will be calculated as the average of the value at randomization 
and the previous visit (Clinic Visit 2).  The end value is the average of the values at 
Weeks 22 and 24 after the start of double-blind treatment. 
• United States (US): Change in pruritus, as indexed by caregiver-reported observed 
scratching, from baseline to Week 24 as measured by the worst scratch of the Albireo 
ObsRO instrument (Appendix 2).  Scores for the 14 days baseline and post-treatment 
intervals will be calculated by averaging the worst scratching scores from each day in 
the interval. 
9.1.2 
Secondary Efficacy Endpoint 
Secondary efficacy endpoints include the following:  
• EU and RoW: Change in pruritus, as indexed by caregiver-reported observed 
scratching, from baseline to Week 24 as measured by the worst scratch of the Albireo 
ObsRO instrument (Appendix 2).  Scores for the 14 days baseline and post-treatment 
intervals will be calculated by averaging the worst scratching scores from each day in 
the interval. 
• US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L 
compared to placebo after 24 weeks of treatment. 
• All Regions: 
• All secondary endpoints are compared to placebo. 
o Change from baseline to Week 24 in fasting s-BA 
o Change from baseline to Week 24 in serum ALT concentration 
o Change in growth from baseline to Week 24, defined as the linear deficit 
(weight for age, and body mass index [BMI]) compared to the national growth 
curves (Z-score, standard deviation [SD] from P50)  
o Proportion of patients achieving meaningful reduction in caregiver-reported 
observed scratching at Week 24 
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o Change from baseline to Week 24 in sleep parameters measured with the 
Albireo PRO and ObsRO instruments 
o Change from baseline to Week 24 in patient-reported itch severity as 
measured by the average daily score of the Albireo PRO instrument 
(Appendix 2); only patients ≥8 years of age will complete the Albireo PRO 
instrument  
o Number of patients undergoing biliary diversion surgery or being listed for 
liver transplantation 
9.1.3 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• Proportion of patients achieving meaningful reduction in patient-reported itch severity at 
Week 24 
• Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, 
caregiver, and clinician via the PGIC items (Appendix 3) 
• Change from baseline in fasting s-BA at Weeks 4 and 12 
• Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
to Weeks 4, 12, and 24 as measured by the worst scratch.  Scores for the 7 worst 
scratching days of the last 14 days at baseline and post-treatment intervals will be selected 
• Change from baseline in patient-reported itch severity and observer-reported scratching at 
Weeks 4 and 12  
• Change from baseline to Week 24 in patient-reported and observer-reported night time 
itching and scratching severity scores, respectively 
• Change from baseline to Week 24 in pooled pruritus score including observer-reported 
scratching for patients <8 years of age and patient-reported itch severity for patients 
≥8 years of age 
• Change from baseline to Week 24 in additional patient-reported and observer-reported 
sleep parameters (e.g., tiredness and number of awakenings) 
• Change from baseline to Week 24 in PedsQL (Appendix 4) 
• Change from baseline in serum ALT concentration at Weeks 4 and 12 
• Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• Change from baseline in AST at Weeks 4, 12, and 24 
• Change from baseline for other biochemical markers and measures of bile acid synthesis 
(autotaxin, p-C4) at Week 24 
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• Change in pediatric end-stage liver disease/model for end-stage liver disease 
(PELD/MELD) score, AST to platelet ratio index (APRI) score, and Fib-4 score from 
baseline to Week 24 
• Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® 
(where available) 
• Liver-related mortality and liver decompensation events 
• All-cause mortality 
9.2 
Efficacy Assessments 
9.2.1 
Serum Bile Acids 
Blood samples for analysis of fasting total s-BA will be drawn at all visits.  Patients will fast 
(water intake only is permissible) for at least 4 hours prior to the collection of samples for 
s-BA.  Exceptions can be made for infants, <12 months of age, if unable to fast for the full 
4 hours.  Any visit at which a bile acid sample result is unreportable, an additional 
unscheduled visit for a repeat sample collection may be scheduled.  All s-BA results during 
the treatment period and follow-up will be blinded.  Samples will be processed and 
transported to a central laboratory per instructions in the laboratory manual. 
9.2.2 
Itching, Scratching and Sleep Score 
Itching, observed scratching, and sleep disturbance will be recorded twice each day via 
eDiary.  Patients and/or caregivers will be instructed to complete the eDiary every day in the 
morning after the patient wakes and in the evening just before the patient goes to sleep 
throughout the study.  Patients and caregivers will receive a formal training session during 
Visit 1. 
The eDiary includes Albireo ObsRO and PRO items.  Patients <8 years of age will not be 
asked to complete the Albireo PRO items; only the Albireo ObsRO will be completed by 
caregivers of patients in this age group.  Older patients, 8 to 17 years of age, will complete 
the Albireo PRO items and the caregiver will complete the Albireo ObsRO items.  The 
Albireo PRO items assess severity of itch, aspects of sleep disturbance (morning diary only), 
and tiredness.  For patients 8 to 12 years of age, the caregiver will read the Albireo PRO 
items along with the child and record the child’s response.  A guide will be provided to the 
caregivers that provides standardized explanations of the Albireo PRO items, in case the 
patient is confused or requires clarification about the meaning of a question.  The Albireo 
ObsRO items assess severity of observed scratching, aspects of observed sleep disturbance 
(morning diary only), and observed signs of tiredness (evening diary only).  The Albireo 
ObsRO and PRO scratching and itch severity items use 0 to 4 response scales, where each 
response is distinguished by a unique facial expression, verbal anchor, number, and color 
code (Appendix 2).  
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A daily score for the Albireo ObsRO scratching item will be calculated by selecting the 
maximum scratching severity score from the 2 ratings for each day.  A daily score will be 
considered missing if both of the daily assessments are missing.  A 14-days score will be 
calculated by averaging the daily scratching scores.  A 14-days score will be considered 
missing if >4 out of 7 days a week of data are missing.  The same approach will be used to 
calculate a patient-reported itch severity score. 
9.2.3 
Growth 
Growth will be measured as height (velocity) and weight (Z-score) using a certified weight 
scale.  BMI will be calculated by weight (kg) / height (m)2.  Change will be defined as linear 
growth deficit (weight and BMI for age) compared to national growth curves (Z-score, SD 
from P50). 
9.2.4 
Quality of Life Questionnaire (PedsQL) 
Patients and caregivers will be asked to complete a QoL questionnaire (PedsQL) at Visit 3 
and Visit 9.  Details of the questions included on the questionnaire are provided in 
Appendix 4. 
9.2.5 
Biomarkers and Other Laboratory Samples 
Blood samples for autotaxin and p-C4 will be drawn at Visits 3, 4, and 9 only for children 
with body weight ≥10 kg.  Samples will be processed and transported to a laboratory per 
instructions in the laboratory manual. 
9.2.6 
PELD/MELD Score 
At randomization and end of treatment, PELD score will be calculated for children up to 
12 years of age.  For children 13 years or older, the MELD score will be calculated. 
PELD Score = 4.80 * ln(total bilirubin in mg/dL) + 18.57 * ln(INR) - 6.87 * ln(albumin in 
g/dL) + 4.36 (if patient <1 year: scores for patients listed for liver transplantation before the 
patient's first birthday continue to include the value assigned for age (<1 year) until the 
patient reaches the age of 24 months) + 6.67 (if the patient has growth failure [≤2 SD]) 
Laboratory values less than 1.0 will be set to 1.0 for the calculation of the PELD score. 
MELD Score = 9.57 * ln(creatinine in mg/dL) + 3.78 * ln(total bilirubin in mg/dL) + 
11.2 * ln(INR) + 6.43 
Laboratory values less than 1.0 will be set to 1.0 and serum creatinine values greater than 
4.0 mg/dL will be set to 4.0 for calculation of the MELD score. 
9.2.7 
Fibroscan® 
Where available, Fibroscan® will be performed as per institution standard practice at Visits 3 
and 9. 
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9.2.8 
Markers of Fibrosis 
APRI score and Fib-4 score will be calculated at randomization and at end of treatment. 
APRI = [(AST in U/L) / (AST ULN in U/L)] / (Platelets in 109/L) 
Fibrosis 4 Score = (Age*AST in U/L) / (Platelets in 109/L * √(ALT in U/L)). 
9.2.9 
Global Impression of Change and Global Impression of Symptom Measures 
Patients (≥8 years of age), caregivers, and clinicians will complete the PGIC and the PGIS 
measures (Appendix 3) at randomization (Visit 3; PGIS only), Visits 4 and 6, and at EOT 
(Visit 9). 
The PGIC items assess change in itch (patient version), scratching (caregiver and clinician 
versions), and sleep (all versions) since starting the study drug.  The PGIS items assess itch 
(patient version), scratching (caregiver and clinician versions), and sleep (all versions) in the 
past week.  Caregivers and clinicians will complete the PGIC and PGIS for all patients; those 
patients ≥8 years of age will complete the patient version. 
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10 
ASSESSMENT OF SAFETY 
The timing and frequency of safety assessments are described in Section 7.1.3 and in Table 1 
above. 
The primary safety analysis for this study will include the incidence of total 
treatment-emergent adverse events (TEAE) and TEAEs categorized by causality, severity, 
and seriousness assessments made by the investigator by comparing study drug exposure to 
placebo. 
Trends in safety will also be evaluated for the following assessments: 
• Physical examinations 
• Concomitant medications 
• Vital signs 
• Laboratory test results (including clinical chemistry, hematology, urinalysis, AFP, and 
fat-soluble vitamins A, D, and E) 
• Abdominal ultrasound 
• Discontinuations due to AEs 
10.1 
Adverse Events 
10.1.1 
Definitions and Investigator Assessments 
An AE is defined as any untoward medical occurrence in an enrolled patient regardless of 
causal relationship with study drug.  An AE can therefore be any clinically significant 
unfavorable and unintended sign, symptom, or disease that occurs once a patient is enrolled 
(ICF is signed) in the study until the patient is discharged from the study, whether or not 
related to the study drug. 
10.1.1.1 
Clinical Significance 
Clinical significance is defined as any abnormal finding that results in further clinical 
investigation(s), treatment(s), or the diagnosis of new condition.  Patient-reported events and 
protocol-mandated laboratory values, vital signs, and physical examination findings can be 
considered clinically significant (i.e., an AE) if there is a deterioration as compared to 
baseline.  Examples of clinically significant worsening from baseline could include, but is not 
limited to, events causing withdrawal from the study and events requiring medical 
intervention outside of the study causing apparent clinical manifestations, or judged relevant 
by the investigator.   
10.1.1.2 
Serious Adverse Events 
Serious criteria are applied by the investigator to each AE as specifically defined below.  
These criteria are used to determine whether an AE is serious or non-serious.  The assessment 
is made independently of severity assessment (Section 10.1.1.3).  For example, the 
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development of a severe rash that occurs after signing of informed consent may not meet 
serious criteria as defined below and therefore would be considered a severe, non-serious AE. 
Any AE that meets any 1 of the following 6 criteria is considered an SAE: 
• The outcome of the AE is death 
• The AE is immediately life-threatening.  Life-threatening means that the patient is, in the 
opinion of the investigator, at immediate risk of death from the reaction as it occurred.  
This does not include an AE that, if more severe, might have caused death 
• The AE results in persistent or significant disability/incapacity.  Disability means a 
substantial disruption of a person's ability to conduct normal life functions 
• The AE requires or prolongs hospitalization  
• The AE results in a congenital anomaly/birth defect 
• The AE is an important medical event.  Important medical events may meet serious 
criteria should the investigator assess that they may significantly jeopardize the patient, 
represent a significant hazard, or requires medical/surgical intervention to prevent one of 
the outcomes listed above.  Examples of potential SAEs based on this criteria include, but 
are not limited to, allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions even if they do not result in inpatient 
hospitalization, or the development of drug dependency and drug abuse. 
10.1.1.3 
Severity Assessment 
Severity assessments are based on the intensity of the event in relation to expectation.  The 
investigator will assess the intensity of AEs based on the following definitions: 
• Mild (awareness of sign or symptom, but easily tolerated) 
• Moderate (discomfort sufficient to cause interference with normal activities) 
• Severe (incapacitating, with inability to perform normal activities) 
Severity is a measure of intensity where seriousness is defined by the criteria outlined in 
Section 10.1.1.2.  An AE of severe intensity need not necessarily be considered serious.  For 
example, nausea that persists for several hours may be considered severe nausea, but not meet 
serious criteria, and therefore would be assessed as a severe AE but not an SAE. 
10.1.1.4 
Causality Assessment 
The investigator determines the causality of all AEs to the study drug using medical judgment 
and considering all relevant factors such as (but not limited to) the underlying study 
indication, coexisting disease, concomitant medication, relevant history, pattern of the AE, 
temporal relationship to the study drug, and de-challenge or re-challenge.  The causality 
assessment of the AE/SAE is to be made as follows. 
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Related to study drug (possibly, probably, or definitely related) 
Based on medical judgment, there is at least a reasonable possibility that the study drug 
caused the event; one or more of the following criteria apply: 
• The event follows a reasonable temporal sequence from administration of study drug 
• The event could not be reasonably attributed to the known characteristics of the patient's 
clinical state, environmental or toxic factors, or other modes of therapy administered to 
the patient 
• The event follows a known pattern of response to study drug 
• The event disappears or decreases on cessation or reduction in dose of the study drug.  (It 
should be noted that in some situations an AE will not disappear or decrease in intensity 
upon discontinuation of study drug despite other clear indications of relatedness) 
• The event reappears or worsens when the study drug is re-administered 
Unrelated to study drug (unlikely or unrelated) 
Based on medical judgment there is no reasonable possibility that the study drug caused the 
event; one or more of the following criteria apply: 
• The event does not follow a reasonable temporal sequence from administration of study 
drug 
• The event could be reasonably attributed to the known characteristics of the patient's 
clinical state, concurrent illness, environment or toxic factors, or other modes of therapy 
administered to the patient 
• The event does not follow a known pattern of response to study drug 
• The event does not disappear or decrease on cessation or reduction in dose of the study 
drug, and it does not reappear or worsen when the study drug is re-administered 
10.1.2 
Recording of Adverse Events 
It is the investigator’s responsibility to assess whether each untoward event is a clinically 
significant worsening from baseline, thereby considered an AE.  For all AEs, the severity, 
seriousness, and causality to study drug for each AE as outlined in Section 10.1.1 will be 
assessed and recorded in the eCRF. 
All serious and non-serious AEs are collected once the caregiver/patient has signed the ICF 
and until the post-treatment follow-up (Visit 10) or 28 days after the last dose of study drug. 
Any AEs or SAEs that are unresolved at the patient’s last AE assessment in the study are 
followed up by the investigator until resolution or stabilization up to the database lock and 
recorded in the eCRF.  Albireo retains the right to request additional information for any 
patient with ongoing AE(s) or SAE(s) at the end of the study. 
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TEAEs are defined as any AE that occurs after randomization (Day 0).  All AEs that occur in 
the Screening Period, i.e., after enrollment and prior to randomization, will be collected on 
the eCRF as non-treatment-emergent AEs. 
If there is a clinically significant deterioration of a laboratory value/vital sign or other routine 
study assessment that is associated with a diagnosis, the clinical diagnosis will be reported as 
an AE and the associated signs and symptoms will be considered additional information 
unless the sign or symptom is more severe than expected given the diagnosis.  For example, if 
an investigator diagnoses a study patient with hepatitis during the study period, hepatitis 
would be considered the AE and the concomitant signs and symptoms of abdominal pain, 
vomiting, and elevated ALT and AST would not be reported separately unless, in the opinion 
of the investigator, one of these signs or symptoms is more severe than expected and 
therefore a separate AE assessment is indicated. 
10.1.3 
Recording and Reporting of Serious Adverse Events 
Every SAE (regardless of severity and causality) that occurs once the caregiver/patient has 
signed the ICF and until the post-treatment follow-up (Visit 10), and 28 days after the final 
dose of study drug, should immediately and not later than within 24 hours of knowledge of 
the event, be reported by the investigator or delegate in the SAE report form.    
Report of a SAE must include at least the following information: 
• Patient identification information (study number, site number, initials, and date of birth 
[as per local country requirements for data protection]) 
• The last study drug administration date 
• The diagnosis of the event with the description (or a brief description of 
signs/symptoms/clinical course if the diagnosis is not available) and the date of onset 
• The action used to treat the event (i.e., treatment medications, temporary discontinuation) 
• The reason(s) for considering the event serious 
• The relationship of the event to the study drug or to the study procedure (e.g., the 
investigator's assessment of causality) 
• A brief narrative of the SAE 
Follow-up reports including all new information obtained of the subsequent course of the 
SAE must be prepared and the information collected in the SAE submitted to 
 
 (Pharmacovigilance and Safety Services Department) by e-mail within 24 hours of 
knowledge of the event. 
The pharmacovigilance manager (PVM) may contact the investigator to obtain further 
information on a reported SAE.  The investigator/investigative staff must respond to any 
request for follow-up information or answers to questions regarding the SAE within the same 
timelines as for initial reports. 
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The PVM reports the occurrence of the SAE and follow-up to the Albireo medical monitor 
for medical assessment of the case. 
Should an outcome of death occur within the study period or within 28 days after the last 
administration of study drug, an SAE form should be completed including the main and 
contributory causes of death. 
All SAE reports must be e-mailed to the following e-mail address within 24 hours: 
 
 
If email is unavailable, SAEs may be transmitted via fax to the following number: 
 
In a study-related medical emergency situation, when the assigned medical monitors for the 
study cannot be reached by a caller, an on call physician can be reached 24 hours per day, 
7 days per week, via the 
 call center.  The following number is a chargeable telephone 
number allowing a global reach from both landlines and mobile phones and the internet page 
indicated below contains a list of country-specific toll-free telephone numbers that is 
provided.  It should be noted that not all countries globally have access to toll-free numbers 
as indicated on the “24/7 Medical Help desk” index.  Countries without toll-free numbers 
need to dial the chargeable number as indicated below.  Furthermore, toll-free numbers might 
not be available from mobile phones. 
 
 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions 
Suspected unexpected serious adverse reaction (SUSAR) is an SAE that occurs in a patient, 
the nature or severity of which is not expected per the applicable product information (e.g., 
the IB for an unauthorized investigational product or summary of product characteristics for 
an authorized product).  
Reporting and tracking of SUSARs will be in accordance to all applicable competent 
authority regulations.  The Ethics Committee and all investigators involved in this study will 
be informed according to national and international requirements. 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments 
10.2.1 
Laboratory Assessments 
Samples will be collected for clinical chemistry, hematology, and urinalysis testing at the 
time points specified in Table 1. The parameters assessed are presented in Table 3. 
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Blood for AFP will be drawn at Visits 3 and 9 and for fat-soluble vitamins at Visits 2, 3, 
and 9. 
A serum pregnancy test will be performed at Visit 1 and Visit 9 if the patient is entering the 
A4250-008 study. A urine pregnancy test will be collected at all other visits for girls who 
have reached menarche.  If a urine pregnancy test is positive, a serum pregnancy test should 
be performed to confirm the pregnancy.  Study drug administration will be held pending the 
confirmatory results.  If the serum pregnancy test is negative, the patient can resume dosing.  
If the serum pregnancy test is positive, the patient should be discontinued (see 
Section 10.2.9). 
All samples will be processed and transported to a laboratory per instructions in the 
laboratory manual. 
The observed values will be recorded and assessed as “normal” or “abnormal not clinically 
significant” or “abnormal clinically significant”. 
Additional safety blood samples may be needed due to follow-up of an abnormal value or 
analysis failure.  The blood samples collected for safety laboratory analysis will be destroyed 
after the analyses have been completed.  
Table 3 
Routine Laboratory Parameters 
Clinical Chemistry 
Hematology 
Urinalysis 
• 
Albumin 
• 
ALT 
• 
Alkaline phosphatase  
• 
AST 
• 
Bilirubin – total and direct 
• 
Calcium 
• 
Chloride 
• 
Creatinine 
• 
Creatine kinase 
• 
Gamma-glutamyl transferase 
• 
Potassium 
• 
Sodium 
• 
Hematocrit 
• 
Hemoglobin 
• 
Platelet count 
• 
Red blood cell count 
• 
White blood cell count 
• 
Blood 
• 
Glucose 
• 
Ketones 
• 
Leukocytes 
• 
Nitrites 
• 
pH 
• 
Protein 
 
10.2.2 
Individual Patient Safety Monitoring 
10.2.2.1 
Liver Monitoring 
Strategies to monitor markers of liver disease throughout the study are outlined below where 
the ULN will be based on central laboratory reference values for age and gender. 
If isolated transaminase elevations are observed, defined as: 
1. Normal bilirubin AND absence of clinical hepatitis symptoms AND 
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• ALT or AST ≥5 × ULN (if normal at baseline) or an absolute threshold of 800 U/L, 
whichever comes first  
• OR ALT or AST ≥3 × baseline (if abnormal at baseline) or an absolute threshold of 
800 U/L, whichever comes first 
Then: 
a) Repeat liver profile (AST, ALT, bilirubin, and prothrombin time [PT] or INR) within 
2 to 3 days 
b) Evaluate creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) 
c) As needed (for example, persistent ALT/total bilirubin elevation or worsening of liver 
function), consider evaluation for alternative etiologies 
Monitor the patient using close observation found in Section 10.2.2.2 
If any ONE of the following criteria are met: 
1. Transaminases (ALT or AST ≥3 × baseline or 800 U/L, whichever comes first) AND 
bilirubin (total bilirubin > 2 × ULN) elevations 
2. Transaminase elevations alone (ALT or AST >10 × ULN or 5 × baseline or absolute 
threshold of 800 U/L, whichever comes first) in presence of normal LDH and CPK 
3. Cholestatic marker elevations (alkaline phosphatase [ALP] or gamma-glutamyl 
transferase >2 × baseline) without alternative explanation 
4. Total bilirubin increased, unrelated to hemolysis (elevated reticulocyte count) or 
established genetic diseases, such as Gilbert’s syndrome 
a. Doubling if total bilirubin was <3 mg/dL at baseline  
b. OR Increase by >3 mg/dL if total bilirubin was ≥3 mg/dL at baseline 
5. INR increase refractory to vitamin K administration 
a. Increase by >1.5 if INR was normal at baseline  
b. OR Increase by >0.4 if INR was abnormal at baseline 
6. Any increase in total bilirubin and transaminases if accompanied by EITHER a symptom 
of clinical hepatitis (e.g., vomiting, nausea, right upper quadrant pain) OR immunological 
reaction (rash or >5% eosinophilia) 
Then: 
a) Interrupt study medication 
b) Initiate drug-induced liver injury work-up for alternative etiologies 
c) Repeat liver profile (AST, ALT, total bilirubin, direct bilirubin, ALP) and PT or INR 
within 48 to 72 hours 
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d) Monitor the patient using close observation found in Section 10.2.2.2 
e) If a patient lives in a remote area, they may be tested locally and the results 
communicated to the investigator site promptly 
10.2.2.2 
Close Observation 
• Repeat liver enzyme and serum bilirubin tests two or three times weekly.  Frequency 
of re-testing can decrease to once a week or less if abnormalities stabilize or the trial 
drug has been discontinued and the subject is asymptomatic  
• Obtain a more detailed history of symptoms and prior or concurrent diseases 
• Obtain a history of concomitant drug use (including non-prescription medications and 
herbal and dietary supplement preparations), alcohol use, recreational drug use, and 
special diets  
• Rule out alternate etiology including acute viral hepatitis types A, B, C, D, and E; 
autoimmune or alcoholic hepatitis; non-alcoholic steatohepatitis; hypoxic/ischemic 
hepatopathy; and biliary tract disease 
• Obtain a history of exposure to environmental chemical agents.  
• Obtain additional tests to evaluate liver function, as appropriate (e.g., INR, direct 
bilirubin) 
• Consider gastroenterology or hepatology consultations. 
10.2.2.3 
De-challenge/Re-challenge for Liver and Clinical Hepatitis Monitoring 
1. Re-challenge is not recommended: 
a. If a patient has had possible/probable drug-induced liver injury 
b. If a decompensation event has occurred (i.e., variceal hemorrhage, ascites, hepatic 
encephalopathy, etc.). 
2. If due to underlying cholestatic liver disease variability or another alternative etiology is 
identified AND liver tests returned to baseline, re-challenge may be considered after 
consultation with the sponsor Medical Monitor. 
3. If the ALT/total bilirubin elevations are observed after re-challenge, then repeat 
re-challenge is discouraged. 
If patient is permanently discontinued, monitoring should be continued as outlined in 
Section 10.2.2.5.  
10.2.2.4 
Diarrhea 
Study drug should be discontinued if a patient develops diarrhea with at least 1 of the 
following concomitant signs or symptoms: grossly bloody stools, vomiting, dehydration 
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requiring treatment with oral or intravenous rehydration and/or electrolyte imbalances, fever 
(≥38 C) and/or the diarrhea persists for 7 or more days. 
If there is gross blood in stool, INR and platelets should be measured.  
Study drug will be reintroduced (re-challenge) when the symptoms have resolved.  If the 
diarrhea re-occurs within 1 week after the re-challenge with no alternate etiology, the patient 
will be permanently discontinued and monitored as outlined in Section 10.2.2.5. 
10.2.2.5 
Monitoring after Permanent Discontinuation of Study Drug Due to Safety 
Once study drug is permanently discontinued for liver, hepatitis, diarrhea, or other severe AE 
related to study drug, the patient will be monitored weekly until the laboratory and clinical 
parameters have normalized/stabilized. 
10.2.3 
Demographics/Medical and Surgical History 
Demographic information per country regulations (age, full date of birth, gender, race, and 
ethnicity), along with medical and surgical history, will be obtained and recorded in the eCRF 
at Visit 1. 
Medical and surgical history will be entered in the eCRF at Visit 1.  This includes date of 
diagnosis of PFIC, prior investigational medications for PFIC, historical (3 months prior to 
Screening) LFT values (AST, ALT, and total bilirubin), ongoing medication, any surgery 
performed, any other diagnosis, and historical biopsy data.   
If a liver biopsy has been performed within 1 year prior to Screening, or during the study, the 
results will be recorded in the eCRF. 
10.2.4 
Clinical Genetic Testing 
If confirmatory clinical genetic testing for PFIC Type 1 or 2 has been performed prior to 
Screening Visit 1, the confirmatory clinical genetic testing laboratory report will be verified 
by a central reader to determine eligibility. 
Regardless of previous genetic testing, a blood sample will be drawn at Visit 1 for all patients 
for DNA extraction.  Genetic testing will only be performed on a patient’s extracted DNA if 
the previous clinical genetic result is equivocal, unavailable, or unobtainable.  In such cases, 
screening for pathologic biallelic variations in the ATP8B1 or ABCB11 genes will be 
performed if patient meets remaining inclusion and exclusion criteria and only after sponsor 
case review.  No other diagnostic genetic testing will be offered. 
10.2.5 
Physical Examination 
A physician or suitably trained qualified assistant will perform a complete physical 
examination, including voluntary photography, at Visits 1, 3, and 9.  A skin examination will 
be conducted at Visits 1, 3, 4, 6, 9, and 10 (see Table 1).  
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A complete physical examination will include assessment of general appearance, eyes, ears, 
nose, throat, head/neck/thyroid, lymph nodes, cardiovascular, lungs/chest, abdomen, 
genitourinary, extremities, skin, musculoskeletal, neurologic, and other.  
Skin will be examined thoroughly and excoriations/scratch marks recorded.  
10.2.6 
Vital Signs 
Evaluation of vital signs will be performed at all visits.  This includes blood pressure (systolic 
and diastolic), pulse, respiratory rate, temperature, height (or length depending on age), and 
body weight (using a certified weight scale) at clinic visits.  BMI will be calculated. 
10.2.7 
Abdominal Ultrasound 
Ultrasound of the liver and spleen will be performed at randomization (Visit 3) and at EOT 
(Visit 9).  Liver size, echogenicity, and presence of masses/nodules as well as spleen size will 
be recorded. 
10.2.8 
Overdose 
A4250 is minimally absorbed and has a very low systemic availability.  Based on toxicology 
data, for study purposes any dose exceeding a total of >3 mg/kg body weight of A4250 taken 
as a single dose or as a cumulative dose within 24 hours is defined as an overdose.   
The no-adverse-effect dose level in the most sensitive species (defined as 20 mg/kg/day) in 
the rat 1 month toxicity study gives a human equivalent dose of approximately 194 mg/day in 
a 60 kg person.  This dose is approximately 10-fold higher than the maximum possible dose 
predicted in human studies (20 mg).  
The investigator/investigative staff should immediately and not later than within 24 hours of 
knowledge of the overdose, be reported by the investigator or delegate on the SAE report 
form.  In the event of an A4250 overdose, the patient should be monitored closely. 
10.2.9 
Pregnancy 
If a pregnancy is discovered in a female patient enrolled in the study before the end of dosing, 
the patient will be immediately discontinued from the study and will attend the same visits as 
a prematurely withdrawn patient.  If the pregnancy is discovered in a female patient enrolled 
in the study after the end of dosing, the patient will continue in the study per protocol.  If a 
pregnancy occurs in a male patient’s partner at any time during the study, the pregnancy 
should also be reported and followed. 
Pregnancy is not considered to be an AE.  However, if the patient has been dosed with the 
study drug, the pregnancy must be reported on the Paper Pregnancy Notification Form 
immediately within 24 hours after recognition to Albireo/Safety Manager (please refer to 
Section 10.1.3).  Date of exposure and as far as possible, details of the period of gestation at 
the time of exposure must be given. 
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The pregnancy should be followed to determine outcome, including spontaneous termination, 
details of birth, and presence of any birth defects, congenital anomalies or newborn or 
maternal complication.  The infant who was exposed in-utero will be followed for up to 
2 years after delivery.  Individual cases with an abnormal outcome in association with the 
study drug should be reported on an expedited basis, i.e., reported rapidly to a competent 
authority. 
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11 
STATISTICAL EVALUATION 
All personnel involved with the analysis of the study will remain blinded until database lock 
for the double-blind Treatment Period and until major protocol violations have been 
identified. 
All statistical analyses will be performed using SAS version 9.3 or higher. 
11.1 
Sample Size and Power 
For the EU and RoW, the primary analysis will relate to the bile acid responder endpoint (at 
the 1-sided 1.25% level).  For the US, the primary analysis will relate to the pruritus endpoint 
(at the 1-sided 1.25% level).  The aim is to enroll 60 to 70 patients in order to get at least 
60 evaluable patients. 
Assuming 60% responders (patients experiencing at least 70% reduction in fasting s-BA 
concentration or reaching ≤70 µmol/L) in the A4250 dose group and 10% responders in the 
placebo group, approximately 20 patients per group are required to achieve a power of 84% 
with a 1.25% 1-sided Type-1 error. 
For pruritus, with approximately 20 patients per treatment group, the power is 83% given an 
assumed treatment effect difference of -1.0 in change from baseline (-1.6 for A4250 
and -0.6 for placebo) on the Albireo ObsRO scratching score with a SD of 0.95 and a Type I 
error of 1.25% (1-sided). 
11.2 
Statistical Methods 
11.2.1 
Statistical Analysis Sets 
Safety Analysis Set 
The safety analysis set will consist of all patients who received at least 1 dose of study drug.  
Patients will be analyzed according to the treatment they actually received.  The safety 
analysis set will be the primary analysis set for safety analyses.  
Full Analysis Set 
The full analysis set (FAS) will consist of all randomized patients who received at least 
1 dose of assigned treatment.  Patients will be analyzed in the treatment group they were 
randomized to even if they received incorrect study drug.  The FAS will be the primary 
analysis set for efficacy analyses. 
Per Protocol Analysis Set 
The per protocol (PP) analysis set is a subset of the FAS, and will consist of all randomized 
patients for whom no major protocol violation (no violation which may affect the study 
efficacy outcome) is documented.  The PP analysis set will provide supportive data for the 
efficacy analyses. 
Allocation of patients to the PP analysis set will be performed before unblinding of the study. 
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11.2.2 
Methods of Statistical Analyses 
11.2.2.1 
General Principles 
The primary objective of this study is to show superiority in efficacy of 
A4250 (120 µg/kg/day and 40 µg/kg/day) compared to placebo in patients with PFIC Types 1 
and 2. 
In the EU and RoW, for the primary efficacy variable fasting bile acid responder endpoint (at 
least a 70% reduction in fasting s-BA concentration or reaching a level ≤70 µmol/L at the end 
of treatment), the null hypothesis will be tested against the alternative hypothesis (α = 0.0125, 
1-sided). 
The test will be performed at a Type I error rate of ≤0.0125 1-sided, as described above, 
based on the least squares means of the treatment difference in mean probability scale. 
To strictly control overall Type I error rate across the dose groups and also across the primary 
and the main secondary endpoint to a 1-sided 2.5% level, a Bonferroni-based parallel 
gate-keeping procedure will be used.  In this procedure the total significance level of 2.5% 
(1-sided) will be split equally between the dose groups.  Only a dose group that is significant 
for the primary endpoint will be tested, at the nominal significance level of 1.25% (1-sided), 
for the main secondary endpoint (here the pruritus endpoint).  If the previous step was not 
successful, the analysis of the following step will be considered descriptive.  If the procedure 
reaches the secondary endpoint for both dose groups, these will be evaluated using the 
Bonferroni Holm procedure.  The multiple comparison procedure is presented in Figure 2. 
Figure 2 
Bonferroni Holm Multiple Comparison Procedure 
 
