Open-Label Pilot Trial to Evaluate the Effects of Ilofotase Alfa on Biomarkers in Adult Patients With Hypophosphatasia
Phase: PHASE1
Conditions: Hypophosphatasia
Sponsor: AM-Pharma

 
 
 
Document Type: 
Final Protocol 
Document Date: 
30 March 2023 
Study Title: 
Open-Label Pilot Trial to Evaluate the Effects of 
Ilofotase Alfa on Biomarkers in Adult Patients 
with Hypophosphatasia 
Protocol Reference Number: 
AP-recAP-HPP-01-01 
NCT Number: 
NCT05890794 
 
 
 
 

Clinical Trial Protocol  
Date: 30 March 2023 
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Version: 1.1 
   
AM-Pharma B.V.                                                       Confidential                       
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TITLE PAGE 
CLINICAL TRIAL PROTOCOL 
 
 
Open-Label Pilot Trial to Evaluate the Effects of Ilofotase Alfa on Biomarkers in Adult Patients with 
Hypophosphatasia 
 
Sponsor code: AP-recAP-HPP-01-01 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
This trial will be performed in compliance with the principles of Good Clinical Practice (GCP). 
 
 
 
 
 
Confidential 
The information in this document is confidential and proprietary to AM-Pharma B.V. No disclosure, dissemination or 
distribution of this document is permitted without the prior written consent of AM-Pharma. 
 
 

Clinical Trial Protocol  
Date: 30 March 2023 
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Version: 1.1 
   
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CLINICAL TRIAL PROTOCOL 
Open-Label Pilot Trial to Evaluate the Effects of Ilofotase Alfa on Biomarkers in Adult Patients with Hypophosphatasia 
 
Protocol number 
 AP-recAP-HPP-01-01 
CT number  
 2023-503186-35-00 
Short title 
Pilot trial of single dose ilofotase alfa in 
hypophosphatasia 
Version 
Version 1.1 
Date of protocol  
30-March-2023 
Investigational product 
Ilofotase alfa 
Principal Investigator 
Lothar Seefried, MD 
Sponsor 
AM-Pharma B.V. 
Stadsplateau 6 WTC Utrecht  
3521 AZ Utrecht  
The Netherlands  
Tel: +31 30 228 9222 
 

Clinical Trial Protocol  
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PROTOCOL SYNOPSIS 
Open-Label Pilot Trial to Evaluate the Effects of Ilofotase Alfa on Biomarkers in Adult Patients with 
Hypophosphatasia 
 
Title (short) 
Pilot trial of single dose ilofotase alfa in hypophosphatasia 
 
Trial code 
Sponsor code: AP-recAP-HPP-01-01  
 
Sponsor 
AM-Pharma BV 
Stadsplateau 6 WTC Utrecht 
3521 AZ Utrecht 
The Netherlands 
 
Trial center 
Clinical Osteology and Clinical Trial Unit 
Konig Ludwig Haus 
Brettreichstraße 11 
D-97074 Wuerzburg 
Germany 
 
 
 
 
Objectives 
Primary objective: 
• 
The change in Inorganic Pyrophosphate (PPi), and Pyridoxal 5’-phosphate (PLP) levels after low, and high dose 
ilofotase alfa in adult hypophosphatasia (HPP) patients. 
Exploratory objectives: 
• 
To determine the safety and pharmacokinetic (PK) profile of a single iv dose of ilofotase alfa in adult HPP patients. 
• 
To determine the pharmacodynamic (PD) profile of a single iv dose of ilofotase alfa in adult HPP patients. 
 
Design and Treatments 
This is a single-center, open-label, randomized, parallel group clinical trial in adult patients with HPP. Two different dose 
levels (0.8 mg/kg and 3.2 mg/kg) will be assessed. Up to twelve patients will be randomized, 6 patients to each trial 
arm. Patients will receive a single dose of 0.8 mg/kg or a single dose of 3.2 mg/kg. Ilofotase alfa will be administered as 
a 1-hour iv infusion on day 1.  
 
Trial period 
The trial period will include a run-in period of 2 days (day -2 to day -1) before administration of ilofotase alfa (day 1), 
followed by 9 days of observation after the ilofotase alfa administration (day 2 to day 10), with an additional follow-
up assessment 14 days after ilofotase alfa administration (day 15).  
 
Patients 
Up to twelve (12) patients ≥ 18 years and ≤ 85 years of age with HPP. 
 
Inclusion criteria 
At screening, the following inclusion criteria must be met: 
1. 
18-85 years old, inclusive.  
2. 
Genetically confirmed variant in the tissue-nonspecific isozyme alkaline phosphatase (ALPL)-Gene. 
3. 
Clinical symptoms of HPP. 
4. 
Medical history with 1) at least two independent measures of Alkaline Phosphatase (ALP) below lower level of 
normal (LLN) and 2) at least one measurement of either PPi or PLP above upper level of normal (ULN). 
5. 
Provision of signed and dated informed consent form (ICF) in accordance with local regulations at screening. 
6. 
Patients must agree not to get pregnant/not to get their partner pregnant, during the trial:  

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• 
Women of Child Bearing Potential must agree to use adequate and effective contraception means (criteria 
CTFG, 2020) for the period between trial drug administration and 14 days thereafter.  
• 
Post-menopausal females not requiring hormonal contraception are defined as: no menses for 12 months 
without an alternative cause, and follicle stimulating hormone level must be used to confirm post-
menopausal state in women not using hormonal contraception or hormonal replacement therapy (CTFG, 
2020).  
• 
Male subjects must agree to wear a condom during sexual intercourse and in case their partner is of 
child bearing potential they must agree to use adequate and effective contraception means (criteria 
CTFG, 2020) for the period between trial drug administration and 14 days thereafter. 
Exclusion criteria 
A patient who meets any of the following exclusion criteria at screening and/or baseline is excluded from participation 
in this trial: 
1. Is unable or unwilling to participate in all scheduled visits and perform all protocol-mandated assessments. 
2. Has a known or suspected hypersensitivity to ilofotase alfa or any components of the formulation used. 
3. Body weight < 40 kilogram and > 120 kilogram. 
4. Patient has a history of clinically significant abnormalities or of any illness that, in the opinion of the trial 
investigator, might confound the results of the trial or pose an additional risk to the patient by their participation 
in the trial. 
5. NSAID use in the past 2 weeks. 
6. Use of corticosteroids in the past 4 weeks. 
7. Use of compounds intended to interfere with bone metabolism (e.g. Denosumab, Teriparatide, Romosozumab, 
Raloxifene) in the past 3 months. 
8. Use of bisphosphonates in the past 2 years. 
9. Participation in a drug trial within 60 days, or five times the half-life of the drug, whichever is longer, prior to 
administration of ilofotase alfa. 
10. Use of asfotase alfa in the previous 3 months. Patients will not be withheld from approved asfotase alfa if medically 
indicated.  
11. A patient who is currently pregnant or lactating. 
12. Use of supplements including Vitamin B6. 
 
Trial Drug, Dosage, and Route of Administration 
Trial drug (ilofotase alfa) is provided in glass vials as a concentrate for infusion (aqueous buffer at a pH of 7.0). Prior to 
administration, the trial drug will be diluted with sterile sodium chloride 0.9% for injection (isotonic saline), United 
States Pharmacopeia (USP) / European Pharmacopoeia (EP) or equivalent, to a final volume of 50 mL and administered 
as an iv infusion using a dosing syringe. Trial drug will be administered as a 1-hour continuous iv infusion via a peripheral 
access point. 
 
Prohibited Medication during the trial period 
1. NSAIDs 
2. Corticosteroids 
3. Compounds intended to interfere with bone metabolism (e.g. Denosumab, Teriparatide, Romosozumab, 
Raloxifene) 
4. Bisphosphonates  
5. Supplements including Vitamin B6 
6. Asfotase alfa 
 
If there is a clinical indication to start any of these above medication, the subject should be withdrawn from the trial.  
Subjects who initiate any of the medication mentioned above during the trial period will be considered dropouts. 
 
Criteria for evaluation 
Primary endpoints 
PPi and PLP serum concentration levels – recorded on day 1 (post dose) trough day 10 (inclusive).  
A composite endpoint of the lowest serum concentration level of each of PPi and PLP will be created.  
 
Exploratory endpoints 
Pharmacokinetics:  
ilofotase alfa serum concentrations measured between day 1 and day 10 (inclusive).  
Safety: 
Adverse events (AE) measured between day 1 and day 15 (inclusive).  

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Vital signs (including supine systolic and diastolic blood pressure, pulse, body temperature, 
respiratory rate) measured at screening, on day 1, and on day 10. 
Clinical laboratory (including clinical chemistry, hematology, and urinalysis) tests, physical 
examination measured on day 1 and day 10 and listed in footnotes 6 and 7 of Table 1. 
 
 
 
Presence of anti-ilofotase antibodies detected on day 10. 
Pharmacodynamics  
Urine phosphoethanolamine (PEA) - measured between day 1 and day 10 (inclusive). 
Immediately consecutive sampling (second morning urine) of urine and blood for 
creatinine, phosphorus, and calcium concentrations to assess phosphate reabsorption 
(ratio of tubular maximum reabsorption of phosphate (TmP) to glomerular filtration rate 
(GFR)(TmP/GFR) and tubular reabsorption of phosphate (TRP)) kidney function and 
Ca/creatinine ratio.  
Blood samples for: Purines, 25 OH Vitamin D, alkaline phosphatase (ALP) activity and 
Isoenzymes, c-Terminal Fibroblast Growth Factor-23 (iFGF-23), parathyroid hormone (PTH), 
C-terminal telopeptide (CTX), procollagen type I N-propeptide  (PINP), Osteocalcin, Vitamin 
B6 VitamerPL, phosphoethanolamine (PEA) in Urine (absolute and relative to sCreat) D 
measured between day 1 and day 10 (inclusive). 
 
Statistical methods 
The final database lock (DBL) will take place after the final patient has reached day 15 and the final PPi and PLP levels 
have been reported.  
 
Primary endpoint 
This analysis will use the intention to treat (ITT) analysis set.  
Biochemical markers of HPP (PPi, and PLP) will be recorded for each patient at baseline (days -2, -1 and 1 before 
randomization) and after treatment, starting on day 1 and ending on day 10. PPi, and PLP levels will be listed and 
summarized for each of day 1 to day 10, overall and by trial arm. The median (minimum, maximum), mean, standard 
deviation (SD) and standard error of the mean (SEM)) PPi, and PLP levels will be described overall and by trial arm. 
These data will also be displayed graphically.  
For both PPi, and PLP the mean change and percentage change from baseline (with SD) will be tabulated per study day 
and by trial arm and shown graphically. The baseline value will be defined as the median value of the measurements 
prior to randomization.   
The maximum change and percentage from baseline (with SD) will be tabulated and shown graphically overall and by 
trial arm. 
 
Exploratory endpoints. 
Endpoints as listed under exploratory objectives will be listed and summarized, overall and by trial arm, using descriptive 
statistics by scheduled time point. No statistical tests will be performed on exploratory endpoints. 
 
Safety 
Safety will be assessed by examining the incidence of adverse events, serious adverse events and will be categorized by 
Medical Dictionary for Regulatory Activities (MedDRA) System Organ Class (SOC) and Preferred Term (PT) and will be 
summarized by trial arm and overall. Physical examination findings will be reported by trial arm and overall using the 
safety analysis set (SAS). For adverse events, seriousness, severity, relationship to trial drug and whether leading to trial 
drug discontinuation or application of any emergency remedy will be displayed. 
 
Anti-ilofotase alfa antibodies 
Any patient who is found to have antibodies against ilofotase alfa measured at day 10 and showing an increase from 
baseline will be considered positive for antibodies. Tables showing the number (and percentage) of patients who were 
positive at baseline (by trial arm and overall) and who were positive by day 10 (by trial arm and overall) will be shown 
using the ITT analysis set. Also, a table showing the number of patients who became positive by day 10 having been 
negative at baseline will be shown (by trial arm and overall). 

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TABLE OF CONTENTS 
1. 
INTRODUCTION ..................................................................................................................... 12 
1.1 
Background ....................................................................................................................... 12 
1.1.1 
Alkaline Phosphatase and Hypophosphatasia ....................................................................... 12 
1.1.2 
Recombinant Human Alkaline Phosphatase (ilofotase alfa) .................................................. 12 
1.1.3 
Clinical Studies with Recombinant Human Alkaline Phosphatase (ilofotase alfa) ................. 12 
1.2 
Rationale ........................................................................................................................... 12 
 
Risk - Benefit .......................................................................................................................... 13 
2. 
OBJECTIVES ........................................................................................................................... 13 
3. 
INVESTIGATIONAL PLAN ......................................................................................................... 13 
3.1 
Trial Design and Plan .......................................................................................................... 13 
3.1.1 
Trial design ............................................................................................................................. 13 
3.1.2 
Screening and Informed consent ........................................................................................... 13 
3.1.3 
Treatment Period ................................................................................................................... 13 
3.1.4 
Follow-up ............................................................................................................................... 13 
3.1.5 
Discussion Trial Design ........................................................................................................... 13 
3.2 
Selection of Trial Population ............................................................................................... 14 
3.2.1 
Inclusion Criteria .................................................................................................................... 14 
3.2.2 
Exclusion Criteria ................................................................................................................... 14 
3.2.3 
Removal of Patients from Therapy or Assessment ................................................................ 15 
3.3 
Treatments ........................................................................................................................ 15 
3.3.1 
Treatments Administered ...................................................................................................... 15 
3.3.2 
Identity of Investigational Product ........................................................................................ 15 
3.3.3 
Trial Drug Preparation ........................................................................................................... 15 
3.3.4 
Trial Drug Administration....................................................................................................... 16 
3.3.5 
Trial Drug Handling and Storage ............................................................................................ 16 
3.3.6 
Drug accountability ................................................................................................................ 16 
3.3.7 
Drug Storage .......................................................................................................................... 16 
3.3.8 
Method of Assigning Patients to Treatment Groups ............................................................. 16 
3.3.9 
Meals during the Trial ............................................................................................................ 17 
3.3.10 Blinding .................................................................................................................................. 17 
3.3.11 Previous / Concomitant Medication and Other Restrictions during the Trial ....................... 17 
3.3.12 Treatment Compliance .......................................................................................................... 17 
3.3.13 Prohibited Medication During the Trial (including the screening period) ............................. 17 
3.3.14 Female subjects ..................................................................................................................... 17 
3.4 
Sample collection ............................................................................................................... 18 
3.4.1 
Primary endpoint samples ..................................................................................................... 18 
3.4.1.1 
PPi....................................................................................................................... 18 
3.4.1.2 
PLP ...................................................................................................................... 18 
3.4.2 
Exploratory assessments ....................................................................................................... 18 
3.5 
Pharmacokinetic and Safety Variables ................................................................................ 18 
3.5.1 
Pharmacokinetic Measurements ........................................................................................... 18 
3.5.1.1 
Ilofotase alfa concentration ............................................................................... 18 
3.5.2 
Safety and Tolerability Measurements .................................................................................. 19 
3.5.2.1 
Adverse Events ................................................................................................... 19 
3.5.2.2 
Vital Signs ........................................................................................................... 19 
3.5.2.3 
Electrocardiogram .............................................................................................. 19 
3.5.2.4 
Physical Examination .......................................................................................... 20 
3.5.2.5 
Anti-Drug Antibodies (ADA) ............................................................................... 20 
3.5.2.6 
Clinical Safety Laboratory ................................................................................... 20 
3.5.2.7 
Pregnancy ........................................................................................................... 20 
3.5.3 
Total of Blood Volume ........................................................................................................... 20 