In the US, for the primary efficacy variable pruritus score endpoint (change from baseline to 
Week 24 in observer-reported scratching), the null hypothesis will be tested against the 
alternative hypothesis (α=0.0125, 1-sided). 
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The test will be performed at a Type I error rate of ≤0.0125 1-sided, as described above, 
based on the least squares means of the treatment difference in mean change. 
To strictly control overall Type I error rate across the dose groups and also across the primary 
and the main secondary endpoint to a one-sided 2.5% level, a Bonferroni-based parallel 
gate-keeping procedure will be used.  In this procedure the total significance level of 2.5% 
(1-sided) will be split equally between the dose groups.  Only a dose group that is significant 
for the primary endpoint will be tested, at the nominal significance level of 1.25% (1-sided), 
for the main secondary endpoint (here the bile acid responder endpoint).  If the previous step 
was not successful, the analysis of the following step will be considered descriptive.  If the 
procedure reaches the secondary endpoint for both dose groups, these will be evaluated using 
the Bonferroni Holm procedure. 
Other secondary and exploratory outcome variables shall provide supportive efficacy and 
safety information regarding the differences between the treatment groups.  No adjustments 
will be performed for multiple dose comparisons when testing the other secondary and 
exploratory efficacy variables. 
A more detailed statistical analysis plan (SAP) will be prepared and finalized before study 
data are locked and unblinded.  
If not otherwise specified, the baseline value of a parameter is defined as the last assessment 
of that parameter on or prior to the first intake of study treatment date. 
11.2.2.2 
Missing Data 
For efficacy variables, with repeated measures, a mixed model will be employed with no 
additional imputation for missing values, i.e., based on “observed cases”, except where 
otherwise specified. 
Follow-up and continued collection of efficacy data for patients who discontinue treatment 
will be made.  These data will be included in the sensitivity analysis for primary endpoints 
estimating a treatment effect taking loss of effect after discontinuation into account.  Because 
complete follow-up of all patients discontinuing treatment may not be possible, a method to 
replace missing data for patients who discontinue the study will be prespecified (in the SAP) 
taking into account loss of effect after discontinuation (for example multiple imputation). 
No other replacement of missing values will be planned. 
11.2.2.3 
Demographic and Baseline Characteristics 
Descriptive summaries of demographics and other baseline characteristics (including medical 
and surgical history) will be presented by treatment group and overall using the FAS.  
Prior medication will be summarized by treatment group and overall using the safety analysis 
set. 
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11.2.2.4 
Subject Disposition 
The following will be summarized (by treatment group and overall where applicable): 
• Patients enrolled (who signed the informed consent) 
• Patients randomized 
• Patients treated 
• Patients completing the study/withdrawing early (including withdrawal reason) 
Additionally, patients enrolled, included in the safety, in the FAS, and in the PP analysis set 
will be summarized by region. 
11.2.2.5 
Evaluation of Primary Efficacy Variables 
The responder endpoint based on change in fasting bile acids will be analyzed in terms of the 
proportion of patients achieving the event of interest.  Patients in need of rescue therapy, i.e., 
biliary diversion will be classified as non-responders after the biliary diversion.  
Generalized linear mixed modelling (GLMM) will be used with a logistic link, to allow for 
repeated assessments over time.  The model will include baseline value, treatment group, 
PFIC class, age class, region, visit, and visit*treatment group interaction.  The treatment by 
visit interaction will remain in the model regardless of significance.  Region will be included 
in the model as a random effect.  The contrasts of primary interest will be the treatment 
difference at Week 24; also, the difference at Week 4, Week 8, Week 12, and Week 18 will 
be estimated and analyzed.  An unstructured covariance matrix will be used to model the 
within-subject error.  Another covariance structure may be chosen if the fit of this structure 
fails to converge.  Model diagnostics checks will be made to ensure appropriate fit. 
The primary analysis of the change from baseline in pruritus score (indexed by 
observer-reported scratching) will be analyzed using a mixed model repeated measures 
(MMRM) analysis.  The contrast of primary interest will be treatment difference at Week 24 
but also the differences at Week 4, Week 8, Week 12, and Week 18 will be estimated and 
analyzed.  Baseline pruritus score will be included as covariate.  The model will include 
baseline value, treatment group, PFIC class, age class, region, visit, and visit*treatment group 
interaction.  The treatment by visit interaction will remain in the model regardless of 
significance.  Region will be included in the model as a random effect and will be defined on 
country level.  An unstructured covariance matrix will be used to model the within-subject 
error.  Another covariance structure may be chosen if the fit of this structure fails to 
converge. 
Non-parametric approach may be considered if more appropriate. 
The primary analyses will be based on the FAS. 
To assess the robustness of the primary efficacy analysis, the following analyses will be 
performed: 
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• Primary efficacy analysis using the PP set  
• Analyses to investigate the influence of missing values, for example, by using pattern 
mixture models and tipping point analysis 
Subgroup analyses will also be performed for each of the 3 age groups (6 months to 5 years, 
6 to 12 years, and 13 to ≤18 years) and for patients who were not included in the previous 
Study A4250-003. 
Several sensitivity analyses will be performed, such as the following: 
• Cochran Mantel Haenzsel test stratified on PFIC class to compare the proportion in 
fasting bile acid responder endpoint at the end of treatment in the two A4250 dose groups 
to placebo group.  Dropouts will be treated as non-responders 
• Based on the primary analysis including the interaction age class*treatment 
• Same as primary analysis in which the random factor region is considered as fixed factor.  
The region*treatment interaction will also be explored 
• Same as primary analysis including the interaction PFIC*treatment  
Other sensitivity analyses may be performed and will be detailed in the SAP. 
11.2.2.6 
Evaluation of Secondary and Exploratory Efficacy Variables 
The change in the secondary and explorative endpoints such as s-BA, total bilirubin, ALT, 
growth and patient-reported itch severity will also be analyzed using the MMRM model as 
specified for the primary analysis.  Nonparametric methods may be applied as an alternative 
approach for some endpoints, if appropriate. Change in growth (weight for age and BMI for 
age) will also be displayed using graphical presentations. 
Proportions of responders (e.g., meaningful reduction in itch severity and scratching at 
Week 24) will be analyzed using the GLMM model as specified for the primary analysis. 
Comparisons of the change from baseline at Week 24 in PedsQL between the treatment 
groups will be conducted using analysis of covariance.  The model will include terms for 
baseline, PFIC class, age class, region, and treatment.  
Undergoing biliary diversion surgery and/or listed for liver transplant, all-cause mortality, 
and liver decompensation events will mainly be analyzed by using descriptive statistics.  
Kaplan-Meier curves will be used when appropriate for time to event data. 
Other exploratory variables (Global Symptom Relief, biochemical markers and measures of 
bile acid synthesis, Albireo PRO and ObsRO sleep parameters, PELD/MELD score, APRI 
score, and Fib-4 score) will be analyzed descriptively for categorical and continuous data, as 
applicable.  For continuous data, the change from baseline will be analyzed in addition to the 
actual visit values.  For categorical data, shift tables or frequency and percentages of patients 
will be presented as appropriate. 
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In addition, subgroup analyses will be performed for each of the 3 age classes (6 months to 
5 years, 6 to 12 years, and 13 to ≤18 years). 
11.2.2.7 
Evaluation of Safety Variables 
Safety data will be analyzed using descriptive statistics and summaries by treatment group of 
SAEs, AEs, vital signs, clinical laboratory tests (hematology, clinical chemistry and 
urinalysis), and concomitant medication.  Analyses will be performed using the safety 
analysis set.  
Summaries of AEs (coded according to the Medical Dictionary for Regulatory Activities 
[MedDRA] system organ class [SOC] and MedDRA preferred term) will include the 
following:  
• Overview of the incidence of TEAEs (TEAEs, drug-related TEAEs, TEAEs leading to 
study discontinuation, and treatment-emergent SAEs) 
• TEAEs by SOC and preferred term 
• Intensity of TEAEs by SOC and preferred term 
• Drug-related TEAEs by SOC and preferred term 
• TEAEs leading to study discontinuation by SOC and preferred term 
• Treatment-emergent SAEs by SOC and preferred term 
Concomitant medication use during the Treatment Period will be summarized by Anatomical 
Therapeutic Chemical (ATC) class and World Health Organization (WHO) preferred name.  
Summaries of vital signs will be presented.  For each visit, the actual results and the change 
from baseline, and the number and percentage of patients with potentially clinically 
significant (PCS) values observed post-baseline will be presented. 
Summaries of clinical safety laboratory data will be presented.  For each visit, the actual 
result and the change from baseline, and the number and percentage of patients with PCS 
values observed post-baseline will be presented. 
Data listings will be provided for each patient for all safety parameters. 
11.2.2.8 
Compliance and Exposure 
Exposure will be analyzed by calculating the number of days with exposure to study drug.  
Results will be presented by treatment group using the safety analysis set.  
The percentage compliance will be described by treatment group and the number of patients 
with a compliance <80%, between 80% and 120%, and >120% will be presented based on the 
safety analysis set. 
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11.2.3 
Blinded Analyses to Confirm Responder Definitions 
A blinded analysis of Albireo ObsRO and PRO eDiary data will be performed after 50% or 
more of the planned sample has completed the Week 24 Follow-Up Period.  The blinded 
analysis will be used to estimate a threshold of clinically meaningful change (i.e., responder 
definition) in Albireo ObsRO and PRO pruritus scores.  The analysis will be performed by a 
group independent from both the study team and Albireo.  Blinded data will be used for this 
analysis; data will be collapsed across treatment groups. 
The analyses will include distribution- and anchor-based analyses to examine a responder 
definition for the Albireo PRO/ObsRO pruritus scores.  Distribution-based analyses will 
include calculation of the SD and standard error of measurement for the baseline Albireo 
PRO and ObsRO scores.  Anchor-based analyses will involve examining the degree of 
change on the Albireo PRO and ObsRO scores from baseline to Week 24 for patients who 
experience change in pruritus according to PGIC and symptom items.  Details of this analysis 
will be included in a stand-alone SAP for assessing the measurement characteristics 
(reliability, validity, sensitivity to change, responder definition) of the Albireo PRO and 
ObsRO items. 
11.2.4 
Data Safety Monitoring Board 
A DSMB consisting of sponsor-independent clinical and statistical experts will be 
established.  The DSMB will periodically meet for the review of accumulating study data, 
including safety (AE and laboratory data) and liver values.  
The DSMB will have access to unblinded data.  The DSMB will make recommendations for 
the remaining part of the study (further details will be provided in the DSMB charter).  The 
DSMB may recommend continuing with the study as planned or stopping the study early for 
safety reasons.  The DSMB will submit its recommendations in writing to Albireo 
Medical/
 who are responsible for responding to the recommendations of the DSMB and 
taking appropriate action.  The investigators will only be informed by Albireo Medical
 
if the study requires a protocol amendment or is stopped.  The DSMB may choose to make 
additional evaluations at any time if they feel this is warranted from a safety point of view.  
The DSMB will act according to its own written standard operating procedure described in a 
charter and will prepare written minutes of its meetings.  The charter of the DSMB will be 
stored in the trial master file.  The DSMB will maintain records of its meetings and these will 
become part of the study file when the study is complete.  In order not to disseminate 
unblinded data and to ensure that all staff involved in the conduct and final analysis of the 
study remain blind to the data, only the members of the DSMB and the unblinded DSMB 
statistician will have access to these data. 
 
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12 
DIRECT ACCESS TO SOURCE DATA/NOTES 
The investigator/institution shall provide direct access to source data/documents for 
study-related monitoring, audits, IEC/Institutional Review Board (IRB) review and 
regulatory inspection. 
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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
13.1 
Conduct of the Study 
Albireo/
 shall implement and maintain quality control and quality assurance procedures 
with written standard operating procedures to ensure that the study is conducted and data are 
generated, documented, and reported in compliance with the protocol, ICH GCP and 
applicable regulatory requirements. 
This study shall be conducted in accordance with the provisions of the Declaration of 
Helsinki (October 1996) and all revisions thereof, and in accordance with United States Food 
and Drug Administration (FDA) regulations (Code of Federal Regulations, Sections 312.50 
and 312.56) and with ICH GCP (CPMP 135/95). 
The investigator may not deviate from the protocol without a formal protocol amendment 
having been established and approved by an appropriate IEC/IRB, except when necessary to 
eliminate immediate hazards to the patient or when the change(s) involve(s) only logistical or 
administrative aspects of the study.  Any deviations may result in the patient having to be 
withdrawn from the study and render that patient non-evaluable. 
13.2 
Study Monitoring 
Before study initiation, at a site initiation visit or at an investigator’s meeting, an Albireo 
representative or designee will review the protocol and eCRF with the investigators and the 
investigative staff.  During the study, the clinical monitor (clinical research associate [CRA]) 
will visit the site regularly to check the completeness of patient records, the accuracy of 
entries on the eCRFs, the adherence to the protocol and to GCP, the progress of enrollment, 
and to ensure that study drug is being stored, dispensed, and accounted for according to 
specifications. 
The investigator must ensure that eCRFs are completed within a timely period of patient 
visits, as per individual site agreements, and must allow the CRA and Albireo representative 
or designee periodic access to patient records and all study-related materials, including 
relevant hospital or clinical records, to confirm their consistency with the eCRF entries.  No 
information in these records about the identity of the patients will leave the study center.  
Albireo monitoring standards require full verification for the presence of the signed ICF, 
adherence to the inclusion/exclusion criteria, documentation of SAEs, and recording of 
primary efficacy and safety variables.  The CRA will review source data compared with the 
eCRFs and will verify source data according to the study-specific monitoring plan.  The 
design of the study, the frequency of patient visits, and the site enrollment rate will determine 
the frequency of monitoring visits.  Upon study completion, the CRA will visit the site to 
conduct a study termination visit, which will include collection of any outstanding 
documentation. 
It is recommended that the investigator/investigative staff log into the eCRF system every 
working day or at minimum twice weekly in order to provide a prompt response to queries.  
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The investigator/investigative staff should respond to queries and make any relevant changes 
to the study data within 3 working days. 
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14 
ETHICS 
14.1 
Independent Ethics Committee/Institutional Review Board 
Prior to the start of the study, the investigator is responsible for ensuring that the protocol and 
consent form have been reviewed and approved by a relevant IEC/IRB.  The IEC/IRB shall 
be appropriately constituted and perform its functions in accordance with FDA ICH GCP and 
local requirements as applicable. 
The IEC/IRB shall approve all protocol amendments (except for logistical or administrative 
changes), written informed consent documents and document updates, patient recruitment 
procedures (e.g., advertisements), written information to be provided to the patients and 
caregivers, IB, available safety information, information about payment and compensation 
available to patients, the investigator's curriculum vitae and/or other evidence of 
qualifications and any other documents requested by the IEC/IRB and regulatory authority 
(competent authority) as applicable. 
14.2 
Written Informed Consents and Assents 
The investigator/investigative staff will explain to each patient (or legally authorized 
representative) the nature of the study, its purpose, the procedures involved, the expected 
duration, alternative treatment, the potential risks and benefits involved, and any discomfort 
that may occur.  Each caregiver/patient will be informed that participation in the study is 
voluntary and that he/she or their child may withdraw from the study at any time and that 
withdrawal of consent will not affect his/her subsequent medical treatment or relationship 
with the treating physician. 
Caregivers/patients will be informed that they/their children may experience side-effects or 
be at risk for symptoms, illnesses, or complications that cannot be foreseen by Albireo.  As 
with other medications, people treated with A4250 may be at risk of developing allergic 
reactions or anaphylaxis.  Caregivers/patients will be advised that study procedures include 
regular blood sampling for measurement of safety parameters and biological markers and that 
some minor risks are associated with these procedures. 
This informed consent should be given by means of a signed ICF, written in non-technical 
language in accordance with ICH GCP, the Declaration of Helsinki, and regulatory 
authorities.  The caregiver/patient should read and consider the statements before signing and 
dating them and should be given a copy of each signed document.  If written consent is not 
possible, oral consent can be obtained if witnessed and followed by a signed statement from 
one or more persons not involved in the study, indicating why the patient was unable to sign 
the form.  No patient can enter the study before his/her or caregiver informed consent has 
been obtained. 
The ICF is part of the protocol and must be submitted by the investigator/investigative staff 
with the protocol for IEC approval.  Albireo will supply an ICF which complies with 
regulatory requirements and is considered appropriate for the study.  Any changes to the ICF 
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suggested by the investigator must be agreed to by Albireo before submission to the IEC and 
a copy of the approved version must be provided to the clinical monitor after IEC approval. 
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15 
DATA HANDLING AND RECORD KEEPING 
15.1 
Case Report Forms/Source Data Handling 
The investigator shall be provided with standardized eCRFs and shall ensure that all data 
from patient visits are promptly entered into the eCRFs in accordance with the specific 
instructions given.  The investigator must sign each eCRF to verify the integrity of the data 
recorded. 
A list of the normal ranges for all laboratory tests to be undertaken forms part of the 
documentation to be collated prior to study start.  As a central laboratory has been selected to 
conduct any or all tests, it is essential that all samples be analyzed at that laboratory. 
The investigator must maintain source documents such as laboratory reports, consultation 
reports, and complete medical history and physical examination reports. 
As no ethnicity data can be collected in the source document for study sites in France, the 
eCRF will be the Source Document for “origines ethniques”,  
15.2 
Retention of Essential Documents 
Essential documents, as defined by ICH GCP, include: the signed protocol and any 
amendment(s); copies of the completed eCRFs (for site archiving, compact discs of eCRF 
data for specific patients will be provided); signed ICFs; hospital records and other source 
documents; IEC approvals and all related correspondence including approved documents; 
drug accountability records; study correspondence; and a list of patients’ names and 
addresses. 
The investigator/investigative staff must retain copies of these essential documents for the 
period specified by ICH GCP and by applicable regulatory requirements.  The 
investigator/investigative staff will inform Albireo of the location where the essential 
documents are stored and must contact Albireo for approval before disposing of any essential 
documents.  The investigator/investigative staff should take measures to prevent accidental or 
premature destruction of these documents. 
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16 
FINANCING AND INSURANCE 
Financing and insurance are addressed in a separate agreement. 
 
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17 
PUBLICATION POLICY 
Albireo will retain the ownership of all data.  When the study is complete, Albireo shall 
arrange the analysis, tabulation of data and preparation of a clinical study report. Albireo may 
also use the data for publication, presentation at scientific meetings and submission to 
regulatory authorities.  All proposed publications based on this study must be subject to the 
sponsor's approval requirements. 
 
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18 
REFERENCE LIST 
1. A4250 Investigator’s Brochure. 
2. Alissa FT, Jaffe R, Shneider BL. Update on progressive familial intrahepatic cholestasis. 
J Pediatr Gastroenterol Nutr. 2008;46(3):241-52. 
3. Davit-Spraul A, Gonzales E, Baussan C, et al. Progressive familial intrahepatic 
cholestasis. Orphanet J Rare Disease 2009;4:1-12. 
4. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and 
functions. Front Biosci (Landmark Ed). 2009 Jan 1;14:2584-98. 
5. Hori T, Justin H, Nguyen JH and Uemoto S. Progressive familial intrahepatic cholestasis. 
Hepatobiliary pancreat Dis Int. 2010; 9(6):570-8. 
6. Jacquemin E. Progressive familial intrahepatic cholestasis. Genetic basis and treatment. 
Clin Liver Dis. 2000;4(4):753-63. 
7. Miethke AG, Zhang W, Simmons J, et al. Pharmacological inhibition of ASBT changes 
bile composition and blocks progression of sclerosing cholangitis in mdr2 knockout mice. 
Hepatology. 2016; 63(2):512–23. 
8. Murray CS, Rees JL. Are subjective accounts of itch to be relied on? The lack of relation 
between visual analogue itch scores and actigraphic measures of scratch. Acta Derm 
Venereol. 2011;91(1):18-23. 
9. Pawlikowska L, Strautnieks S, Jankowska I, et al. Differences in presentation and 
progression between severe FIC1 and BSEP deficiencies. J Hepatol. 2010;53(1):170-8. 
10. Suchy FJ, Sokol RJ, Balistreri WF, eds. Liver Disease in Children. 3rd Edition. New 
York, NY: Cambridge University Press; 2007:310–4. 
11. Whitington PF, Whitington GL. Partial external diversion of bile for the treatment of 
intractable 
pruritus 
associated 
with 
intrahepatic 
cholestasis. 
Gastroenterology. 
1988;95(1):130-6. 
 
 
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19 
APPENDICES 
Appendix 1 
Concomitant Medications Prohibited During the Study 
 
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Appendix 2 
Diary Questions including Albireo Patient-Reported Outcomes/Observer-
Reported Outcome Instrument 
 
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Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) 
 
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Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaire 
 
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CLINICAL STUDY PROTOCOL A4250-005 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
 
IND Number:   
130591 
Text Product: 
A4250 
Indication: 
Progressive Familial Intrahepatic Cholestasis (PFIC) 
Sponsor: 
Albireo AB 
Development Phase: 
Phase 3 
Sponsor Signatory: 
 
Sponsor Medical Monitor: 
 
Principal Investigator: 
 
Date of the Protocol: 
20 September 2017 
Version of the Protocol: 
Final 
 
 
 
 
Confidential 
The information in this document contains trade secrets and commercial information that are 
privileged or confidential and may not be used, divulged, published, or otherwise disclosed 
without the written consent of Albireo AB.  These restrictions on disclosure apply equally to 
all future information supplied to you or generated by you in connection with the study. 


 
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SPONSOR SIGNATURE PAGE 
PROTOCOL TITLE:  A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children with Progressive 
Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
 
PROTOCOL NUMBER:  A4250-005 
 
Albireo AB 
 
 
 
 
 
 
 
 
 
 
 
Date (day/month/year) 




 
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Page 4 
 
INVESTIGATOR SIGNATURE PAGE 
 
PROTOCOL TITLE:  A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children with Progressive 
Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
 
PROTOCOL NUMBER:  A4250-005 
I have read this protocol and agree that it contains all necessary details for performing this 
study.  I will conduct the study as outlined herein and will complete the study within the time 
designated, in accordance with all stipulations of the protocol and in accordance with Good 
Clinical Practices (GCPs), local regulatory requirements, and the Declaration of Helsinki. 
I will provide copies of the protocol and all pertinent information to all individuals 
responsible to me who assist in the conduct of this study.  I will discuss this material with 
them to ensure that they are fully informed regarding the study drug and the conduct of the 
study. 
I will use only the informed consent approved by the Independent Ethics Committee (IEC) 
and will fulfil all responsibilities for submitting pertinent information to the IEC responsible 
for this study. 
I agree that the sponsor (Albireo AB) shall have access to any source documents from which 
case report form information may have been generated. 
I further agree not to originate or use the name of Albireo AB, A4250 in any publicity, news 
release, or other public announcement, written or oral, whether to the public, press, or 
otherwise, relating to this protocol, to any amendment to the protocol, or to the performance 
of this protocol without the prior written consent of Albireo AB. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature 
 
Date (day/month/year) 


 
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1 
ADMINISTRATIVE INFORMATION 
A Double-blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate 
Efficacy and Safety of A4250 in Children with Progressive Familial Intrahepatic 
Cholestasis Types 1 and 2 (PEDFIC 1) 
Protocol No.: 
A4250-005 
Date of the Protocol: 
20 September 2017 
Date and Number of Amendment(s): 
Not applicable 
Sponsor: 
Albireo AB 
 
Arvid Wallgrens backe 20 
 
413 46 Gӧteborg, Sweden 
Clinical Research Organization: 
 
 
 
 
 
 
 
Sponsor Signatory: 
 
 
   
 
  
 
  
 
 
 
  
Sponsor Medical Monitor: 
 
 
 
 
 
 
 
 
Principal Investigator: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Clinical Laboratory: 
 
 
 
 
 
 
  
 
 
 
 
 
 


 
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STUDY SYNOPSIS 
Name of Sponsor/Company: 
Albireo 
Individual Study 
Table Referring to Part of the 
Dossier: 
 
 
Volume: 
 
Page: 
(For National Authority Use Only) 
Name of Product: 
A4250 
Name of Active Ingredient: 
((Insert name))   
Title of Study: 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate Efficacy and Safety of A4250 
in Children with Progressive Familial Intrahepatic Cholestasis Types 1 and 2 (PEDFIC 1) 
Principal Investigator: 
 
Study Center(s): 
Up to 45 sites will be initiated for this study in the United States (US), Canada, Europe (EU), Middle East, and 
Australia. 
Publication(s): 
None. 
Planned Study Period: 
Q4 2017 to Q2 2019 
Development Phase: 
Phase 3 
Objectives: 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day A4250 in children with 
PFIC Types 1 and 2, as determined by the following: 
• 
Proportion of patients experiencing at least a 70% reduction in fasting s-BA levels or reaching a 
level ≤70 µmol/L compared to placebo after 24 weeks of treatment 
• 
Change from baseline to Week 24 in pruritus compared to placebo  
Secondary Objectives 
• 
To evaluate the effect of A4250 based on alanine aminotransferase (ALT)  
• 
To evaluate the effect of A4250 on insulin-like growth factor 1 (IGF-1) levels 
• 
To evaluate the effect of A4250 on pruritus-related sleep disturbance 
• 
To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion and liver 
transplantation) 
• 
To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 
Methodology: 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the efficacy and safety of 
40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in children with progressive familial 
intrahepatic cholestasis (deficiencies of familial intrahepatic cholestasis -1 (FIC1) or bile salt export pump 
(BSEP).  
The study includes a 14- to 56-day Screening Period followed by a 24-week Treatment Period and a 4-week 
Follow-up Period.  After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of A4250, or a 
matching placebo.  All patients completing Day 168 (Visit 9) in this study will be invited to participate in a 
72-week open-label extension study (A4250-008) in which all patients will receive active treatment. 
There will be 10 visits during the study; 4 of them can be performed, where permitted by local regulations, by a 
study nurse visiting the patient at home, and 1 scheduled telephone contact. 


 
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Name of Sponsor/Company: 
Albireo 
Individual Study 
Table Referring to Part of the 
Dossier: 
 
 
Volume: 
 
Page: 
(For National Authority Use Only) 
Name of Product: 
A4250 
Name of Active Ingredient: 
((Insert name))   
Number of Patients: 
Approximately 60 patients diagnosed with PFIC-1 or PFIC-2 will be enrolled. 
Diagnosis and Main Criteria for Inclusion: 
1. A male or female patient, with clinical diagnosis of PFIC 1 or 2, between the ages of ≥6 months 
and ≤18 years at Visit 1 with a body weight above 5 kg 
2. Patient must have genetic confirmation of PFIC-1 or PFIC-2 through identification of biallelic 
pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated serum bile acid (s-BA) levels, specifically measured to be ≥100 µmol/L, 
taken as the average of 2 samples 7 to 28 days apart (Visits 1 and 2) prior to randomization  
4. Patient must have history of significant pruritus as indexed by caregiver-reported observed scratching 
in the eDiary, average ≥2 (on 0 to 4 scale) in the week prior to randomization 
5. Patient and/or legal guardian must sign informed consent (and assent) as appropriate.  Patients who 
turn 18 years of age (or legal age per country) during the study will be required to re-consent in order 
to remain in the study 
6. Patients are expected to have a consistent caregiver(s) for the duration of the study 
7. Caregivers and age-appropriate patients (≥8 years of age) must be willing and able to use an eDiary 
device as required by the study 
Test Product, Dose and Mode of Administration: 
A4250, 40 µg/kg/day or 120 µg/kg/day, orally administered 
Reference Therapy, Dose and Duration of Administration: 
Matching placebo, orally administered 
Duration of Treatment: 
24 weeks 
Variables 
Efficacy:  
Primary Efficacy Endpoints 
The primary efficacy endpoints are: 
• 
EU and rest of world (RoW): Proportion of patients experiencing at least a 70% reduction in fasting 
serum bile acid (s-BA) levels from baseline to the end of treatment or reaching a level ≤70 µmol/L 
compared to placebo. 
Fasting s-BA baseline will be calculated as the average of the value at randomization and the previous 
screening (Visit 1).  The end value is the average of the values at Weeks 22 and 24 after the start of 
double-blind treatment. 
• 
US: Change in pruritus, indexed as caregiver-reported observed scratching score from baseline to 
Week 24 as measured by the average daily score of the ObsRO instrument.  The average daily score 
will be calculated using the 7 days pre-treatment for baseline, and the last 7 days of treatment for 
Week 24 for the end value. 
Secondary Efficacy Endpoints 
Secondary efficacy endpoints include the following: 
• 
EU and RoW: Change in pruritus, as indexed by caregiver-reported observed scratching score as 
measured by the average daily score of the Albireo ObsRO instrument compared to placebo.  The 
average daily score will be calculated using the 7 days pretreatment for baseline, and the last 7 days of 


 
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Name of Sponsor/Company: 
Albireo 
Individual Study 
Table Referring to Part of the 
Dossier: 
 
 
Volume: 
 
Page: 
(For National Authority Use Only) 
Name of Product: 
A4250 
Name of Active Ingredient: 
((Insert name))   
treatment for Week 24. 
• 
US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA levels or reaching a 
level ≤70 µmol/L from baseline to the end of treatment compared to placebo. 
All Regions: 
All secondary endpoints will be compared to placebo. 
• 
Change from baseline to Week 24 in fasting s-BA 
• 
Change from baseline to Week 24 in patient-reported itch severity as measured by the average daily 
score of the Albireo PRO instrument (only patients ≥8 years of age will complete the PRO instrument). 
• 
Change from baseline to Week 24 in ALT 
• 
Change from baseline to Week 24 in IGF-1 
• 
Proportion of patients achieving meaningful reduction in caregiver-reported observed scratching at 
Week 24 
• 
Change from baseline to Week 24 in sleep parameters measured with the Albireo PRO and ObsRO 
instruments 
• 
Number of patients undergoing biliary diversion surgery or being listed for liver transplantation 
• 
Change from baseline to Week 24 in PedsQL 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• 
Proportion of patients achieving meaningful reduction in patient-reported itch severity at Week 24 
• 
Assessment of global symptom relief at Weeks 4, 12, and 24 as measured by patient, caregiver, and 
clinician via the patient global impression of change (PGIC) items 
• 
Change from baseline in fasting s-BA at Weeks 4 and 12 
• 
Change from baseline in patient-reported itch severity and observer-reported scratching at Weeks 4 and 
12 
• 
Change from baseline to Week 24 in patient-reported and observer-reported night time itching and 
scratching severity scores, respectively 
• 
Change from baseline to Week 24 in pooled pruritus score including observer-reported scratching for 
patients <8 years of age and patient-reported itch severity for patients ≥8 years of age 
• 
Change from baseline to Week 24 in additional patient-reported and observer-reported sleep parameters (e.g., 
tiredness, number of awakenings) 
• 
Change from baseline in ALT at Weeks 4 and 12 
• 
Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• 
Change from baseline in AST at Weeks 4, 12, and 24 
• 
Change from baseline for other biochemical markers and measures of bile acid synthesis (autotaxin, 
p-C4, LDL-C) at Week 24 
• 
Change in PELD/MELD score, AST to platelet ratio index (APRI) score, and Fib-4 score from baseline 
to Week 24 
• 
Liver-related mortality and liver decompensation events. 
• 
All-cause mortality 
Safety:   
Safety criteria are as follows: 
• 
The incidence of treatment-emergent SAEs, based upon information from patient reports, including the 


 
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Name of Sponsor/Company: 
Albireo 
Individual Study 
Table Referring to Part of the 
Dossier: 
 
 
Volume: 
 
Page: 
(For National Authority Use Only) 
Name of Product: 
A4250 
Name of Active Ingredient: 
((Insert name))   
description, incidence, and severity of an SAE 
• 
Assessments based upon information in patients' diary reports as reviewed by the study nurse and on 
data collected at the study visits 
• 
Occurrence of treatment-emergent AEs (TEAE) including severity and relatedness to study drug at all 
visits 
• 
Physical examinations 
• 
Concomitant medications at all visits 
• 
Vital signs at all clinic visits 
• 
Laboratory test results (including hematology, clinical chemistry, and urinalysis) 
Statistical Methods: 
The primary objective of this study is to show superiority in efficacy of A4250 (120 µg/kg/day and 
40 µg/kg/day) compared to placebo in patients with PFIC Types 1 and 2. 
In the EU and RoW, for the primary efficacy variable fasting bile acid responder endpoint (at least a 70% 
reduction in fasting s-BA levels or reaching a level ≤70 µmol/L at the end of treatment), the null hypothesis will 
be tested against the alternative hypothesis (α=0.025, 1-sided).  Generalized linear mixed modelling (GLMM) 
will be used with a logistic link, to allow for repeated assessments over time.  The model will include baseline 
value, treatment group, PFIC class, age class, region, visit and visit*treatment group interaction.  The contrasts 
of primary interest will be the treatment difference at Week 24 but also the difference at Week 4, Week 8, 
Week 12, and Week 18 will be estimated and analyzed. 
A fixed-sequence multiple testing approach will be used.  In the first step, the high dose (120 µg/kg/day) versus 
placebo will be tested at 2.5% 1-sided significance level.  If significant, the low dose (40 µg/kg/day) versus 
placebo will also be tested, in a second step, at 2.5% 1-sided significance level.  The second step will only be 
considered confirmatory providing the previous step was successful. 
In the US, for the primary efficacy variable pruritus score endpoint (change from baseline to Week 24 in 
observer-reported scratching), the null hypothesis will be tested against the alternative hypothesis (α=0.025, 
1-sided).  The primary analysis of the change from baseline in pruritus score (indexed by observer-reported 
scratching) will be analyzed using a mixed model repeated measures (MMRM) analysis.  The contrasts of 
primary interest will be the treatment difference at Week 24 but also the difference at the other weeks will be 
estimated and analyzed.   
In the first step, the high dose (120 µg/kg/day) versus placebo will be tested at 2.5% 1-sided significance level.  
If significant, the low dose (40 µg/kg/day) versus placebo will also be tested, in a second step, at 2.5% 1-sided 
significance level.  The second step will only be considered confirmatory providing the previous step was 
successful.  
Secondary and exploratory outcome variables shall provide supportive efficacy and safety information regarding 
the differences between the treatment groups.  For secondary efficacy variables, supportive statistical tests will 
be made and P-values will be reported in an exploratory way (if not otherwise specified).  No adjustments will 
be performed for multiple-dose comparisons when testing the secondary and exploratory efficacy variables. 
 
Date of the Protocol:  20 September 2017 


 
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TABLE OF CONTENTS 
1 
ADMINISTRATIVE INFORMATION ........................................................................ 5 
2 
STUDY SYNOPSIS ...................................................................................................... 6 
3 
TABLE OF CONTENTS ............................................................................................ 10 
3.1 
List of Tables .................................................................................................. 13 
3.2 
List of Figures................................................................................................. 13 
3.3 
List of Appendices .......................................................................................... 13 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ................................ 14 
5 
INTRODUCTION ....................................................................................................... 17 
5.1 
Investigational Medicinal Product .................................................................. 17 
5.2 
Background..................................................................................................... 17 
5.2.1 
Progressive Familial Intrahepatic Cholestasis ........................................... 17 
5.2.2 
Summary of Clinical and Nonclinical Studies ........................................... 19 
5.3 
Rationale ......................................................................................................... 20 
6 
STUDY OBJECTIVES ............................................................................................... 21 
6.1 
Primary Objectives ......................................................................................... 21 
6.2 
Secondary Objectives ..................................................................................... 21 
7 
INVESTIGATIONAL PLAN ...................................................................................... 22 
7.1 
Overall Study Design and Plan....................................................................... 22 
7.1.1 
Description ................................................................................................. 22 
7.1.2 
Schedule of Assessments ........................................................................... 24 
7.1.3 
Schedule of Blood Samples ....................................................................... 25 
7.1.4 
Study Procedures and Assessments ........................................................... 30 
7.1.4.1 
Screening Period (Days -56 to 0) ............................................... 30 
7.1.4.2 
Treatment Period (Day 0 to Day 168) ........................................ 31 
7.1.4.3 
Follow-Up Period ....................................................................... 33 
7.2 
Discussion of Study Design............................................................................ 34 
7.2.1 
Risk/Benefit and Ethical Assessment ........................................................ 34 
7.2.2 
Early Termination ...................................................................................... 34 
7.3 
Selection of Study Population ........................................................................ 34 
7.3.1 
Inclusion Criteria ....................................................................................... 35 
7.3.2 
Exclusion Criteria ...................................................................................... 35 
7.3.3 
Withdrawal of Patients............................................................................... 37 
8 
TREATMENT OF PATIENTS ................................................................................... 39 
8.1 
Identity of Study Drug .................................................................................... 39 
8.2 
Administration of Study Drug ........................................................................ 39 
8.3 
Study Treatment Packaging and Labeling ...................................................... 40 


 
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8.3.1 
Packaging and Labeling ............................................................................. 40 
8.3.2 
Storage ....................................................................................................... 40 
8.3.3 
Blinding and Randomization of Study Drug ............................................. 40 
8.4 
Procedure for Breaking the Randomization Code .......................................... 41 
8.5 
Patient Compliance ......................................................................................... 42 
8.6 
Study Drug Accountability ............................................................................. 42 
8.7 
Concomitant Therapy and Prohibited Medications ........................................ 42 
9 
ASSESSMENT OF EFFICACY ................................................................................. 43 
9.1 
Efficacy Endpoints ......................................................................................... 43 
9.1.1 
Primary Efficacy Endpoint ........................................................................ 43 
9.1.2 
Secondary Efficacy Endpoint .................................................................... 43 
9.1.3 
Exploratory Efficacy Endpoints ................................................................. 44 
9.2 
Efficacy Assessments ..................................................................................... 45 
9.2.1 
Serum Bile Acids ....................................................................................... 45 
9.2.2 
Itching and Sleep Score ............................................................................. 45 
9.2.3 
Growth and IGF-1 ...................................................................................... 46 
9.2.4 
Quality of Life (PedsQL) Questionnaire.................................................... 46 
9.2.5 
Biomarkers and Other Laboratory Samples ............................................... 46 
9.2.6 
PELD/MELD Score ................................................................................... 46 
9.2.7 
Liver Ultrasonography ............................................................................... 46 
9.2.8 
Markers of Fibrosis .................................................................................... 46 
9.2.9 
Global Impression of Change and Global Impression of Symptom 
Measures .................................................................................................... 47 
10 
ASSESSMENT OF SAFETY...................................................................................... 48 
10.1 
Adverse Events ............................................................................................... 48 
10.1.1 
Definitions and Investigator Assessments ................................................. 48 
10.1.1.1 Clinical Significance .................................................................. 48 
10.1.1.2 Serious Adverse Events .............................................................. 48 
10.1.1.3 Severity Assessment ................................................................... 49 
10.1.1.4 Causality Assessment ................................................................. 49 
10.1.2 
Recording of Adverse Events .................................................................... 50 
10.1.3 
Recording and Reporting of Serious Adverse Events ................................ 51 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions .............. 52 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments .................................................................................................... 53 
10.2.1 
Laboratory Assessments ............................................................................ 53 
10.2.2 
Liver Monitoring ....................................................................................... 54 
10.2.3 
Demographics/Medical and Surgical History ............................................ 55 


 
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10.2.4 
Clinical Genetic Testing ............................................................................ 55 
10.2.5 
Physical Examination ................................................................................ 55 
10.2.6 
Vital Signs ................................................................................................. 55 
10.2.7 
Overdose .................................................................................................... 56 
10.2.8 
Pregnancy .................................................................................................. 56 
11 
STATISTICAL EVALUATION ................................................................................. 57 
11.1 
Sample Size and Power .................................................................................. 57 
11.2 
Statistical Methods ......................................................................................... 57 
11.2.1 
Statistical Analysis Sets ............................................................................. 57 
11.2.2 
Methods of Statistical Analyses ................................................................. 58 
11.2.2.1 General Principles ...................................................................... 58 
11.2.2.2 Missing Data............................................................................... 59 
11.2.2.3 Demographic and Baseline Characteristics ................................ 59 
11.2.2.4 Subject Disposition..................................................................... 59 
11.2.2.5 Evaluation of Primary Efficacy Variables .................................. 60 
11.2.2.6 Evaluation 
of 
Secondary 
and 
Exploratory 
Efficacy 
Variables ..................................................................................... 61 
11.2.2.7 Evaluation of Safety Variables ................................................... 61 
11.2.2.8 Compliance and Exposure .......................................................... 62 
11.2.3 
Blinded Analyses to Confirm Responder Definitions ............................... 62 
11.2.4 
Data Safety Monitoring Board ................................................................... 63 
12 
DIRECT ACCESS TO SOURCE DATA/NOTES ..................................................... 64 
13 
QUALITY CONTROL AND QUALITY ASSURANCE .......................................... 65 
13.1 
Conduct of the Study ...................................................................................... 65 
13.2 
Study Monitoring............................................................................................ 65 
14 
ETHICS ....................................................................................................................... 67 
14.1 
Independent Ethics Committee/Institutional Review Board .......................... 67 
14.2 
Written Informed Consents and Assents ........................................................ 67 
15 
DATA HANDLING AND RECORD KEEPING ....................................................... 69 
15.1 
Case Report Forms/Source Data Handling..................................................... 69 
15.2 
Retention of Essential Documents .................................................................. 69 
16 
FINANCING AND INSURANCE .............................................................................. 70 
17 
PUBLICATION POLICY ........................................................................................... 71 
18 
REFERENCE LIST ..................................................................................................... 72 
19 
APPENDICES ............................................................................................................. 73 
 


 
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3.1 
List of Tables 
Table 1 
Schedule of Assessments ................................................................................... 26 
Table 2 
Schedule and Volumes of Blood Sampling........................................................ 29 
Table 3 
Dosing and Capsule Strength ............................................................................. 40 
Table 4 
Routine Laboratory Parameters .......................................................................... 54 
 
3.2 
List of Figures 
Figure 1 
Study Design ...................................................................................................... 24 
 
3.3 
List of Appendices 
Appendix 1 Concurrent Medications Prohibited During the Study ....................................... 73 
Appendix 2 Diary Questions including Albireo Patient-Reported Outcomes/Observer-
Reported Outcome Instrument ........................................................................... 74 
Appendix 3 Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) ....................................................................... 75 
Appendix 4 Pediatric Quality of Life Inventory (PedQL) Questionnaire .............................. 76 
 


 
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4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
ADME 
absorption, distribution, metabolism, and excretion 
AE(s) 
adverse event(s) 
AFP 
alfa-fetoprotein 
ALT 
alanine aminotransferase 
ANCOVA 
analysis of covariance 
APRI 
AST to platelet ratio index 
ASBT 
apical sodium-dependant bile transporter (also known as IBAT) 
AST 
aspartate aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
AUC 
area under the curve 
BMI 
body mass index 
BP 
blood pressure 
BRIC 
benign recurrent intrahepatic cholestasis 
BSEP 
bile salt export pump 
Cmax 
maximum drug concentration 
CRA 
clinical research associate 
CRF(s) 
case report form(s) 
CRO 
clinical research organization 
CRP 
c-reactive protein 
CYP450 
cytochrome P450 
DSMB 
Data and Safety Monitoring Board 
eCRF 
electronic case report form 
eDiary 
electronic diary 
EDTA 
ethylenediaminetetraacetic acid 
EOT 
end of treatment 
EU 
European Union 
EUDRACT 
European Union drug regulatory agency clinical trial 
EVCTM 
Eudravigilance Clinical Trial Module 
FAS 
full analysis set 
FDA 
United States Food and Drug Administration 
FIC1 
familial intrahepatic cholestasis-1 
GCP 
Good Clinical Practice 
GFR 
glomerular filtration rate 
GI 
gastrointestinal 
GLMM 
generalized linear mixed modelling 
HDPE 
high-density polyethylene 