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3.5.4 
Timing of Assessments ........................................................................................................... 21 
3.6 
Statistical Procedures ......................................................................................................... 21 
3.6.1 
Trial Populations .................................................................................................................... 21 
3.6.2 
Statistical Evaluations ............................................................................................................ 21 
3.6.2.1 
Primary endpoint ............................................................................................... 21 
3.6.2.2 
Exploratory endpoints. ....................................................................................... 22 
3.6.2.3 
Pharmacokinetic Evaluation ............................................................................... 22 
3.6.2.4 
Evaluation of Safety and Tolerability Parameters .............................................. 22 
3.6.2.5 
Adverse Events ................................................................................................... 22 
3.6.2.6 
Vital signs ........................................................................................................... 22 
3.6.2.7 
Physical Examination .......................................................................................... 22 
3.6.2.8 
Anti-Drug Antibodies .......................................................................................... 22 
3.6.2.9 
Clinical Laboratory Test Values .......................................................................... 22 
3.6.3 
Determination of Sample Size ............................................................................................... 22 
3.7 
Data Quality Assurance ...................................................................................................... 23 
4. 
ETHICS ................................................................................................................................... 24 
4.1 
Regulatory and Ethical Considerations ................................................................................ 24 
4.2 
Patient Information and Consent ........................................................................................ 24 
4.3 
Data Protection ................................................................................................................. 25 
4.4 
Patient Confidentiality ....................................................................................................... 25 
5. 
DOCUMENTATION ................................................................................................................. 26 
5.1 
Archiving ........................................................................................................................... 26 
5.2 
Recording of Data in Source Documents and eCRFs ............................................................. 26 
6. 
CONFIDENTIALITY AND PUBLICATION POLICY ......................................................................... 27 
7. 
APPENDICES .......................................................................................................................... 28 
7.1 
Adverse Events and Serious Adverse Events: Definitions and Procedures for Recording, 
Evaluating, Follow-up, and Reporting ................................................................................. 28 
8. 
REFERENCES .......................................................................................................................... 32 
LIST OF TABLES 
Table 1. Schedule of Assessments ...................................................................................................................................... 7 
Table 2. Trial Drug Administered ...................................................................................................................................... 15 
Table 3. PK sample schedule for determination of ilofotase alfa ..................................................................................... 19 
Table 4. Number and Volume of Blood Samples and Total Blood Volume Collected per Patient.................................... 20 
 

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LIST OF ABBREVIATIONS 
ADA 
Anti-drug antibodies 
AE 
Adverse event  
AKI 
Acute kidney injury 
ALP 
Alkaline Phosphatase 
ALPL 
tissue-nonspecific isozyme alkaline phosphatase 
ALT (SGPT) 
Alanine aminotransferase (serum glutamic-pyruvic transaminase) 
AST (SGOT) 
Aspartate aminotransferase (serum glutamic-oxaloacetic transaminase) 
AUC 
Area under the concentration-time curve 
BMI 
Body Mass Index 
CIOMS 
Council for International Organizations of Medical Sciences 
CL 
Clearance 
CT 
Clinical Trial 
CTFG 
Clinical Trial Facilitation Group 
CTX 
C-terminal telopeptide 
CRO 
DBL 
Clinical Research Organization 
Database lock 
DMC 
Data Monitoring Committee 
DNA 
Deoxyribonucleic acid 
EC 
Ethical Committee 
ECC 
ECG 
Endogenous Creatinine Clearance 
Electrocardiogram 
eCRF 
ELISA 
electronic Case Report Form 
Enzyme Linked Immunosorbent Assay  
EP 
EU 
European Pharmacopoeia 
European Union 
GCP 
Good Clinical Practice 
Gamma-GT 
Gamma glutamyl transferase 
GPI 
Glycosylphosphatidylinositol 
GMP 
Good Manufacturing Process 
FSH 
Follicle Stimulating Hormone 
HPP 
Hypophosphatasia 
IB 
ICF 
Investigator Brochure 
Informed Consent Form 
ICH 
International Conference on Harmonization 
IEC 
Independent Ethics Committee 
iFGF-23 
c-Terminal Fibroblast Growth Factor-23 
IMP 
Investigational Medicinal Product 
ITT 
Intent to Treat 
IRB 
IV 
Institutional Review Board 
Intravenous 
LDH 
Lactate dehydrogenase 
LLN 
Lower Level of Normal 
MAD 
Multiple Ascending Dose 
MCH 
Mean corpuscular hemoglobin 
MCHC 
Mean corpuscular hemoglobin concentration 
MCV 
Mean corpuscular volume 
MedDRA 
Medical dictionary for regulatory activities 
MSD 
Meso Scale Discovery 
NSAID 
Non-Steroidal Anti-inflammatory Drug 
PEA 
Phosphoethanolamine 
PINP 
procollagen type I N-propeptide  
PD 
PK 
Pharmacodynamic 
Pharmacokinetic(s) 
PKS 
PK analysis sample 
PLP 
Pyridoxal 5’-phosphate  
PopPK 
Population PK 

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PPi 
Inorganic Pyrophosphate 
PT 
Preferred Term 
PTH 
Parathyroid Hormone 
ULN 
Upper Level of Normal 
SA-AKI 
SAE 
Sepsis Associated Acute Kidney Injury 
Serious adverse event 
SAD 
Single Ascending Dose 
SAS 
Population for Safety Analysis 
SD 
Standard Deviation 
SEM 
Standard Error of the Mean 
SOC 
System Organ Class 
TmP/GFR 
ratio of tubular maximum reabsorption of phosphate (TmP) to glomerular filtration rate (GFR) 
TRP 
tubular reabsorption of phosphate 
USP 
United States Pharmacopeia 
VD 
Volume of distribution 