 
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HIV 
human immunodeficiency virus 
hsCRP 
high sensitivity c-reactive protein 
IB 
Investigator’s Brochure 
IBAT 
ileal bile acid transporter (also known as ASBT) 
ICF 
informed consent form 
ICH 
International Conference on Harmonisation 
 
 
ICSR 
individual case safety reports 
IEC 
Independent Ethics Committee 
IGF-1 
insulin-like growth factor 1 
INR 
international normalized ratio 
IRB 
Institutional Review Board 
IWRS 
Interactive Web Response System 
LDL-C 
low-density lipoprotein cholesterol 
LFT(s) 
liver function test(s) 
MDR3 
multidrug-resistant 3 
MedDRA 
Medical Dictionary for Regulatory Activities 
MELD 
model for end-stage liver disease 
mFAS 
modified full analysis set 
MMRM 
mixed model repeated measures 
ObsRO 
observer-reported outcome 
PBC 
primary biliary cholangitis 
p-C4 
serum 7α-hydroxy-4-cholesten-3-one concentration 
PCS 
potentially clinically significant 
PD 
pharmacodynamics(s) 
PEBD 
partial external biliary diversion 
PedsQL 
Pediatric Quality of Life Inventory 
PELD 
pediatric end-stage liver disease 
PFIC 
progressive familial intrahepatic cholestasis 
PGIC 
patient global impression of change 
PGIS 
patient global impression of symptoms 
PP 
per protocol 
PRO 
patient-reported outcome 
PVM 
pharmacovigilance manager 
QoL 
quality of life 
RoW 
rest of world 
RR 
respiratory rate 


 
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SAE(s) 
serious adverse event(s) 
SAP 
statistical analysis plan 
s-BA 
serum bile acid 
s-FGF19 
serum fibroblast growth factor 19 concentration 
s-IGF-1 
serum insulin-like growth factor 1 concentration 
SOC 
system organ class 
SST 
serum separating tube 
SUSAR 
suspected unexpected serious adverse reaction 
TEAE 
treatment-emergent adverse events 
ULN 
upper limit of normal 
US 
United States 
VAS 
visual analogue scale 
WHO 
World Health Organization 
 


 
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5 
INTRODUCTION 
5.1 
Investigational Medicinal Product 
A4250 is a small molecule and a potent selective inhibitor of the ileal bile acid transporter 
(IBAT), also known as the apical sodium-dependent bile acid transporter (ASBT).  IBAT is 
an integral brush border membrane glycoprotein that co-transports sodium and bile acids and 
appears to be a critical component in the regulation of the bile acid pool size in animals and 
man.  This transporter, expressed in the distal ileum, is a key element in the enterohepatic 
circulation of bile acids since it facilitates the high-affinity, high-capacity reabsorption of bile 
acids.  Indeed, 95% of secreted bile acids are reabsorbed via IBAT [Hofmann 2009; 
Miethke 2016].  A4250 is orally administered and acts locally in the gut where it binds 
reversibly to IBAT to decrease the reuptake of bile acids.  A4250 has minimal systemic 
exposure at the predicted therapeutic dose ranges. 
No safety signals were found with A4250 in preclinical safety and toxicity studies.  In the 
first clinical study of A4250 in healthy volunteers, A4250 was given as a single oral dose up 
to 10 mg or daily oral doses up to 3 mg/day for 7 days.  A4250 was well tolerated and the 
only findings were gastrointestinal (GI) related effects, such as diarrhea, at higher doses. 
In an exploratory Phase 2 study in children with cholestatic pruritus, A4250 was given as oral 
doses of 0.01 to 0.2 mg/kg/day for 4 weeks.  A4250 reduced mean levels of serum bile acids 
(s-BA) in all cohorts.  In 11 of 13 progressive familial intrahepatic cholestasis 
(PFIC) patients, substantial reductions (ranging from 43% to 98%) were observed.  Patient 
reported diary data documented improvement of pruritus during treatment.  A4250 was well 
tolerated.  No serious adverse events (SAEs) were deemed to be related to study drug, and 
most adverse events (AEs), including some increased transaminases, were considered 
unrelated to study drug and transient in nature. 
5.2 
Background 
5.2.1 
Progressive Familial Intrahepatic Cholestasis 
PFIC is a rare autosomal recessive cholestatic liver disease estimated to affect between 1 in 
every 50,000 to 100,000 children born worldwide.  PFIC represents 10% to 15% of causes of 
cholestasis in children and 10% to 15% of liver transplantation indications in children.  All 
types of PFIC exist worldwide and both sexes appear to be equally affected. 
The common underlying pathogenesis of PFIC is disruption of bile formation and bile 
transport through the liver [Jacquemin 2000].  The classification of PFIC has evolved over 
the years.  The most commonly used sub-classifications are PFIC Types 1, 2, and 3, which 
are based on the associated affected gene and described in more detail below. 
• PFIC, Type 1:  also referred to as “Byler disease” or “familial intrahepatic 
cholestasis-1 (FIC1) protein deficiency.” FIC1 protein is located on the canalicular 
membrane of hepatocytes and facilitates movement of aminophospholipids from the outer 
to inner leaflet of the plasma membrane of the hepatocyte.  The ATP8B1 gene encodes 


 
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FIC1 protein.  Biallelic pathologic variants in the ATP8B1 gene are associated with FIC1 
dysfunction and classified clinically as PFIC Type 1 disease. 
• PFIC, Type 2:  also referred to as “Byler syndrome” or “bile salt export pump (BSEP) 
deficiency.”  BSEP is a transporter protein that is expressed at the canalicular membrane 
of hepatocytes, and is the primary exporter of bile acids.  The ABCB11 gene encodes the 
BSEP protein.  Biallelic pathologic variations in the ABCB11 gene are associated with 
BSEP dysfunction and classified clinically as PFIC Type 2 disease. 
• PFIC, Type 3:  is caused by a deficiency of the multidrug-resistant 3 protein (MDR3) due 
to mutations in the ABCB4 gene.  MDR3 is a phospholipid translocase critical for 
phospholipid secretion. 
Severe pruritus is common in children diagnosed with PFIC.  Itching (and subsequent 
scratching) is a significant morbidity for these patients and their families [Suchy 2007].  A 
more severe degree of pruritus is experienced compared to patients with other forms of liver 
disease and to other pruritic conditions such as atopic dermatitis [Murray 2011].  In patients 
with PFIC, liver biopsy reveals canalicular cholestasis and, later, the appearance of portal 
fibrosis.  Serum biochemistry indicates cholestasis with hyperbilirubinemia, elevated alanine 
aminotransferase (ALT) and aspartate aminotransferase (AST).  The concentrations of bile 
acids in serum are very high, while serum γ-GT activity (exception being MDR3 variants) 
and cholesterol are normal [Hori 2010].  Symptoms of portal hypertension and liver failure 
will develop during the course of the disease [Davit-Spraul 2009; Alissa 2008].  Symptoms 
develop early; median age at onset of symptoms is 2 months, and 78% of PFIC patients 
present with jaundice [Pawlikowska 2010].  The life-threatening and debilitating nature of 
PFIC is reflected by the fact that survival in those not resorting to surgery is 50% at 10 years 
of age and almost zero at 20 years of age.  Approximately half of PFIC patients undergo liver 
transplantation [Davit-Spraul 2009] and treatment-resistant pruritus is the leading indication 
for the surgical procedure partial external biliary diversion (PEBD), mostly in PFIC Type 1 
and 2 patients. 
PFIC is life-threatening and debilitating.  Currently, the treatment for PFIC is palliative and 
there is no pharmaceutical treatment approved for use in PFIC.  The therapeutic choices are 
restricted to non-specific therapy of the symptoms and signs of the disease such as nutritional 
support, preventing vitamin deficiencies, and treatment of extrahepatic features.  Medical 
treatment options include off-label use of ursodeoxycholic acid, rifampin, antihistamines, and 
naltrexone.  A minority of patients respond nominally and transiently to these interventions.  
Biliary diversion is used to decrease systemic bile acids through interruption of the 
enterohepatic circulation and so avoid transplantation.  Liver transplantation is typically only 
viewed as an option when patients have failed medical treatment and/or biliary diversion and 
have liver failure or persistent uncontrolled pruritus. 


 
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5.2.2 
Summary of Clinical and Nonclinical Studies 
This is a summary of nonclinical and clinical studies.  More detail is provided in the 
Investigator’s Brochure (IB; see A4250 IB).  
A4250 is a potent, selective IBAT inhibitor with no safety signals seen in the safety 
pharmacological studies.  In addition, no safety signals were seen in repeat-dose oral toxicity 
studies in rats with up to 26 weeks of daily dosing and in dogs with up to 39 weeks of daily 
dosing.  No safety signals were identified when A4250 was administered daily to juvenile 
rats from age 14 days to 63 days. 
A4250 is minimally absorbed with very low systemic exposure in both humans and other 
species.  In blood, protein binding is high (>99.7%).  Systemically absorbed A4250 is slowly 
metabolized in humans and in preclinical studies by hydroxylation only, a metabolite that 
retains IBAT activity.  A4250 did not show any significant inhibition of cytochrome 
P450 (CYP450) enzymes in the rat.  A4250 inhibited human CYP3A4 and 
CYP2D6 (IC50: 16 µmol/L for both enzymes), and CYP2C9 (IC50:1.2 µmol/L).  
To date, A4250 has been studied in 3 Albireo-sponsored clinical studies: a study in healthy 
volunteers evaluating single and multiple administration of A4250; a single-dose absorption, 
distribution, metabolism, and excretion (ADME) study; and a Phase 2 study in children with 
cholestatic pruritus.  In addition, an investigator-sponsored study has been conducted in 
patients with primary biliary cholangitis (PBC).  In total, 98 subjects have been exposed to 
A4250. 
In the first clinical study, A4250 was given to healthy volunteers as a single oral dose up to 
10 mg or daily oral doses up to 3 mg/day for 7 days.  Following a single oral dose of 10 mg, 
the median maximum drug concentration (Cmax) was 0.2 nmol/L and the area under the curve 
(AUC) was 0.8 nmol × h/L (n=6).  Pharmacodynamic (PD) evaluation of bile acid synthesis 
biomarkers (serum fibroblast growth factor 19 [s-FGF19] and plasma 7α Hydroxy 
4-cholesten-3-one [p-C4]) identified the expected decrease in s-FGF19 and an increase in p-
C4.  In addition, total plasma bile acids decreased and total fecal bile acids increased in 
A4250-treated patients compared to placebo. A4250 was generally well tolerated.  The only 
findings were GI-related effects, such as diarrhea at higher doses. 
In the ADME study, healthy subjects were administered a single oral dose of 3 mg 
[14C]-A4250 capsule.  The main excretion route was determined to be fecal.  There were no 
quantifiable concentrations of A4250 in plasma or total radioactivity in plasma or whole 
blood.  A4250 was well tolerated.  AEs were reported and most were mild and not related to 
A4250. The 3 AEs experienced by 2 subjects reported as related to study drug were GI in 
nature, including diarrhea (2 AEs; 2 subjects) and abdominal pain (1 AE; 1 subject). 
In an exploratory Phase 2 study in children with cholestatic pruritus, A4250 was administered 
as oral daily doses of 0.01 to 0.2 mg/kg for 4 weeks.  Twenty patients (8 females, 12 males) 
aged 1 to 17 years were enrolled in this open-label dose-escalation study.  Four patients were 
re-enrolled into a second dose level.  No treatment-related SAEs were reported, and most 


 
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AEs, including some increased transaminases, were transient.  Four-week, once-daily 
treatment with A4250 reduced mean levels of s-BA in all cohorts.  Substantial s-BA 
reductions in 11 of 13 PFIC patients (ranging from 43% to 98%) were observed.  A4250 also 
reduced s-BA in the 11 non-PFIC patients, but to a lesser extent than in PFIC patients.  
Patient-reported diaries documented improvement of the visual analogue scale (VAS)-itch 
score in 17 of 24 patients during treatment.  The dose with the greatest improvement showed 
a mean decrease (±SEM) of 2.9 ±1.1 from baseline.  The results from this trial form the basis 
of the current (A4250-005) study design. 
5.3 
Rationale 
In this investigational study, A4250 is under development for treatment of PFIC and is also 
being considered for other orphan cholestatic liver diseases. 
There is currently no medical treatment approved for PFIC.  In PFIC patients with severe 
pruritus, biliary diversion surgery is used to decrease systemic bile acids through interruption 
of the enterohepatic circulation and thereby reducing pruritus [Whitington 1988].  Liver 
transplantation is typically used as an option when patients have failed medical treatment 
and/or biliary diversion and have liver failure or persistent uncontrolled pruritus. 
By inhibiting IBAT with high selectivity and potency, A4250 has the potential to relieve 
cholestasis and improve liver function without surgical intervention in patients with PFIC.  
Since A4250 is minimally absorbed and achieves a substantial reduction of s-BA, it has the 
potential to be a safer and less invasive alternative to surgical biliary drainage.  The rationale 
for using A4250 is to decrease s-BA levels, and to reduce the major morbidity of pruritus, 
improving the health and wellbeing of patients affected with PFIC.  Ultimately, this 
noninvasive therapeutic approach has the potential to not only improve quality of life (QoL), 
but also reduce the need for biliary diversion and subsequent liver failure and liver 
transplantation. 
The doses selected for this study, 40 and 120 µg/kg/day, were chosen based on the efficacy 
(reduction of s-BA and pruritus) and safety data generated from patients with PFIC and other 
types of cholestasis dosed in the exploratory Phase 2 study (A4250-003).  
This study will be conducted in compliance with the protocol and with the International 
Conference on Harmonisation (ICH) guidelines on Good Clinical Practice (GCP). 
Patients completing 24 weeks of double-blind treatment will be offered to enter Study 
A4250-008 and receive open-label treatment with A4250. 


 
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6 
STUDY OBJECTIVES 
6.1 
Primary Objectives 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day 
A4250 in children with PFIC Types 1 and 2, as determined by the following: 
• The proportion of patients experiencing at least a 70% reduction in fasting s-BA 
levels or reaching a level ≤70 µmol/L compared to placebo after 24 weeks of 
treatment 
• A change from baseline to Week 24 in pruritus compared to placebo  
6.2 
Secondary Objectives 
• To evaluate the effect of A4250 based on ALT 
• To evaluate the effect of A4250 on insulin-like growth factor 1 (IGF-1) levels 
• To evaluate the effect of A4250 on pruritus-related sleep disturbance 
• To evaluate the effect of A4250 on the need for surgical treatment (biliary diversion and 
liver transplantation)  
• To assess the safety and tolerability of repeated daily doses of A4250 for 24 weeks 


 
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7 
INVESTIGATIONAL PLAN 
7.1 
Overall Study Design and Plan 
7.1.1 
Description 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the 
efficacy and safety of 40 µg/kg/day and 120 µg/kg/day of A4250 compared to placebo in 
children with PFIC (deficiencies of FIC1 or BSEP). 
Study data will be periodically reviewed by an unblinded Data Safety and Monitoring Board 
(DSMB). 
The study includes a 14- to 56-day Screening Period followed by a 24-week Treatment 
Period and a 4-week Follow-Up Period. 
After completion of the Screening Period, eligible patients (20 per treatment group) will be 
randomized on Day 0 (Visit 3) in a 1:1:1 fashion to receive 40 µg/kg/day or 120 µg/kg/day of 
A4250, or a matching placebo. 
All patients completing Day 168 (Visit 9) in this study will be invited to participate in a 
72-week open-label extension study (A4250-008) in which all patients will receive active 
treatment. 
There will be 10 visits during the study (Figure 1); 4 of them can be performed, where 
permitted by local regulations, by a study nurse visiting the patient at home, and 1 scheduled 
telephone contact, as follows: 
• Visit 1: Screening Period start. Patient is considered enrolled after signing the informed 
consent form (ICF) (Days -56 to -14) 
• Visit 2: Screening Visit 2 (Days -28 to -7) 
• Visit 3: Randomization Visit (Day 0) 
• Telephone Contact 1: (Day 14) 
• Visit 4: Clinic Visit (Day 28) 
• Visit 5: Clinic or Home Visit (Day 56) 
• Visit 6: Clinic Visit (Day 84) 
• Visit 7: Clinic or Home Visit (Day 126) 
• Visit 8: Clinic or Home Visit (Day 154) 
• Visit 9: Clinic Visit, End of Treatment (Day 168) 
• Visit 10: Follow-Up Visit, 28 days after last dose of study drug, or if a patient 
discontinues prematurely (Day 196).  Patients continuing in Study A4250-008 will not 
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Additional clinical visits may be required for patients who need direct site assistance, e.g., 
due to AE monitoring, to fulfil screening requirements, and/or for safety maintenance. 
Screening Period 
As soon as a patient is considered for this study and prior to any other study procedures being 
conducted by the investigator/investigative staff, the caregiver/patient will have the nature of 
the study explained to him/her and be asked to sign an ICF.  After signing the form, patients 
will be evaluated for study eligibility.  Informed consent must be obtained prior to any study 
procedures that do not form a part of the patient’s normal care.  
Prior to entering the Screening Period, patients who complete the consenting process and 
require medication washout will have the appropriate washout period for prohibited 
medications (see Appendix 1) before the screening visit (Clinic Visit 1) is performed.  This 
visit can be conducted immediately for patients who have signed the ICF and do not require 
drug washout. 
If the patients have had a liver biopsy performed the results from the biopsy should be 
recorded in the electronic case report form (eCRF).  Daily recording of pruritus using an 
electronic diary (eDiary) to determine confirmation of the magnitude and the baseline 
pruritus symptoms before the study will be started.  Patients failing inclusion due to 
laboratory values or pruritus scores, as indexed by caregiver-reported observed scratching, 
can be rescreened after 2 months.  Patients not fulfilling inclusion/exclusion criteria after 
3 attempts are not allowed to rescreen. 
Caregivers/patients will be instructed on the daily use of the eDiary (see Appendix 2).  The 
eDiary will include patient-reported outcome (PRO) and observer-reported outcome (ObsRO) 
items for evaluation of itching (PRO), scratching (ObsRO), and sleep disturbance (PRO and 
ObsRO) during the Screening Period (Study Days -56 to 0) and throughout the 24-week 
Treatment Period (Study Days 0 to 168).  Additionally, caregivers/patients will be requested 
to use the diary to report the date and time each dose of study drug is administered during the 
Treatment Period. 
There will be a ±42 day window for the Screening Period; therefore, up to 56 days of diary 
entries are possible.  However, only the most recent 7 days before randomization will be used 
for eligibility and baseline calculations.  Observer-reported outcomes in patients of all ages 
will be recorded by a caregiver (or caregiver’s designee).  If possible, the same caregiver will 
complete the ObsRO items throughout the study.  If there is more than one primary caregiver 
(e.g., if parents are divorced and share custody), multiple caregivers will be included in the 
initial training. 
At a second screening visit (Visit 2) that can be performed at the clinic or in the patient’s 
home, additional laboratory samples will be taken for eligibility assessments. 


 
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Treatment Period 
Eligibility for enrollment will be determined at Visit 3 using the pruritus (scratching reports 
from the ObsRO) data in the week before Visit 3 and the liver biochemistry evaluations from 
the 2 previous screening visits.  Two weeks after Visit 3, there will be a telephone contact 
with the clinic to evaluate any AEs. 
The patient will return to the clinic at Visit 4, Visit 5, Visit 6, Visit 7, Visit 8, and Visit 9 for 
assessments.  Visit 5, Visit 7, and Visit 8 may, alternatively, be performed in the patient’s 
home by a study nurse, if permitted by local regulations.  If a new caregiver will begin to 
complete the ObsRO during the course of the trial, they will be provided with training during 
a clinic visit before they begin to use the eDiary. 
The last dose of study drug will be administered the day before Visit 9.  At Study Visit 9, the 
patient has the option to enroll in an open-label extension study (A4250-008).  If the patient 
is continuing into Study A4250-008 this will be the last visit in this study and the first visit in 
Study A4250-008.  This visit should also be performed if the patient is prematurely 
withdrawn from the study. 
Clinic Visit 10 will take place 28 days after Visit 9 for patients not continuing into Study 
A4250-008.  All patients prematurely withdrawn will have this visit 28 days after the last 
dose of study drug. 
Figure 1 
Study Design 
 
T: telephone contact; V: study visit 
7.1.2 
Schedule of Assessments 
The schedule of assessments is presented in Table 1. 


 
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7.1.3 
Schedule of Blood Samples 
The schedule and volumes of blood samples are presented in Table 2. 
 


 
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Table 1 
Schedule of Assessments 
Study Activity 
Screening 
Screening 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-14)  
±2a 
-28 – (-7)  
±2a 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic  
(or Home)  
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
(or Home)  
Visit 5 
Clinic  
Visit 6 
Clinic  
(or Home)  
Visit 7 
Clinic  
(or Home) 
Visit 8 
Clinic 
Visit 9/EOTb 
Clinic 
Visit 10c 
Informed consent d 
X 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
Concomitant medication 
documentede 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical and surgical history 
X 
 
X 
 
 
 
 
 
 
 
 
Blood sampling for genetic 
testingf 
X 
 
 
 
 
 
 
 
 
 
 
Physical examinationg 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
Vital signsh 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
eDiary: itching, scratching, and 
sleep scoresi 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Clinical chemistry and 
hematology testsj 
 
X 
X 
 
X 
X 
X 
X 
 
X 
X 
Serum bile acids 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
INR 
 
X 
 
 
X 
 
X 
 
X 
 
X 
Growth marker (s-IGF-1) 
 
 
X 
 
 
 
 
 
 
X 
 


 
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Study Activity 
Screening 
Screening 
Treatment Period 
Follow-
Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-14)  
±2a 
-28 – (-7)  
±2a 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic  
(or Home)  
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
(or Home)  
Visit 5 
Clinic  
Visit 6 
Clinic  
(or Home)  
Visit 7 
Clinic  
(or Home) 
Visit 8 
Clinic 
Visit 9/EOTb 
Clinic 
Visit 10c 
Biochemical markersk 
 
Xl 
Xm 
 
X 
 
 
 
 
X 
 
Liver ultrasonography 
 
 
X 
 
 
 
 
 
 
X 
 
QoL questionnaire (PedsQL) 
 
 
X 
 
 
 
 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of Change 
 
 
X 
 
X 
 
X 
 
 
X 
 
Patient/Caregiver/Clinician 
Global Impression of Symptoms 
 
 
X 
 
X 
 
X 
 
 
X 
 
Pregnancy testn 
X 
X 
X 
 
X 
X 
X 
X 
 
X 
X 
Study drug dispensedo 
 
 
X 
 
X 
X 
X 
X 
X 
 
 
AEs documented 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Study drug compliance 
 
 
 
X 
X 
X 
X 
X 
X 
X 
 
AE: adverse event; BMI: body mass index; BP: blood pressure; CRP: c-reactive protein; ICF: informed consent form; INR: international normalized ratio; LDL-C: 
low-density lipoprotein cholesterol; LFT: liver function test; PedsQL: Pediatric Quality of Life Inventory; RR: respiratory rate; s-IGF-1: serum insulin-like growth factor 1 
concentration. 
a 
Blood samples will be collected at both screening visits.  A duration of 28 days is required between visits for small children <10 kg, 14 days for children 10 to 15 kg, and 
7 days for children ≥15 kg.  Thus, the total Screening Period will be 14 to 56 days. 
b 
Must also be performed at the time a patient is prematurely withdrawn from the study.  This visit is the same as Visit 1 in the extension protocol (Study A4250-008). 
c 
Assessments must be performed 28 days following premature withdrawal (28 days following the final dose of study drug).  This visit is omitted if patients are entering 
Study A4250-008. 
d 
Adverse event information will be collected from the time of enrollment (signing of the informed consent) to study discontinuation.  AEs that occur after enrollment and 
prior to randomization will be considered non-treatment emergent AEs.  The ICF must be signed before any medication withdrawal required by the protocol is 


 
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performed.  The consent may therefore be signed before Visit 1. 
e 
Includes past (within 1 month prior to randomization) and current medication.  Specific question regarding stopping or reducing anti-pruritus treatment. 
f 
If unable to genetically confirm PFIC due to inaccessible or unavailable clinical genetic testing results, screening for pathologic variations of the ATP8B1 and ABCB11 
genes will be performed at the sponsor’s discretion if patient meets inclusion and exclusion criteria and after sponsor review. 
g 
At Visits 1, 3 and 9, a complete physical examination will be performed.  At Visits 4, 6 and 10, a skin exam (only) will be conducted. 
h 
Includes BP (measured after the patient has been in a seated position for ≥3 minutes of rest), pulse, RR, and temperature.  Height and body weight will be measured 
using a certified weight scale.  BMI will be calculated. 
i 
Includes training on use of the device at screening (Visit 1) and review of patient daily entries during 7 days prior to clinic Visit 3 to determine eligibility.  
j 
Laboratory parameters include: CRP, hematology, clinical chemistry (including LFTs) and urinalysis assessments.  Blood will be drawn predose from patients in a fasted 
state for ≥4 hours for assessments to be performed in the morning. 
k 
Samples are taken for the analysis of alfa-fetoprotein and the pharmacodynamic biochemistry markers (autotaxin, p-C4, and LDL-C). 
l 
Only alfa-fetoprotein and LDL-C. 
m Only autotaxin and p-C4. 
n 
For girls who have reached menarche.  Serum test at Visit 2; urine test at other visits.  A serum pregnancy test will also be conducted at Visit 9 for patients entering 
Study A4250-008. 
o 
Study drug to be taken once daily on Day 1 through Day 168 as described in Section 8.2.   


 
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Table 2 
Schedule and Volumes of Blood Sampling 
 
Screening 
Screening 
Random
ization 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
FU 
Total 
blood 
volume 
Study Days 
-56 – (-14)  
±2 
-28 – (-7)  
±2 
0 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Study Visits 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
Visit 9/EOT 
Visit 10 
Hematology test (EDTA) 
 
1 
1 
1 
1 
1 
1 
 
1 
1 
8 
Clinical chemistry test (SST)a 
 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
 
2.5 
2.5 
20 
Pregnancy 
 
0 
 
 
 
 
 
 
 
 
0 
INR 
 
2 
 
2 
 
2 
 
2 
 
2 
10 
Serum bile acid 
2.5 
0 
0 
0 
0 
0 
0 
2.5 
0 
0 
5 
Blood sample for genetic testing 
3 
 
 
 
 
 
 
 
 
 
3 
Serum growth marker (IGF-1) 
 
 
2 
 
 
 
 
 
2 
 
4 
Autotaxin 
 
 
1a 
1a 
 
 
 
 
1a 
 
3 
p-C4 (7α-hydroxy-4-cholesten-3-one) 
 
 
2a 
2a 
 
 
 
 
2a 
 
6 
hsCRP 
 
0 
0 
0 
0 
0 
0 
 
0 
0 
0 
LDL-C 
 
0 
 
0 
 
 
 
 
0 
 
0 
Alfa-fetoprotein (AFP) 
 
0 
 
0 
 
 
 
 
0 
 
0 
Total amount of blood for children 
>5 kg 
5.5 
5.5 
5.5 
5.5 
3.5 
5.5 
3.5 
4.5 
5.5 
5.5 
50 
Total amount of blood for children 
>10 kg 
5.5 
5.5 
8.5 
8.5 
3.5 
5.5 
3.5 
4.5 
8.5 
5.5 
59 
Note: All blood volume presented in mL.  All samples of 0 mL are included in the clinical chemistry test (SST). 
CRP: c-reactive protein; EDTA: ethylenediaminetetraacetic acid; EOT: end of treatment; FU; Follow-up; INR: international normalized ratio; SST: serum-separating tube 
a 
Not collected for children ≤ 10 kg. 


 
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7.1.4 
Study Procedures and Assessments 
7.1.4.1 
Screening Period (Days -56 to 0) 
Day -56 to Day -14/Clinic Visit 1 
Patients will undergo a screening visit up to 56 days prior to the planned first day of study 
treatment.  Screening procedures and assessments are as follows: 
• Obtain written informed consent 
• Assess inclusion/exclusion criteria (Section 7.3) 
• Record demographics (age, gender, race) 
• Document concomitant medications 
• Medical and surgical history (date of diagnosis of PFIC Types 1 or 2, historical liver 
function test [LFT] values, any surgery performed, any other diagnosis, historical biopsy 
data, and date of menarche where applicable for female subjects) 
• Blood sample for genetic testing 
• Perform or collect past confirmatory clinical genetic laboratory report for PFIC1/PFIC2 
and send to central reader for review.  If clinical confirmatory genetic testing results are 
not attainable, contact sponsor for consideration 
• Physical examination (Section 10.2.5) and vital signs (Section 10.2.6) 
• eDiary training and compliance requirements; patients begin daily recording of pruritus 
using the eDiary (Appendix 2) 
• s-BA (Section 9.2.1) 
• Urine pregnancy test for girls of who have reached menarche 
• AE monitoring 
Day -28 to Day -7/Clinic or at-Home Visit 2 
A second screening visit, which can take place either at the clinic or by a study nurse in the 
patient’s home, will be performed.  Since blood samples will be collected at both screening 
visits, a minimum duration of 28 days is required between visits for small children <10 kg, 
14 days for children 10 to 15 kg, and 7 days for children ≥15 kg.  Thus, the total Screening 
Period could range from 14 to 56 days.  Screening procedures and assessments are as follows: 
• Review concomitant medications 
• PRO/ObsRO eDiary review for itching, scratching, and sleep scores and compliance 
• Clinical chemistry and hematology tests (Table 4) 
• s-BA (Section 9.2.1) 
• International normalized ratio (INR) 


 
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• Biomarkers (alfa-fetoprotein [AFP] and LDL-C) 
• Serum pregnancy test for girls of who have reached menarche 
• AE monitoring 
7.1.4.2 
Treatment Period (Day 0 to Day 168) 
Study Day 0/Clinic Visit 3 (Randomization) 
At the randomization visit, the following assessments will be conducted: 
• Review inclusion/exclusion criteria (Section 7.3) 
• Review concomitant medications, medical and surgical history 
• Physical examination (Section 10.2.5) and vital signs (Section 10.2.6) 
• PRO/ObsRO eDiary review for compliance, entrance criteria requirement (see Inclusion 
Criteria 4, Section 7.3.1), and itching/scratching/sleep scores 
• Clinical chemistry and hematology tests 
• s-BA (Section 9.2.1)  
• Serum IGF-1 growth marker 
• Biomarkers (autotaxin and p-C4) 
• Liver ultrasonography 
• Administer QoL (Pediatric Quality of Life Inventory [PedsQL]) questionnaire 
• Urine pregnancy test for girls who have reached menarche 
• Patient/caregiver/clinician complete the patient global impression of change (PGIC) and 
the patient global impression of symptoms (PGIS) 
• AE monitoring 
• Study drug is dispensed and patients are instructed to administer daily from Day 1 
Study Day 14 (Telephone Contact) 
A study nurse will contact the patient/caregiver by telephone for monitoring of the following:  
• Concomitant medications 
• AEs 
• Study drug compliance 
• PRO/ObsRO eDiary review for itching, scratching, and sleep scores and compliance 
Study Day 28 and Day 84/Clinic Visits 4 and 6  
The following procedures and assessments will be performed: 
• Review of concomitant medications 


 
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• Skin exam (Section 10.2.5) and vital signs (Section 10.2.6) 
• PRO/ObsRO eDiary review for itching, scratching, and sleep scores and compliance  
• Clinical chemistry and hematology tests 
• s-BA (Section 9.2.1) 
• INR 
• Biomarkers (AFP and LDL-C; autotaxin and p-C4), at Visit 4 only 
• Urine pregnancy test for girls who have reached menarche 
• Patient/caregiver/clinician complete the PGIC and PGIS 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 56 and Day 126/Clinic or at-Home Visits 5 and 7 
Visits 5 and 7 may be conducted in the patient’s home by a study nurse.  The following 
procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• PRO/ObsRO eDiary review for itching, scratching, and sleep scores and compliance  
• Clinical chemistry and hematology tests 
• s-BA (Section 9.2.1) 
• Urine pregnancy test for girls who have reached menarche 
• AE monitoring 
• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 154/Clinic or at-Home Visit 8  
The following procedures and assessments will be performed: 
• Review of concomitant medications 
• Vital signs (Section 10.2.6) 
• PRO/ObsRO eDiary review for itching, scratching, and sleep scores and compliance 
• s-BA (Section 9.2.1) 
• INR 
• AE monitoring 


 
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• Evaluation of study drug compliance 
• Study drug is dispensed 
Study Day 168/Clinic Visit 9 
The last dose of study drug is taken in the morning the day before Visit 9, and the following 
procedures and assessments will be performed:  
• Review of concomitant medications 
• Physical examination (Section 10.2.5) and vital signs (Section 10.2.6) 
• PRO/ObsRO eDiary review for itching, scratching, and sleep scores and compliance 
• Clinical chemistry and hematology tests 
• s-BA (Section 9.2.1) 
• Serum IGF-1 growth marker 
• Biomarkers (AFP and LDL-C; autotaxin and p-C4) 
• Liver ultrasonography 
• Administer PedsQL questionnaire 
• Urine pregnancy test for girls who have reached menarche 
• Patient/caregiver/clinician complete the PGIC and PGIS 
• AE monitoring 
• Evaluation of study drug compliance 
7.1.4.3 
Follow-Up Period 
Study Day 196/Clinic Visit 10 
Patients not continuing into Study A4250-008 and those patients who are prematurely 
withdrawn, will return to the study site 28 days after Visit 9 or the last dose of study drug for 
the following assessments: 
• Review of concomitant medications 
• Skin exam (Section 10.2.5) and vital signs (Section 10.2.6) 
• PRO/ObsRO eDiary review for itching, scratching, and sleep scores and compliance 
• Clinical chemistry and hematology tests 
• s-BA (Section 9.2.1) 
• INR 
• Urine pregnancy text for girls who have reached menarche 
• AE monitoring 


 
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7.2 
Discussion of Study Design 
The results from Study A4250-003 have informed the current (A4250-005) study design. 
7.2.1 
Risk/Benefit and Ethical Assessment 
A4250 has been evaluated in 3 Albireo-sponsored clinical studies: 1 double-blind 
placebo-controlled study in healthy volunteers, 1 single-dose ADME study, and 1 Phase 2 
study in children with cholestatic pruritus.  In addition, an investigator-sponsored study has 
been conducted in patients with PBC. 
A total of 98 subjects/patients have been exposed to A4250.  Healthy subjects have been 
exposed to up to 10 mg as a single dose and up to 3 mg daily as part of a multiple-dose 
evaluation.  Children with cholestatic liver disease have been treated with up to 
0.2 mg/kg/day (200 µg/kg/day) for 4 weeks.  In total, 2 SAEs have been reported; both were 
judged by the physician to be not related to the study drug. 
Patients with cholestatic liver diseases suffer from excess bile acids in the liver resulting in 
tissue damage.  A commonly used treatment in these patients is bile diversion surgery, 
whereby approximately 50% to 100% of the enterohepatic circulation of bile acids is 
interrupted.  Inhibition of IBAT with A4250, thereby interrupting the enterohepatic 
circulation of bile acids, is therefore a potential medical alternative to surgery which could be 
of benefit to these patients if shown to be effective and safe.  
Based on the mode of action of an IBAT inhibitor, loose stools or diarrhea could be expected.  
However, in the pediatric Phase 2 study with 4 weeks daily treatment, only 1 patient had mild 
transient diarrhea after a single dose that did not recur on multiple dosing. 
7.2.2 
Early Termination 
This study may be terminated at any time by Albireo if serious side effects should occur, if 
the investigator does not adhere to the protocol; or if, in the sponsor's judgment, there are no 
further benefits to be achieved from the study.  In this event, Albireo/designee will inform the 
study investigators, institutions, and all regulatory authorities. 
Albireo may temporarily or permanently discontinue the study at an investigative site at any 
time for safety, ethical, compliance, or other reasons.  If this is necessary, Albireo will 
endeavor to provide advance notification to the site.  If a site or the study is suspended or 
discontinued, the investigator/investigative staff will be responsible for promptly informing 
the Independent Ethics Committee (IEC).  If required by local regulations, Albireo/designee 
will be responsible for informing the IEC and the regulatory authority of study or site 
discontinuation.  In such an event, all study data and unused study drug must be returned to 
Albireo. 
7.3 
Selection of Study Population 
A total of 60 patients with clinical diagnosis of PFIC 1 or 2 that has been genetically 
confirmed will be randomized.  