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1. 
INTRODUCTION 
Recombinant human alkaline phosphatase (ilofotase alfa) is a full-length human chimeric alkaline 
phosphatase (ALP) that could benefit patients with hypophosphatasia (HPP), which is characterized by 
low activity of tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). 
1.1 
Background 
1.1.1 
Alkaline Phosphatase and Hypophosphatasia  
Hypophosphatasia is a rare, inherited, metabolic disease characterized by low activity of the tissue-
nonspecific isoenzyme of alkaline phosphatase (TNSALP) biochemically characterized by an excess of 
extracellular inorganic pyrophosphate (PPi) and pyridoxal 5'-phosphate (PLP) 2. Treatment of HPP with a 
recombinant TNSALP (asfotase alfa) has demonstrated to be very effective in the infantile form 3–6 and is 
currently in clinical investigation for the treatment of adults with juvenile onset HPP 1,7. 
1.1.2 
Recombinant Human Alkaline Phosphatase (ilofotase alfa) 
Ilofotase alfa is a full-length human chimeric ALP derived by recombinant technology and produced in 
Chinese hamster ovary cells. Ilofotase alfa is encoded by a human intestinal ALP sequence wherein the 
sequence encoding the crown domain has been substituted with the corresponding human placental ALP 
sequence. The glycosylphosphatidylinositol (GPI) anchor has been removed resulting in a form that can 
be secreted. Ilofotase alfa has a projected mass of approximately 105 kDa based on the amino acid 
sequence derived from the deoxyribonucleic acid (DNA) sequence and approximately 130 kDa as a fully 
glycosylated molecule. 
1.1.3 
Clinical Studies with Recombinant Human Alkaline Phosphatase (ilofotase alfa) 
In a Phase 1 single ascending dose (SAD) and multiple ascending dose (MAD) clinical trial in 51 healthy 
volunteers, all reported adverse events (AEs) were classified as mild or moderate and most were evenly 
distributed over the active and the placebo groups. The trial did not raise any safety concerns and single 
intravenous (iv) infusion of ilofotase alfa in the range of 0.32 mg/kg to 3.2 mg/kg (200 to 2000 U/kg), as 
well as daily iv infusions of ilofotase alfa at doses of 0.8 and 1.6 mg/kg (500 and 1000 U/kg) for three 
consecutive days, were well tolerated by healthy male and female patients. 
A Phase 2a/2b Proof-of-Concept and Dose-Finding trial (STOP-AKI) with ilofotase alfa in 301 patients with 
SA-AKI (Sepsis Associated Acute Kidney Injury) has been completed. In Part 1 of STOP-AKI, patients were 
randomized to receive 0.4, 0.8, or 1.6 mg/kg of ilofotase alfa or placebo once a day for three consecutive 
days to identify the optimal dose. At the interim analysis, 1.6 mg/kg ilofotase alfa was selected by the 
Data Monitoring Committee (DMC) as the optimal ilofotase alfa dose for Part 2. In Part 2, 1.6 mg/kg 
ilofotase alfa was compared to placebo. The primary endpoint, the mean daily endogenous creatinine 
clearance (ECC) on Days 1 – 7 (area under the curve [AUC] 1-7 ECC), was not significant. However, ilofotase 
alfa showed a significant relative reduction in 28-day mortality of more than 40%. A positive effect on 
mortality was also seen in the 0.8 mg/kg dose. Overall, ilofotase alfa showed a long-term effect on the 
kidney function with significant improvement of ECC on day 21 and day 28 compared to day 1 and over 
the 28-day period. Non-fatal serious adverse events (SAEs) were comparable in the ilofotase alfa and 
placebo groups, indicating that the drug is well tolerated and safe.  
A randomized, double-blind, placebo-controlled, two-arm parallel group, multi-center Phase 3 pivotal 
trial to investigate the efficacy and safety of ilofotase alfa for the treatment of patients with SA-AKI has 
recently been prematurely terminated (NCT04411472), in this trial no safety concerns were raised by the 
DMC.  
A more detailed description of ilofotase alfa and results from non-clinical studies are provided in the 
Investigator Brochure (IB). 
1.2 
Rationale 
Ilofotase alfa is a human recombinant enzyme replacement therapy that is hypothesized to be able to 
supplement deficient TNSALP and has demonstrated efficacy in animal models of HPP 8. Ilofotase alfa has 
demonstrated to be effective and without safety concerns in adults with sepsis and acute kidney injury 
9,10. This is a pilot trial for a potential Phase 2/3 trial aimed at identifying whether treatment with ilofotase 
alfa can normalize circulating levels of PPi, PLP and other biochemical markers of TNSALP deficiency along 
with the safety/tolerability of different doses of ilofotase alfa. 

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1.2.1 
Risk – Benefit 
No specific benefit to the participants in this trial can be expected, however the study results are intended 
to improve the understanding of ilofotase alfa and its potential as a possible additional future treatment 
option.  
Ilofotase alfa has been assessed in healthy volunteers and in adults with sepsis and acute kidney injury, 
without serious safety or tolerability observations. In addition, the risks to subjects in this study will be 
minimized by an adequate selection of the eligibility criteria and diligent close medical monitoring, 
including both clinical symptoms as laboratory assessments and wider parameters of the subjects. 
Overall, risks are considered justified by the benefit of a potential additional future treatment option in 
HPP. 
 
2. 
OBJECTIVES 
Primary objective: 
• 
The change in Inorganic Pyrophosphate (PPi) and Pyridoxal 5’-phosphate (PLP) levels after low and 
high dose of ilofotase alfa  in adult HPP patients. 
Exploratory objective: 
• 
To determine the safety and pharmacokinetic (PK) profile of a single intravenous dose of ilofotase 
alfa in adult HPP patients. 
• 
To determine the pharmacodynamic (PD) profile of a single intravenous dose of ilofotase alfa in adult 
HPP patients. 
 
3. 
INVESTIGATIONAL PLAN 
3.1 
Trial Design and Plan 
3.1.1 
Trial design 
This is a single-center, open-label, randomized, parallel group clinical trial in adult patients with HPP. Two 
different dose levels (0.8 mg/kg and 3.2 mg/kg) will be assessed. Up to twelve (12) patients will be 
randomized to one of two trial arms, 6 in each trial arm.  
Patients will receive a single dose of 0.8 mg/kg or a single dose of 3.2 mg/kg. Ilofotase alfa will be 
administered as a 1-hour intravenous infusion on day 1. 
3.1.2 
Screening and Informed consent 
Patients will report for the eligibility screening according to the inclusion and exclusion criteria defined in 
Section 3.2 within 6 weeks prior to the first drug administration.  
Patients will voluntarily sign the trial specific Informed Consent Form (ICF) prior to any trial specific 
screening procedures being performed. The written informed consent will be obtained for all patients, 
regardless of their eligibility for the trial; the signed ICFs will be retained and archived at the trial center 
and another copy will be provided to the patient. 
Eligibility screening will consist of assessments as presented in the Schedule of assessments Table 1. 
3.1.3 
Treatment Period 
The patients will arrive at the clinical research center two days (day -2) before the drug administration 
(day 1 is the day of drug administration). Patients will leave the research center at the end of the 
observation period on day 10 after completion of all assessments (see Schedule of Assessments Table 1). 
Assessments will be performed during the treatment period as presented in the Schedule of Assessments 
(see Table 1). 
3.1.4 
Follow-up 
The follow-up will be performed at day 15. The specific procedures that will be performed are presented 
in the Schedule of Assessments (see Table 1). 
3.1.5 
Discussion Trial Design 
A randomized, open-label design was chosen to assist the medical assessment of whether there is a 
change in PPi, and PLP between the two different doses. HPP patients may have a history of elevated 

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serum levels in either PPi or PLP, or both. The doses selected, single doses of 0.8 mg and 3.2 mg, were 
also assessed in the single ascending dose studies in healthy volunteers AP-recAP-AKI-01-01 and AP-
recAP-AKI-01-02 previously conducted. This dosing regimen has been administered to both healthy 
volunteers and SA-AKI patients without relevant safety observations. Since both doses administered in 
the current trial have been administered previously in other clinical trials, the groups can be dosed in 
parallel.  
3.2 
Selection of Trial Population 
Up to 12 patients should be enrolled. 
3.2.1 
Inclusion Criteria 
At screening, the following inclusion criteria must be met: 
1. 
18-85 years old, inclusive, at screening.  
2. 
Genetically confirmed variant in the ALPL-Gene. 
3. 
Clinical symptoms of HPP. 
4. 
Medical history with 1) at least two independent measures of Alkaline Phosphatase (ALP) below 
lower level of normal (LLN) and 2) at least one measurement of PPi or PLP above upper level of 
normal (ULN). 
5. 
Provision of signed and dated informed consent form (ICF) in accordance with local regulations at 
baseline. 
6. 
Patients must agree not to get pregnant/not to get their partner pregnant, during the trial   
• 
Women of Child Bearing Potential must agree to use adequate and effective 
contraception means (criteria CTFG, 202011) for the period between trial drug 
administration and 14 days thereafter. 
• 
Post-menopausal females not requiring hormonal contraception are defined as: no 
menses for 12 months without an alternative cause, and follicle stimulating hormone level 
must be used to confirm post-menopausal state in women not using hormonal 
contraception or hormonal replacement therapy (CTFG, 2020).  
• 
Male subjects must agree to wear a condom during sexual intercourse and in case their 
partner is of child bearing potential they must agree to use adequate and effective 
contraception means (criteria CTFG, 2020) for the period between trial drug 
administration and 14 days thereafter. 
3.2.2 
Exclusion Criteria 
A patient who meets any of the following exclusion criteria at screening and/or baseline is excluded from 
participation in this trial: 
1. 
Is unable or unwilling to participate in all scheduled visits and perform all protocol-mandated 
assessments. 
2. 
Has a known or suspected hypersensitivity to ilofotase alfa or any components of the formulation 
used. 
3. 
Body weight < 40 kilogram and > 120 kilogram. 
4. 
Patient has a history of clinically significant abnormalities or of any illness that, in the opinion of the 
trial investigator, might confound the results of the trial or pose an additional risk to the patient by 
their participation in the trial. 
5. 
NSAID use in the past 2 weeks. 
6. 
Use of corticosteroids in the past 4 weeks. 
7. 
Compounds intended to interfere with bone metabolism (e.g. Denosumab, Teriparatide, 
Romosozumab, Raloxifene) in the past 3 months. 
8. 
Use of bisphosphonates in the past 2 years. 
9. 
Participation in a drug trial within 60 days, or five times the half-life of the drug, whichever is longer, 
prior to administration of ilofotase alfa. 
10. 
Use of asfotase alfa in the previous 3 months. Patients will not be withheld from approved asfotase 
alfa if medically indicated. 
11. 
A patient who is currently pregnant or lactating. 
12. 
Use of Supplements including Vitamin B6. 

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3.2.3 
Removal of Patients from Therapy or Assessment 
Participation in the trial is strictly voluntary. A patient has the right to withdraw from the trial at any time 
for any reason. 
The Principal Investigator has the right to terminate participation of a patient for any of the following 
reasons: difficulties in obtaining blood samples, violation of the protocol, severe or serious adverse events 
(SAEs), or for any other reason relating to the patient's safety or integrity of the trial data. 
In the event a patient is withdrawn from the trial, the Sponsor will be informed immediately. If there is a 
medical reason for withdrawal, the patient will remain under the supervision of the Principal Investigator 
until satisfactory health has returned. 
If the Principal Investigator withdraws a patient for a trial drug related reason (according to the judgement 
of the Principal Investigator) he/she is considered a dropout. 
The site will use best effort to ensure that non-completers and dropouts who have received trial drug 
complete the follow up assessments. 
On withdrawal after dosing and prior to the scheduled Day 10 visit, the assessments of the Day 10 visit 
should be done accordingly.  
3.3 
Treatments 
3.3.1 
Treatments Administered 
1-hour iv infusions of 0.8 mg/kg, or 3.2mg/kg ilofotase alfa. 
3.3.2 
Identity of Investigational Product 
Specifications for trial drug administered are summarized in Table 2. 
 
Table 2. Trial Drug Administered 
 
Trial Drug Name 
ilofotase alfa 
Dosage Formulation 
IMP is a clear, colorless to slightly yellow concentrate solution for 
infusion.  
Unit Dose Strength(s)/Dosage Level(s) 
Ilofotase alfa has an activity of approximately 5,000 U/mL (units of 
ALP activity) at 8.0 mg/mL (protein concentration) in an aqueous 
buffer at a pH of 7.0 containing: 
• 
20 mM citrate 
• 
250 mM sorbitol 
• 
2 mM MgCl2 
• 
50 µM ZnCl2 
The solution will be diluted with sterile sodium chloride 0.9% for 
injection (isotonic saline), USP/EP or equivalent and administered as 
an iv infusion to the intended doses. The intended dose will be in a 
volume of 50 mL. 
Route of Administration 
Intravenous infusion 
Packaging and Labeling 
 
For preparation of the trial drug, vials with 4 mL extractable volume 
of 8.0 mg/mL ilofotase alfa will be provided and labeled according to 
EU requirements.  
Supplier 
Almac Clinical Services Ltd.  
 
3.3.3 
Trial Drug Preparation  
For preparation of the trial drug, vials with 4 mL extractable volume of 8.0 mg/mL ilofotase alfa will be 
provided. Prior to administration, the trial drug will be diluted with sterile sodium chloride 0.9% for 
injection (isotonic saline), USP/EP or equivalent, and administered as an iv infusion using an infusion line 
with an in-line filter and dosing syringe. The intended dose is 0.8 mg (500 U) per kg of body weight 
ilofotase alfa or 3.2 mg (2,000 U) per kg of body weight ilofotase alfa The intended dose will be in a volume 
of 50 mL. 