 
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An attempt will be made to enroll a minimum of 10 patients from each age category 
(6 months to 5 years, 6 to 12, and 13 to ≤18 years).  
A maximum of 5 patients that were treated in Study A4250-003 will be allowed to participate 
in this study. 
7.3.1 
Inclusion Criteria 
1. A male or female patient, with clinical diagnosis of PFIC 1 or 2, between the ages of 
≥6 months and ≤18 years at Visit 1 with a body weight above 5 kg. 
2. Patient must have genetic confirmation of PFIC-1 or PFIC-2 through identification of 
biallelic pathogenic variants in either the ATP8B1 or ABCB11 genes 
3. Patient must have elevated s-BA, specifically measured to be ≥100 µmol/L, taken as the 
average of 2 samples 7 to 28 days apart (Visits 1 and 2) prior to randomization 
4. Patient must have history of significant pruritus as indexed by caregiver-reported 
observed scratching in the eDiary, average ≥2 (on 0-4 scale) in the week prior to 
randomization 
5. Patient and/or legal guardian must sign ICF (and assent) as appropriate.  Patients who 
turn 18 years of age (or legal age per country) during the study will be required to 
re-consent in order to remain in the study 
6. Patients will be expected to have a consistent caregiver(s) for the duration of the study. 
7. Caregivers and age appropriate patients (≥8 years of age) must be willing and able to use 
an eDiary device as required by the study 
7.3.2 
Exclusion Criteria 
1. Patient with pathologic variations of the ABCB11 gene that predict complete absence of 
the BSEP function 
2. Patient with past medical history or ongoing presence of other types of liver disease 
including, but not limited to, the following: 
a) Biliary atresia of any kind 
b) Benign recurrent intrahepatic cholestasis (BRIC), indicated by a history of normal 
s-BAs 
c) Suspected or proven liver cancer or metastasis to the liver on imaging studies 
d) Histopathology on liver biopsy is suggestive of alternate non-PFIC related etiology of 
cholestasis 
3. Patient with a past medical history or ongoing presence of any other disease or condition 
known to interfere with the absorption, distribution, metabolism (specifically bile acid 
metabolism), or excretion of drugs in the intestine, including but not limited to, 
inflammatory bowel disease 


 
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4. Patient with past medical history or ongoing chronic (i.e., >3 months) diarrhea requiring 
intravenous fluid or nutritional intervention for treatment of the diarrhea and/or its 
sequelae 
5. Patient has a confirmed past diagnosis of infection with human immunodeficiency virus 
(HIV) or other present and active, clinically significant, acute, or chronic infection, or 
past medical history of any major episode of infection requiring hospitalization or 
treatment with parenteral anti-infective treatment within 4 weeks of treatment start (Study 
Day 1) or completion of oral anti-infective treatment within 2 weeks prior to start of 
Screening Period. 
6. Any patient with suspected or confirmed cancers except for basal cell carcinoma, and 
non-liver cancers treated at least 5 years prior to Screening with no evidence of 
recurrence 
7. Patient with a past medical history of chronic kidney disease with an impaired renal 
function and a glomerular filtration rate (GFR) <70 mL/min/1.73 m2  
8. Patient with surgical history of disruption of the enterohepatic circulation excluding those 
who have undergone a successful reversal procedure that has permanently restored flow 
of bile acids from the liver to the duodenum 
9. Patient has had a liver transplant or listed for liver transplant 
10. Decompensated liver disease, coagulopathy, history or presence of clinically significant 
ascites, variceal hemorrhage, and/or encephalopathy 
11. INR >1.4 (the patient may be treated with Vitamin-K intravenously, and if INR is <1.4 at 
resampling the patient may be included) 
12. ALT >15 × upper limit of normal (ULN) 
13. ALT >20 × ULN at any time point during the last 6 months 
14. Serum bilirubin >5 × ULN 
15. Patient suffers from uncontrolled, recalcitrant pruritic condition other than PFIC.  
Examples include, but not limited to, refractory atopic dermatitis or other primary pruritic 
skin diseases 
16. Any patient who is pregnant or lactating or who is planning to become pregnant within 
24 weeks of randomization 
17. Sexually active females who are not using a reliable contraceptive method with ≤1% 
failure rate, such as barrier protection, hormonal contraception, intrauterine device, or 
abstinence) throughout the duration of the study 
18. Patient with a past medical history of alcohol or substance abuse will be excluded.  
Patient must agree to refrain from illicit drug and alcohol use during the study 


 
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19. Administration of bile acid or lipid binding resins within 30 days prior to baseline/Day 0 
and throughout the trial 
20. Patient has had investigational exposure to a drug, biologic agent, or medical device 
within 30 days prior to screening, or 5 half-lives of the study agent, whichever is longer 
21. Any other conditions or abnormalities which, in the opinion of the investigator or medical 
monitor, may compromise the safety of the patient, or interfere with the patient 
participating in or completing the study 
7.3.3 
Withdrawal of Patients 
Patients/caregivers will be informed that they have the right to withdraw from the study at 
any time, without prejudice to their medical care, and that they are not obliged to state their 
reasons. 
Any withdrawal, and reasons for withdrawal, must be fully documented in the eCRF and 
source documents and the patient followed by the investigator/investigative staff.  Even after 
the study is completed at the follow-up visit, the investigator/investigative staff will, 
regardless of reason for withdrawal, record any patient data they receive concerning SAEs, 
and all drug-related non-serious AEs, and subsequently report these to Albireo.  Withdrawn 
patients will not be replaced. 
Patients will be withdrawn in the following circumstances: 
• A patient’s/caregiver’s desire for withdrawal for any reason 
• Lost to follow-up (every effort must be made to contact the patient/caregiver; a certified 
letter must be sent) 
• An AE that, in the opinion of the investigator, necessitates withdrawal 
• A worsening of the symptoms related to diarrhea occurs 
• Death 
• Patient/caregiver estimation of lack of efficacy 
• A patient’s/caregiver’s substantial noncompliance (eDiary and study drug compliance) or 
protocol violation 
• An investigator’s opinion that continuing the patient in the study is not appropriate.  The 
investigator may withdraw a patient at any time, if it is considered to be in the patient’s 
best interest 
A patient who prematurely withdraws due to lack of efficacy, and has completed at least 
12 weeks treatment in the double-blind study, will be offered the opportunity to enter 
open-label Study A4250-008.  A patient who withdraws from treatment prematurely and will 
not enter Study A4250-008 will be requested to complete all visits and safety assessments per 
the current protocol (A4250-005).  As a minimum, patients will have end of treatment 


 
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(Visit 9) assessments at the time of withdrawal and a follow-up assessment (Visit 10) 28 days 
following the last dose of study drug, and the patient will be recorded as withdrawn. 


 
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8 
TREATMENT OF PATIENTS 
8.1 
Identity of Study Drug 
A4250 and placebo will be supplied as capsules for oral administration.  White opaque 
capsules filled with pellets containing A4250 or placebo will be provided.  Two different 
capsule sizes will be available, as follows: 
• Capsule size 0 that can be opened 
• Capsule size 3 that can be opened or swallowed intact 
The content of the A4250 and placebo capsules will be identical in appearance.  Capsule 
filling weight will also be identical for high dose, low dose, and placebo.  Bottles with 
34 capsules will be given to the patient at each visit.  A patient who requires 2 or more 
capsules per day will be given multiple bottles. 
8.2 
Administration of Study Drug 
Patients will be dosed with A4250 at a dose of 40 µg/kg/day, 120 µg/kg/day, or placebo for 
24 weeks.  Study drug will be dispensed to the patient at Visits 3, 4, 5, 6, 7, and 8, together 
with instructions on how to store and take the drug.  Study drug administration data, 
including whether each patient took each dose or partial doses of study drug, whether there 
were any delayed or missed doses, and whether the capsule was opened or swallowed whole 
will be documented through the eDiary and transferred to the study database. 
A4250 should be taken in the morning, together with food.  On clinic visits days when 
laboratory assessments are conducted (Visits 4, 5, 6, 7, and 9) study drug should be taken 
after the visit and after laboratory samples are taken.  Patients should not crush or chew the 
capsule(s).  When swallowing the capsule intact, the patient should administer the dose with a 
glass of water. 
For a patient unable to swallow capsule intact, the capsule can be opened and the contents 
sprinkled and mixed in a small amount of soft room-temperature yogurt, apple sauce, or fruit 
purée.  The amount of food should be less than what the patient/infant is expected to eat and 
should be followed by water.  
1. Hold the capsule over the prepared food, gently twist open and allow the content to fall 
into food 
2. Mix the entire capsule content with 1 or 2 tablespoons (15 to 30 mL) of soft food (apple 
sauce, fruit purée, or yogurt) 
3. Administer the mixture immediately after mixing. 
The number of capsules provided to the patient will be based on body weight at Visit 3. At 
Visit 6, if a patient’s weight has shifted to another weight category (identified in Table 3), the 
number of capsules per day may be adjusted.   
 


 
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Table 3 
Dosing and Capsule Strength 
Body Weight 
(kg) 
Capsule 
Size 
Number of 
Capsules 
per Day 
Capsule 
Strength, 
Low Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Strength, 
High Dose 
(µg) 
Total 
Dose (µg) 
Capsule 
Size, 
Placebo 
(µg) 
5.0 to <7.5 
0 
1 
200 
200 
600 
600 
0 
7.5 to <12.5 
0 
2 
200 
400 
600 
1200 
0 
12.5 to <17.5 
0 
3 
200 
600 
600 
1800 
0 
17.5 to <19.5 
0 
4 
200 
800 
600 
2400 
0 
19.5 to <25.5 
3 
2 
400 
800 
1200 
2400 
3 
25.5 to <35.5 
3 
3 
400 
1200 
1200 
3600 
3 
35.5 to <45.5 
3 
4 
400 
1600 
1200 
4800 
3 
45.5 to 55.5 
3 
5 
400 
2000 
1200 
6000 
3 
>55.5 
3 
6 
400 
2400 
1200 
7200 
3 
8.3 
Study Treatment Packaging and Labeling 
8.3.1 
Packaging and Labeling 
The capsules will be packed in high-density polyethylene (HDPE) containers, with 
child-proof polypropylene caps.  Study drug capsules containing A4250 in the strength 
specified in Table 3 above will be manufactured. 
Packaging and labeling will be prepared to comply with applicable regulatory requirements. 
8.3.2 
Storage 
Treatment packs containing A4250 capsules should be stored and dispensed in accordance 
with regulations in their original containers.  The storage facility at the investigative site 
should be locked and the storage temperature should be between 15°C and 25°C. 
Patients/caregivers should be informed of appropriate storage conditions (i.e., room 
temperature, between 15°C and 25°C). 
Any deviations from the recommended storage conditions should be immediately reported to 
Albireo and the study drug should not be used until authorization has been given by Albireo. 
8.3.3 
Blinding and Randomization of Study Drug 
A double-blind design is employed so that both the investigators and the patients will be 
unaware of the treatment assignment during the study.  
After written informed consent is obtained from an eligible patient, an 8-digit patient 
identification number will be assigned.  The first 2 digits will denote the country, followed by 
a 3-digit site number and a 3-digit patient sequence number.  This number will be created and 
allocated by the Interactive Web Response System (IWRS), when the patient first enters the 
system at screening.  The randomization codes will be computer-generated by 
 
Biostatistics, and kept by a statistician independent from the project team.  The 


 
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randomization will be done in blocks and stratified according to PFIC class (Type 1 and 2) 
and age class (6 months to 5 years, 6 to 12 years, and 13 to ≤18 years) to ensure approximate 
balance between dose schemes (1:1:1).  A separate randomization list will be prepared for the 
maximum 5 patients from Study A4250-003 regardless of stratification.  Randomization 
codes will be assigned sequentially as patients become eligible for randomization. 
Patients who are eligible for randomization will be assigned a unique 4-digit randomization 
number by IWRS.  This randomization number identifies which treatment will be allocated to 
the patient.  Patients who withdraw from the study after randomization visit are not to be 
replaced and their randomization number will not be reused.   
To ensure blinding, the study drug and the matching placebo have the same shape and size.  
Patients will receive capsule(s) of A4250 according to their dose group or capsule(s) of 
matching placebo once a day during the double-blind Treatment Period.  Labels on the study 
drug containers will not identify the treatment that a patient is randomized to.  Traceability of 
the treatment is ensured by the study drug number. 
The 5-digit study drug number will identify study drug packs and will be detailed on the 
study drug label.  Dispensing of study drug will be coordinated by IWRS.  
The system will assign a study drug number corresponding to the randomization arm.  The 
randomization number will be used in the background only to ensure no unblinding and will 
not be displayed to end-users in the IWRS. 
8.4 
Procedure for Breaking the Randomization Code 
Should a situation arise where unblinding is urgently required (i.e., knowledge of treatment 
codes is required to adequately manage a life-threatening situation), the investigator at that 
study site may perform immediate unblinding through IWRS without the need for 
communication with the 
 Medical Monitor. 
The study site investigator and appropriate 
 project team members will be authorized to 
access the emergency unblinding functionality within the IWRS.  The system will require the 
user to enter an authorization key number to complete the emergency unblinding transaction.  
The exact description of the treatment assigned to the individual patient then will be 
accessible.  Emergency unblinding can thus be made for any patient without affecting the 
double-blind nature of the study.  Patient treatment information may only be accessed in the 
event of an emergency and out of necessity to know the identity of the allocated study drug in 
order to institute appropriate therapeutic management.  The investigator should make every 
effort to discuss the rationale for emergency unblinding with the 
 Medical Monitor 
prior to unblinding the individual patient.  Once the randomization code is broken for a 
patient, he/she must be withdrawn from the study and all assessments and procedures at 
Visit 9 should be performed.  As a follow-up, Visit 10 will be performed according to the 
time schedule.  In the event that emergency unblinding is performed, the investigator can 
view and must print the unblinding confirmation document from IWRS.  The investigator 
must record on the confirmation document printout the reason for the emergency unblinding, 


 
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and sign the document.  The confirmation document must then be kept in a safe place until 
the end of the study.  Once a randomization code has been broken, the investigator must 
inform the 
 Medical Monitor in writing within 24 hours. 
8.5 
Patient Compliance 
The study nurse will monitor eDiary compliance by daily review of the CRF health website.  
If both diary entries on a day are missing during the baseline week or Week 24, the study 
nurse will call the patient/caregiver to remind them to complete all scheduled entries.  Any 
noncompliance will be documented and explained in the source documents and in the eCRF. 
Study drug compliance will also be checked through diary responses.  Additionally, patients 
will return all unused study drug at Visits 4, 5, 6, 7, 8, and 9.  The study site staff will count 
all returned drug, assess compliance, and record details in the eCRF. 
Treatment Compliance = 100 × ((Number of study drug dispensed – number of study drug 
returned)/number of study drug that should be taken). 
Treatment compliance between 80% and 120% will be acceptable. 
8.6 
Study Drug Accountability 
Records shall be maintained of the delivery of study treatment(s) to the study site(s), of the 
inventory at the study site(s), of each use of the study treatment(s) for each patient, and of the 
return of unused study treatment(s) to Albireo. 
These records shall include dates, quantities, batch numbers, expiry dates, and the unique 
code numbers assigned to the study drug and to the study patients. 
The investigator will be responsible for ensuring that the records adequately document that 
the patients were provided the quantities specified in the protocol and that all study drug 
received from Albireo is reconciled.  
8.7 
Concomitant Therapy and Prohibited Medications 
The investigator will note all ongoing medication and any medication recently (within 
1 month) stopped in the eCRF at Visit 1.  At Visits 2 through 10, all changes in medication 
(stopping or starting new medication and changes in dose) will be recorded in the eCRF.  All 
treatment for pruritus (prescribed or not) and changes in the treatment will also be recorded. 
All medications taken by a patient within 1 month prior to the first intake of study drug are 
regarded as prior medication. 
All medications taken by a patient on or after the first intake of study drug, and which 
continue to be taken during the study, are regarded as concomitant medication.  
Prohibited medications are listed in Appendix 1. 
 


 
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9 
ASSESSMENT OF EFFICACY 
9.1 
Efficacy Endpoints 
9.1.1 
Primary Efficacy Endpoint 
The primary efficacy endpoints are as follows: 
• Europe (EU) and rest of the world (RoW): Proportion of patients experiencing at 
least a 70% reduction in fasting s-BA levels from baseline to the end of treatment or 
reaching a level ≤70 µmol/L compared to placebo 
Fasting s-BA baseline will be calculated as the average of the value at randomization and the 
previous screening (Visit 2).  The end value is the average of the values at Weeks 22 and 24 
after the start of double-blind treatment. 
• United States (US): Change in pruritus, as indexed by caregiver-reported observed 
scratching score from baseline to Week 24 as measured by the average daily score of 
the Albireo ObsRO instrument (Appendix 2) compared to placebo.  The average daily 
score will be calculated using the 7 days pretreatment for baseline, and the last 7 days 
of treatment for Week 24 for the end value 
9.1.2 
Secondary Efficacy Endpoint 
Secondary efficacy endpoints include the following:  
• EU and RoW: Change in pruritus, as indexed by caregiver-reported observed 
scratching score as measured by the average daily score of the Albireo ObsRO 
instrument (Appendix 2) compared to placebo.  The average daily score will be 
calculated using the 7 days pretreatment for baseline, and the last 7 days of treatment 
for Week 24 
• US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA 
levels from baseline to the end of treatment or reaching a level ≤70 µmol/L compared 
to placebo 
• All Regions: 
• All secondary endpoints are compared to placebo 
o Change from baseline to Week 24 in fasting s-BA 
o Change from baseline to Week 24 in patient-reported itch severity as 
measured by the average daily score of the Albireo PRO instrument 
(Appendix 2; only patients ≥8 years of age will complete the PRO instrument)  
o Change from baseline to Week 24 in ALT 
o Change from baseline to Week 24 in IGF-1  
o Proportion of patients achieving meaningful reduction in caregiver-reported 
observed scratching at Week 24 


 
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o Change from baseline to Week 24 in sleep parameters measured with the 
Albireo PRO and ObsRO instruments  
o Number of patients undergoing biliary diversion surgery or being listed for 
liver transplantation 
o Change from baseline to Week 24 in PedsQL (Appendix 4) 
9.1.3 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are compared to placebo and include the following: 
• Proportion of patients achieving meaningful reduction in patient-reported itch severity at 
Week 24 
• Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, 
caregiver, and clinician via PGIC items (Appendix 3) 
• Change from baseline in fasting s-BA at Weeks 4 and 12 
• Change from baseline in patient-reported itch severity and observer-reported scratching at 
Weeks 4 and 12 
• Change from baseline to Week 24 in patient-reported and observer-reported night time 
itching and scratching severity scores respectively 
• Change from baseline to Week 24 in pooled pruritus score including observer-reported 
scratching for patients <8 years of age and patient-reported itch severity for patients 
≥8 years of age 
• Change from baseline to Week 24 in additional patient-reported and observer-reported 
sleep parameters (e.g., tiredness, number of awakenings) 
• Change from baseline in ALT at Weeks 4 and 12 
• Change from baseline in total bilirubin at Weeks 4, 12, and 24 
• Change from baseline in AST at Weeks 4, 12, and 24 
• Change from baseline for other biochemical markers and measures of bile acid synthesis 
(autotaxin, p-C4, LDL-C) at Week 24 
• Change in pediatric end-stage liver disease/model for end-stage liver disease 
(PELD/MELD) score, AST to platelet ratio index (APRI) score, and Fib-4 score from 
baseline to Week 24 
• Liver-related mortality and liver decompensation events 
• All-cause mortality 


 
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9.2 
Efficacy Assessments 
9.2.1 
Serum Bile Acids 
Blood samples for analysis of fasting total s-BA will be drawn at all visits (in clinic and at 
patient’s home).  Patients will fast for at least 4 hours prior to the taking of samples for s-BA, 
except for water intake.  However, less than a 4-hour fast prior to the blood draw will be 
accepted in infants who are unable to fast for 4 hours.  Samples will be treated and 
transported to a central laboratory per instructions in the laboratory manual. 
9.2.2 
Itching and Sleep Score 
Itching, observed scratching, and sleep disturbance will be assessed twice each day via 
eDiary.  Patients and/or caregivers will be instructed to complete the eDiary every day in the 
morning after the patient wakes and in the evening just before the patient goes to sleep 
throughout the study.  Patients and caregivers will receive a formal training session during 
Visit 1.  Training materials describing the ObsRO and PRO items are included in 
Appendix 2. 
The eDiary includes ObsRO and PRO items.  Patients <8 years of age will not be asked to 
complete the PRO items; only the ObsRO will be completed by caregivers of patients in this 
age group.  Older patients, 8 to 17 years of age, will complete the PRO items and the 
caregiver will complete the ObsRO items.  The PRO items assess severity of itch, aspects of 
sleep disturbance (morning diary only), and tiredness.  For patients 8 to 12 years of age, the 
caregiver will read the PRO items along with the child and record the child’s response.  A 
guide will be provided to the caregivers that provides standardized explanations of the PRO 
items, in case the patient is confused or requires clarification about the meaning of a question.  
The ObsRO items assess severity of observed scratching, aspects of observed sleep 
disturbance (morning diary only), and observed signs of tiredness (evening diary only).  The 
ObsRO and PRO scratching and itch severity items use 0 to 4 response scales, where each 
response is distinguished by a unique facial expression, verbal anchor, number, and color 
code (Appendix 2).  
A daily score for the ObsRO scratching item will be calculated by selecting the maximum 
scratching severity score from the 2 ratings for each day.  A daily score will be considered 
missing if both of the daily assessments are missing.  A weekly score will be calculated by 
averaging the daily scratching scores.  A weekly score will be considered missing if >4 out of 
7 days of data are missing.  The same approach will be used to calculate a patient-reported 
itch severity score. 
Additionally, patients/caregivers will be requested to report in the diary the date and time that 
each dose of study drug was administered during the Treatment Period.  Details of the 
questions included in the diary, including the PRO/ObsRO tool used in the study, are located 
in Appendix 2. 


 
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9.2.3 
Growth and IGF-1 
Growth will be measured as height (velocity) and weight (z-score) using a certified weight 
scale (i.e., stadiometer). 
Blood samples for IGF-1 will be drawn at randomization (Visit 3), and at end of treatment 
(Visit 9).  Samples will be handled and transported to a central laboratory according to 
instructions in the laboratory manual. 
9.2.4 
Quality of Life (PedsQL) Questionnaire 
Patients will be asked to fill out a QoL questionnaire (PedsQL) at Visit 3 and Visit 9.  Details 
of the questions included on the questionnaire are provided in Appendix 4. 
9.2.5 
Biomarkers and Other Laboratory Samples 
Blood samples for AFP and LDL-C will be drawn at Visits 2, 4, and 9.  Blood samples for 
autotaxin and p-C4 will be drawn at Visits 3, 4, and 9 (only for children with body 
weight ≥10 kg).  Samples will be handled and transported to a central laboratory per 
instructions in the laboratory manual. 
9.2.6 
PELD/MELD Score 
At randomization and end of treatment, PELD score will be calculated for children up to 
12 years of age.  For children 13 years or older, the MELD score will be calculated. 
PELD Score = 4.80 * ln(bilirubin in mg/dL) + 18.57 * ln(INR) - 6.87 * ln(albumin in g/dL) + 
4.36 (if patient <1 year: scores for patients listed for liver transplantation before the patient's 
first birthday continue to include the value assigned for age (<1 year) until the patient reaches 
the age of 24 months) + 6.67 (if the patient has growth failure [≤2 standard deviation]) 
Laboratory values less than 1.0 will be set to 1.0 for the calculation of the PELD score. 
MELD Score = 9.57 * ln(creatinine in mg/dL) + 3.78 * ln(bilirubin in mg/dL) + 
11.2 * ln(INR) + 6.43 
Laboratory values less than 1.0 will be set to 1.0 and serum creatinine values greater than 
4.0 mg/dL will be set to 4.0 for calculation of the MELD score. 
9.2.7 
Liver Ultrasonography 
Ultrasonography of liver and spleen will be performed at randomization (Visit 3) and at end 
of treatment (Visit 9).  Liver and spleen size, masses, and nodules should be recorded. 
9.2.8 
Markers of Fibrosis 
APRI score and Fib-4 score will be calculated at randomization and at end of treatment. 
APRI = [(AST in U/L) / (AST ULN in U/L)] / (Platelets in 109/L) 
Fibrosis 4 Score = (Age*AST in U/L) / (Platelets in 109/L * √(ALT in U/L). 


 
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9.2.9 
Global Impression of Change and Global Impression of Symptom Measures 
Patients, caregivers, and clinicians will complete the patient global impression of change 
(PGIC) and the patient global impression of symptom (PGIS) measures (Appendix 3) at 
randomization (Visit 3), Visits 4 and 6, and at end of treatment (Visit 9). 
The PGIC items assess change in itch (patient version), scratching (caregiver and clinician 
versions), and sleep (all versions) since starting the study medication.  The PGIS items assess 
itch (patient version), scratching (caregiver and clinician versions), and sleep (all versions) in 
the past week.  Caregivers and clinicians will complete the PGIC and PGIS for all patients; 
those patients ≥8 years of age will complete the patient version. 


 
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10 
ASSESSMENT OF SAFETY 
The timing and frequency of safety assessments are described in Section 7.1.4 and in Table 1 
above. 
The primary safety analysis for this study will include the incidence of treatment-emergent 
adverse events (TEAE), study drug exposure compared to placebo, all safety assessments 
including AEs from patient reports, and the patient diaries.  The analysis includes causality, 
severity, and seriousness assessments made by the investigator. 
Trends in safety will also be assessed for the following assessments: 
• Physical examinations 
• Concomitant medications at all visits 
• Vital signs at all clinic visits 
• Laboratory test results (including hematology, clinical chemistry, liver monitoring, and 
urinalysis) 
• SAEs including the description, incidence, severity, and causality from signing the ICF to 
completion of the study 
• Discontinuations due to AEs 
10.1 
Adverse Events 
10.1.1 
Definitions and Investigator Assessments 
An AE is defined as any untoward medical occurrence in an enrolled patient regardless of 
causal relationship with study drug.  An AE can therefore be any clinically significant 
unfavorable and unintended sign, symptom, or disease that occurs once a patient is enrolled 
(ICF is signed) in the study until the patient is discharged from the study, whether or not 
related to the study drug. 
10.1.1.1 
Clinical Significance 
Clinical significance is defined as any abnormal finding that results in further clinical 
investigation(s), treatment(s), or the diagnosis of new condition.  Patient-reported events and 
protocol-mandated laboratory values, vital signs, and physical examination findings can be 
considered clinically significant (i.e., an AE) if there is a deterioration as compared to 
baseline.  Examples of clinically significant worsening from baseline could include, but is not 
limited to, events causing withdrawal from the study and events requiring medical 
intervention outside of the study causing apparent clinical manifestations, or judged relevant 
by the investigator.   
10.1.1.2 
Serious Adverse Events 
Serious criteria are applied by the investigator to each AE as specifically defined below.  
These criteria are used to determine whether an AE is serious or non-serious.  The assessment 


 
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is made independently of severity assessment (Section 10.1.1.3).  For example, the 
development of a severe rash that occurs after signing of informed consent may not meet 
serious criteria as defined below and therefore would be considered a severe, non-serious AE. 
Any AE that meets any 1 of the following 6 criteria is considered an SAE: 
• The outcome of the AE is death 
• The AE is immediately life-threatening.  Life-threatening means that the patient is, in the 
opinion of the investigator, at immediate risk of death from the reaction as it occurred.  
This does not include an AE that, if more severe, might have caused death 
• The AE results in persistent or significant disability/incapacity.  Disability means a 
substantial disruption of a person's ability to conduct normal life functions 
• The AE requires or prolongs hospitalization  
• The AE results in  a congenital anomaly/birth defect 
• The AE is an important medical event.  Important medical events may meet serious 
criteria should the investigator assess that they may significantly jeopardize the patient, 
represent a significant hazard, or requires medical/surgical intervention to prevent one of 
the outcomes listed above.  Examples of potential SAEs based on this criteria include, but 
are not limited to, allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions even if they do not result in inpatient 
hospitalization, or the development of drug dependency and drug abuse. 
10.1.1.3 
Severity Assessment 
Severity assessments are based on the intensity of the event in relation to expectation.  The 
investigator will assess the intensity of AEs based on the following definitions: 
• Mild (awareness of sign or symptom, but easily tolerated) 
• Moderate (discomfort sufficient to cause interference with normal activities) 
• Severe (incapacitating, with inability to perform normal activities) 
Severity is a measure of intensity where seriousness is defined by the criteria outlined in 
Section 10.1.1.2.  An AE of severe intensity need not necessarily be considered serious.  For 
example, nausea that persists for several hours may be considered severe nausea, but not meet 
serious criteria, and therefore would be assessed as a severe AE but not an SAE. 
10.1.1.4 
Causality Assessment 
The investigator determines the causality of all AEs to the study drug using medical judgment 
and considering all relevant factors such as (but not limited to) the underlying study 
indication, coexisting disease, concomitant medication, relevant history, pattern of the AE, 
temporal relationship to the study medication, and de-challenge or re-challenge.  The 
causality assessment of the AE/SAE is to be made as follows. 
Related to study drug (i.e., possibly, probably, or definitely related) 


 
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Based on medical judgment, there is at least a reasonable possibility that the study drug 
caused the event; one or more of the following criteria apply: 
• The event follows a reasonable temporal sequence from administration of study drug 
• The event could not be reasonably attributed to the known characteristics of the patient's 
clinical state, environmental or toxic factors, or other modes of therapy administered to 
the patient 
• The event follows a known pattern of response to study drug 
• The event disappears or decreases on cessation or reduction in dose of the study drug.  (It 
should be noted that in some situations an AE will not disappear or decrease in intensity 
upon discontinuation of study drug despite other clear indications of relatedness) 
• The event reappears or worsens when the study drug is re-administered 
Unrelated to study drug (i.e., unlikely, probably not, or definitely not related) 
Based on medical judgment there is no reasonable possibility that the study drug caused the 
event; one or more of the following criteria apply: 
• The event does not follow a reasonable temporal sequence from administration of study 
drug 
• The event could be reasonably attributed to the known characteristics of the patient's 
clinical state, concurrent illness, environment or toxic factors, or other modes of therapy 
administered to the patient 
• The event does not follow a known pattern of response to study drug 
• The event does not disappear or decrease on cessation or reduction in dose of the study 
drug, and it does not reappear or worsen when the study drug is re-administered 
10.1.2 
Recording of Adverse Events 
It is the investigator’s responsibility to assess whether each untoward event is a clinically 
significant worsening from baseline, thereby considered an AE.  For all AEs, the severity, 
seriousness, and causality to study drug for each AE as outlined in Section 10.1.1 will be 
assessed and recorded in the eCRF. 
All serious and non-serious AEs are collected once the caregiver/patient has signed the ICF 
and until the post-treatment follow-up (Visit 10), 28 days after the final dose of study drug or 
up until premature discontinuation. 
Any AEs or SAEs that are unresolved at the patient’s last AE assessment in the study are 
followed up by the investigator for as long as medically indicated, but without further 
recording in the CRF.  Albireo retains the right to request additional information for any 
patient with ongoing AE(s) or SAE(s) at the end of the study. 


 
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Investigators must follow patients with AEs and SAEs until the event has resolved or the 
condition has stabilized.  If the patient dies, this should be captured as the outcome of the AE 
unless no link can be established, in which case death will be reported as a separate event.  
If a patient is lost to follow-up, this should be captured accordingly within the AE eCRF and 
on a follow-up SAE report for tracking purposes. 
TEAEs are defined as any AE that occurs after randomization (Day 0).  All AEs that occur in 
the Screening Period, i.e., after enrollment and prior to randomization, will be collected on 
the eCRF as non-treatment emergent AEs. 
If there is a clinically significant deterioration of a laboratory value/vital sign or other routine 
study assessment that is associated with a diagnosis, the clinical diagnosis will be reported as 
an AE and the associated signs and symptoms will be considered additional information 
unless the sign or symptom is more severe than expected given the diagnosis.  For example, if 
an investigator diagnoses a study patient with hepatitis during the study period, hepatitis 
would be considered the AE and the concomitant signs and symptoms of abdominal pain, 
vomiting, and elevated ALT and AST would not be reported separately unless, in the opinion 
of the investigator, one of these signs or symptoms is more severe than expected and 
therefore a separate AE assessment is indicated. 
10.1.3 
Recording and Reporting of Serious Adverse Events 
Every SAE (regardless of severity and causality) that occurs once the caregiver/patient has 
signed the ICF and until the post-treatment follow-up (Visit 10), and 28 days after the final 
dose of study drug, should immediately and not later than within 24 hours of knowledge of 
the event, be reported by the investigator or delegate in the eCRF SAE Report Form.  The 
clinical research organization (CRO) pharmacovigilance manager (PVM) should at the same 
time be notified of the event by e-mail.  A confirmation of receipt must be sent to the 
investigator within 1 working day.   
Report of a SAE must include at least the following information: 
• Patient identification information (study number, site number, initials, and date of birth) 
• The last study drug administration date 
• The diagnosis of the event with the description (or a brief description of 
signs/symptoms/clinical course if the diagnosis is not available) and the date of onset 
• The action used to treat the event (i.e., treatment medications, temporary discontinuation) 
• The reason(s) for considering the event serious 
• The relationship of the event to the study drug or to the study procedure (e.g., the 
investigator's assessment of causality) 
• A brief narrative of the SAE 


 
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Follow-up reports including all new information obtained of the subsequent course of the 
SAE must be prepared and the information collected in the SAE eCRF submitted to the CRO 
PVM by e-mail within 24 hours of knowledge of the event. 
The PVM may contact the investigator to obtain further information on a reported SAE.  The 
investigator/investigative staff must respond to any request for follow-up information or 
answers to questions regarding the SAE within the same timelines as for initial reports. 
The PVM reports the occurrence of the SAE and follow-up to the Albireo medical monitor 
for medical assessment of the case. 
Should an outcome of death occur within the study period or within 60 days after the last 
administration of study drug, an AE form and an SAE form should be completed, detailing 
the AE that resulted in the death.  (Note: death is an outcome, not an event.) 
The report should contain a comment regarding the co-involvement of progression of disease, 
if appropriate, and should assign main and contributory causes of death. 
All SAE reports must be e-mailed to the following number within 24 hours: 
 
 
If email is unavailable, SAEs may be transmitted via fax to the following number: 
 
In a study-related medical emergency situation, when the assigned medical monitors for the 
study cannot be reached by a caller, an on call physician can be reached 24 hours per day, 
7 days per week, via the 
 call center.  The following number is a chargeable telephone 
number allowing a global reach from both landlines and mobile phones and the internet page 
is a list of country-specific toll-free telephone numbers that is provided.  It should be noted 
that not all countries globally have access to toll-free numbers as indicated on the 
“24/7 Medical Help desk” index.  Countries without toll-free numbers need to dial the 
chargeable number as indicated above.  Furthermore, toll-free numbers might not be available 
from mobile phones. 
 
 
The 
 representative will work with the investigator to compile all the necessary 
information and ensure that the appropriate sponsor representative receives a report within 
1 day (24 hours) for all fatal and life-threatening cases and within 5 days for all other SAEs. 
10.1.4 
Reporting of Suspected Unexpected Serious Adverse Reactions 
Suspected unexpected serious adverse reaction (SUSAR) is an SAE that occurs in a patient 
due to exposure to an investigational product that may or may not be dose-related and is 
unexpected per sponsor assessment. 


 
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For this clinical study there is an established reporting system for SUSARs (definitions 
cf. Directive 2001/20/EC), which shall ensure that all relevant information about SUSARs is 
recorded and reported to the competent authority and the Ethics Committee according to the 
requirements of Article 17(1)(a), (b) and (d) of Directive 2001/20/EC, i.e., in the case of fatal 
or life-threatening SUSARs as soon as possible, and in any case no later than 7 days with 
relevant follow-up information within an additional 8 days, and all other SUSARs to be 
reported as soon as possible but within a maximum of 15 days of first knowledge by the 
sponsor.  The investigator will be kept informed by Albireo without undue delay.  The setup 
of the SUSAR reporting system is described in written standard operating procedures to 
ensure compliance with the necessary quality standards at every stage of reporting and 
follow-up, which is in line with the communication from the commission: detailed guidance 
on the collection, verification, and presentation of AE/reaction reports arising from clinical 
trials on medicinal products for human use (CT-3: O.J. 2011/C 172/01 of 11.6.2011). 
It is the sponsor’s responsibility to report SUSARs to appropriate regulatory authorities and 
ethics committees, which cumulatively have a reasonable possibility of a causal relationship 
to the investigational product, are serious, and are unexpected.  The minimum information 
will include the valid European Union drug regulatory agency clinical trial (EUDRACT) 
number, study number, the identifiable coded patient, the identifiable reporter, the SUSAR, 
the investigational product (including active substance name and code), and the causality 
assessment. 
Reporting of SUSARs as individual case safety reports (ICSR), including the required 
administrative information, will be performed through the Eudravigilance Clinical Trial 
Module (EVCTM) to the competent authorities in Europe and through the FDA Adverse 
Event Reporting System and ICH E2B(R3) message standard.  The Ethics Committee and all 
investigators involved in this study will be informed according to national and international 
requirements.  Albireo may delegate the SUSAR reporting to 
 
10.2 
Laboratory Values/Vital Signs/Physical Examinations and Other Safety 
Assessments 
10.2.1 
Laboratory Assessments 
Blood samples will be drawn for clinical chemistry, high sensitivity c-reactive protein 
(hsCRP), international normalized ratio (INR), and hematology analyses as shown in Table 1.  
Blood will be drawn with patient in a fasting state for ≥4 hours.  Samples will be handled and 
transported to a central laboratory per instructions in the laboratory manual. 
Routine laboratory parameters are listed in Table 4. 
The observed values will be recorded and assessed as “normal” or “abnormal not clinically 
significant” or “abnormal clinically significant”. 
The total amount of blood drawn during the whole study period is estimated to be 
approximately 59 mL (11 mL for children with body weight <10 kg over a 4-week period; 
Table 2 above).  Additional blood may also be drawn at unscheduled visits for bile acids if 


 
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needed due to analysis failure; if other safety blood samples are needed due to follow-up of 
an abnormal value or analysis failure, the blood volume will increase accordingly.  The blood 
samples collected for safety laboratory analysis will be destroyed after the analyses have been 
completed.  
Table 4 
Routine Laboratory Parameters 
Clinical Chemistry 
Hematology 
Urinalysis 
• 
Albumin 
• 
ALT 
• 
Alkaline phosphatase  
• 
AST 
• 
Bilirubin – total and conjugated 
• 
Calcium 
• 
Chloride 
• 
Creatinine 
• 
Creatine kinase 
• 
Gamma-glutamyl transferase 
• 
Potassium 
• 
Sodium 
• 
Hematocrit 
• 
Hemoglobin 
• 
Platelet count 
• 
Red blood cell count 
• 
White blood cell count 
• 
Blood 
• 
Glucose 
• 
Ketones 
• 
Leukocytes 
• 
Nitrites 
• 
pH 
• 
Protein 
A serum pregnancy test will be performed at Visit 2, and a urine pregnancy test will be 
collected at all other visits for girls who have reached menarche.  
10.2.2 
Liver Monitoring 
Strategies to monitor markers of liver disease throughout the study are outlined below where 
the ULN will be based on central laboratory reference values for age and sex.  
If ALT >3 × individual baseline or ALT >15 × ULN during treatment 
AND 
bilirubin >2 × individual baseline or bilirubin >2 × ULN (if baseline <ULN) 
Weekly monitoring should be initiated 
If ALT continues to increase AND ALT >5 × individual baseline 
AND 
bilirubin >3 × individual baseline OR bilirubin >3 × ULN (if baseline <ULN) 
OR 
ALT >20 × ULN irrespective of bilirubin 
Study medication should be stopped and weekly monitoring continued. 
If ALT and bilirubin returns to 1.5 × individual baseline or <2 × ULN study medication could 
be restarted. 
ULN will be based on central laboratory reference values for age and sex. 


 
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10.2.3 
Demographics/Medical and Surgical History 
Demographic information (age, birth year, gender, race, and ethnicity), along with medical 
and surgical history, will be obtained and recorded in the eCRF at Visit 1. 
Medical and surgical history will be entered in the CRF at Visit 1.  This includes date of 
diagnosis of PFIC, historical (3 months prior to screening) LFT values (AST, ALT, 
bilirubin), ongoing medication, any surgery performed, any other diagnosis, and historical 
biopsy data.  If a biopsy is performed during the study, this will be recorded in the eCRF at 
the visit closest after the biopsy.  Patients with biopsy data not older than 2 years at study 
start will be given the opportunity to participate in a sub-study to evaluate the effect of A4250 
on liver histology. 
10.2.4 
Clinical Genetic Testing 
If clinical genetic testing for verification of PFIC diagnosis has been performed prior to 
enrollment of patients in the study, the results will be entered in the eCRF.  Verification of 
the clinical laboratory report will be performed by a central reader to determine eligibility.  
Eligibility confirmation will be returned to the site within 7 days after receipt of the clinical 
genetic test result laboratory form. 
Regardless of previous testing, a blood sample will be drawn at Visit 1 on all patients for 
DNA extract and freeze.  Genetic testing will only be performed on this sample if clinical 
testing is equivocal, unavailable, or unobtainable.  In such cases, screening for pathologic 
biallelic variations in the ATP8B1 or ABCB11 genes will be performed if patient meets 
remaining inclusion and exclusion criteria and only after sponsor case review.  No other 
diagnostic genetic testing will be offered. 
10.2.5 
Physical Examination 
A physician or suitably trained qualified assistant will perform a complete physical 
examination at Visits 1, 3, and 9.  A skin exam (only) will be conducted at Visits 4, 6, and 10 
(see Table 1).  
A complete physical examination will include assessment of general appearance, eyes, ears, 
nose, throat, head/neck/thyroid, lymph nodes, cardiovascular, lungs/chest, abdomen, 
genitourinary, extremities, skin, musculoskeletal, neurologic, and other.  
Skin will be examined thoroughly and excoriations/scratch marks recorded.  
10.2.6 
Vital Signs 
Evaluation of vital signs will be performed at all visits.  This includes blood pressure (systolic 
and diastolic, measured after the patient has been in a seated position for ≥3 minutes of rest), 
pulse, respiratory rate, temperature, height and body weight (using a certified weight scale 
and consistent site-specific guidelines; e.g., removal of outer coat, shoes) at clinic visits.  
Body mass index (BMI) will be calculated. 


 
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10.2.7 
Overdose 
A4250 is minimally absorbed and has a very low systemic availability.  Based on toxicology 
data, for study purposes any dose exceeding a total of >3 mg/kg body weight of A4250 taken 
as a single dose or as a cumulative dose within 24 hours is defined as an overdose.   
The no-adverse-effect dose level in the most sensitive species (defined as 20 mg/kg/day) in 
the rat 1 month toxicity study gives a human equivalent dose of approximately 194 mg/day in 
a 60 kg person.  This dose is approximately 10-fold higher than the maximum possible dose 
predicted in human studies (20 mg).  
The investigator/investigative staff should immediately and not later than within 24 hours of 
knowledge of the overdose, be reported by the investigator or delegate in the eCRF SAE 
report form.  Any overdose, according to the definition above, must also be recorded in the 
study drug section of the patient’s eCRF.  In the event of an A4250 overdose, the patient 
should be monitored closely.  Hospitalization should be considered as an alternative for 
observation and for appropriate supportive treatment. 
If the investigator and/or Albireo consider the overdose event to be a medically important 
condition, then it should be reported as an SAE in the same expedited manner as an SAE 
(please refer to Section 10.1.3). 
10.2.8 
Pregnancy 
If a pregnancy is discovered before the end of dosing the patient will be immediately 
discontinued from further dosing in the study.  If the pregnancy is discovered after the end of 
dosing the patient will continue in the study per protocol. 
Pregnancy is not considered to be an AE.  However, if the patient has been dosed with the 
study drug, the pregnancy must be reported on the Paper Pregnancy Notification Form 
immediately within 24 hours after recognition to Albireo/Safety Manager (please refer to 
Section 10.1.3).  Date of exposure and as far as possible, details of the period of gestation at 
the time of exposure must be given. 
The pregnancy should be followed up to determine outcome, including spontaneous 
termination, details of birth, and presence of any birth defects, congenital anomalies or 
newborn or maternal complication.  Individual cases with an abnormal outcome in 
association with the study drug should be reported on an expedited basis, i.e., reported rapidly 
to a competent authority. 
 


 
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11 
STATISTICAL EVALUATION 
All personnel involved with the analysis of the study will remain blinded until database lock 
for the double-blind Treatment Period and until major protocol violations have been 
identified. 
All statistical analyses will be performed using SAS version 9.3 or higher. 
11.1 
Sample Size and Power 
For the EU and RoW, the primary analysis will relate to the bile acid responder endpoint (at 
the 1-sided 2.5% level).  For the US, the primary analysis will relate to the pruritus endpoint 
(at the 1-sided 2.5% level). 
Assuming 60% responders (patients experiencing at least 70% reduction in fasting s-BA 
levels or reaching ≤70 µmol/L) in the A4250 dose group and 10% responders in the placebo 
group, 20 patients per group are needed to achieve a power of 90% with a 2.5% 1-sided 
Type-1 error. 
For pruritus, with 20 patients per treatment group, the power is approximately 90% given an 
assumed treatment effect difference of -1.0 in change from baseline (-1.6 for A4250 
and -0.6 for placebo) on the ObsRO scratching score with a standard deviation of 0.95 and a 
Type I error of 2.5% (1-sided). 
This gives an overall power of approximately 80% for both endpoints. 
11.2 
Statistical Methods 
11.2.1 
Statistical Analysis Sets 
Safety Analysis Set 
The safety analysis set will consist of all patients who received at least 1 dose of study drug.  
Patients will be analyzed according to the treatment they actually received.  The safety 
analysis set will be the primary analysis set for safety analyses.  
Full Analysis Set 
The full analysis set (FAS) will consist of all patients randomized with a valid baseline and at 
least 1 valid post-baseline measurement for at least 1 efficacy variable, such as fasting s-BA, 
pruritus score, ALT, IGF-1, sleep disturbance (PRO/ObsRO), and PedsQL.  Patients will be 
analyzed in the treatment group they were randomized to even if they received incorrect 
study drug.  The FAS will be the primary analysis set for efficacy analyses. 
Modified Full Analysis Set 
The modified full analysis set (mFAS) is a subset of the FAS and will consist of all 
randomized patients who were not included in Study A4250-003. 


 
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Per Protocol Analysis Set 
The per protocol (PP) analysis set is a subset of the FAS, and will consist of all randomized 
patients for whom no major protocol violation (no violation which may affect the study 
efficacy outcome) is documented.  The PP analysis set will provide supportive data for the 
efficacy analyses. 
Allocation of patients to the PP analysis set will be performed before unblinding of the study. 
11.2.2 
Methods of Statistical Analyses 
11.2.2.1 
General Principles 
The primary objective of this study is to show superiority in efficacy of 
A4250 (120 µg/kg/day and 40 µg/kg/day) compared to placebo in patients with PFIC Types 1 
and 2. 
In the EU and RoW, for the primary efficacy variable fasting bile acid responder endpoint (at 
least a 70% reduction in fasting s-BA levels or reaching a level ≤70 µmol/L at the end of 
treatment), the null hypothesis will be tested against the alternative hypothesis (α = 0.025, 
1-sided). 
The test will be performed at a Type I error rate of ≤0.025 1-sided, as described above, based 
on the least squares means of the treatment difference in mean probability scale with 
corresponding 95% confidence intervals. 
The Type I error will be protected by performing a hierarchical structure of analysis with 
closed tests of hypotheses. 
A fixed-sequence multiple testing approach will be used.  In the first step, the high dose 
(120 µg/kg/day) versus placebo will be tested at 2.5% 1-sided significance level.  If 
significant, the low dose (40 µg/kg/day) versus placebo will also be tested, in a second step, 
at 2.5 % 1-sided significance level.  The second step will only be considered confirmatory 
providing the previous step was successful.  If the previous step was not successful, the 
analysis of the following step will be considered descriptive. 
In the US, for the primary efficacy variable pruritus score endpoint (change from baseline to 
Week 24 in observer-reported scratching), the null hypothesis will be tested against the 
alternative hypothesis (α=0.025, 1-sided). 
The test will be performed at a Type I error rate of ≤0.025 1-sided, as described above, based 
on the least squares means of the treatment difference in mean change with corresponding 
95% confidence intervals. 
The Type I error will be protected by performing a hierarchical structure of analysis with 
closed tests of hypotheses using a fixed-sequence multiple testing approach.  In the first step, 
the high dose (120 µg/kg/day) versus placebo will be tested at 2.5% 1-sided significance 
level.  If significant, also the low dose (40 µg/kg/day) versus placebo will be tested, in a 
second step, at 2.5 % 1-sided significance level.  The second step will only be considered 


 
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confirmatory providing the previous step was successful.  If the previous step was not 
successful, the analysis of the following step will be considered descriptive. 
Secondary and exploratory outcome variables shall provide supportive efficacy and safety 
information regarding the differences between the treatment groups.  For secondary efficacy 
variables, supportive statistical tests will be made and P-values will be reported in 
exploratory way (if not otherwise specified).  No adjustments will be performed for multiple 
dose comparisons when testing the secondary and exploratory efficacy variables. 
A more detailed statistical analysis plan (SAP) will be prepared and finalized before study 
data are locked and unblinded.  
If not otherwise specified, the baseline value of a parameter is defined as the last assessment 
of that parameter on or prior to the first intake of study treatment date. 
11.2.2.2 
Missing Data 
For efficacy variables, with repeated measures, a mixed model will be employed with no 
additional imputation for missing values, i.e., based on “observed cases”, except where 
otherwise specified. 
Follow-up and continued collection of efficacy data for patients who discontinue treatment 
will be made.  These data will be included in the sensitivity analysis for primary endpoints 
estimating a treatment effect taking loss of effect after discontinuation into account.  Because 
complete follow-up of all patients discontinuing treatment may not be possible, a method to 
replace missing data for patients who discontinue the study will be prespecified (in the SAP) 
taking into account loss of effect after discontinuation (for example multiple imputation). 
No other replacement of missing values will be planned. 
11.2.2.3 
Demographic and Baseline Characteristics 
Descriptive summaries of demographics and other baseline characteristics (including medical 
and surgical history) will be presented by treatment group and overall using the FAS.  
Prior medication will be summarized by treatment group and overall using the safety analysis 
set. 
11.2.2.4 
Subject Disposition 
The following will be summarized (by treatment group and overall where applicable): 
• Patients enrolled (who signed the informed consent) 
• Patients randomized 
• Patients treated 
• Patients completing the study/withdrawing early (including withdrawal reason) 
Additionally, patients enrolled, included in the safety, in the FAS, and in the PP analysis set 
will be summarized by region. 


 
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11.2.2.5 
Evaluation of Primary Efficacy Variables 
The responder endpoint based on change in fasting bile acids will be analyzed in terms of the 
proportion of patients achieving the event of interest.  Patients in need of rescue therapy, i.e., 
biliary diversion will be classified as non-responders after the biliary diversion.  
Generalized linear mixed modelling (GLMM) will be used with a logistic link, to allow for 
repeated assessments over time.  The model will include baseline value, treatment group, 
PFIC class, age class, region, visit, and visit*treatment group interaction.  The treatment by 
visit interaction will remain in the model regardless of significance.  Region will be included 
in the model as a random effect.  The contrasts of primary interest will be the treatment 
difference at Week 24; also, the difference at Week 4, Week 8, Week 12, and Week 18 will 
be estimated and analyzed.  An unstructured covariance matrix will be used to model the 
within-subject error.  Another covariance structure may be chosen if the fit of this structure 
fails to converge. 
The primary analysis of the change from baseline in pruritus score (indexed by 
observer-reported scratching) will be analyzed using a mixed model repeated measures 
(MMRM) analysis.  The contrast of primary interest will be treatment difference at Week 24 
but also the differences at Week 4, Week 8, Week 12, and Week 18 will be estimated and 
analyzed.  Baseline pruritus score will be included as covariate.  The model will include 
baseline value, treatment group, PFIC class, age class, region, visit, and visit*treatment group 
interaction.  The treatment by visit interaction will remain in the model regardless of 
significance.  Region will be included in the model as a random effect and will be defined on 
country level.  An unstructured covariance matrix will be used to model the within-subject 
error.  Another covariance structure may be chosen if the fit of this structure fails to 
converge. 
Non-parametric approach may be considered if more appropriate. 
The primary analyses will be based on the FAS. 
To assess the robustness of the primary efficacy analysis, the following analyses will be 
performed: 
• Primary efficacy analysis using the PP set  
• Primary efficacy analysis using the mFAS 
• Analyses to investigate the influence of missing values, for example, by multiple 
imputation technique 
Subgroup analyses will also be performed for each of the 3 age groups (6 months to 5 years, 
6 to 12 years, and 13 to ≤18 years). 
Several sensitivity analyses will be performed, such as the following: 


 
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• Cochran Mantel Haenzsel test stratified on PFIC class to compare the proportion in 
fasting bile acid responder endpoint at the end of treatment in the 2 A4250 dose groups to 
placebo group 
• Based on the primary analysis including the interaction age class*treatment 
• Same as primary analysis in which the random factor region is considered as fixed factor.  
The region*treatment interaction will also be explored 
• Same as primary analysis including the interaction PFIC*treatment  
Other sensitivity analyses may be performed and will be detailed in the SAP. 
11.2.2.6 
Evaluation of Secondary and Exploratory Efficacy Variables 
The change in the secondary and explorative endpoints such as s-BA, total bilirubin, ALT, 
IGF-1, and patient-reported itch severity will also be analyzed using the MMRM model as 
specified for the primary analysis.  Nonparametric methods may be applied as an alternative 
approach for some endpoints, if appropriate. 
Proportions of responders (e.g., meaningful reduction in itch severity and scratching at 
Week 24) will be analysed using the GLMM model as specified for the primary analysis. 
Comparisons of the change from baseline at Week 24 in PedsQL between the treatment 
groups will be conducted using analysis of covariance (ANCOVA).  The model will include 
terms for baseline, PFIC class, age class, region, and treatment.  
Undergoing biliary diversion surgery and/or listed for liver transplant, all-cause mortality, 
and liver decompensation events will mainly be analyzed by using descriptive statistics.  
Kaplan-Meier curves will be used when appropriate for time to event data. 
Other exploratory variables (global symptom relief, biochemical markers and measures of 
bile acid synthesis, PRO and ObsRO sleep parameters, PELD/MELD score, APRI score, and 
Fib-4 score) will be analyzed descriptively for categorical and continuous data, as applicable.  
For continuous data, the change from baseline will be analyzed in addition to the actual visit 
values.  For categorical data, shift tables or frequency and percentages of patients will be 
presented as appropriate. 
In addition, subgroup analyses will be performed for each of the 3 age classes (6 months to 
5 years, 6 to 12 years, and 13 to ≤18 years). 
11.2.2.7 
Evaluation of Safety Variables 
Safety data will be analyzed using descriptive statistics and summaries by treatment group of 
SAEs, AEs, vital signs, clinical laboratory tests (hematology, clinical chemistry and 
urinalysis), and concomitant medication.  Analyses will be performed using the safety 
analysis set.  


 
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Summaries of AEs (coded according to the Medical Dictionary for Regulatory Activities 
[MedDRA] system organ class [SOC] and MedDRA preferred term) will include the 
following:  
• Overview of the incidence of TEAEs (TEAEs, drug-related TEAEs, TEAEs leading to 
study discontinuation, and treatment-emergent SAEs) 
• TEAEs by SOC and preferred term 
• Intensity of TEAEs by SOC and preferred term 
• Drug-related TEAEs by SOC and preferred term 
• TEAEs leading to study discontinuation by SOC and preferred term 
• Treatment-emergent SAEs by SOC and preferred term 
Concomitant medication use during the Treatment Period will be summarized by Anatomical 
Therapeutic Chemical (ATC) class and World Health Organization (WHO) preferred name.  
Summaries of vital signs will be presented.  For each visit, the actual results and the change 
from baseline, and the number and percentage of patients with potentially clinically 
significant (PCS) values observed post-baseline will be presented. 
Summaries of clinical safety laboratory data will be presented.  For each visit, the actual 
result and the change from baseline, and the number and percentage of patients with PCS 
values observed post-baseline will be presented. 
Data listings will be provided for each patient for all safety parameters. 
11.2.2.8 
Compliance and Exposure 
Exposure will be analyzed by calculating the number of days with exposure to study drug.  
Results will be presented by treatment group using the safety analysis set.  
The percentage compliance will be described by treatment group and the number of subjects 
with a compliance <80%, between 80% and 120%, and >120% will be presented based on the 
safety analysis set. 
11.2.3 
Blinded Analyses to Confirm Responder Definitions 
A blinded analysis of ObsRO and PRO eDiary data will be performed after 50% or more of 
the planned sample has completed the Week 24 Follow-Up Period.  The blinded analysis will 
be used to estimate a threshold of clinically meaningful change (i.e., responder definition) in 
ObsRO and PRO pruritus scores.  The analysis will be performed by a group independent 
from both the study team and Albireo.  Blinded data will be used for this analysis; data will 
be collapsed across treatment groups. 
The analyses will include distribution- and anchor-based analyses to examine a responder 
definition for the PRO/ObsRO pruritus scores.  Distribution-based analyses will include 
calculation of the standard deviation and standard error of measurement for the baseline PRO 
and ObsRO scores.  Anchor-based analyses will involve examining the degree of change on 


 
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the PRO and ObsRO scores from baseline to Week 24 for patients who experience change in 
pruritus according to PGIC and symptom items.  Details of this analysis will be included in a 
stand-alone SAP for assessing the measurement characteristics (reliability, validity, 
sensitivity to change, responder definition) of the PRO and ObsRO items. 
11.2.4 
Data Safety Monitoring Board 
A DSMB consisting of sponsor-independent clinical and statistical experts will be 
established.  The DSMB will periodically meet for the review of accumulating study data, 
including safety (AE and laboratory data) and liver values.  
The DSMB will have access to unblinded data.  The DSMB will make recommendations for 
the remaining part of the study (further details will be provided in the DSMB charter).  The 
DSMB may recommend continuing with the study as planned or stopping the study early for 
safety reasons.  The DSMB will submit its recommendations in writing to Albireo 
Medical/
 who are responsible for responding to the recommendations of the DSMB and 
taking appropriate action.  The investigators will only be informed by Albireo Medical/
 
if the study requires a protocol amendment or is stopped.  The DSMB may choose to make 
additional evaluations at any time if they feel this is warranted from a safety point of view.  
The DSMB will act according to its own written standard operating procedure described in a 
charter and will prepare written minutes of its meetings.  The charter of the DSMB will be 
stored in the trial master file.  The DSMB will maintain records of its meetings and these will 
become part of the study file when the study is complete.  In order not to disseminate 
unblinded data and to ensure that all staff involved in the conduct and final analysis of the 
study remain blind to the data, only the members of the DSMB and the unblinded DSMB 
statistician will have access to these data. 
 


 
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12 
DIRECT ACCESS TO SOURCE DATA/NOTES 
The investigator/institution shall provide direct access to source data/documents for 
study-related monitoring, audits, IEC/IRB review and regulatory inspection. 


 
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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
13.1 
Conduct of the Study 
Albireo/
 shall implement and maintain quality control and quality assurance procedures 
with written SOPs to ensure that the study is conducted and data are generated, documented, 
and reported in compliance with the protocol, ICH GCP and applicable regulatory 
requirements. 
This study shall be conducted in accordance with the provisions of the Declaration of 
Helsinki (October 1996) and all revisions thereof, and in accordance with FDA regulations 
(CFR, Sections 312.50 and 312.56) and with ICH GCP (CPMP 135/95). 
The investigator may not deviate from the protocol without a formal protocol amendment 
having been established and approved by an appropriate IEC/IRB, except when necessary to 
eliminate immediate hazards to the patient or when the change(s) involve(s) only logistical or 
administrative aspects of the study.  Any deviations may result in the patient having to be 
withdrawn from the study and render that patient non-evaluable. 
13.2 
Study Monitoring 
Before study initiation, at a site initiation visit or at an investigator’s meeting, an Albireo 
representative will review the protocol and eCRF with the investigators and the investigative 
staff.  During the study, the clinical monitor (clinical research associate [CRA]) will visit the 
site regularly to check the completeness of patient records, the accuracy of entries on the 
CRFs, the adherence to the protocol and to GCP, the progress of enrollment, and to ensure 
that study drug is being stored, dispensed, and accounted for according to specifications. 
The investigator must ensure that CRFs are completed within 5 days of patient visits and 
must allow the Albireo medical monitor or CRA periodic access to patient records and all 
study-related materials, including relevant hospital or clinical records, to confirm their 
consistency with the CRF entries.  No information in these records about the identity of the 
patients will leave the study center.  Albireo monitoring standards require full verification for 
the presence of the signed ICF, adherence to the inclusion/exclusion criteria, documentation 
of SAEs, and recording of primary efficacy and safety variables.  The CRA will review 
source data compared with the CRFs and will verify source data according to the 
study-specific monitoring plan.  The design of the study, the frequency of patient visits, and 
the site enrollment rate will determine the frequency of monitoring visits.  Upon study 
completion, the CRA will visit the site to conduct a study termination visit, which will 
include collection of any outstanding documentation. 
It is recommended that the investigator/investigative staff log into the system every working 
day or at minimum twice weekly in order to provide a prompt response to queries.  The 
investigator/investigative staff should log into the system the day after submitting an SAE 
report to answer any queries.  This practice should continue until all questions regarding the 
SAE have been answered.  The investigator/investigative staff should respond to queries and 


 
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make any relevant changes to the study date within 3 working days.  The 
investigator/investigative staff should respond as quickly as possible to queries received 
concerning an SAE. 


 
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14 
ETHICS 
14.1 
Independent Ethics Committee/Institutional Review Board 
Prior to the start of the study, the investigator is responsible for ensuring that the protocol and 
consent form have been reviewed and approved by a relevant IEC/IRB.  The IEC/IRB shall 
be appropriately constituted and perform its functions in accordance with FDA ICH GCP and 
local requirements as applicable. 
The IEC/IRB shall approve all protocol amendments (except for logistical or administrative 
changes), written informed consent documents and document updates, patient recruitment 
procedures (e.g., advertisements), written information to be provided to the patients, IB, 
available safety information, information about payment and compensation available to 
patients, the investigator's curriculum vitae and/or other evidence of qualifications and any 
other documents requested by the IEC/IRB and regulatory authority (competent authority) as 
applicable. 
14.2 
Written Informed Consents and Assents 
The investigator/investigative staff will explain to each patient (or legally authorized 
representative) the nature of the study, its purpose, the procedures involved, the expected 
duration, alternative treatment, the potential risks and benefits involved, and any discomfort 
that may occur.  Each caregiver/patient will be informed that participation in the study is 
voluntary and that he/she or their child may withdraw from the study at any time and that 
withdrawal of consent will not affect his/her subsequent medical treatment or relationship 
with the treating physician. 
Caregivers/patients will be informed that they/their children may experience side-effects or 
be at risk for symptoms, illnesses, or complications that cannot be foreseen by Albireo.  As 
with other medications, people treated with A4250 may be at risk of developing allergic 
reactions or anaphylaxis.  Caregivers/patients will be advised that study procedures include 
regular blood sampling for measurement of safety parameters and biological markers and that 
some minor risks are associated with these procedures. 
This informed consent should be given by means of a signed ICF, written in non-technical 
language in accordance with ICH GCP, the Declaration of Helsinki, and regulatory 
authorities.  The caregiver/patient should read and consider the statements before signing and 
dating them and should be given a copy of each signed document.  If written consent is not 
possible, oral consent can be obtained if witnessed and followed by a signed statement from 
one or more persons not involved in the study, indicating why the patient was unable to sign 
the form.  No patient can enter the study before his/her or caregiver informed consent has 
been obtained. 
The ICF is part of the protocol and must be submitted by the investigator/investigative staff 
with the protocol for IEC approval.  Albireo will supply an ICF which complies with 
regulatory requirements and is considered appropriate for the study.  Any changes to the ICF 


 
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suggested by the investigator must be agreed to by Albireo before submission to the IEC and 
a copy of the approved version must be provided to the clinical monitor after IEC approval. 


 
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15 
DATA HANDLING AND RECORD KEEPING 
15.1 
Case Report Forms/Source Data Handling 
The investigator shall be provided with standardized CRFs and shall ensure that all data from 
patient visits are promptly entered into the CRFs in accordance with the specific instructions 
given.  The investigator must sign each CRF to verify the integrity of the data recorded. 
A list of the normal ranges for all laboratory tests to be undertaken forms part of the 
documentation to be collated prior to study start.  If a central laboratory has been selected to 
conduct any or all tests, it is essential that all samples be analyzed at that laboratory. 
The investigator must maintain source documents such as laboratory reports, consultation 
reports, and complete medical history and physical examination reports. 
Data to be recorded directly on the CRFs (i.e., no prior written or electronic record of data) 
and considered to be source data must be identified in the protocol. 
15.2 
Retention of Essential Documents 
Essential documents, as defined by ICH GCP, include: the signed protocol and any 
amendment(s); copies of the completed CRFs (for site archiving, CDs of CRF data for 
specific patients will be provided); signed ICFs; hospital records and other source documents; 
IEC approvals and all related correspondence including approved documents; drug 
accountability records; study correspondence; and a list of patients’ names and addresses. 
The investigator/investigative staff must retain copies of these essential documents for the 
period specified by ICH GCP and by applicable regulatory requirements.  The 
investigator/investigative staff will inform Albireo of the location where the essential 
documents are stored and must contact Albireo for approval before disposing of any essential 
documents.  The investigator/investigative staff should take measures to prevent accidental or 
premature destruction of these documents. 


 
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16 
FINANCING AND INSURANCE 
Financing and insurance are addressed in a separate agreement. 
 


 
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17 
PUBLICATION POLICY 
Albireo will retain the ownership of all data.  When the study is complete, Albireo shall 
arrange the analysis, tabulation of data and preparation of a clinical study report. Albireo may 
also use the data for publication, presentation at scientific meetings and submission to 
regulatory authorities.  All proposed publications based on this study must be subject to the 
sponsor's approval requirements. 
 


 
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18 
REFERENCE LIST 
1. A4250 Investigator’s Brochure. 
2. Alissa FT, Jaffe R, Shneider BL. Update on progressive familial intrahepatic cholestasis. 
J Pediatr Gastroenterol Nutr. 2008;46(3):241-52. 
3. Davit-Spraul A, Gonzales E, Baussan C, Jaquemin E. Progressive familial intrahepatic 
cholestasis. Orphanet J Rare Disease 2009;4:1-12. 
4. Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and 
functions. Front Biosci (Landmark Ed). 2009 Jan 1;14:2584-98. 
5. Hori T, Justin H, Nguyen JH and Uemoto S. Progressive familial intrahepatic cholestasis. 
Hepatobiliary pancreat Dis Int. 2010; 9(6):570-578. 
6. Jacquemin E. Progressive familial intrahepatic cholestasis. Genetic basis and treatment. 
Clin Liver Dis. 2000 Nov;4(4):753-63. 
7. Miethke et al. Pharmacological inhibition of ASBT changes bile composition and blocks 
progression of sclerosing cholangitis in mdr2 knockout mice. Hepatology. 2016 February; 
63(2):512–523. 
8. Murray CS, Rees JL. Are subjective accounts of itch to be relied on? The lack of relation 
between visual analogue itch scores and actigraphic measures of scratch. Acta Derm 
Venereol. 2011;91(1):18-23. 
9. Pawlikowska L, Strautnieks S, Jankowska I. et al. Differences in presentation and 
progression between severe FIC1 and BSEP deficiencies, J Hepatol. 2010; 53(1):170-178. 
10. Suchy FJ, Sokol RJ, Balistreri WF, eds. Liver Disease in Children. 3rd Edition. New 
York, NY: Cambridge University Press; 2007:310–314. 
11. Whitington PF, Whitington GL. Partial external diversion of bile for the treatment of 
intractable 
pruritus 
associated 
with 
intrahepatic 
cholestasis. 
Gastroenterology. 
1988;95(1):130-136. 
 
 


 
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19 
APPENDICES 
Appendix 1 
Concurrent Medications Prohibited During the Study 
 


 
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Appendix 2 
Diary Questions including Albireo Patient-Reported Outcomes/Observer-
Reported Outcome Instrument 
 


 
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Appendix 3 
Patient Global Impression of Change (PGIC) and Patient Global 
Impression of Symptoms (PGIS) 
 


 
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Appendix 4 
Pediatric Quality of Life Inventory (PedQL) Questionnaire 
 

SAP_001.pdf: 
 
 
 
 
Statistical Analysis Plan 
Protocol Number: A4250-005 
Version: Final v1.2, Date: 25Aug2020
Page 1 of 65
 
 
 
 
 
Statistical Analysis Plan (SAP) 
 
 
 
 
 
 
Protocol Title: 
A Double-Blind, Randomized, Placebo-Controlled, Phase 3 
Study to Demonstrate Efficacy and Safety of A4250 in Children 
with Progressive Familial Intrahepatic Cholestasis Types 1 and 
2 (PEDFIC 1) 
Protocol Number: 
A4250-005 
IND number 
130591 
EudraCT number 
2017-002338-21 
Protocol Version, Date 
Final Amendment 06, 24 June 2019 
Document Version, Date: 
Final v1.2, 25-Aug-2020  
Prepared by: 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
 
Statistical Analysis Plan 
Protocol Number: A4250-005 
Version: Final v1.2, Date: 25Aug2020
Page 2 of 65
 
 
SIGNATURE PAGE 
 
 
 
 
Prepared by: 
  
 
  
 
  
 
 
 Date 
 
  
 
  
Reviewed by: 
  
 
  
 
  
 
 
 Date 
 
  
 
  
Reviewed and Approved by: 
  
 
  
 
  
 
 Date 
 
  
 
  
 
 Date 


 
 
 
 
Statistical Analysis Plan 
Protocol Number: A4250-005 
Version: Final v1.2, Date: 25Aug2020
Page 3 of 65
 
 
REVISION HISTORY 
 
Version/Date 
Modified by 
Reason for Modifications 
Final v1.0/23Oct2019 
NA 
 
Final v1.1/03Aug2020 
 
1. To add the handling for patients 
whose baseline s-BA ≤70 µmol/L and 
for patients whose baseline pruritus 
score is ≤ 1. 
2. To add responder analysis based on 
the clinically meaningful threshold by 
using monthly scores. To add the result 
of clinically meaningful change from 
the blinded psychometric analysis. 
3. To clarify off-treatment pruritus 
assessments will be included for 
analysis. For completers, the 
denominator to calculate US primary 
endpoint will be based on the last 
eDiary report day. 
4. To update dictionary version. 
5. To clarify the worst scenario will be 
used for tipping point analysis of EU 
and ROW primary endpoint. 
6. To clarify the growth analysis will 
be based on derived growth data using 
the software or methods from the 
Centers for Disease Control and 
Prevention (CDC) website for patients 
with age ≥ 2 years old and from the 
WHO website for patients with age < 2 
years old. Also, to add a MMRM 
analysis. 
7. To clarify the counting of number of 
adverse events. To add the adverse 
events of interest and their definitions. 
8. To clarify that local laboratory data 
will be used when central laboratory 
data are not available. 
9. To clarify the calculation of change 
from baseline for AM&PM pruritus 
scores (daily, weekly, bi-weekly, 
monthly) for analysis purposes. 
10. To update the analysis windows of 
assessments. 


 
 
 
 
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Protocol Number: A4250-005 
Version: Final v1.2, Date: 25Aug2020
Page 4 of 65
 
Final v1.2/25Aug2020 
 
1, To remove completer analysis set 
and its analysis.  
2. To add MMRM analysis for s-BA 
and ALT. 
3. To clarify the handling for treatment 
completers in responder analyses. 
 
 
 
 


 
 
 
 
Statistical Analysis Plan 
Protocol Number: A4250-005 
Version: Final v1.2, Date: 25Aug2020
Page 5 of 65
 
TABLE OF CONTENTS 
 
SIGNATURE PAGE .................................................................................................................. 2 
TABLE OF CONTENTS ........................................................................................................... 5 
LIST OF ABBREVIATIONS .................................................................................................... 7 
1 INTRODUCTION ................................................................................................................ 9 
2 STUDY OBJECTIVES ...................................................................................................... 10 
2.1 Primary objectives ..................................................................................................... 10 
2.2 Secondary objectives ................................................................................................. 10 
3 STUDY DESIGN ............................................................................................................... 11 
3.1 General study design ................................................................................................. 11 
3.2 Randomization and blinding ..................................................................................... 12 
3.3 Study treatments and assessments ............................................................................. 13 
4 STUDY ENDPOINTS ....................................................................................................... 20 
4.1 Primary efficacy endpoints ........................................................................................ 20 
4.2 Secondary efficacy endpoints .................................................................................... 20 
4.3 Exploratory endpoints ............................................................................................... 22 
4.4 Safety ......................................................................................................................... 23 
5 SAMPLE SIZE AND POWER .......................................................................................... 24 
6 ANALYSIS SETS .............................................................................................................. 26 
6.1 Full Analysis Set (FAS) ............................................................................................ 26 
6.2 Safety Analysis Set (SAS) ......................................................................................... 26 
6.3 Per-Protocol Analysis Set (PP) ................................................................................. 26 
7 Statistical Considerations and analysis .............................................................................. 27 
7.1 Handling of missing data and/or invalid data and outliers ........................................ 27 
7.1.1 Handling of Missing data for efficacy endpoints .......................................... 27 
7.1.1.1 Pruritus Endpoint .............................................................................................. 27 
7.1.1.2 Serum bile acid responder endpoint ................................................................. 27 
8 STATISTICAL METHODS .............................................................................................. 28 
8.1 General statistical conventions .................................................................................. 28 
8.2 Subject disposition .................................................................................................... 29 
8.3 Protocol deviations .................................................................................................... 29 
8.4 Demographics and baseline characteristics ............................................................... 30 
8.4.1 Demographics ................................................................................................ 30 
8.4.2 Baseline and disease characteristics .............................................................. 30 
8.4.3 Medical and surgical history ......................................................................... 31 
8.4.4 Prior and concomitant medications ............................................................... 31 
8.5 Extent of exposure ..................................................................................................... 31 
8.5.1 Treatment duration ........................................................................................ 31 
8.5.2 Study drug compliance .................................................................................. 32 
8.6 Efficacy analyses ....................................................................................................... 32 
8.6.1 Analyses methods .......................................................................................... 33 


 
 
 
 
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8.6.1.1  Cochran Mantel Haenszel (CMH) Test ........................................................... 33 
8.6.1.2  Analysis of Covariance (ANCOVA) model .................................................... 33 
8.6.1.3  Multiplicity ...................................................................................................... 34 
8.6.2 Treatment by center interaction analysis (multi-center study) ...................... 34 
8.6.3 Analyses of primary efficacy endpoints ........................................................ 34 
8.6.3.1  Sensitivity and supportive analyses of primary efficacy endpoints ................ 35 
8.6.4 Analyses of secondary efficacy endpoints .................................................... 37 
8.6.5 Analyses of exploratory endpoints ................................................................ 39 
8.6.6 Analyses based on exit survey data ............................................................... 39 
8.7 Safety analyses .......................................................................................................... 40 
8.7.1 Adverse events .............................................................................................. 41 
8.7.1.1 
Definition for TEAEs of Fat-Soluble Vitamin Deficiency, Diarrhoea, and 
Hepatotoxicity ..................................................................................................... 43 
8.7.1.2 
Definitions and Determination of Hepatic Events ........................................ 43 
8.7.2 Clinical laboratory evaluations ...................................................................... 45 
8.7.3 Vital signs ...................................................................................................... 46 
8.7.4 Physical examinations ................................................................................... 47 
8.7.5 Pharmacokinetics (PK) .................................................................................. 47 
8.8 Subgroup analyses ..................................................................................................... 47 
8.9 Blinded analysis ........................................................................................................ 48 
8.9.1 Pruritus Responder Definitions ..................................................................... 48 
8.9.2 Data Safety Monitoring Board ...................................................................... 48 
9 CHANGES TO PLANNED ANALYSIS FROM STUDY PROTOCOL.......................... 49 
10 REFERENCES ................................................................................................................... 52 
11 APPENDICES .................................................................................................................... 53 
 
 


 
 
 
 
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LIST OF ABBREVIATIONS 
The following abbreviations will be used within this SAP. 
Abbreviation or 
special term 
Explanation 
E 
Adverse Event 
 
AFP 
Alfa-fetoprotein 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Aminotransferase 
APRI 
AST to Platelet Ratio Index 
ANCOVA 
Analysis of Covariance 
AST 
Aspartate Aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BMI 
Body Mass Index 
BSEP 
Bile Salt Export Pump 
CAP 
Controlled Attenuation Parameter 
CDC 
Centers for Disease Control and Prevention 
CI 
Confidence Interval 
CMH 
Cochran-Mantel-Haenszel 
COVID-19 
Coronavirus Disease 2019 
CRO 
Contract Research Organization 
CSR 
Clinical Study Report 
DSMB 
Data Safety and Monitoring Board 
eCRF 
Electronic Case Report Form 
EOT 
End of Treatment 
EU 
European Region 
FAS 
Full Analysis Set 
eDiary 
Electronic Diary 
GGT 
Gamma Glutamyl Transferase 
GIC 
Global Impression of Change 
GIS 
Global Impression of Symptoms 
ICF 
Informed Consent Form 
ICH  
International Conference on Harmonisation 
 
 
INR 
International Normalized Ratio 
IWRS 
Interactive Web Response System 


 
 
 
 
Statistical Analysis Plan 
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Page 8 of 65
 
Abbreviation or 
special term 
Explanation 
kg 
Kilogram 
MAR 
Missing at Random  
MCMC 
Markov Chain Monte Carlo 
MedDRA 
Medical Dictionary for Regulatory Activities 
MELD 
Model for End-Stage Liver Disease 
MI 
Multiple Imputation 
MMRM 
Mixed-Effect Model for Repeated Measures 
MNAR 
Missing Not at Random  
ObsRO 
Observer-Reported Outcome 
p-C4 
Plasma 7α-hydroxy-4-cholesten-3-one Concentration 
PD 
Protocol Deviation 
PFIC 
Progressive Familial Intrahepatic Cholestasis 
PELD 
Pediatric End-Stage Liver Disease 
PedsQL 
Pediatric Quality of Life Inventory 
PK 
Pharmacokinetics 
PMM 
Pattern Mixture Model 
PP 
Per Protocol  
PRO 
Patient-Reported Outcome 
PT 
Preferred Term 
QoL 
Quality of Life  
RoW 
Rest of World 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SAF 
Safety Analysis Set 
SAS 
Statistical Analysis System 
s-BA 
Serum Bile Acid 
SD 
Standard Deviation 
SI 
International System of Unit 
SOC 
System Organ Class 
TEAE 
Treatment Emergent Adverse Event 
TFLs 
Tables, Figures and Listings 
ULN 
Upper Limit of Normal 
US 
United States  


 
 
 
 
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1 
INTRODUCTION 
The purpose of this Statistical Analysis Plan (SAP) is to provide detailed descriptions of the 
statistical methods, data derivations, and data displays for study protocol A4250-005 “A 
Double-Blind, Randomized, Placebo-Controlled, Phase 3 Study to Demonstrate Efficacy and 
Safety of A4250 in Children with Progressive Familial Intrahepatic Cholestasis Types 1 and 
2 (PEDFIC 1)”. The table of contents and templates for the Tables, Figures and Listings 
(TFLs) will be produced in a separate document. 
 