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Reconstituted trial drug can be stored at 2 to 8°C for up to 24 hours or for a maximum of 8 hours at room 
temperature and protected from light. 
Details will be described in the Pharmacy Manual. 
3.3.4 
Trial Drug Administration  
Only patients enrolled and randomized in the trial may receive trial drug. Start and stop time and date of 
each trial drug administration will be collected in the electronic case report form (eCRF). Trial drug is 
administered as an iv infusion using an infusion line with an in-line filter and dosing syringe. Infusion will 
take place at a constant rate of 50 mL/hour for 60 minutes. 
3.3.5 
Trial Drug Handling and Storage  
Receipt and handling of trial drug will be in accordance with procedures at the site. Storage will be in a 
lockable, monitored storage facility with access limited to authorized staff only. Trial drug vials are to be 
stored at 2°C to 8°C and protected from light and moisture until preparation for use. 
Full details of trial drug packaging, storage, and shipment will be provided in the Pharmacy Manual. Trial 
drug will be packaged and labeled in accordance with Good Manufacturing Practice (GMP), Good Clinical 
Practice (GCP) and local requirements. 
3.3.6 
Drug accountability 
Current ICH GCP guidelines require the investigator to ensure that trial drug deliveries from the Sponsor 
are received by a responsible person (e.g. a pharmacist or delegate), and 
• 
that such deliveries are recorded, 
• 
that trial drug is handled and stored safely and properly, 
• 
that trial drug is only dispensed to trial patients in accordance with the protocol, 
• 
that any unused trial drug is returned to the Sponsor or standard procedures for the alternative 
disposition of unused trial drug are followed. 
• 
Drug inventory and accountability records for the trial drugs will be kept by the 
investigator/pharmacist. Trial drug accountability throughout the trial must be documented. The 
following guidelines are therefore pertinent: 
i. The investigator agrees not to supply trial drugs to any person except the patients in this 
trial. 
ii. The investigator/pharmacist will keep the trial drugs in a pharmacy or other locked and 
secure storage facility under controlled storage conditions, accessible only to those 
authorized by the investigator to dispense these test drugs. 
iii. A trial drug inventory will be maintained by the investigator/pharmacist. The inventory will 
include details of materials received and a clear record of when they were dispensed and to 
which patient. 
iv. At the conclusion or termination of this trial, the investigator/pharmacist agrees to conduct 
a final drug supply inventory and to record the results of this inventory on the Drug 
Accountability Record. It must be possible to reconcile delivery records with those of used 
and returned medication. Any discrepancies must be accounted for. Appropriate forms of 
deliveries and returns must be signed by the person responsible. 
v. Unused drug will be locally destroyed according to standard institutional procedures after 
drug accountability has been conducted by the Sponsor or representative at the end of the 
trial. If such is not possible, unused drug will be returned to the Sponsor (or its designee).  
3.3.7 
Drug Storage 
Storage at 2°C to 8°C, protected from light and will be indicated on the label. 
The clinical supplies storage area at the site must be monitored by the site staff for temperature 
consistency with the acceptable storage temperature range specified by the Sponsor. Documentation of 
temperature monitoring should be maintained. 
3.3.8 
Method of Assigning Patients to Treatment Groups 
After obtaining oral and written informed consent, patients will be screened according to the inclusion 
and exclusion criteria. Patients who have met all eligibility criteria receive a screening number upon 
inclusion in the trial. Replacement patients, for those patients who fail screening but were not 
randomized, will receive the screening number of the patient to be replaced, increased by 1000. Patients 

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who complete screening will be randomized to one of two trial arms, just prior to dosing, and will be given 
a subject number which will ensure identification throughout the trial. 
 
Patients who dropout or withdraw for any reason before completing all screening evaluations 
successfully, will be considered as “screening failures“. Such patients will not receive a subject number, 
but will receive a screening number, only applicable data will be entered in the eCRFs. The Principal 
Investigator or authorized designee will keep a screening and enrollment log of all patients screened to 
document patient numbers and passing screening status of enrolled patients into the treatment and 
patients who fail screening and the reasons for their exclusion.  
3.3.9 
Meals during the Trial 
There are no special requirements related to food and beverage intake during the trial. Patients should 
abstain from alcohol from day -2 to day 10 of the trial. 
3.3.10 
Blinding 
The trial is open label. Neither patients, clinicians, trial staff nor pharmacists will be blind to the trial drug.  
3.3.11 
Previous / Concomitant Medication and Other Restrictions during the Trial 
The use of acetaminophen (paracetamol) is allowed. Other medication to treat AEs may only be 
prescribed after consultation of the Sponsor unless there is an emergency that does not allow discussion. 
In the event medication is used, the name of the drug, the dose and dosage regimen will be recorded in 
the eCRF.  
Strenuous activity (activity resulting in muscle pains or perspiration) is not allowed from day-2 to day 10 
of the trial.  
3.3.12 
Treatment Compliance 
To ensure treatment compliance, medication dosing will be supervised by the Principal Investigator or 
his/her deputy. The exact times of medication dosing will be recorded in the eCRF. The actual volume dosed 
will be checked. 
3.3.13 
Prohibited Medication During the Trial (including the screening period) 
1. NSAIDs 
2. Corticosteroids 
3. Compounds intended to interfere with bone metabolism (e.g. Denosumab, Teriparatide, 
Romosozumab, Raloxifene). 
4. Bisphosphonates  
5. Supplements including Vitamin B6 
6. Asfotase alfa 
 
If there is a clinical indication to start any of these above medication, the subject should be withdrawn 
from the trial.  Subjects who initiate any of the medication mention above during the trial period will be 
considered dropouts. 
3.3.14 
Female subjects 
Women of Child Bearing Potential (i.e. fertile, following menarche and until becoming post-menopausal 
unless permanently sterile) must have adequate and effective contraception means. Methods that are 
considered as highly effective birth control methods are: 1) Combined (estrogen and progestogen 
containing) hormonal contraception associated with inhibition of ovulation (oral, intravaginal or 
transdermal), 2) progestogen-only hormonal contraception associated with inhibition of ovulation (oral, 
injectable, implantable), 3) intrauterine device (IUD), 4) intrauterine hormone-releasing system (IUS), 5) 
bilateral tubal occlusion, 6) vasectomised partner, 7) sexual abstinence. Contraception methods 3, 4, 5 and 
6 have lower user dependency and should preferably be used, in particular when contraception is 
introduced as a result of participation in the clinical trial. (CTFG, 202011) 
 
For post-menopausal female, with post-menopause defined as no menses for 12 months without an 
alternative medical cause, a high follicle stimulating hormone (FSH) level in the postmenopausal range may 
be used to confirm a post-menopausal state in women not using hormonal contraception or hormonal 

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replacement therapy. However, in the absence of 12 months of amenorrhea, a single FSH measurement is 
insufficient. (CTFG, 202011) 
3.4 
Sample collection  
The Schedule of Assessments is presented in Table 1. All samples will be pseudonymized, collected and 
analyzed according to the instructions in the lab manual. Left over samples and samples for exploratory 
biomarkers will be bio-banked and stored at -80 °C in an appropriate facility. Unused samples will be 
retained for up to 10 years after completion of the trial and may be used for research purposes relating to 
HPP and/or the effects of ilofotase alfa on HPP disease parameters. 
3.4.1 
Primary endpoint samples 
All samples to be obtained while fasting in the morning. There will be 3 samples obtained to determine 
baseline (day -2, day -1, and day 1 before randomization and dosing). There will be 12 samples obtained 
to determine changes in response to treatment (3 samples on day 1 (2, 4, and 8 hours after start dosing), 
and daily samples from day 2 until day 10. 
3.4.1.1 
PPi 
All samples to be obtained while fasting in the morning and processed following the laboratory manual. 
Briefly, plasma samples will be centrifuged within 15 minutes of collection to yield platelet-depleted 
plasma, after which they will be stored at -80 °C until assessment according to the kit manufacturer 
instructions. 
3.4.1.2 
PLP 
All samples to be obtained while fasting in the morning and processed following the laboratory manual. 
Briefly, all blood samples to be obtained while fasting in the morning, plasma samples will be centrifuged 
within 15 minutes of collection to yield platelet-depleted plasma, after which they will be stored at -80 °C 
until assessment according to the kit manufacturer instructions. 
3.4.2 
Exploratory assessments  
Immediately consecutive sampling (second morning urine) of urine and blood for creatinine, phosphorus, 
and calcium concentrations to assess phosphate reabsorption (TmP/GFR and TRP) kidney function and 
Ca/creatinine ratio. 
 