Any deviations from this SAP will be described and justified in the Clinical Study Report 
(CSR). The preparation of this SAP has been based on International Conference on 
Harmonisation (ICH) E3 and E9 guidelines [1, 2]. All data analyses and generation of TFLs 
will be performed using SAS® version 9.4 or higher. The SAP will be finalized and signed off 
prior to locking the database and unblinding. 
 
 
 


 
 
 
 
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2 
STUDY OBJECTIVES 
2.1 
Primary objectives 
 
To demonstrate the efficacy of repeated daily doses of 40 µg/kg/day and 120 µg/kg/day 
odevixibat (A4250) in children with progressive familial intrahepatic cholestasis (PFIC) 
Types 1 and 2, as determined by the following: 
 
• The proportion of patients experiencing at least a 70% reduction in fasting serum bile 
acid (s-BA) concentration from Baseline to end of treatment or reaching a level 
≤70 µmol/L after 24 weeks of treatment 
 
• The proportion of positive pruritus assessments at the subject level over the 24-week 
treatment period. A positive pruritus assessment is defined as a scratching score of ≤1 
or at least a one-point drop from baseline on the Albireo Observer-Reported Outcome 
(ObsRO) instrument  
 
 
2.2 
Secondary objectives 
 
• To evaluate the effect of odevixibat on serum alanine aminotransferase (ALT) 
concentration  
• To evaluate the effect of odevixibat on growth 
• To evaluate the effect of odevixibat on sleep disturbance 
• To evaluate the effect of odevixibat on the need for surgical treatment (biliary 
diversion or liver transplantation)  
• To assess the safety and tolerability of repeated daily doses of odevixibat for 24 
weeks 
 
 
 


 
 
 
 
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3 
STUDY DESIGN  
3.1 
General study design 
 
This is a double-blind, randomized, placebo-controlled, Phase 3 study to investigate the 
efficacy and safety of 40 µg/kg/day and 120 µg/kg/day of odevixibat compared to placebo in 
children with PFIC Type 1 and 2. 
 
The study includes a 35- to 56-day Screening Period followed by a 24-week Treatment Period 
and a 4-week Follow-Up Period. After completion of the Screening Period, eligible 
patients (20 per treatment group) will be randomized on Day 0 (Visit 3) in a 1:1:1 fashion to 
receive 40 µg/kg/day or 120 µg/kg/day of odevixibat, or a matching placebo. Study data will 
be periodically reviewed by an unblinded Data Safety and Monitoring Board (DSMB). 
A total of approximately 60 patients with a clinical diagnosis of PFIC Type 1 or 2 that have 
been genetically confirmed will be randomized. The randomization will be done in blocks and 
stratified according to PFIC type (Type 1 and 2) and age category (6 months to 5 years, 6 to 
12 years, and 13 to ≤18 years) to ensure approximate balance between dose schemes (1:1:1). 
 
The study will target 15% of enrolled patients to be PFIC-1 patients. If enrollment of all 
PFIC-2 patients is completed and the 15% enrollment of PFIC-1 patients has not yet been 
achieved, then enrollment of PFIC-2 patients will continue to reach the total study target 
enrollment. An attempt will be made to randomize a minimum of 10 patients from each age 
category (6 months to 5 years, 6 to 12 years, and 13 to ≤18 years). A maximum of 5 patients 
that were treated in Study A4250-003 will be allowed to participate in this study. 
 
Patients who complete the Treatment Period and Visit 9 (Day 168, Week 24) in this study will 
be invited to participate in a 72-week open-label extension study (A4250-008, EudraCT 
Number 2017-002325-38, subject to a separate study approval) in which all patients will 
receive odevixibat 120 µg/kg/day. Prior to protocol amendment 6, patients who had 
completed at least 12 weeks of the Treatment Period and had either no improvement or 
intolerable symptoms were eligible to complete the End of Treatment (EOT) Visit and were 
offered the opportunity to enter Study A4250-008.  
 
There will be up to 10 clinic visits during the study and one scheduled telephone contact, as 
follows: 
 
• Visit 1: Screening Period start. Patient is considered enrolled after signing the informed 
consent form (ICF) (Days -56 to -35) 
• Visit 2: Screening Visit 2 (Days -28 to -7) 
• Visit 3: Randomization Visit (Day 0) 
• Telephone Contact 1: (Day 14 - Week 2) 
• Visit 4: Clinic Visit (Day 28 - Week 4) 
• Visit 5: Clinic Visit (Day 56 - Week 8) 
• Visit 6: Clinic Visit (Day 84 - Week 12) 
• Visit 7: Clinic Visit (Day 126 - week 18) 
• Visit 8: Clinic Visit (Day 154 - Week 22) 


 
 
 
 
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• Visit 9: Clinic Visit, End of Treatment (Day 168 - Week 24) 
• Visit 10: Follow-Up Visit, 28 days after last dose of study drug (Day 196).  Patients 
continuing in Study A4250-008 will not have this follow-up visit. 
 
Additional clinical visits may be required for patients who need direct site assistance, e.g., due 
to adverse event (AE) monitoring, to fulfill screening requirements, and/or for safety 
maintenance. 
 
Caregivers/patients will be instructed on the daily use of the eDiary. The eDiary will include 
patient-reported outcome (PRO) for patients ≥8 years of age and observer-reported outcome 
(ObsRO) instruments for evaluation of itching (PRO), scratching (ObsRO), and sleep 
disturbance (PRO and ObsRO) during the Screening Period (study Days -56 to 0), throughout 
the 24-week Treatment Period (study Days 0 to 168), and Follow-up (Day 196). Additionally, 
caregivers will be requested to use the eDiary to report the time each dose of study drug is 
administered during the Treatment Period. All entries made in the ediary are transmitted 
overnight to the database which can be accessed via Trial Manager. 
 
The study nurse will monitor eDiary compliance by routine review of the Trial Manager 
website. If both diary entries on a day are missing, the study nurse will call the 
patient/caregiver to remind them to complete all scheduled entries. Any noncompliance will 
be documented and explained in the source documents. 
 
3.2 
Randomization and blinding 
 
A double-blind design is employed so that both the investigators and the patients will be 
unaware of the treatment assignment during the study. The sponsor and contract research 
organization (CRO) personnel (except CRO statisticians generating the list of randomization 
codes) involved in this study will also not be aware of the treatment assignment during the 
study. 
 
After written informed consent/assent is obtained from an eligible patient/legal guardian, an 
8-digit patient identification number will be assigned.  The first 2 digits will denote the 
country, followed by a 3-digit site number and a 3-digit patient sequence number.  This 
number will be created and allocated by the Interactive Web Response System (IWRS), when 
the patient first enters the system at screening.  The randomization codes will be computer-
generated by an 
 biostatistician and kept by an unblinded statistician 
 
independent from the project team. The randomization will be done in blocks and stratified 
according to PFIC type (Type 1 and 2) and age category (6 months to 5 years, 6 to 12 years, 
and 13 to ≤18 years) to ensure approximate balance between dose schemes (1:1:1).  A 
separate randomization list will be prepared for the maximum of 5 patients from Study 
A4250-003 regardless of stratification.  Randomization codes will be assigned sequentially as 
patients become eligible for randomization. 
 
Patients who are eligible for randomization will be assigned a unique 4-digit randomization 
number by IWRS.  This randomization number identifies which treatment will be allocated to 


 
 
 
 
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the patient.  Patients who withdraw from the study after the randomization visit will not be 
replaced and their randomization number will not be reused.  
   
To ensure blinding, the study drug and the matching placebo have the same shape and size.  
Patients will receive capsule(s) of odevixibat according to their assigned dose group or 
capsule(s) of matching placebo once a day during the double-blind Treatment Period.  Labels 
on the study drug containers will not identify the treatment to which a patient is randomized.  
Traceability of the treatment is ensured by the study drug number. 
 
The IWRS system will assign a study drug number(s) corresponding to the randomization arm 
at each dispensing visit.  The 5-digit study drug number will identify study drug packs and 
will be detailed on the study drug label.  Dispensing of study drug will be coordinated by 
IWRS. 
 
3.3 
Study treatments and assessments 
 
There will be three treatment groups in this study.  
 
• Odevixibat 40 µg/kg/day group 
• Odevixibat 120 µg/kg/day group 
• Placebo group 
 
Odevixibat and placebo will be supplied as capsules for oral administration. Study drug is to 
be taken once daily on Day 1 through Day 168 as described in Protocol Section 8.2.   
Study drug will be dispensed to the patient at Visits 3, 4, 5, 6, 7, and 8, together with 
instructions on how to store and take the drug.  Study drug administration data, including 
whether each patient took each dose or partial doses of study drug, whether there were any 
delayed or missed doses, and whether the capsule was opened or swallowed whole will be 
documented through the eDiary and transferred to the clinical study database. 
 
The number of capsules provided to the patient will be based on body weight at Visit 3. At 
Visit 6, if a patient’s weight has shifted to another weight category (identified in 
Protocol Table 2), the number of capsules per day may be adjusted. 
 
Odevixibat should be taken in the morning, together with food. On clinic visit days when 
laboratory assessments are conducted, study drug should be taken after the visit and after 
laboratory samples are taken. The last dose of study drug will be administered the day before 
Visit 9.  At Study Visit 9, the patient has the option to enroll in an open-label extension study 
(A4250-008) should they meet the required entry criteria.  If the patient is continuing into 
Study A4250-008 this will be the last visit in this study and the first visit in Study A4250-008.  
Visit 9/EOT should also be performed if the patient is prematurely withdrawn from the study. 
 
Clinic Visit 10 will take place 28 days after Visit 9 for patients not continuing into Study 
A4250-008.  A patient who withdraws from treatment prematurely and does not enter Study 
A4250-008 will have EOT (Visit 9) assessments at the time of withdrawal and a follow-up 


 
 
 
 
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assessment (Visit 10) 28 days following the last dose of study drug, and the patient will be 
recorded as withdrawn. 
 
Efficacy assessments and daily recording of pruritus using an electronic diary (eDiary) in this 
study are briefly introduced as follows. 
 
Serum Bile Acids 
 
Blood samples for analysis of fasting total s-BA will be drawn at all visits. Samples will be 
processed and transported to a central laboratory per instructions in the laboratory manual. 
Patients will fast (water intake only is permissible) for at least 4 hours prior to the collection 
of samples for s-BA.  Exceptions can be made for infants, <12 months of age, if unable to fast 
for the full 4 hours.  For any visit at which a bile acid sample result is unreportable, an 
additional unscheduled visit for a repeat sample collection may be scheduled.  All s-BA 
results during the treatment period and follow-up will be blinded.   
 
Itching, Scratching and Sleep Score 
 
Itching, observed scratching, and sleep disturbance will be recorded twice each day via 
eDiary.  Patients and/or caregivers will be instructed to complete the eDiary every day 
throughout the study in the morning after the patient wakes up and, in the evening, just before 
the patient goes to sleep. Patients and caregivers will receive a formal training session during 
Visit 1.   
 
The eDiary includes Albireo ObsRO and PRO items.  Patients <8 years of age will not be 
asked to complete the Albireo PRO items; only the Albireo ObsRO will be completed by 
caregivers of patients in this age group.  Older patients, 8 to 18 years of age, will complete the 
Albireo PRO items and the caregiver will complete the Albireo ObsRO items.  The Albireo 
PRO items assess severity of itch, aspects of sleep disturbance (morning diary only), and 
tiredness.  For patients 8 to 12 years of age, the caregiver will read the Albireo PRO items 
along with the child and record the child’s response.  A guide will be provided to the 
caregivers that provides standardized explanations of the Albireo PRO items, in case the 
patient is confused or requires clarification about the meaning of a question.  The Albireo 
ObsRO items assess severity of observed scratching, aspects of observed sleep disturbance 
(morning diary only) and observed signs of tiredness (evening diary only).  The Albireo 
ObsRO and PRO scratching and itch severity items use 0 to 4 response scales, where each 
response is distinguished by a unique facial expression, verbal anchor, number, and color 
code (Protocol Appendix 2).  
 
Growth 
 
Growth will be measured as height (velocity) and weight (Z-score) using a certified weight 
scale at Visits 1, 3, 6 and 9.  BMI will be calculated by weight (kg) / height (m2).  Change will 
be defined as linear growth deficit (weight and BMI for age) compared to standard growth 
curve (Z-score, standard deviation from the 50th percentile [SD from P50]). 
 


 
 
 
 
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Quality of Life Questionnaire (PedsQL) 
 
Patients and caregivers will be asked to complete a QoL questionnaire (PedsQL) at Visit 3 
and Visit 9.  Details of the questions included on the questionnaire are provided in 
Protocol Appendix 4. 
 
Biomarker  
 
Blood samples for autotaxin and p-C4 will be drawn at Visits 3, 4, and 9 for children with 
body weight >10 kg. 
 
PELD/MELD Score 
 
At randomization and EOT, pediatric end-stage liver disease (PELD) score will be calculated 
for children under 12 years of age.  For children 12 years or older, the (model for end-stage 
liver disease) MELD score will be calculated.  
 
PELD Score = 4.80 * ln(total bilirubin) + 18.57 * ln(INR) - 6.87 * ln(albumin) + 4.36 (if 
patient <1 year: scores for patients listed for liver transplantation before the patient's first 
birthday continue to include the value assigned for age (<1 year) until the patient reaches the 
age of 24 months) + 6.67 (if the patient has growth failure [< -2SD]) 
 
Laboratory values less than 1.0 will be set to 1.0 for the calculation of the PELD score. 
 
MELD Score = 9.57 * ln(creatinine) + 3.78 * ln (total bilirubin) + 11.2 *ln(INR) + 6.43 
Laboratory values less than 1.0 will be set to 1.0 and serum creatinine values greater than 
4.0 mg/dL (equivalent of 353.6 µmol/L) will be set to 4.0 for calculation of the MELD score. 
 
For calculation of the PELD/MELD score [3] laboratory parameters will be converted (if 
necessary) into the following units: 
 
- Total Bilirubin in mg/dL 
- Albumin in g/dL 
- Creatinine in mg/dL 
 
Fibroscan® 
 
Where available, Fibroscan® will be performed as per institution standard practice at Visits 3 
and 9. The data collected on the Fibroscan form (liver stiffness measured in kPa and 
controlled attenuation parameter [CAP] measured in dB/m) will be converted to determine 
stage of fibrosis and grade of steatosis, respectively, using a score card [4,5] as outlined in the 
table below. 
 
 
 


 
 
 
 
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Fibroscan Scoring for Cholestatic Liver Disease (Fibrosis Score): 
 
Score 
F0 to F1 
No scarring/ 
Mild fibrosis 
F2 
Moderate 
fibrosis 
F3 
Severe fibrosis 
F4 
Cirrhosis or 
Advanced 
fibrosis 
FibroScan 
Result 
2 to 7 kPa 
>7 to 9 kPa 
>9 to 17 kPa 
>17 kPa 
 
CAP Score & Steatosis Grading: 
CAP Score 
Amount of Liver 
with Fatty Change 
Steatosis Grade 
 
<238 dB/m 
 
S0 
238 to 260 dB/m 
11 to 33% 
S1 
260 to 290 dB/m 
34 to 66% 
S2 
> 290 dB/m 
≥67% 
S3 
 
Markers of Fibrosis 
 
APRI score and Fib-4 score will be calculated at randomization and at end of treatment. 
APRI = [(AST in U/L) / (AST ULN in U/L)] × 100 / (Platelets in 109/L) 
Fibrosis 4 Score = (Age*AST in U/L) / (Platelets in 109/L * √ (ALT in U/L)). 
 
Global Impression of Change and Global Impression of Symptom Measures 
 
Patients ≥8 years of age, caregivers, and clinicians will complete the global impression of 
change (GIC) and the global impression of symptoms (GIS) measures at randomization (Visit 
3; GIS only), Visits 4 and 6, and at EOT (Visit 9). The GIC items assess change in itch 
(patient version), scratching (caregiver and clinician versions), and sleep (all versions) since 
starting the study drug.  The GIS items assess itch (patient version), scratching (caregiver and 
clinician versions), and sleep (all versions) in the past week.   
 
A detailed description of procedures and assessments to be conducted during this study is 
summarized in the schedule of study assessments in Table 1 below. 
 
 


 
 
 
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Table 1: Schedule of Study Assessments 
Study Activity 
Screening 
Period 
Treatment Period 
Follow-Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
Informed consent c 
X 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
Concomitant medicationd 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical and surgical history 
X 
 
X 
 
 
 
 
 
 
 
 
Physical examination including 
voluntary photography 
X 
 
X 
 
 
 
X 
 
 
X 
 
Skin examination 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
Vital signse 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
eDiary: itching, scratching, and sleep 
scoresf 
Daily diary entry 
Clinical chemistryg 
X 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Hematologyg 
 
 
X 
 
X 
X 
X 
X 
 
X 
X 
Urinalysisg 
 
X 
 
 
 
 
X 
 
 
X 
 
Serum bile acidsh 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
INR  
 
X 
 
 
X 
 
X 
 
X 
 
X 
Autotaxini 
 
 
X 
 
X 
 
 
 
 
X 
 


 
 
 
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Study Activity 
Screening 
Period 
Treatment Period 
Follow-Up 
Random- 
ization 
Phone  
Contact 
4 weeks 
8 weeks 
12 weeks 
18 weeks 
22 weeks 
24 weeks 
Study Days 
-56 – (-35)  
±2 
-28 – (-7)  
±2 
0 
14 ±3 
28 ±3 
56±3 
84±3 
126 ±3 
154 ±3 
168 ±3 
196 ±3 
Visits 
Clinic  
Visit 1 
Clinic 
Visit 2 
Clinic 
Visit 3 
n/a 
Clinic  
Visit 4 
Clinic  
Visit 5 
Clinic  
Visit 6 
Clinic  
Visit 7 
Clinic  
Visit 8 
Clinic 
Visit 9/EOTa 
Clinic 
Visit 10b 
p-C4i 
 
 
X 
 
X 
 
 
 
 
X 
 
AFP 
 
 
X 
 
 
 
 
 
 
X 
 
Vitamins A & Eh 
 
X 
 
 
 
 
X 
 
 
X 
 
25-hydroxy vitamin D 
 
 
X 
 
 
 
X 
 
 
X 
 
Blood sample for A4250 PKi 
 
 
 
 
X 
 
 
 
 
X 
 
Abdominal Ultrasound 
 
 
X 
 
 
 
 
 
 
X 
 
Fibroscan® (where available) 
 
 
X 
 
 
 
 
 
 
X 
 
QoL questionnaire (PedsQL) 
 
 
X 
 
 
 
 
 
 
X 
 
Patient/Caregiver/Clinician Global 
Impression of Change 
 
 
 
 
X 
 
X 
 
 
X 
 
Patient/Caregiver/Clinician Global 
Impression of Symptoms 
 
 
X 
 
X 
 
X 
 
 
X 
 
Pregnancy testj 
Xj 
X 
X 
 
X 
X 
X 
X 
X 
Xj 
X 
Study drug dispensedk 
 
 
X 
 
X 
X 
X 
X 
X 
 
 
Adverse eventsl 
Continuous collection 
Study drug compliance 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 


 
 
 
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AFP: alfa-fetoprotein; BMI: body mass index; eCRF: electronic case report form; eDiary: electronic diary; EOT: End of Treatment; ICF: informed consent form; INR: international normalized ratio; p-C4: plasma 7α 
Hydroxy 4-cholesten-3-one; PedsQL: Pediatric Quality of Life Inventory; QoL: quality of life; RR: respiratory rate; s-BA: serum bile acid; PK: pharmacokinetics. 
 
a 
Must also be performed at the time a patient is prematurely withdrawn from the study.  This visit is the same as Visit 1 in the extension protocol (Study A4250-008). 
b 
Assessments must be performed 28 days following the final dose of study drug.  This visit is omitted if patients are entering Study A4250-008. 
c 
The ICF (and assent) must be signed before any study procedures are performed. 
d 
Past (within 1 month prior to Visit 1) and current medication will be documented in the eCRF. 
e 
Includes blood pressure, pulse, RR, temperature, height (or length depending on age), and body weight (using a certified weight scale).  BMI will be calculated. 
f 
Includes training on use of the device at Screening (Visit 1) and review of patient daily entries during 14 days prior to Clinic Visit 3 to determine eligibility.  
g 
See protocol Table 3 for detailed parameters. 
h 
Patients will fast (water intake only is permissible) for at least 4 hours prior to the collection of samples for s-BA and vitamin A.  Exceptions can be made for infants, less than 12 months of age, if unable to fast for the 
full 4 hours.  All s-BA results during the treatment period and follow-up will be blinded. 
i 
Samples will not be collected for patients ≤10 kg.  In the event of an SAE, a PK sample should be collected as close to the onset of the event as possible. 
j 
For girls who have reached menarche.  Serum test at Visit 1; urine test at other visits.  A serum pregnancy test will also be conducted at Visit 9 for patients entering Study A4250-008.  If a urine pregnancy test is positive, 
a serum pregnancy test should be performed to confirm the pregnancy. 
k 
Study drug to be taken once daily on Day 1 through Day 167 as described in protocol Section 8.2 
l 
Adverse event information will be collected from the time of signing of the ICF to study discontinuation. 
 


 
 
 
 
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4 
STUDY ENDPOINTS 
4.1 
Primary efficacy endpoints 
 
The primary efficacy endpoints are as follows: 
 
• Europe (EU) and rest of the world (RoW): Proportion of patients experiencing at least 
a 70% reduction in fasting s-BA concentration from baseline to the end of treatment or 
reaching a level ≤70 µmol/L after 24 weeks of treatment. 
 
Fasting s-BA baseline will be calculated as the average of the last 2 values prior to the 
first dose. The end value is the average of the values at Weeks 22 and 24 after the start of 
double-blind treatment. If a patient’s baseline value is ≤70 µmol/L, then only the criterion 
of at least 70% reduction in fasting s-BA concentration will be used to determine whether 
the patient is a responder or not for the primary endpoint analysis. 
 
• United States (US): Proportion of positive pruritus assessments at the subject level over 
the 24-week treatment period based on the Albireo ObsRO instrument.  
 
A positive pruritus assessment is defined as a scratching score of ≤1 or at least a one-
point drop from baseline on the Albireo ObsRO instrument. At each assessment, the AM 
score will be compared to the baseline AM average, and the PM score will be compared 
to the baseline PM average. Both AM and PM pruritus assessments will be included in 
the analysis of this endpoint. AM scores from the period of 14 days prior to or on the first 
dose day of study medication will be averaged as AM baseline. PM scores from the 
period of 14 days prior to the first dose day of study medication will be averaged as PM 
baseline. If a patient’s baseline average score is ≤ 1, then only the criterion of a one-point 
drop from baseline on the Albireo ObsRO instrument will be used to determine whether a 
pruritus assessment is positive or not for the primary endpoint analysis.  
 
4.2 
Secondary efficacy endpoints 
The secondary efficacy endpoints include the following:  
• EU and RoW: Proportion of positive pruritus assessments at the subject level over the 
24-week Treatment Period based on the Albireo ObsRO instrument. 
 
• US: Proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to the end of treatment or reaching a level ≤70 μmol/L 
compared to placebo after 24 weeks of treatment. 
 


 
 
 
 
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• All Regions: 
            All other secondary endpoints are compared to placebo. 
o Change from baseline to Week 12 and to Week 24 in fasting s-BA 
 
o Change from baseline to Week 12 and to Week 24 in serum ALT concentration 
 
o Change in growth from baseline to Week 12 and to Week 24, defined as the linear 
deficit (height for age, weight for age and MBI for age) compared to a standard 
growth curve (Z-score, SD from P50) 
 
o Proportion of responders for pruritus scores at Weeks 12 and 24 based on the 
ObsRO instrument. For pruritus scores, a responder is defined as a patient 
achieving a reduction in score equivalent to or exceeding the clinically 
meaningful threshold established from the blinded psychometric analysis 
(Pruritus Evidence Dossier).  
 
o Change in sleep parameters measured with the Albireo PRO and ObsRO 
instruments by each 4-week interval over the 24-week treatment period 
 
o Proportion of individual assessments meeting the definition of a positive pruritus 
assessment at the subject level over the 24-week treatment period as reported on 
the Albireo PRO instrument. A positive pruritus assessment includes an itch score 
≤1, or at least a one-point drop from baseline based on the Albireo PRO 
instrument; only patients ≥8 years of age will complete the Albireo PRO 
instrument 
 
o Proportion of individual assessments meeting the definition of a positive pruritus 
assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 
0-18, Weeks 0-24, respectively, and the proportion of positive pruritus 
assessments at each 4-week interval as reported on the Albireo ObsRO 
instrument. A positive pruritus assessment is defined as a scratching score of ≤1 
or at least a one-point drop from baseline on the Albireo ObsRO instrument. At 
each assessment, the AM or PM score will be compared to the baseline AM or 
PM average, respectively. 
 
o Proportion of individual AM assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, 
Weeks 0-18, Weeks 0-24, respectively, and the proportion of positive pruritus 
assessments at each 4-week interval as reported on the Albireo ObsRO 
instrument. A positive pruritus assessment is defined as a scratching score of ≤1 
or at least a one-point drop from baseline on the Albireo ObsRO instrument. At 
each assessment, the AM score will be compared to the baseline AM average. 
 
o Proportion of individual PM assessments meeting the definition of a positive 
pruritus assessment at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, 


 
 
 
 
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Weeks 0-18, Weeks 0-24, respectively, and the proportion of positive pruritus 
assessments at each 4-week interval as reported on the Albireo ObsRO 
instrument. A positive pruritus assessment is defined as a scratching score of ≤1 
or at least a one-point drop from baseline on the Albireo ObsRO instrument. At 
each assessment, the PM score will be compared to the baseline PM average. 
 
o Number of patients undergoing biliary diversion surgery or liver transplantation 
 
o Number and percent of patients achieving positive pruritus assessment for more 
than 50% of the time during the 24-week treatment period 
 
4.3 
Exploratory endpoints 
Exploratory efficacy endpoints include the following: 
• Assessment of Global Symptom Relief at Weeks 4, 12, and 24 as measured by patient, 
caregiver, and clinician via the global impression of change (GIC) item  
 
• Change from baseline in fasting s-BA at Week 4 
 
• Change in pruritus, as indexed by caregiver-reported observed scratching, from baseline 
by each 4-week interval of the Treatment Period as measured by the Albireo ObsRO 
instrument 
 
• Change from baseline by each 4-week interval of the Treatment Period in patient-
reported and observer-reported night time itching and scratching severity scores, 
respectively, as measured by the Albireo PRO and ObsRO instruments. 
 
• Change from baseline by each 4-week interval of the Treatment Period in patient-
reported and observer-reported morning time itching and scratching severity scores, 
respectively, as measured by the Albireo PRO and ObsRO instruments. 
 
• Change from baseline by each 4-week interval of the Treatment Period in pooled pruritus 
score including observer-reported scratching for patients <8 years of age and patient-
reported itch severity for patients ≥8 years of age, as measured by the Albireo PRO and 
ObsRO instruments, respectively.  
 
• Change from baseline by each 4-week interval of the Treatment Period in additional 
patient-reported and observer-reported sleep parameters (e.g. tiredness and number of 
awakenings), as measured by the Albireo PRO and ObsRO instruments, respectively. 
 
• Change from baseline to Week 24 in PedsQL 
 
• Change from baseline in serum ALT concentration at Week 4 
 


 
 
 
 
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• Change from baseline in total bilirubin at Weeks 4, 12, and 24 
 
• Change from baseline in AST and gamma-glutamyl transferase at Weeks 4, 12, and 24 
 
• Change from baseline for other biochemical markers and measures of bile acid synthesis 
(autotaxin, plasma 7α-hydroxy-4-cholesten-3-one concentration [p-C4]) at Week 24 
 
• Change in paediatric end-stage liver disease/model for end-stage liver disease 
(PELD/MELD) score, AST to platelet ratio index (APRI) score, and Fib-4 score from 
baseline to Week 24 
 
• Change from baseline to Week 24 in stage of liver fibrosis as assessed by Fibroscan® 
(where available) 
 
• For patients who answered Yes to Questions 1 and 2 in the exit survey, changes from 
baseline in the average scratching score based on the Albireo ObsRO instrument in the 
last 2 weeks before treatment termination  
 
• For patients who answered No to Questions 1 or 2 in the exit survey, changes from 
baseline in the average scratching score based on the Albireo ObsRO instrument in the 
last 2 weeks before treatment termination  
 
4.4 
Safety  
Safety criteria are as follows: 
 
• Primary safety analysis is the incidence of treatment-emergent adverse events (TEAEs) and 
TEAEs categorized by causality, severity, and seriousness assessments made by the 
investigator by comparing active arms vs. placebo. This includes liver-related mortality and 
liver decompensation events (protocol Section 10.2.2.3) and all-cause mortality. 
 
• Safety will also be evaluated by the following assessments: 
 
- Physical examinations 
- Concomitant medications 
- Vital signs 
- Laboratory test results (including clinical chemistry, hematology, urinalysis, AFP, vitamins 
  A and E, 25-hydroxy vitamin D, and INR) 
- Abdominal ultrasound 
- Discontinuations due to AEs 
 
 


 
 
 
 
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5 
SAMPLE SIZE AND POWER 
For the EU and RoW, the primary analysis will be related to the serum bile acid responder 
endpoint. For the US, the primary analysis will be related to the pruritus endpoint. The CMH test 
stratified by randomization strata will be used to test the serum bile acid responder endpoint.  
The individual null hypothesis is that the odds ratio of the response in an active arm vs. placebo 
is 1.  The alternative hypothesis is that the odds ratio is greater than 1.  An ANCOVA will be 
used for treatment comparisons for the proportion of positive pruritus assessments at subject 
level.  The individual null hypothesis is that the average proportion of positive assessments is the 
same between the active and placebo arms.  The alternative hypothesis is that the average 
proportion is larger in the active arm. 
 
Since each active arm will be compared with the placebo, there will be two individual null 
hypotheses for each endpoint.  In each endpoint, a pooled analysis for the closed testing 
procedure will be applied to control the 1-sided overall type I error rate for the two odevixibat 
treatment comparisons vs. placebo at the 0.025 level [6,7,8]. The significance of an active arm 
will be claimed if both 1-sided p-values in the pooled analysis and individual treatment 
comparison are less than or equal to 0.025.  
 
The study will enroll 60 to 70 patients in order to obtain at least 20 evaluable patients in each 
arm. For each primary endpoint, simulations with 5,000 iterations using 20 patients per arm were 
conducted to estimate the power after multiplicity adjustment, resulting in a standard error (SE) 
of <0.7% for each estimated power. 
   
Based on the Phase 2 study (A4250-003) data, both low and high dose groups are assumed to 
have the same positive treatment effects in both the serum bile acid and pruritus endpoints in the 
simulation. For serum bile acids, binomial distributions were used to simulate the proportion of 
responders to estimate the power. The simulated proportions were analyzed using the Cochran-
Mantel-Haenszel (CMH) test to generate the following 1-sided p-values: both odevixibat arms 
pooled vs. placebo, low dose vs. placebo, and high dose vs. placebo. Assuming 60% responders 
in the odevixibat arms and 10% responders in the placebo arm, the power to claim significance 
for a particular odevixibat arm after multiplicity adjustment is approximately 94%. The 
probability to claim significance for at least one arm, and for both arms are approximately 99% 
and 91%, respectively. If the response rates are 50% in the odevixibat arms and 10% in the 
placebo arm, with 20 patients per arm, the probability to claim significance for a particular 
odevixibat arm after multiplicity adjustment is approximately 82%. The probability to claim 
significance for at least one arm, and for both arms is approximately 91% and 73%, respectively. 
 
For the proportion of positive pruritus assessments at the subject level in pruritus scores, beta-
binomial distributions were used for power simulations since the binary results of the individual 
assessments within a subject are likely correlated [9]. The effect size was 1.0526 from the 
original sample size calculation using change from baseline as the endpoint.  The same effect 
size was assumed for the current endpoint for the low and high dose vs. the control.  A difference 
of 15%, 20%, 25%, and 30% in proportion of positive pruritus assessments were considered in 
power simulation.  Within each difference, proportions of positive assessments in the placebo 
arm ranging from 15% to 35% were considered.  Subsequently, the proportion of positive 


 
 
 
 
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assessments in an active arm, the standard deviation, and the corresponding beta binomial 
parameters were calculated to satisfy the assumed effect size.  These parameters were used to 
simulate correlated binary results within each subject. The simulated proportions were analyzed 
using analysis of variance (ANOVA) to generate the following 1-sided p-values: both odevixibat 
arms pooled vs. placebo, low dose vs. placebo, and high dose vs. placebo. The simulation in each 
scenario was repeated for 5,000 iterations using the current sample size of 20 patients per arm. 
The simulated power to claim significance for a particular arm after multiplicity adjustment is 
quite consistent under different scenarios and is approximately 89%. The probability to claim 
significance for at least one arm, and for both arms is approximately 95% and 83%, respectively.   
 
 


 
 
 
 
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6 
ANALYSIS SETS 
 
6.1 
Full Analysis Set (FAS) 
 
The full analysis set (FAS) will consist of all randomized patients who received at least 1 dose of 
study treatment.  Patients will be analyzed in the treatment group they were randomized to even 
if they received incorrect study drug.  The FAS will be the primary analysis set for efficacy 
analyses. 
 
6.2 
Safety Analysis Set (SAS) 
 
The safety analysis set will consist of all randomized patients who received at least 1 dose of 
study drug.  Patients will be analyzed according to the treatment they actually received.  In case 
of wrong study drug dispensed to a patient in error, the patient will be assigned to the treatment 
arm that the actual drug was mostly taken. The safety analysis set will be used for safety 
analyses.  
 
6.3 
Per-Protocol Analysis Set (PP) 
 
The per protocol (PP) analysis set is a subset of the FAS and will consist of all randomized 
patients for whom no important protocol deviation (no deviation which may affect the study 
efficacy outcome) is documented.  In identifying this population, important protocol deviations 
are defined as: 
• Failure to meet the following inclusion criteria 
o Inclusion criteria (2) Patient must have a clinical genetic confirmation of PFIC-1 or 
PFIC-2 through identification of biallelic pathogenic variants in either the ATP8B1 or 
ABCB11 genes 
o  Inclusion criteria (3) Patient must have elevated s-BA concentration, specifically 
measured to be ≥100 μmol/L, taken as the average of 2 samples at least 7 days apart 
(Visits 1 and 2) prior to randomization 
o  Inclusion criteria (4) Patient must have history of significant pruritus and a caregiver-
reported observed scratching in the eDiary average of ≥2 (on 0 to 4 scale) in the 2 
weeks prior to randomization 
• Enrollment in spite of meeting the following exclusion criteria 
o  Exclusion criteria (1) Patient with pathologic variations of the ABCB11 gene that 
predict complete absence of the BSEP protein 
o Exclusion criteria (15) Patient suffers from uncontrolled, recalcitrant pruritic 
condition other than PFIC.  Examples include, but not limited to, refractory atopic 
dermatitis or other primary pruritic skin diseases 
• Compliance rate of <80% with study drug administration estimated based on CRF data 
 
Important protocol deviations will be also identified by study team during protocol deviation 
review. The PP analysis set will provide supportive data for the efficacy analyses of primary and 
selected secondary endpoints. 
 


 
 
 
 
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7 
STATISTICAL CONSIDERATIONS AND ANALYSIS 
 
7.1 
Handling of missing data and/or invalid data and outliers 
 
7.1.1 Handling of Missing data for efficacy endpoints 
7.1.1.1 Pruritus Endpoint 
In the primary analysis of the pruritus endpoint, all intermittently missing assessments will be 
classified as negative pruritus assessments (that is, not meeting the definition of a positive 
assessment as defined in Section 4.1). Similarly, all missing planned assessments after premature 
treatment discontinuation will be counted as negative pruritus assessments. All planned 
assessments after death or initiation of rescue treatments such as biliary diversion surgery or 
liver transplantation will be counted as negative pruritus assessments. Both on-treatment and off-
treatment pruritus assessments up to Day 168 will be included for analysis.  
 
In general, all 336 (=168 x 2) assessments planned over the 24-week treatment period will be 
included in the denominator of the endpoint definition. However, if the last pruritus assessment 
for a patient who completes treatment (i.e., completes Week 24 assessment) is reported prior to 
Day 168, then the denominator used for analysis will be (the last report day x 2). The last report 
day will be determined for ObsRO and PRO instruments separately. 
 
For the analysis of the pruritus endpoint, an additional method will also be used to calculate the 
proportion of positive pruritus assessments up to the last report day before or on Day 168. In 
other words, the denominator to calculate the proportion will be (the last report day x 2). All 
missing data before or on the last report day will be considered as negative assessments. 
 
 
In addition, a multiple imputation method will be performed for pruritus assessments as a 
continuous endpoint by considering the impact of intermittently missing data as well as 
intercurrent events (Protocol Section 11.2.2.2). Moreover, a tipping point analysis will be 
performed to further evaluate the influence of missing data. The details of this are addressed in 
the Section 8.6.3.1. 
7.1.1.2 Serum bile acid responder endpoint 
In the primary analysis of the fasting bile acid responder endpoint, dropouts and treatment 
completers with a missing average at the end of the treatment will be treated as non-responders. 
Patients in need of surgical rescue (i.e., biliary diversion and/or liver transplant) will be classified 
as non-responders.  
  
As a sensitivity analysis, a tipping point analysis of fasting bile acid responder endpoint will be 
performed to evaluate the strength of evidence if the result is significant. The details of this are 
addressed in the Section 8.6.3.1. 
 


 
 
 
 
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8 
STATISTICAL METHODS 
 
8.1 
General statistical conventions 
 
All personnel involved with the analysis of the study will remain blinded until database lock. For 
DSMB analyses see Section 8.9.2. All statistical procedures will be completed using SAS® 
version 9.4 or higher. 
 
Statistical testing for each primary endpoint analysis will be performed with a 1-sided overall 
Type I error rate of 0.025, adjusting for 2 comparisons between each active arm vs. the control. 
All other secondary endpoints for treatment comparisons will not be adjusted for multiplicity and 
nominal p values will be provided. Two-sided 95% confidence intervals (CI) will be provided 
when relevant. 
 
For analysis adjusting for stratification factors (PFIC type and age category), if a patient is under 
6 months due to imputed birth date (Appendix A), then the patient will be grouped to the age 
category ‘6 months to 5 years’.  
 
Continuous variables will be summarized using descriptive statistics, including the number of 
patients with non-missing value (n), mean, median, SD, minimum and maximum. “n” will be 
presented without a decimal point, minimum and maximum values will be presented in the same 
precision as in the database, mean and median will be presented in one more decimal place than 
the minimum and maximum, and SD will be presented in one more decimal place than the mean 
and median. 
 
For categorical variables, summaries will include counts of patients (frequencies) and 
percentages. Percentages will be rounded to one decimal place. Descriptive summaries of change 
from baseline in categorical variables will be provided using shift tables, if applicable.  
 
For summary purposes, if not otherwise specified, the baseline value of a parameter is defined as 
the last non-missing assessment of that parameter prior to the first intake of study drug 
(Appendix A). Derived variables used for the analyses are provided in Appendix A. All 
summaries will be presented by treatment group, unless otherwise specified. 
 
For summary tables, the following 2 presentations will be used: 
 
• For baseline related tables and protocol deviations: odevixibat 40 µg/kg/day, odevixibat 120 
µg/kg/day, odevixibat 2 doses combined, placebo, and overall (3 treatment groups combined) 
will be presented. 
• For the safety and efficacy related tables: odevixibat 40 µg/kg/day, odevixibat 120 
µg/kg/day, odevixibat 2 doses combined, and placebo will be presented. 
 
The visit window details for the derivation of study weeks and time points are outlined in 
Appendix B. 
 
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listings will be sorted by patient ID, treatment group, date/time and visit. Each patient’s sex and 
age will be stated on each listing. Data listings will be based on all randomized patients. 
 
8.2 
Subject disposition 
 
All patients who provided informed consent will be included in a summary of patient 
accountability.   
 
The following categories will be summarized (by treatment group and overall where applicable): 
 
• Patients screened 
• Screening failures 
• Patients randomized 
• Patients randomized from Study A4250-003 
• Patients randomized and dosed 
• Patients completing treatment 
• Patients completing the study 
• Patients discontinuing treatment (including reasons for treatment discontinuation) 
• Patients withdrawing early from study (including withdrawal reasons) 
• Patients entering the extension study (A4250-008) 
 
Additionally, patients included in the FAS analysis set will be summarized by region 
(Appendix C). 
 
 
8.3 
Protocol deviations 
 
Important protocol deviations will be identified prior to database lock. All exclusions of patients 
from the per-protocol (PP) analysis sets will be performed programmatically using the criteria of 
important protocol deviations (Section 6.3) in a blinded way and reviewed at the final protocol 
deviations review meeting prior to the final database lock, through clinical input provided by the 
sponsor, using the following sources of information:  
 
• Supportive subject listings, provided by the 
lead statistician ahead of the protocol 
deviation review meeting, based on data recorded on the eCRF. 
• Protocol deviation logs, provided by 
 Clinical, Pharmacovigilance, Medical and 
Data Management. 
 
The number of patients with identified important protocol deviations will be summarized by 
treatment group and overall (on randomized patients). Details on important protocol deviations 
will be provided in a listing on all randomized patients.  
 
In addition, a listing will be provided with all protocol deviations identified based on data 
recorded on the eCRF and/or protocol deviation logs from 
 Medical and Data Management 
(on randomized patients) prior to database lock and documented in the CSR.  


 
 
 
 
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8.4 
Demographics and baseline characteristics 
 
8.4.1 Demographics 
 
Age, height, weight and BMI at baseline and other demographic variables e.g., age category (6 
months to 5 years, 6 to 12 years, and 13 to 18 years), sex, race, ethnicity, country, and region 
(see Appendix C) will be summarized by treatment group and overall descriptively using the 
FAS analysis set.  
 
8.4.2 Baseline and disease characteristics 
 
The following disease characteristics will be summarized by treatment group and overall: years 
since PFIC diagnosis, type of PFIC, presence of significant pruritus per investigator report, at 
least one s-BA level >100 umol/L within 6 months prior to screening visit, reason for 
discontinuation of historical PFIC related investigational medications, and diagnostic genetic 
laboratory test as well as Child-Pugh classification and hepatic impairment classification per 
NCI Organ Dysfunction Working Group for the FAS analysis set. 
 
Child-Pugh classification: mild (Class A), moderate (Class B), or severe hepatic impairment 
(Class C) is based on the FDA Guidance for Pharmacokinetics in Patients with Impaired Hepatic 
Function. Determination of the classification is made based on medical review of baseline 
laboratory data and medical history. 
 
Hepatic impairment will also be classified per the NCI Organ Dysfunction Working Group 
classification as mild, moderate or severe (Mansfield et al. 2016 [10]). Both the Child-Pugh and 
NCI classification will be included in the baseline characteristics and both will be applied in the 
subgroup analysis based on hepatic impairment status. 
 
The following continuous parameters will also be summarized: estimated glomerular filtration 
rate (eGFR); baseline Z-score (weight, height, and BMI); and baseline values of alanine 
aminotransferase (ALT); aspartate aminotransferase (AST); gamma-glutamyl transferase (GGT); 
total bilirubin; alkaline phosphatase; INR; and vitamins A, E, and 25 hydroxy D. 
 
eGFR will be calculated based on the modified/bedside Schwartz equation. For patients <18 year 
of age, Bedside Schwartz Equation will be used:  
GFR (mL/min/1.73 m²) = (36.2 × Height in cm) / Creatinine in µmol/L 
 
For patients ≥ 18 years, the isotope dilution mass spectrometry (IDMS)-traceable Modification 
of Diet in Renal Disease (MDRD) Study equation will be used: 
GFR (mL/min/1.73 m2) = 175 × (Creatinine in µmol/L/88.4)-1.154 × (Age)-0.203 × (0.742 if female) 
× (1.212 if African American) 
 
 
 


 
 
 
 
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A listing will be created to present all the demographic and baseline characteristics for the FAS 
analysis set. In addition, an individual listing on liver biopsies performed within 1 year prior to 
screening will be produced. 
 
The results of pathologic variants identified for ATP8B1 or ABCB11 genes will be listed for all 
patients for the FAS analysis set. 
 
8.4.3 Medical and surgical history 
 
The frequencies and percentages of patients with reported medical and surgical history will be 
presented by system organ class (SOC) and preferred term (PT) by treatment group and overall 
for the SAF analysis set. Medical and surgical history will be coded using MedDRA version 23.0 
throughout the study. The summary table will be sorted alphabetically for SOC and PT. Medical 
and surgical history will also be listed. 
 
8.4.4 Prior and concomitant medications 
 
Prior and concomitant medications used in this study will be coded using the version of the 
World Health Organization Drug Global Dictionary March 2020 throughout the study.  
 
Prior medications: All medications taken by a patient within 3 months prior to the first intake 
of study drug with a recorded medication stop date prior to the first intake of study drug are 
regarded as prior medication. This will not include historical PFIC related investigational 
medications collected separately on CRFs. 
 
Concomitant medications: All medications taken by a patient on or after the first intake of 
study drug and medications with start date before the first intake of study drug without a 
recorded stop date prior to the first intake of study drug. 
 
Prior and concomitant medication use during the study will be summarized descriptively using 
frequency tables by Anatomical Therapeutic Chemical (ATC) class 4 and preferred term (PT) by 
treatment group for the SAF analysis set. ATC class and PT will be presented alphabetically. All 
prior and concomitant medication will be listed. Details for imputing missing or partial start 
and/or stop dates of non-study medication are described in Appendix D. 
 
8.5 
Extent of exposure 
 
8.5.1 Treatment duration 
 
Exposure will be summarized with descriptive statistics (n, mean, SD, minimum, median, and 
maximum) and presented by treatment group using the SAF analysis set. The exposure of study 
drug will be derived as follows: 
 
Duration of exposure (in weeks) = (Date of last study drug intake – Date of first study drug 
intake + 1)/7. 
  


 
 
 
 
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Investigators are allowed to interrupt the study drug to allow adverse events to resolve, if 
necessary.  Any drug interruption will not be considered when calculating the treatment duration. 
 
8.5.2 Study drug compliance 
 
Study drug compliance will be assessed by maintaining adequate study drug dispensing records. 
The study drug compliance over the treatment period will be calculated using 2 sources of data.  
 
Source Data from CRFs 
 
Treatment Compliance = 100 × [(Number of capsules dispensed – number of capsules 
returned)/number of capsules that should have been taken]. 
 
The number of capsules that should have been taken is calculated as the number of days that the 
patient was in the treatment period (exposure as above) multiplied by the number of prescribed 
capsules to be taken (based on patient’s body weight, protocol Table 2) during the treatment 
period. The total number of capsules actually taken is the total number of capsules recorded as 
taken based on the CRF (number of capsules dispensed minus returned) summed over the 
treatment period. If the number of capsules not returned is confirmed as missing and the study 
drug is confirmed as not having been returned, the derivation will not be done.  
 
Source Data from eDiary 
 
Treatment Compliance =100 x (total number of capsules taken/total number of capsules planned 
to be taken). The number of capsules planned to be taken will be estimated based on patient’s 
body weight at Visit 3 and Visit 6 per protocol Table 2. The number of capsules planned to be 
taken before Visit 6 will be the same as the number estimated at Visit 3, and after Visit 6 will be 
the same as the number estimated at Visit 6.  
 
The calculated compliance rates will be summarized and the one based on CRFs data will be 
considered the primary compliance rate.  The compliance rates reported by visit on the CRFs 
will be summarized also. 
 
Descriptive summary statistics will be used to summarize study drug compliance by treatment 
group. The number and percentage of patients with compliance <80%, between 80% and 120%, 
and >120% will also be presented based on the SAF analysis set. 
 
 
8.6 
Efficacy analyses 
 
This section addresses the analyses to be conducted on the primary, secondary, and exploratory 
efficacy variables. The primary objective of this study is to demonstrate the efficacy of repeated 
daily doses of 40 μg/kg/day and 120 μg/kg/day of odevixibat in children with progressive 
familial intrahepatic cholestasis (PFIC) Types 1 and 2, as determined by the following: 
 
• Proportion of patients experiencing at least a 70% reduction in fasting serum bile acid (s-BA) 


 
 
 
 
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concentration from baseline to end of treatment or reaching a level ≤70 μmol/L compared to 
placebo after 24 weeks of treatment 
 
• The proportion of positive pruritus assessments at the subject level over the 24-week Treatment 
Period 
 
The efficacy analysis will be carried out using the patients from the FAS and/or PP analysis set, 
unless otherwise specified. Figures for selected efficacy endpoints, such as the primary pruritus 
endpoint, change in s-BA, change in other patient reported and caregiver reported outcomes, will 
be provided by treatment group and by visit.  
 
For efficacy endpoints derived from central laboratory data, if central laboratory data are not 
available due to COVID-19 or other reasons, local laboratory data will be used. 
 
8.6.1 Analyses methods 
8.6.1.1  Cochran Mantel Haenszel (CMH) Test 
The CMH test is used in the analysis of stratified categorical data. It tests the association 
between treatment and a binary outcome such as responder while considering the stratification 
factors. For the proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L after 24 
weeks of treatment, the CMH test will be used for treatment comparisons. CMH stratified by 
PFIC type and age category will be performed to compare the proportion of the responders in 
fasting bile acid at the end of treatment (based on average of Weeks 22 and 24). The testing 
procedure is addressed in the Section 8.6.1.3. 
 
In the CMH test, data in a stratum will not be used in the calculation of the p-value if a row sum 
or column sum is 0 in the contingency table.  To ensure that all data are used when this occurs, 
data will be pooled with the adjacent age stratum that has a smaller number of subjects.  If a row 
sum or column sum is still 0 after pooling data, all age groups will be pooled (i.e. the CMH test 
will be stratified by PFIC type only).  If a row sum or column sum is still 0, the CMH test will 
not be stratified. Pooling strategy will also be conducted for a stratum with <4 patients.  
 
The proportion together with corresponding Clopper-Pearson exact 95% CI, odds ratio and 
corresponding 95% CI, and p-value for the CMH test will be presented. The proportion 
difference with corresponding exact unconditional 95% CI without adjusting stratification 
factors will be presented. Miettinen-Nurminen (score) CI with adjusting stratification factors will 
be reported for common risk difference (i.e., proportion difference). The exact CI will be 
reported for the common odds ratio by using an algorithm based on Vollset, Hirji, and Elashoff 
(1991) [11]. SAS code will be found in Appendix E. 
8.6.1.2  Analysis of Covariance (ANCOVA) model 
The ANCOVA model is usually used to test the main and interaction effects of categorical 
variables on a continuous dependent variable, controlling for the covariates. The ANCOVA 
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at subject level over the 24-week treatment period between the treatment groups.  
8.6.1.3  Multiplicity 
For EU and RoW, the primary analysis will be related to the serum bile acid responder endpoint. 
For US, the primary analysis will be related to the pruritus endpoint. For each primary endpoint, 
a pooled analysis for the closed testing procedure will be applied to control the 1-sided overall 
type I error rate for two treatment comparisons vs. the placebo at the 0.025 level as specified 
below: 
 
In the closed testing procedure, the low and high dose groups are pooled to compare with the 
placebo group first. If the 1-sided p-value is ≤0.025, the 1-sided p-values for low dose vs. 
placebo and high dose vs. placebo, respectively will be calculated. If both individual p-values are 
≤0.025, a significant treatment effect will be declared on both dose groups. If only one of them is 
≤0.025, a significant treatment effect will be declared on the corresponding dose group.  
 
In testing the primary endpoint for US, treatment arms will be separated in the pooled analysis 
and the “lsmestimate" statement in SAS Proc Mixed will be used to compare pooled active arms 
vs. the placebo.  See Appendix E. 
 
Adjusted p-values will also be reported when unadjusted p-values are presented directly from the 
model. The adjusted p-value for an individual dose is calculated as the maximum value of the 
unadjusted p-value for the pooled low and high doses and the unadjusted p-value for the 
individual doses.  
 
Analyses of secondary and exploratory endpoints will provide supportive efficacy and safety 
information regarding the differences between the treatment groups. No adjustments will be 
performed for multiple comparisons when testing these secondary and exploratory endpoints. 
 
8.6.2 Treatment by center interaction analysis (multi-center study) 
 
There will be only a few patients per center, so no direct exploration of treatment-by-center 
interaction effect is planned. Additional treatment-by-covariate interactions may be explored. 
 
8.6.3 Analyses of primary efficacy endpoints 
 
The primary analyses on the primary endpoints will be based on the FAS. Additional analyses on 
the primary endpoints will be conducted for PP analysis set. 
 
For EU and RoW, proportion of patients experiencing at least a 70% reduction in fasting s-BA 
concentration from baseline to the end of treatment or reaching a level ≤70 µmol/L after 24 
weeks of treatment will be analyzed using the CMH test.  Patients with missing data at the end of 
treatment will be classified as non-responders. CMH stratified by PFIC type and age category 
will be performed to compare the proportion of the responders in fasting bile acid at the end of 
treatment (based on average of Weeks 22 and 24). The testing procedure is addressed in the 
Section 8.6.1.3. The contrasts of primary interest will be the treatment differences at end of 
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be estimated and analyzed. 
 
For US, the primary endpoint is the proportion of positive pruritus assessments at the subject 
level over the 24-week Treatment Period. A positive pruritus assessment is defined as a 
scratching score of ≤1 or at least a one-point drop from baseline on the Albireo ObsRO 
instrument. At each assessment, the AM score will be compared to the baseline AM average, and 
the PM score will be compared to the baseline PM average. Both AM and PM pruritus 
assessments will be included in the analysis of this endpoint. For analysis purposes, diary entries 
will be assigned to a study day based on the recorded date regardless of recorded time. AM 
scores from the period of 14 days prior to or on the first dose day of study medication will be 
averaged as AM baseline. PM scores from the period of 14 days prior to the first dose day of 
study medication will be averaged as PM baseline. Baseline score will be rounded to an integer 
to evaluate the positive pruritus assessments for the primary analysis. Additional analysis will be 
conducted using unrounded baseline score.  
 
 
An ANCOVA model will be used for treatment comparisons. The model will include treatment 
arm, AM baseline pruritus score, PM baseline pruritus score, and stratification factors, i.e., PFIC 
type and age category. The testing procedure is addressed in the section 8.6.1.3. 
 
LS mean (SE) by treatment arm and LS mean difference (SE), 95% confidence intervals, and p-
values (where applicable) between treatments vs. placebo will be provided (see Section 8.6.1.3 
below). SAS code will be found in Appendix E. If there are concerns on model assumptions, 
normality will be checked based on Shapiro-Wilk test and homogeneity of variances will be 
checked based on Levene’s test. For normality testing, Shapiro-Wilk test will be performed for 
each arm. The p-value from each arm will be combined to get an overall p-value based on 
Fisher's combined probability test.  
 
A non-parametric ANCOVA based on the rank scores will be used as a sensitivity analysis to 
calculate the p-values. The proportion of positive pruritus assessments at subject level over 24-
week treatment period will be ranked ignoring treatment group. The value of midranks will be 
used in the case of ties.  The baseline pruritus scores will also be ranked in the same manner.  
8.6.3.1  Sensitivity and supportive analyses of primary efficacy endpoints 
 
EU and RoW primary endpoint 
 
• A sensitivity analysis will be performed by excluding patients whose baseline value is ≤70 
µmol/L using the same approach as for the primary analysis. 
 
• Logistic regression will be performed as a supportive analysis for this endpoint. The model 
will include treatment arm, baseline value, and stratification factors, i.e., PFIC type and age 
category. If the logistic model cannot converge, then the age category with small sample size 
may be pooled to handle the convergence of the modeling. 
 


 
 
 
 
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• A tipping point analysis will be conducted to evaluate the influence of missing data at the 
end of treatment. To start, one patient on placebo arm with a missing value at the end of 
treatment is assumed as a responder and the primary analysis will be re-run to see if the 
active treatment group is still significantly better compared to the placebo arm. This will be 
performed for each patient on placebo arm with a missing value at the end of treatment to see 
how the result changes, and the worst result will be reported for this step (i.e. the worst 
scenario). If the result is still significant then this process will continue by assuming any 2 
patients on placebo arm with missing values as responders until the result becomes non-
significant. 
 
US primary endpoint 
 
• An additional method is used to calculate the proportion of positive pruritus assessments up 
to the last report day before or on Day 168. In other words, the denominator to calculate the 
proportion will be (the last report day x 2). All missing data before or on the last report day 
will be considered as negative assessments. This estimated proportion of positive pruritus 
assessments at the patient level over the 24-week treatment period based on the Albireo 
ObsRO instrument will be analyzed using the same approach as for the primary analysis. 
 
• To address the concern that results from subjects with a baseline AM or baseline PM average 
score less than 2 may be difficult to interpret, additional supportive analysis will exclude 
patients from the primary endpoint analysis who have a baseline AM or baseline PM average 
score that rounded to 0 or 1. 
 
• A supportive analysis of pruritus assessments as a continuous endpoint will be conducted. 
The analysis will be performed by considering the impact of intermittently missing data as 
well as intercurrent events.  
 
The monthly (28-day) average in change from baseline for AM and PM, respectively will be 
calculated. There is a total of 6 monthly averages for AM assessments and 6 monthly 
averages for PM assessments over the 24-week treatment period.  A mixed-effect model for 
repeated measures (MMRM) will be used to compare treatment effects at the last 28 days for 
AM and PM, respectively.  Since missing not at random (MNAR) may occur, missing 
monthly average will be imputed with data from control arm completers using multiple 
imputation (MI).  If there are subjects in the active arms who discontinued the study due to, 
e.g., relocation, their missing values may be imputed using active arm completers.  To 
simplify the MI procedure, the description below only pertains to imputation using placebo 
completers. The MMRM model will include baseline score, treatment group, time (in 
month), treatment-by-baseline interaction, treatment-by-time interaction and stratification 
factors (i.e., PFIC type and age category). Unstructured covariance matrix will be used. 
 
The following procedures in MI will be followed: 
 
1. Intermediate missing values are imputed by the Markov Chain Monte Carlo (MCMC) 
imputation model, so only monotone missing values exist after imputation.  200 imputed data 


 
 
 
 
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sets will be created. 
 
2. Subjects are classified to 3 subgroups: a. Completers in the control arm without a missing 
average at the last 4 weeks. b. Subjects with a missing average at the last 4 weeks in any arm, 
and c. Completers in the active arm without a missing average in the last 4 weeks.  Subjects 
in subgroup ‘a’ will be used to impute missing values for subjects in subgroup ‘b.’ 
 
3. The monotone missing values are imputed chronologically using SAS Proc MI.  This is 
done by each imputed data set from step 1. 
 
4. After the imputation, all subjects in the 3 subgroups are pooled and the data are sorted by 
the imputation ID from step 1.  
 
5. MMRM is used to compare treatment effect in the last 4 weeks.  This is done by the 
imputation IDs from step 1. 
 
6. Proc MIAnalyze is used to summarize and conclude the results from step 5. 
 
• If the comparison result from the analysis of MMRM with multiple imputation is significant, 
a tipping point analysis will be performed by reducing the imputed scores in placebo patients 
by 0.1 each time until the result is non-significant. In addition, the imputed scores in active 
patients will be increased by 0.1 each time till the result is non-significant. 
 
• The MMRM with multiple imputation will be conducted for monthly average of change from 
baseline in AM and PM scores, and for monthly average of change from baseline in daily 
worst score. 
 
 
8.6.4 Analyses of secondary efficacy endpoints 
 
All secondary analyses will be based on the FAS. The analysis based on the PP populations will 
be performed for the 3 selected efficacy endpoints as specified below.  
 
The change in the secondary endpoints such as serum bile acids, ALT, growth, Albireo PRO and 
ObsRO sleep parameters will be summarized by visit using descriptive statistics. Change in 
growth (weight for age and BMI for age) will also be displayed using graphical presentations.  
The summary of change in the secondary endpoints such as serum bile acids, ALT and growth 
will also be provided based on the PP analysis set.  
 
Change in serum bile acids, change in ALT, change in growth will also be analyzed using a 
MMRM model. The model will include terms for baseline, PFIC type, age category, treatment, 
visit, treatment-by-baseline interaction and treatment-by-visit interaction. Unstructured 
covariance matrix will be used. The analysis of serum bile acids may be performed based on log 
transformed values of serum bile acids if deemed appropriate. 
 


 
 
 
 
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The analysis of growth data will be based on calculated values using the software or methods 
from the Centers for Disease Control and Prevention (CDC) website for patients with age ≥ 2 
years old and from the WHO website for patients with age < 2 years old. CRF collected growth 
data will be listed. 
 
The proportion of responders at Weeks 12 and 24 based on the ObsRO instrument will be 
analyzed using the same model as specified for the primary analysis for EU and RoW. The 
definition of the responder is a patient who reports a decrease in pruritus score from unrounded 
baseline equivalent to or greater than the threshold of meaningful change estimated from the 
blinded psychometric analysis; the results of the blinded psychometric analysis across all 
anchors support a threshold of a 1.0 point for AM, PM and AM and PM scratching scores. 
Section 8.9.1 includes more details about the blinded psychometric analysis. Specifically, the 
averaged pruritus score during weeks 11 and 12 and during weeks 23 and 24 will be used to 
calculate the proportion of patients achieving meaningful reduction at Week 12 and Week 24 
from the baseline score. Similarly, the responder analysis will be conducted based on monthly 
scores at Week 12 and Week 24. Moreover, the analysis will be performed by AM, PM 
separately and AM & PM combined. For treatment completers whose last eDiary report day is 
before Week 24, if there is not enough eDiary data to calculate bi-weekly or monthly average 
scores at week 24, they will be excluded from analysis.  
 
The proportion of individual assessments meeting the definition of a positive pruritus assessment 
at the subject level from Weeks 0-4, Weeks 0-8, Weeks 0-12, Weeks 0-18, Weeks 0-24, 
respectively, and the proportion of positive pruritus assessments at each 4-week interval over the 
24-week treatment period for AM or PM, respectively will be analyzed using the same model as 
specified for the primary analysis for US.  This analysis will be performed for PRO/ObsRO 
instruments separately. 
 
The number and percent of patients achieving positive pruritus assessment for more than 50% of 
the time will be summarized by treatment group. Both AM and PM pruritus assessments will be 
included in the analysis of this endpoint. The proportion of patients achieving positive pruritus 
assessment for more than 50% of the time will be analyzed using the same model as specified for 
the primary analysis for EU and RoW. This analysis will also be performed separately for AM 
pruritus assessments only and PM pruritus assessments only. 
 
A cumulative distribution function (CDF) plot showing proportions of patients achieving 
positive pruritus assessment for more than X% of the time, for X from 0 to 100%, by treatment 
group will be provided.   
 
Number and percent of patients undergoing biliary diversion surgery and/or liver transplant will 
be summarized by using descriptive statistics. Kaplan-Meier curves will be used when 
appropriate for time to event data. Median event-free times and associated 95% confidence 
intervals will be calculated for each treatment group using Brookmeyer and Crowley 
methodology and a log-log transformation for constructing confidence intervals. 
 
 


 
 
 
 
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8.6.5 Analyses of exploratory endpoints 
 
The exploratory efficacy variables are listed in Section 4.3. All the exploratory analyses will be 
based on the FAS.  
 
Exploratory variables including Global Symptom Relief, biochemical markers and measures of 
bile acid synthesis, Albireo PRO and ObsRO itching/scratching severity scores, additional 
Albireo PRO and ObsRO sleep parameters, PELD/MELD score, APRI score, and the Fib-4 score 
will be analyzed descriptively for categorical and continuous data. For continuous data, the 
change from baseline will be analyzed in addition to the presentation of actual visit values. For 
Albireo PRO and ObsRO itching/scratching severity scores, unrounded baseline will be used to 
calculate change from baseline. For categorical data, shift tables or frequency and percentages of 
patients will be presented as appropriate. A line graph of itching/scratching daily severity scores 
of Albireo PRO and ObsRO over time for each patient will be provided.  
 
For continuous data, such as PELD/MELD score, APRI score, and Fib-4 score, biochemical 
markers and measures of bile acid synthesis, the change from baseline to Week 24 will be 
analyzed descriptively in addition to the actual visit values. Global Symptom Relief data at 
Weeks 4, 12, and 24 will be summarized as categorical variables. Time points (visit window) are 
specified in Appendix B. 
 
Comparisons of the change from baseline at Week 24 in the PedsQL total score (calculated as 
average score of all answered items) between the treatment groups will be conducted using 
analysis of covariance (ANCOVA). The model will include terms for baseline, PFIC type, age 
category, and treatment. The analysis will be conducted based on the total scores reported by 
child (>=5 years of age) and by parent (only including patients >=5 years of age) separately. If 
the sample size based on child reported data (>=5 years of age) is less than 10, then ANCOVA 
will not be conducted. The PedsQL total score of the family impact module will be analyzed 
similarly. The total score and the domain scores will be summarized descriptively. 
 
Change from baseline to Week 24 in stage of liver fibrosis measured by Fibroscan® will be 
analyzed descriptively. The frequencies of stages may be summarized by shift tables. Steatosis 
grade analysis will be performed in a similar fashion. 
 
 
8.6.6 Analyses based on exit survey data 
  
The exit survey in Appendix F contains 3 questions and will be done by patients and caregivers 
separately. The answer Yes in Questions 1 and 2 will be verified with Question 3 for a positive 
and meaningful change. 
 
The exit survey has the following questions: 
 
1. Have you or your child experienced change from the study drug at the end of the study? 
Yes/No;  
 


 
 
 
 
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2. Was the change meaningful? Yes/No;  
 
3. In what way was the change meaningful? 
 
The exit survey was implemented in the middle of the trial so data from the exit survey will not 
be available for all patients. Analyses on exit survey are listed below. Analyses will be repeated 
considering missing data as No to Questions 1 and 2, and considering missing data as Yes to 
Questions 1 and 2. 
 
The number and percent of patients with the following categories will be provided by treatment 
and overall:  
• Q1 answer=Yes and Q2 answer=Yes 
• Q1 answer=No and Q2 answer=No 
• Q1 answer=Yes and Q2 answer=No  
 
The exit surveys will be answered by both patients (≥8 yrs) and caregivers. Exit survey data will 
be provided in a data listing.  
 
The following analyses on the exit survey will be performed using the descriptive summary 
based on pruritus assessments of Albireo ObsRO instrument in the last 2 weeks while patients 
are still on the study medications.  
 
• For patients who answered Yes to Questions 1 and 2 in the exit survey, changes from 
baseline in the average scratching score based on the Albireo ObsRO instrument in the last 2 
weeks before treatment termination 
 
• For patients who answered No to Questions 1 or 2 in the exit survey, changes from baseline 
in the average scratching score based on the Albireo ObsRO instrument in the last 2 weeks 
before treatment termination 
 
The summary based on the pruritus assessments of Albireo PRO instrument will also be 
provided. 
 
 
8.7 
Safety analyses 
 
All definitions relative to safety endpoints are detailed in Section 4.4. 
 
All safety analyses will be based on the Safety Analyses Set (as treated patients) and will be 
performed for all safety variables specified below and summarized by treatment group 
(odevixibat 40 µg/kg/day, odevixibat 120 µg/kg/day, odevixibat 2 doses combined, and placebo).  
 
For each safety variable, the last value collected prior to first dose of study drug will be used as 
the baseline for all analyses. 
 
No inferential statistical analysis of safety endpoints will be performed. 


 
 
 
 
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8.7.1 Adverse events 
 
All adverse events (AEs) will be classified by Primary System Organ Class (SOC) and Preferred 
Term (PT) according to MedDRA version 23.0 throughout the study. 
 
AEs will be classified as treatment emergent adverse events (TEAEs), defined as follows:  
An AE (classified by preferred term) that occurs during the Treatment Period will be considered 
a TEAE if it has a start date on or after the first dose date of study drug, or it has a start date 
before the date of the first dose date of study drug, but worsened in severity on or after the date 
of the first dose date of study drug.   
 
AEs with missing start dates, but with stop dates either overlapping with the treatment period or 
missing, will be considered TEAEs. A TEAE with missing drug-relationship will be considered 
as related. A TEAE with missing severity will be considered as severe. Details for imputing 
missing or partial start dates of adverse events are described in Appendix D. 
 
For counting of the number of AEs, if 2 AE records have the same preferred term, and the start 
date of the 2nd AE is the same as or next day after the end date of the 1st AE, then they will be 
counted as one AE only. 
 
An overall summary of the incidence of TEAEs (number of patients with any events and number 
of events, if applicable) will include the following: 
• All TEAEs  
• Drug-related TEAEs (AE will be defined as drug-related if causality is either probable, 
possibly or definitely)  
• Severe TEAEs 
• TEAEs leading to study discontinuation 
• Serious TEAEs 
• Drug-related serious TEAEs 
• TEAEs leading to death 
• Any hepatic TEAE 
a. Any liver-related TEAE (events that, per the Investigator, are considered related to 
PFIC)  
b. Any suspected drug-induced liver injury (DILI) TEAEs (as adjudicated by the Data 
Safety Monitoring Board [DSMB] and defined in Section 8.7.1.2) 
c. Any TEAE of liver decompensation (as defined in Section 8.7.1.2)  
d. Any TEAE in the standardised MedDRA query (SMQ) of Drug Related Hepatic 
Disorders; Severe Events Only 
• Any Fat-Soluble Vitamin Deficiency TEAEs refractory to clinically recommended 
vitamin supplementation 


 
 
 
 
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• Any Clinically Significant Diarrhea TEAEs 
 
TEAEs (number of patients with any events and number of events, if applicable) by SOC and PT 
in each treatment group will be tabulated for the following:  
• TEAEs by SOC and PT including fat-soluble vitamin deficiencies, diarrhea, hepatoxicity 
(as defined in Section 8.7.1.1). Note that hepatotoxicity will also be included with the 
tabulation of hepatic events 
• All TEAEs by preferred term by descending incidence in Odevixibat All Doses column 
• TEAEs leading to study discontinuation by SOC and PT 
• Serious TEAE by SOC and PT 
• Drug-related serious TEAE by SOC and PT  
• TEAEs leading to death by SOC and PT 
• Common TEAEs (≥10% in any treatment group) 
• Hepatic TEAEs 
a. Liver-related TEAEs (events that, per the Investigator, are considered related to 
PFIC) by SOC and preferred term 
b. Suspected DILI TEAEs (as adjudicated by the DSMB and defined in Section 8.7.1.2) 
c. TEAEs of liver decompensation (as defined in Section 8.7.1.2) by SOC and preferred 
term 
d. Any TEAE in the standardised MedDRA query (SMQ) of Drug Related Hepatic 
Disorders; Severe Events Only 
 
Summary tables for number of patients with any TEAEs by SOC and PT by severity (mild, 
moderate, severe) and by causality (related [possibly, probably and definitely] vs unrelated 
[unlikely and unrelated]), will also be provided during the treatment period. AEs with the worst 
severity will be used in the by-severity summaries. Similarly, AEs with the worst causality (most 
related to treatment) will be used in the by-causality summaries. If severity or causality is 
missing, data will be imputed to the worst category. 
 
When a patient has the same adverse event, based on preferred terminology, reported multiple 
times in the treatment period, the patient will only be counted once at the preferred terminology 
level in adverse event frequency tables. Where a patient has multiple adverse events within the 
same SOC in the treatment period, the patient will only be counted once at the SOC level in 
adverse event frequency tables. 
 
In the AE summaries, AEs will be sorted by alphabetically for SOC and PT. In addition, the 
numbers of patients with liver-related mortality, liver decompensation events, and all-cause 
mortality will be presented using descriptive statistics. Kaplan-Meier curves may be used for 
time to event data (time taken to experience liver decompensation event, all-cause mortality, and 
biliary diversion surgery in weeks). 
 


 
 
 
 
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All AEs (including pre-treatment and post-treatment AEs), SAEs and deaths will be listed. 
Separate listings for AEs leading to dose interruption, and AEs of interest (Fat-Soluble Vitamin 
Deficiency, Diarrhea, and Hepatic AEs as defined below) will also be provided. 
8.7.1.1 
Definition for TEAEs of Fat-Soluble Vitamin Deficiency, Diarrhoea, and 
Hepatotoxicity  
The following TEAEs have been defined based on the population under study: 
• New or worsening of fat-soluble vitamin deficiency refractory to clinically recommended 
vitamin supplementation. 
All Investigator-reported verbatim terms related to decreases in vitamin levels or vitamin 
deficiency (e.g. preferred term of hypovitaminosis, Vitamin A decreased, Vitamin A 
deficiency) with the relevant concomitant medication records reviewed by the Medical 
Monitor, queried as needed, and the appropriate MedDRA preferred terms reported. 
• Clinically significant diarrhoea, defined as any of the following: 
o Diarrhoea that persists for 21 or more days without any other aetiology based on 
medical review of other concurrent AEs for possible other causes of the diarrhoea or 
diagnostic testing (e.g. viral infections) 
o Reported by the Investigator as severe in intensity or reported as an SAE due to the 
requirement for hospitalisation or as an important medical event 
o Diarrhoea with concurrent dehydration requiring treatment with oral or intravenous 
rehydration and/or other treatment intervention based on medical review of AEs and 
concomitant medications 
• Hepatoxicity: based on the SMQ of Drug Related Hepatic Disorders; Severe Events Only  
 
For each of these categories, tabular summaries will be presented by MedDRA SOC and 
preferred term; the tables will also include the overall incidence of these AEs. Listings of 
patients with each of the events as defined will be provided. 
8.7.1.2 
Definitions and Determination of Hepatic Events 
A DSMB was formed to independently assure the safety of patients enrolled in Study A4250 
008, as well as to evaluate the integrity of study conduct and the data generated. The DSMB 
reviews the safety data at regular, pre-defined intervals and on an ad hoc basis as needed and 
makes recommendations regarding patient safety and study continuance (continuation, 
modification, or termination of the study). The DSMB is comprised of three paediatric 
hepatologists and an unblinded biostatistician. 
 