Blood samples for: 25 OH Vitamin D,  ALP activity and Isoenzymes, iFGF-23, PTH, CTX, PINP, Osteocalcin,  
PL, and processed following the laboratory manual.  
Blood sample for purine measurement processed following the laboratory manual. 
 
For serum collection, blood samples will be allowed to clot for 30-60 minutes and then centrifuged for 15 
min to separate the serum. For plasma sample collection, blood samples will be centrifuged within 15 
minutes of collection to yield plasma. After collection of the serum/plasma in new tubes the samples will 
be stored at -80 °C until assessment. 
 
Urine samples (for PEA, absolute and relative to Creatinine) will be collected as fresh as possible and 
transferred specimen from standard urine collection cup into the pre- labeled transfer tubes. After 
collection the urine samples will be stored at -80 °C until assessment. 
3.5 
Pharmacokinetic and Safety Variables 
The Schedule of Assessments is presented in Table 1. 
3.5.1 
Pharmacokinetic Measurements  
3.5.1.1 
Ilofotase alfa concentration 
Blood samples of 3 mL will be collected via an indwelling iv catheter or by a direct venipuncture at the 
time points indicated in Table 3 (see also Schedule of Assessments presented in Table 1).  
The time clock (zero hours) starts at commencement of the iv dose of ilofotase alfa. The 60 minutes, 2-, 
4- and 8-hours post dose blood samples may be collected within ±5 minutes of the scheduled times, 

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within ±60 minutes for all the other scheduled times. The exact times of blood sampling will be recorded 
in the eCRF.  
 
Table 3. PK sample schedule for determination of ilofotase alfa 
 
Pre-dose 
day 1 
Pre-dose (2 samples) 
Post infusion 
day 1 
60 min (immediately post dose) 
2h 
4h 
8 h  
day 2 
24 h 
day 3 
48 h 
day 4 
72 h 
day 5 
96 h 
day 6 
120 h 
day 7 
144h 
day 8 
168 h 
day 9 
192 h 
day 10 
216 h 
 
Note: The 60 minutes sample is to be taken from the arm NOT used for infusion. 
 
PK samples will be analyzed by a central laboratory. Processing, storage, and shipment of the samples are 
described in detail in the Laboratory Manual. 
3.5.2 
Safety and Tolerability Measurements 
Safety and tolerability assessments will consist of AEs, and results of vital signs, clinical laboratory tests, 
physical examinations, and anti-drug antibodies (ADA). Assessments will be performed in accordance with 
the schedule of assessments. The actual times of all assessments will be recorded in the eCRF. 
3.5.2.1 
Adverse Events 
An AE is defined as any untoward medical occurrence in a patient administered a trial drug and which 
does not necessarily have a causal relationship with this treatment. An AE can therefore be any 
unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a trial drug, whether related to the trial drug. 
At several time points before and after drug administration, patients will be asked non-leading questions 
to determine the occurrence of AEs. Patients will be asked in general terms about any AEs at regular 
intervals during the trial period. In addition, all AEs reported spontaneously during the trial will be 
recorded. 
 
All answers will be interpreted by the Principal Investigator using the Medical Dictionary for Regulatory 
Activities (MedDRA), an internal validated medication dictionary for AEs and will be recorded in the AEs 
Record. Details on the rating of the intensity of the (S)AEs and relationship to the trial treatment are given 
in Appendix 7.1. 
3.5.2.2 
Vital Signs 
During the trial, blood pressure and pulse will be recorded after 5 minutes of rest in the supine position 
and after 2 minutes of rest in the standing position. These assessments will be made using an automated 
device. Body temperature and respiratory rate will be measured subsequently or at the same time as 
blood pressure and pulse.  
3.5.2.3 
Electrocardiogram 
At screening a standard 12 lead ECG will be recorded after 5 minutes of rest in the supine position using 
an ECG machine. The following ECG parameters will be recorded: heart rate, PR-interval, QRS-duration, 

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QT-interval, and QTc-interval (Fridericia's formula) together with the Principal Investigator’s conclusion 
on the ECG profile. This ECG will serve as reference in case additional ECG are made on clinical indication. 
3.5.2.4 
Physical Examination 
Physical examination will be performed according to the procedures of the trial center.  
3.5.2.5 
Anti-Drug Antibodies (ADA) 
Blood samples of 4 mL will be taken at baseline (day 1) and at the end of the observation period (day 10) 
to determine the presence of antibodies against ilofotase alfa. The date and time of blood collection will 
be documented in the eCRF. 
Immunological testing will be performed by a central laboratory by using an Enzyme Linked 
Immunosorbent Assay (ELISA) on a Meso Scale Discovery (MSD) platform. The assay is validated in human 
and septic serum samples. Processing, storage, and shipment of the samples are described in detail in the 
Laboratory Manual. 
3.5.2.6 
Clinical Safety Laboratory 
Blood samples for clinical laboratory assessments will be collected at screening, day 1, and day 10, and 
analyzed locally according to the procedures of the trial center. 
The following parameters will be measured: 
 
Clinical chemistry (serum quantitatively): 
 
total bilirubin, gamma glutamyl transferase (gamma-GT), aspartate aminotransferase (AST) (serum 
glutamic-oxaloacetic transaminase [SGOT]), alanine aminotransferase (ALT) (serum glutamic-pyruvic 
transaminase [SGPT]), lactate dehydrogenase (LDH), creatinine, blood urea, total protein (albumin 
and total), inorganic phosphate, sodium, potassium, calcium, glucose. 
 
Hematology (blood quantitatively): 
 
leucocytes, erythrocytes, hemoglobin, hematocrit, thrombocytes, partial automated differentiation: 
lymphocytes, monocytes, eosinophils, basophils, neutrophils, mean corpuscular volume (MCV), 
mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC). 
 
Urinalysis (urine qualitatively): 
 
hemoglobin, urobilinogen, ketones, glucose, protein. 
 
In the event of unexplained or unexpected clinical laboratory test values, the test(s) will be repeated as 
soon as possible and followed up until the results have returned to the normal range and/or an adequate 
explanation for the abnormality is found. The clinical laboratory will clearly mark all laboratory test values 
that are outside the normal range and the Principal Investigator will indicate which of these deviations 
are clinically significant. These clinically significant deviating laboratory results will then be recorded as 
AEs and the relationship to the treatment will be indicated (see also Appendix 7.1). 
3.5.2.7 
Pregnancy 
Pregnancies will be recorded during the whole trial. Females who become pregnant during the trial must 
stop participation in the trial. In case of pregnancy the subject or female partners of male subjects will be 
followed-up until the pregnancy outcome is known. The Investigator must notify the Sponsor of any 
pregnancy. 
The Investigator must actively follow-up, document and report on the outcome of all pregnancies, even 
if the subjects are withdrawn from the study. 
 
3.5.3 
Total of Blood Volume 
Table 4 presents the number and volume of blood samples and the total volume of blood that will be 
collected throughout the trial per patient and trial part. 
 