 
 
 
 
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As requested by the FDA, the DSMB has been chartered to review data from patients with 
hepatic AEs, including cases of suspected DILI and patients with liver decompensation events as 
defined below. The DSMB also reviews events that are reported in the SMQ of Drug Related 
Hepatic Disorders; Severe Events Only. The DSMB reviews each case and provides their expert 
opinion on the aetiology of the event. 
 
For this review, Albireo prepares slides for presentation at the DSMB meeting on each patient 
who meets the criteria for hepatic event adjudication. Relevant liver-related laboratory values 
over time and a narrative summary of relevant information is provided to the DSMB. During the 
open session of the meeting, an Albireo physician reviews each case with the DSMB members, 
responds to questions, and/or obtains any additional information requested by the DSMB. 
Albireo’s assessment of the hepatotoxicity aetiology is documented for each event. During the 
closed session, the DSMB independently assesses the event aetiology which is documented on a 
Hepatic Event Adjudication Form that is attached to the meeting minutes. If the DSMB requests 
follow-up information, this is provided at the next scheduled meeting. Treatment assignment 
remains blinded for this review. The Adjudication Process Document outlines the events that 
will be adjudicated.  
8.7.1.2.1 Suspected Drug-Induced Liver Injury 
As outlined in the protocols for Study A4250 008, patients with laboratory criteria that meet any 
of the following are considered suspected events of DILI and undergo review and adjudication of 
the event aetiology by the DSMB: 
 
• ALT or AST ≥5 × upper limit of normal (ULN) if ALT or AST is normal at Baseline, or 
an absolute threshold of 800 U/L, whichever comes first 
• ALT or AST ≥3 × Baseline if ALT or AST is abnormal at Baseline, or an absolute 
threshold of 800 U/L, whichever comes first 
• ALT or AST ≥3 × ULN or 800 U/L, whichever comes first, and total bilirubin >2 × ULN 
• Doubling of total bilirubin if total bilirubin was <3 mg/dL at Baseline 
• Increase in total bilirubin by >3 mg/dL if total bilirubin was ≥3 mg/dL at Baseline 
• INR increase >1.5 if INR was normal at Baseline and increase is refractory to Vitamin K 
administration 
• INR increase by >0.4 if INR was abnormal at Baseline and increase is refractory to 
Vitamin K administration 
• Any increase in total bilirubin and transaminases if accompanied by either a symptom of 
clinical hepatitis (vomiting, nausea, right upper quadrant pain) or immunological reaction 
(rash or 5% eosinophilia) 
8.7.1.2.2 Liver Decompensation Adverse Events 
Events either identified by Investigators or meeting the Albireo definition of liver 
decompensation will undergo review and adjudication by the DSMB. Patients who meet either of 
the following criteria undergo review and adjudication of the event aetiology by the DSMB for 
liver decompensation: 
 


 
 
 
 
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• INR elevation >1.5 that is refractory to vitamin K administration 
• In a patient with portal hypertension and cirrhosis, transition to decompensated cirrhosis 
evidenced by any of the following: 
o Presence of ascites 
o Hepatorenal syndrome 
o Portopulmonary hypertension 
o Hepatopulmonary syndrome 
o Variceal haemorrhage 
o Hepatic encephalopathy 
8.7.1.2.3 SMQ Drug-Related Hepatic Disorder AEs: 
Events in the Standardised MedDRA query SMQ Drug Related Hepatic Disorders; Severe 
Events Only (SMQ No. 20000007) that are not captured in any of the above parameters will be 
presented to the DSMB for review. 
 
The data of all adjudicated hepatic events provided by the DSMB will also be listed. 
 
 
8.7.2 Clinical laboratory evaluations 
 
Descriptive statistics for clinical laboratory values (in SI units for all tests, and in conventional 
units for selected tests (Appendix H)) and absolute changes from baseline at each post-baseline 
visit (Appendix B) will be presented. The change to the last visit will also be summarized. A 
shift table from baseline to the highest or lowest value for quantitative variables will be 
presented by treatment group for the clinical laboratory parameters listed in Table 2. 
 
Central laboratory data will be used for the summary. If central laboratory data are not available 
due to COVID-19 or other reasons, local laboratory data will be used. 
 
Figures for ALT will be prepared as follows:  
 
• Mean change from baseline over time; all the treatment groups; one line per treatment 
group. 
• Individual change from baseline over time; treatment group 40 µg/kg/day odevixibat; one 
line per patient. 
• Individual change from baseline over time; treatment group 120 µg/kg/day odevixibat; 
one line per patient. 
• Individual change from baseline over time; treatment group placebo; one line per patient. 
 
 


 
 
 
 
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The clinical laboratory test results will be listed.  
 
Table 2:  Laboratory Parameters 
 Clinical Chemistry 
Hematology 
Urinalysis 
Other Labs 
• 
Albumin 
• 
ALT 
• 
Alkaline phosphatase 
(ALP) 
• 
AST 
• 
Bilirubin – total and direct 
• 
Calcium 
• 
Chloride 
• 
Creatinine 
• 
Creatine kinase 
• 
Gamma-glutamyl 
transferase (GGT) 
• 
Potassium 
• 
Sodium 
• 
Hematocrit 
• 
Hemoglobin 
• 
Platelet count 
• 
Red blood cell count 
• 
White blood cell count 
and differential 
(neutrophils, 
eosinophils, basophils, 
lymphocytes, and 
monocytes) 
• 
Blood 
• 
Glucose 
• 
Ketones 
• 
Leukocytes 
• 
Nitrites 
• 
pH 
• 
Protein 
• 
Vitamin A and 
E 
• 
25-Hydroxy 
vitamin D 
• 
International 
normalized 
ratio (INR) 
• 
Alfa-fetoprotein 
(AFP) 
• 
Prothrombin 
Time 
 
Furthermore, a listing of patients meeting the following criteria for liver monitoring will be 
provided: 
 
• (ALT or AST ≥ 3x Baseline or >= 800 U/L) and TB > 2 ULN at the same visit 
• ALT or AST >= 10 ULN or >= 5x Baseline or >= 800 U/L in presence of normal LDH 
and CPK 
• ALT or AST >= 5 ULN (Normal Baseline) or >= 3x Baseline (Abnormal Baseline) or >= 
800 U/L 
• INR > 1.5 (Normal Baseline) or increased by > 0.4 relative to Baseline (Abnormal 
Baseline) 
• Total bilirubin (TB) >= 2x Baseline (Baseline TB < 3 mg/dL) or increased by >= 3 
mg/dL relative to Baseline (Baseline TB >= 3 mg/dL) 
 
8.7.3 Vital signs 
 
Descriptive statistics for vital signs (temperature, systolic blood pressure, diastolic blood 
pressure, heart rate, respiratory rate, weight, height and BMI) and their changes from baseline at 
each post-baseline visit will be presented by treatment group. The change to the last visit will 
also be summarized. A shift table for the number of patients with changes from baseline to the  
highest or lowest value (Appendix G) will be provided by treatment group during the treatment 
period. 
 


 
 
 
 
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Vital signs data will be listed. 
 
8.7.4 Physical examinations 
 
All physical examination data (general appearance, eyes, ears, nose, throat, head/neck/thyroid, 
lymph nodes, cardiovascular, lungs/chest, abdomen, genitourinary, extremities, skin, 
musculoskeletal, neurological, and other body systems) will be summarized at each visit with 
percentages and frequencies along with abnormalities by treatment group.  
 
Abdominal ultrasound assessments for the liver, including Riedel’s lobe, the portal vein and the 
spleen will be also summarized descriptively for each visit. 
 
Skin assessments on a 5-point scale (0-4 from no evidence of scratching to cutaneous bleeding, 
haemorrhage, scabbing) for face, right arm, left arm, right leg, left leg and torso will be 
summarized at each visit with percentages and frequencies by treatment group.  
 
Physical examination and skin assessment data will be listed (including pre-treatment and post-
treatment results). 
 
8.7.5 Pharmacokinetics (PK) 
 
PK samples will only be drawn at visits 4 and 9 (only for children with body weight >10 kg) and 
if an SAE occurs, as close to the onset of an SAE as possible and if volume limitations permit.  
Samples will be processed and transported to a laboratory per instructions in the laboratory 
manual. Plasma concentrations will be listed. No PK parameters will be derived.  
 
8.8 
Subgroup analyses 
 
Subgroup analyses will be performed for each of the 3 age groups (6 months to 5 years, 6 to 
12 years, and 13 to 18 years), PFIC 1 and 2, region (US, EU and RoW), sex, race, ethnicity, 
baseline serum bile acid (≥250 and <250 µmol/L), Child-Pugh classification, Bile Salt Export 
Pump (BSEP) type of PFIC 2 patients, and the use of UDCA, rifampicin (alone or either one). 
Subgroup analyses may be conducted for hepatic impairment classification per NCI Organ 
Dysfunction Working Group (ODWG) if appropriate. Statistical analysis will be performed only 
when the sample size is ≥10 in each treatment group. If the sample size is <10 in either treatment 
group, only summary statistics will be provided; the p-value will not be reported. Due to small 
sample size in subgroups, all subgroup analysis will not be stratified by the stratification factors.  
 
Analyses will be provided for the following parameters: 
- The proportion of positive pruritus assessments at the subject level over the 24-week treatment 
period (primary endpoint)  
- s-BA responder (primary endpoint)  
- Change from baseline in s-BA, ALT, growth (secondary endpoints)  
 


 
 
 
 
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8.9 
Blinded analysis 
 
8.9.1 Pruritus Responder Definitions  
 
Blinded analyses of Albireo ObsRO and PRO eDiary data were to be performed after 50% or 
more of the planned patients have completed the Week 24 Treatment Period. The actual number 
of 24-week treatment completers included in the analyses was 45 (~73%). The blinded analyses 
were used to estimate a threshold of clinically meaningful change (i.e., the responder definition) 
in Albireo ObsRO and PRO pruritus scores.  Please refer to the psychometric analysis 
(Pruritus Evidence Dossier) for details. The analyses were performed by a group 
, 
independent from both the study team and the sponsor.   
 
 
8.9.2 Data Safety Monitoring Board 
 
The DSMB will receive data in the form of tables, figures and listings (provided by unblinded 
DSMB statistician from 
 who is independent to the 
 study team).  The requirement 
for blinded or unblinded data is defined in the DSMB charter.  The data provided to the DSMB 
will include, but is not limited to, demographics, baseline characteristics, medical and surgical 
history, prior and concomitant medications, AE and SAE data (by SOC and PT and by maximum 
intensity), laboratory data, liver monitoring, vital signs, abdominal ultrasound and patient 
disposition data.  
 


 
 
 
 
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9 
CHANGES TO PLANNED ANALYSIS FROM STUDY 
PROTOCOL 
 The changes to the planned analysis in the protocol are listed below. 
 
1. Endpoints 
 
(a) Added one secondary efficacy endpoint. 
 
• Number of patients achieving positive pruritus assessment for more than 50% of the time 
during 24-week treatment period 
 
(b) Modified one secondary efficacy endpoint 
 
Protocol: Proportion of responders for pruritus scores at Weeks 12 and 24 based on the Albireo 
PRO and ObsRO instruments. 
 
SAP: Proportion of responders for pruritus scores at Weeks 12 and 24 based on the ObsRO 
instrument.  
 
 
(c) Added the following exploratory efficacy endpoints. 
• For patients who answered Yes in Questions 1 and 2 in the exit survey, changes from 
baseline in the average scratching score based on the Albireo ObsRO instrument in the 
last 2 weeks before treatment termination  
 
• For patients who answered No in Questions 1 or 2 in the exit survey, changes from 
baseline in the average scratching score based on the Albireo ObsRO instrument in the 
last 2 weeks before treatment termination 
 
(d) Removed the following exploratory efficacy endpoints. 
• Liver-related mortality and liver decompensation events 
• All-cause mortality 
 
(e) Added the following safety endpoints. 
• Liver-related mortality and liver decompensation events 
• All-cause mortality 
 
 
2. Efficacy Analyses 
 
(a) Changed the definition of fasting s-BA baseline  
Protocol: Fasting s-BA baseline will be calculated as the average of the value at randomization 
and the previous visit (Clinic Visit 2). 
 


 
 
 
 
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SAP: Fasting s-BA baseline will be calculated as the average of the last 2 values prior to the first 
dose. The reason to change is that some patients may have unscheduled assessments between 
Clinic Visit 2 and Randomization Visit. 
 
(b) Added the details of strata pooling strategy for the CMH test  
 
(c) Added rounded baseline score which will be used for the analysis of pruritus assessments 
 
(d) Added analyses for the number of patients achieving positive pruritus assessment for more 
than 50% of the time during 24-week treatment period 
 
(f) Added the following analyses for the US primary endpoint of pruritus assessments 
• An additional method to calculate the proportion of positive pruritus assessments up to 
the last report day before or on Day 168. In other words, the denominator to calculate the 
proportion will be (the last report day x 2). All missing data before or on the last report 
day will be considered as negative assessments. 
• Additional supportive analysis will exclude patients from the primary endpoint analysis 
who have a baseline AM or baseline PM average score that rounded to 0 or 1. 
• Multiple imputation and tipping point analysis for pruritus assessments as a continuous 
endpoint. 
 
(g) Added the following analyses for the EU and RoW primary endpoint of fasting s-BA 
responder endpoint 
• Tipping point analysis to evaluate the influence of missing data at the end of treatment 
 
(h) Added the following analysis for secondary endpoints 
• Change in serum bile acids, change in ALT, change in growth will be analyzed using a 
MMRM model 
 
(i) Changes in Subgroup Analysis 
• Removed a subgroup: for patients who were not included in the previous Study 
A4250-003 
• Added subgroups by region (US, EU and RoW), sex, race, ethnicity, baseline serum bile 
acid (≥250 and <250 µmol/L), Child-Pugh classification, Bile Salt Export Pump (BSEP) 
type of PFIC 2 patients, and the use of UDCA, rifampicin (alone or either one). Subgroup 
analyses may be conducted for hepatic impairment classification per NCI Organ 
Dysfunction Working Group (ODWG) if appropriate. 
 
(j) Corrected Formula of PELD Score and APRI Score 
 
Protocol: PELD Score = 4.80 * ln(total bilirubin) + 18.57 * ln(INR) - 6.87 * ln(albumin) + 
4.36 (if patient <1 year: scores for patients listed for liver transplantation before the patient's first 
birthday continue to include the value assigned for age (<1 year) until the patient reaches the age 
of 24 months) + 6.67 (if the patient has growth failure [≤2 SD]) 
 
SAP: PELD Score = 4.80 * ln(total bilirubin) + 18.57 * ln(INR) - 6.87 * ln(albumin) + 4.36 (if 


 
 
 
 
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patient <1 year: scores for patients listed for liver transplantation before the patient's first 
birthday continue to include the value assigned for age (<1 year) until the patient reaches the age 
of 24 months) + 6.67 (if the patient has growth failure [< -2SD]) 
 
Protocol: APRI = [(AST in U/L) / (AST ULN in U/L)] / (Platelets in 109/L) 
 
SAP: APRI = [(AST in U/L) / (AST ULN in U/L)] × 100/ (Platelets in 109/L) 
 
(k) Changed the definition of negative pruritus assessments 
 
Protocol: All planned assessments after the intercurrent events (premature treatment 
discontinuation, death or initiation of rescue treatments such as biliary diversion surgery or liver 
transplantation) will be counted as negative pruritus assessments. 
 
SAP: All missing planned assessments after premature treatment discontinuation will be counted 
as negative pruritus assessments. All planned assessments after death or initiation of rescue 
treatments such as biliary diversion surgery or liver transplantation will be counted as negative 
pruritus assessments. Both on-treatment and off-treatment pruritus assessments up to Day 168 
will be included for analysis. 50% rule will be applied to the calculation. Please see details in 
Appendix A. 
 


 
 
 
 
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10 
REFERENCES 
 
1. ICH Topic E3: Structure and Content of Clinical Study Reports (CPMP/ICH/137/95- 
adopted December 1995). 
2. ICH Topic E9: Statistical Principles for Clinical Trials (CPMP/ICH/363/96 – adopted 
March 1998). 
3. https://www.unos.org/wp-
content/uploads/unos/MELD_PELD_Calculator_Documentation.pdf; accessed on 25-Jul-
2018 
4. Memorial Sloan-Kettering Cancer Center (Understanding your Fibroscan Results) 
https://www.mskcc.org/cancer-care/patient-education/understanding-your-fibroscan-
results 
5. Washington University, Louisville Kentucky (Jewish Hospital Fibroscan Interpretation 
Assessment SOP). 
https://louisville.edu/medicine/departments/medicine/divisions/gimedicine/physician-
resources/calculators-and-tools-files/fibroscan-interpretation-sheet 
6. Kane JM, Skuban A, Ouyang J, et al. A multicenter, randomized, double-blind, 
controlled phase 3 trial of fixed-dose brexpiprazole for the treatment of adults with acute 
schizophrenia, Schizophr Res. 2015: 164(1-3):127-35.  
7. Lehmacher W, Wassmer G, Reitmeir P. Procedures for Two-Sample Comparisons with 
Multiple Endpoints Controlling the Experimentwise Error Rate. Biometrics 1991:47, 
511-521. 
8. Marcus R, Peritz E, Gabriel KR. On closed testing procedures with special reference to 
ordered analysis of variance. Biometrika 1976:63:655-660. 
9. Kadane JB. Sums of Possibly Associated Bernoulli Variables: The Conway-Maxwell-
Binomial Distribution. Bayesian Analysis. 2016; 11(2):403-420. 
10. Aaron S. Mansfield, Michelle A. Rudek, Diana Vulih, Gary L. Smith, Pamela Jo Harris, 
and S. Percy Ivy (2016). “The effect of hepatic impairment on outcomes in phase 1 
clinical trials in cancer subjects”. 
11. Vollset, S. E., Hirji, K. F., and Elashoff, R. M. (1991). “Fast Computation of Exact 
Confidence Limits for the Common Odds Ratio in a Series of Tables.” Journal of the 
American Statistical Association 86:404–409  
12. Peds cases - Pediatrics for Medical Students - Developed by Chris Novak and Peter Gill 
for PedsCases.com April 21, 2016 (http://www.pedscases.com/pediatric-vital-
signsreference-Chart)  
 
 


 
 
 
 
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11 
APPENDICES 


 
 
 
 
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Protocol Number: A4250-005 
Version: Final v1.2, Date: 25Aug2020
Page 54 of 65
 
Appendix A: Derived Variables 
 
The table below provides the list of derived variables for demographic and baseline 
characteristics, various duration derivations, drug compliance, baseline derivations and other 
important derivations applicable for this study. 
 
Variables 
Formula 
Demographic and Baseline characteristics 
Age (in years) 
For patients not from France and Germany, age will be calculated 
based on date of birth. For patients from France and Germany, only 
birth year was collected, and July 1 was imputed in the eDC 
system. The CRF collected age months and age years are based on 
the imputed date of birth. For analysis purpose, age will be 
calculated based on collected age months and age years on CRF or 
the external file (primary source). 
Body mass index (BMI) (kg/ m2)   
weight (kg)/[height (m2) 
PFIC diagnosis (in years) 
Will be derived by SAS YRDIF function 
yrdif (date of diagnosis of PFIC, Date of informed consent, 
'ACT/ACT');  
 
 
Derivation of Duration 
Study day at any visit 
Date of interest – date of first dose of study drug. One day is added 
if this difference is ≥ 0 
Extent of Exposure (days) 
Date of last study drug intake – Date of first study drug intake + 1 
 
 
Overall Study Drug Compliance 
Compliance based on CRF data 
100 × [(total number of capsules dispensed – total number of 
capsules returned) /(total number of capsules planned to be taken)] 
Compliance based on eDiary data 
100 x (total number of capsules taken/total number of capsules 
planned to be taken). This will be considered as the primary 
compliance rate. 
The number of capsules planned to be taken will be estimated 
based on patient’s body weight at Visit 3 and Visit 6 per Table 2 in 
the protocol. The number of capsules planned to be taken before 
Visit 6 will be the same as the one estimated at Visit 3, and after 
Visit 6 will be the same as the one estimated at Visit 6. 
 
 
Derivations for Efficacy Parameters 
Baseline (general) 
The baseline value of a parameter is defined as the last assessment 
of that parameter prior to the first intake of study treatment, except 
as indicated below. 


 
 
 
 
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Baseline for proportion of patients 
experiencing at least a 70% reduction 
in fasting s-BA and for s-BA change 
from baseline 
Fasting s-BA baseline will be calculated as the average of the last 2 
values prior to the first dose (see also Appendix B). If only one 
non-missing value is available, it will be used as baseline.  
End value for proportion of patients 
experiencing at least a 70% reduction 
in fasting s-BA and for s-BA change 
from baseline 
The end value is the average of the values at Weeks 22 and 24 after 
the start of double-blind treatment. (Appendix B). If one value is 
missing, then the non-missing value will be used as the end value. 
If both values are missing, then the end value is missing.  
Baseline, daily, weekly and monthly 
score by AM and PM respectively 
(scratching, itch severity) 
For both the Albireo ObsRO scratching item and the Albireo PRO 
itch severity score: 
All non-missing AM scores from the period of 14 days prior to or 
on the first dose day of study medications will be averaged as 
baseline.  
All non-missing PM scores from the period of 14 days prior to the 
first dose day of study medications will be averaged as baseline.  
Baseline score will be considered missing if ≥8 out of 14 
assessments in the 14 days are missing (i.e. 50% rule is applied 
based on the number of planned assessments). 
A weekly AM (PM) score will be calculated by averaging all non-
missing AM (PM) scores in a week.  A weekly score will be 
considered missing if ≥4 out of 7 AM (PM) scores in a week are 
missing. 
A bi-weekly AM (PM) score will be calculated by averaging all 
non-missing AM (PM) scores in 2 weeks.  A bi-weekly score will 
be considered missing if ≥8 out of 14 AM (PM) scores in 2 weeks 
are missing. 
A monthly AM (PM) score will be calculated by averaging all non-
missing AM (PM) scores in a month (28 days).  A monthly score 
will be considered missing if ≥15 out of 28 AM (PM) scores a 
week are missing. 
AM & PM baseline, daily, weekly 
and monthly score (scratching, itch 
severity) 
For both the Albireo ObsRO scratching item and the Albireo PRO 
itch severity score: 
A daily AM & PM score will be averaged from the 2 ratings for 
each day. A daily score will be considered missing if both 
assessments are missing.   
A weekly score will be calculated by averaging all non-missing 
AM and PM scores in a week.  A weekly score will be considered 
missing if ≥8 out of 14 assessments in a week are missing. 
A bi-weekly score will be calculated by averaging all non-missing 
AM and PM scores in 2 weeks.  A bi-weekly score will be 
considered missing if ≥15 out of 28 assessments in 2 weeks are 
missing. 
A monthly score will be calculated by averaging all non-missing 
AM and PM scores in a month (28 days).  A monthly score will be 
considered missing if ≥29 out of 56 assessments in a month are 
missing. 


 
 
 
 
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All non-missing AM scores from the period of 14 days prior to or 
on the first dose day of study medications, and all non-missing PM 
scores from the period of 14 days prior to the first dose day of 
study medications will be averaged as the AM & PM baseline 
score. Baseline score will be considered missing if ≥15 out of 28 
assessments in the 14 days are missing. One thing of note: 
Inclusion criteria #4:  
Patient must have history of significant pruritus and a caregiver-
reported observed scratching in the eDiary average of ≥2 (on 0 to 4 
scale) in the 2 weeks prior to randomization. 
The pruritus score for purposes of establishing eligibility was 
calculated by taking the average of the worse of the two scores for 
each day.  The values for week one were averaged and the values 
for week 2 were averaged.  The two weekly averages were then 
averaged to provide a single score. If this score was ≥2 the pruritus 
eligibility requirement was met.  The calculation of pruritus 
eligibility score is different from the calculation of the baseline 
pruritus score defined in the SAP. 
Average of change from baseline 
from each AM and PM score 
(scratching, itch severity) 
 
The calculation below is based on the change from baseline in each 
AM and PM score. The values based on this calculation will be 
used for data analysis. 
Change from baseline (daily) will be calculated by averaging all 
non-missing values of change from baseline (AM and PM) in a 
day. A daily change from baseline will be considered missing if 
both AM and PM change from baseline in a day are missing. 
Change from baseline (weekly) will be calculated by averaging all 
non-missing values of change from baseline (AM and PM) in a 
week. A weekly change from baseline will be considered missing if 
≥8 out of 14 change from baseline values in a week are missing. 
Change from baseline (bi-weekly) will be calculated by averaging 
all non-missing values of change from baseline (AM and PM) in a 
week. A weekly change from baseline will be considered missing if 
≥15 out of 28 change from baseline values in a week are missing. 
Change from baseline (monthly) will be calculated by averaging all 
non-missing values of change from baseline (AM and PM) in a 
month (28 days). A monthly change from baseline will be 
considered missing if ≥29 out of 56 assessments in a month are 
missing. 
Baseline, daily, weekly and monthly 
(sleep parameters, such as difficulty 
of falling asleep and staying asleep, 
tiredness, the number of awakenings) 
For patient- and observer reported outcome scores of sleep 
parameters, the same approach above will be used as for 
scratching/itch severity by AM and PM respectively since there is 
just 1 rating per day. 
Proportion of positive pruritus 
assessments 
In the primary analysis of the pruritus endpoint, all intermittently 
missing assessments will be classified as negative pruritus 
assessments. Similarly, all missing planned assessments after 
discontinuation of study drug will be counted as negative pruritus 
assessments. All planned assessments after death or initiation of 
rescue treatments such as biliary diversion surgery or liver 
transplantation will be counted as negative pruritus assessments. 
Both on-treatment and off-treatment pruritus assessments up to 
Day 168 will be included for analysis. 


 
 
 
 
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Protocol Number: A4250-005 
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Rounded baseline score will be used for the primary analysis. 
Additional analysis will be conducted based on unrounded baseline 
score. 
 
In general, all 336 assessments planned over the 24-week treatment 
period will be included in the denominator of the endpoint 
definition. However, if a completer’s last pruritus assessment is 
reported prior to Day 168, then (the last report day x 2) will be 
used as the denominator. The last report day will be determined for 
ObsRO and PRO instruments separately. 
Regarding the proportion of positive pruritus assessments for each 
4-week interval, the proportion is calculated up to the last interval 
with at least 50% of planned assessments. 
 
Derivations for Safety Parameters 
Adverse Events (AE) duration (days) 
AE end date – AE start date +1 
Treatment Emergent Adverse Event 
(TEAE) 
An AE (classified by preferred term) that occurs during the 
Treatment Period will be considered a TEAE if it has a start date 
on or after the first dose date of study drug, or it has a start date 
before the date of the first dose date of study drug, but worsened in 
severity on or after the date of the first dose date of study drug. 


 
 
 
 
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Protocol Number: A4250-005 
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Appendix B: Visit Window 
 
The observation closest to the target day is the measurement used in the analysis for each visit. 
The following 3 tables of analysis visit windows will apply for laboratory parameters, 
questionnaires, vital signs, physical measurements and other efficacy parameters in the study, if 
not otherwise specified. 
 
Table 3.1: Analysis Visit Windows (General) 
Timing of assessment 
(days relative to Treatment) 
Visit Name to display for 
Analysis 
 
Target day 
Study Day (Relative Day) 
Screening (Days -56 to -1) 
Baseline  
 
<= -1 
Day 1 Week 1 
Baseline  
 
1 (pre-dose) 
Week 4 (± 3 days) 
Week 4 
28 
Post-baseline - 42 
Week 8 (± 3 days) 
Week 8 
56 
43 - 70 
Week 12 (± 3 days) 
Week 12 
84 
71 - 105 
Week 18 (± 3 days) 
Week 18 
126 
106 - 140 
Week 22 (± 3 days) 
Week 22 
154 
141 - 161 
Week 24/EOT (± 3 days) 
Week 24 
168 
162 - (last dose day + 14)  
4 Weeks Post Last Dose of 
Study Drug (± 3 days) 
Follow-up  
 
28 post last dose 
≥Last dose day + 15 
 
Table 3.2: Analysis Visit Windows for GIC/GIS, Physical Measurements and Selected Lab Tests 
Timing of assessment 
(days relative to Treatment) 
Visit Name to display for 
Analysis 
 
Target day 
Study Day (Relative Day) 
Screening (Days -56 to -1) 
Baseline  
 
<= -1 
Day 1 Week 1 
Baseline  
 
1 (pre-dose) 
Week 4 (± 3 days) 
Week 4 
28 
Post-baseline - 56 
Week 12 (± 3 days) 
Week 12 
84 
57 - 126 
Week 24/EOT (± 3 days) 
Week 24 
168 
127 - (last dose day + 14)  
4 Weeks Post Last Dose of 
Study Drug (± 3 days) 
Follow-up  
 
28 post last dose 
≥Last dose day + 15 
Note: Physical measurements include physical examination, voluntary photography and skin examination. Selected 
lab tests include urinalysis, autotaxin, p-C4, Vitamins A & E and 25-hydroxy vitamin D. 
 
Table 3.3: Analysis Visit Windows for PedsQL, Fibroscan, Abdominal Ultrasound and AFP 
Timing of assessment 
(days relative to Treatment) 
Visit Name to display for 
Analysis 
 
Target day 
Study Day (Relative Day) 
Screening (Days -56 to -1) 
Baseline  
 
<= -1 
Day 1 Week 1 
Baseline  
 
1 (pre-dose) 
Week 24/EOT (± 3 days) 
Week 24 
168 
71 - (last dose day + 14)  
4 Weeks Post Last Dose of 
Study Drug (± 3 days) 
Follow-up  
 
28 post last dose 
≥Last dose day + 15 
 
 
For laboratory and non-laboratory parameters, if a patient has more than one measurement 
included within a window, the assessment closest to the target day will be used. In case of ties 
between observations located on different sides of the target day, the earlier assessment will be 
used. For laboratory parameters, central laboratory results will be used over local laboratory 
results if both are in a same analysis window. 
 


 
 
 
 
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Derivations for pruritus and other itching, scratching, sleep parameters measured by the Albireo 
ObsRO and PRO instruments will be derived based on the following analysis window. For 
analysis purposes, diary entries will be assigned to a study day based on the recorded date 
regardless of recorded time. Please also refer to the derivations in the table of derived variables 
in Appendix A for details.  
 
Analysis Window 
Treatment Period 
Start Study Day 
End Study Day 
Baseline 
Week 0 (randomization)+ 
Day -14,  
PM assessment 
Day 1,  
AM assessment 
Week 4 
Week 4 
Day 1,  
PM assessment 
Day 29,  
AM assessment 
Week 8 
Week 8 
Day 29,  
PM assessment 
Day 57,  
AM assessment 
Week 12 
Week 12 
Day 57,  
PM assessment 
Day 85,  
AM assessment 
Week 16 
Week 16 
Day 85,  
PM assessment 
Day 113,  
AM assessment 
Week 20 
Week 20 
Day 113,  
PM assessment 
Day 141,  
AM assessment 
Week 24  
Week 24 (EOT) 
Day 141,  
PM assessment 
Day 169,  
AM assessment 
Follow-up 
Week 28 (Follow-up) 
Date of EOT  
PM assessment 
Date of EOT + 28 days  
AM assessment 
 
 
 
 


 
 
 
 
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Protocol Number: A4250-005 
Version: Final v1.2, Date: 25Aug2020
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Appendix C: Definition of Region Variable 
 
Country  
Country code 
Region variable 
Australia 
AUS 
RoW 
Belgium 
BEL 
EU 
Canada 
CAN 
RoW 
France 
FRA 
EU 
Germany 
DEU 
EU 
Israel 
ISR 
RoW 
Italy 
ITA 
EU 
Netherlands 
NLD 
EU 
Poland 
POL 
EU 
Saudi Arabia 
SAU 
RoW 
Spain 
ESP 
EU 
Sweden 
SWE 
EU 
Turkey 
TUR 
RoW 
United Kingdom 
GBR 
EU 
United States 
USA 
US 


 
 
 
 
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Protocol Number: A4250-005 
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Appendix D: Handling of Missing or Incomplete Dates 
 
Global statement: 
If the imputed date is prior to the date of birth, then impute the missing date as date of birth. 
 
Imputation rules for missing or partial AE start date are defined below: 
 
If only Day of AE start date is missing:  
If the AE start year and month are the same as that for the first dose date, then: 
 If the full (or partial) AE end date is NOT before the first dose date or AE end date is 
missing, then impute the AE start day as the day of first dose date;  
 If AE end date is prior to first dose date, then impute the AE start day as 1. 
 
 
If Day and Month of AE start date are missing: 
If AE start year = first dose year, then: 
 If the full (or partial) AE end date is NOT before the first dose date or AE end date is 
missing, then impute the AE start month and day as the month and day of first dose date;  
 If AE end date is prior to first dose date, then impute the AE start month as January and 
the day as 1. 
 
 
If Year of AE start date is missing: 
If the year of AE start is missing or AE start date is completely missing, then query the site with 
no imputation. Also compare the full (or partial) AE end date to the first dose date. If the AE end 
date is before the first dose date, then the AE should be considered as a pre-treatment AE. 
Otherwise, the AE will be considered as TEAE. 
 
Imputation rules for missing or partial non-study medication start/stop dates are defined below: 
 
Missing or partial non-study medication start date: 
 If only day is missing, use the first day of the month.  
 If day and month are both missing, use the first day of the year.  
 If day, month and year are all missing, use the date of the day before the first dose date. 
 
Missing or partial non-study medication stop date: 
 If only day is missing, use the last day of the month. 
 If day and month are both missing, use the last day of the year. 
 If day, month and year are all missing, assign ‘continuing’ status to stop date 
 
 
 
 
 
 
 


 
 
 
 
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Appendix E: SAS Codes 
 
1. Cochran-Mantel-Haenszel test 
 
Proc freq data=< >; 
  tables pfic*ageclass*trt*responder/cmh commonriskdiff; 
  exact comor; 
  ods output cmh=cmh (where=(AltHypothesis="General Association")); 
run; 
 
2. ANCOVA model 
 
Proc mixed data=< >; 
  class trt(ref=’Placebo’) pfic ageclass; 
  model proportion of positive assessment = trt baseline pfic ageclass / solution; 
  lsmeans trt / cl diff alpha=0.05; 
  lsmestimate trt ‘total odevixibat dose’ 0.5 0.5 0 ; 
  lsmestimate trt ‘avg odevixibat dose effect - placebo’ 0.5 0.5 -1/ cl alpha=0.05; 
run; 
 
 
 
 
 


 
 
 
 
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Appendix F: Exit Survey 
 
 
 
 
 


 
 
 
 
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Protocol Number: A4250-005 
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Appendix G: Normal Reference Ranges for Vital Signs [12] 
 
Heart Rate by Age (beats/minute) reference 
Age  
Awake Rate  
Infant (<1 y)  
100-190  
Toddler (1-2 y)  
98-140  
Preschool (3-5 y)  
80-120  
School-age (6-11 y)  
75-118  
Adolescent (12-15 y)  
60-100  
> 15 y 
60-100 
 
 
Normal Respiratory Rate by Age (breaths/minute) reference:  
Age  
Respiratory Rate  
Infants (<1 y)  
30-53  
Toddler (1-2 y)  
22-37  
Preschool (3-5 y)  
20-28  
School-age (6-11 y)  
18-25  
Adolescent (12-15 y)  
12-20  
> 15 y 
12-20 
 
Normal Blood Pressure by Age (mm Hg) reference:  
Age  
Systolic Pressure  
Diastolic Pressure  
Infant (<1 y)  
72-104  
37-56  
Toddler (1-2 y)  
86-106  
42-63  
Preschooler (3-5 y)  
89-112  
46-72  
School-age (6-9 y)  
97-115  
57-76  
Preadolescent (10-11 y)  
102-120  
61-80  
Adolescent (12-15 y)  
110-131  
64-83  
 > 15 y 
90-120 
50-80 
 
Normal Temperature Range by Method:  
 
Method  
Temperature (°C)  
 
Rectal  
36.6-38  
 
Ear  
35.8-38  
 
Oral  
35.5-37.5  
 
Axillary  
36.5-37.5  
 
Temporal/Core* 
35.8-38 
 
*In the eCRF core and temporal temperature measurements can be ticked as methods. Rectal & 
Tympanic (Ear) fall in this group. Additionally, temporal temperature measurements (using a 
temperature scanner on the forehead) – approximate closely to core temperature measurements 
and therefore can be classified in this category. For that reason, the normal temperature range for 
core and temporal temperature measurements are defined as described. 


 
 
 
 
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Protocol Number: A4250-005 
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Appendix H: SI and US Conventional Units of Clinical Laboratory Values 
SERUM CHEMISTRY 
SI UNIT 
CONVENTIONAL UNIT 
Analyte 
    Alpha Fetoprotein 
IU/mL 
ng/mL 
    Direct bilirubin 
µmol/L 
mg/dL 
    Calcium 
mmol/L 
mg/dL 
    Chloride 
mmol/L 
mEq/L 
    Creatinine 
µmol/L 
mg/dL 
    Potassium 
mmol/L 
mEq/L 
    Sodium 
mmol/L 
mEq/L 
    Serum bile acid 
µmol/L 
mg/dL 
    Total bilirubin 
µmol/L 
mg/dL 
Haematology 
SI Unit 
Conventional Unit 
Analyte 
    Haematocrit 
ratio (L/L) 
% 
    Haemoglobin 
g/L 
g/dL 
    Red blood count (RBC) 
x10ˆ12/L 
x10ˆ6/µL 
    Platelet count 
x10ˆ9/L 
x10ˆ3/µL 
    White blood cell count 
x10ˆ9/L 
x10ˆ3/µL 
Fat Soluble Vitamins  
SI Unit 
Conventional Unit 
    Vitamin A 
µmol/L 
ug/dL 
    Vitamin E 
µmol/L 
mg/L 
    Vitamin D (25-dihydroxy) 
nmol/L 
ng/mL 
    Vitamin K 
nmol/L 
ng/mL 
Urinalysis 
SI Unit 
Conventional Unit 
Analyte 
  
  
    Glucose 
mmol/L 
mg/dL 
    Ketones 
mmol/L 
mg/dL 
    Protein 
mmol/L 
mg/dL