Table 4. Number and Volume of Blood Samples and Total Blood Volume Collected per Patient 

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Assessment 
Maximum # samples 
mL of blood per 
sample 
Total volume of 
blood (mL) 
Clinical laboratory 
3 
8 
24 
Clinical Chemistry 
 
 
 
Hematology  
 
 
 
ADA 
2 
4 
8 
Pharmacokinetics 
ilofotase alfa 
15 
 
3 
 
45 
 
Pharmacodynamics 
15 
14 
210 
Total volume of blood drawn 
 
 
287 
 
3.5.4 
Timing of Assessments 
The actual times of all assessments will be recorded in the eCRF. 
For PK blood sampling, post dose blood samples may be collected within ±5 minutes of the scheduled 
time for samples the first 8 hours post dose, within ±60 minutes of the scheduled time for all other 
samples.  
For safety assessments, pre-dose assessments on day 1 will also be performed between waking up and 
dosing. Sampling for clinical laboratory assessments at screening and post-dose at day 10 does not have 
a time-window. 
In the event assessments are planned for the same scheme time, the order of the assessments should be 
arranged in such a way that PK blood sampling is done exactly on time, in practice generally vital signs 
will be done first, followed by blood sampling (exactly on time), followed by safety laboratory sampling. 
Baseline is defined as measurements taken on day -2, day -1 and day 1. Measurements taken on day 1 
must be taken at any time up to immediately before randomization to be considered as baseline 
measurements. Measurements taken at or after randomization are not considered baseline 
measurements. 
3.6 
Statistical Procedures 
3.6.1 
Trial Populations 
Intention to Treat population (ITT) 
All randomized patients will be included in the ITT population.  
 
Population for the safety analysis (SAS) 
All patients who have received trial medication. 
 
Population for the PK analyses (PKS) 
All patients who have received ilofotase alfa and for whom sufficient bioanalytical data are available to 
calculate reliable estimates of the PK parameters. 
3.6.2 
Statistical Evaluations 
3.6.2.1 
Primary endpoint 
This analysis will use the intention to treat (ITT) analysis set.  
Biochemical markers of HPP (PPi, and PLP) will be recorded for each patient at baseline (days -2, -1 and 1 
before randomization) and after treatment, starting on day 1 and ending on day 10. PPi, and PLP levels will 
be listed and summarized for each of day 1 to day 10, overall and by trial arm. The median (minimum, 
maximum), mean, standard deviation (SD) and standard error of the mean (SEM)) PPi, and PLP levels will 
be described overall and by trial arm. These data will also be displayed graphically.  
For both PPi, and PLP the mean change and percentage change from baseline (with SD) will be tabulated 
per study day and by trial arm and shown graphically. The baseline value will be defined as the median 
value of the measurements prior to randomization.   

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The maximum change from baseline  will be tabulated (median, minimum, maximum, mean (including SD, 
SEM and 95% confidence interval)) and shown graphically (mean, SD and 95% confidence interval) overall 
and by trial arm. 
3.6.2.2 
Exploratory endpoints. 
Endpoints as listed under exploratory objectives will be listed and summarized, overall and by trial arm, 
using descriptive statistics by scheduled time point. No statistical tests will be performed on exploratory 
endpoints. 
3.6.2.3 
Pharmacokinetic Evaluation 
The PK serum concentration will be plotted versus time by treatment (individually, combined individual 
data and geometric mean). All individual data will be listed and described descriptively. The PK 
parameters and their statistical evaluation will be separate from the Clinical Trial Report of this trial. A 
separate analysis may include data of this trial in the existing population PK (PopPK) models to evaluate 
clearance (CL) and volume of distribution (Vd), which will be reported separately. 
3.6.2.4 
Evaluation of Safety and Tolerability Parameters 
The Principal Investigator will evaluate the safety and tolerability of the treatment applied during the trial. 
This evaluation will consider the recorded AEs, vital signs, clinical laboratory, physical examination 
findings, and any other parameter that is relevant for safety assessment. 
3.6.2.5 
Adverse Events 
All AEs reported will be coded and classified according to MedDRA. A listing will be given of all individual 
AEs. Summary tables of treatment emergent AEs (TEAEs) will be presented per treatment by system organ 
class and preferred term based on the MedDRA terminology list: one containing the number of TEAEs 
(frequency of occurrence, number and percentage of patients experiencing the event) by treatment and 
severity (intensity) and another containing the number of treatment related TEAEs (frequency of 
occurrence, number and percentage of patients experiencing the event) per treatment and severity. 
Additional total counts by treatment and relationship, and by treatment and severity will be given. 
3.6.2.6 
Vital signs  
Vital signs will be listed, and they will be presented descriptively, where applicable. 
3.6.2.7 
Physical Examination 
Changes from baseline for physical examination will be described and listed. 
3.6.2.8 
Anti-Drug Antibodies 
ADA data will be listed. Any patient who is found to have antibodies against ilofotase alfa measured at 
day 10 and showing an increase from baseline will be considered positive for antibodies. Tables showing 
the number (and percentage) of patients who were positive at baseline (by trial arm and overall) and who 
were positive by day 10 (by trial arm and overall) will be shown using the ITT analysis set. Also, a table 
showing the number of patients who became positive by day 10 having been negative at baseline will be 
shown (by trial arm and overall). 
3.6.2.9 
Clinical Laboratory Test Values 
Summary statistics and plots will be generated for the change from baseline values, as deemed clinically 
appropriate. Summary statistics for the raw laboratory safety tests, may also be computed, as deemed 
clinically appropriate. 
3.6.3 
Determination of Sample Size 
No formal sample size calculation is provided as the purpose of this trial is exploratory. Up to twelve 
patients will be randomized, 6 to each trial arm. If this randomization rate is not achieved in a reasonable 
timeframe, the Sponsor will review the required sample size.  

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3.7 
Data Quality Assurance 
The clinical research center will be monitored by the trial monitor to ensure correct performance of the 
trial procedures and assure that the trial will be conducted according to the relevant regulatory 
requirements.  
By signing this protocol, the Sponsor agrees to be responsible for implementing and maintaining quality 
control and quality assurance systems with written procedures to ensure that trials are conducted, and 
data are generated, documented, and reported in compliance with the protocol, accepted standards of 
GCP and all applicable federal state and local laws, rules and regulations relating to the conduct of the 
clinical trial. 
Regulatory authorities, the IRB/IEC and/or the Sponsor may request access to all source documents, 
eCRFs, and other trial documentation for on-site audit or inspection. Direct access to these documents 
must be guaranteed by the Investigator, who must always provide support for these activities. 

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4. 
ETHICS 
4.1 
Regulatory and Ethical Considerations 
The Investigator must promptly supply the Sponsor or its designee, the IRB/ IEC, competent authorities 
and, where applicable, the Trial Center, with written reports on any changes significantly affecting the 
conduct of the trial or increasing the risk to patients. 
This trial will be conducted in accordance with the protocol and: 
• 
Consensus ethical principles derived from international guidelines including the Declaration of 
Helsinki and Council for International Organizations of Medical Sciences (CIOMS) International 
Ethical Guidelines. 
• 
Applicable International Conference on Harmonization (ICH) GCP Guidelines. 
• 
Regulation (EU) No 536/2014. 
• 
Applicable laws and regulations. 
The protocol, substantial protocol amendments, ICF, IB, and other relevant documents (e.g., any other 
written information regarding this trial to be provided to the patient or the patient’s legal representative) 
must be submitted to the competent authorities and IRB/IEC and reviewed and approved by both 
institutions prior to being used in the trial, except for changes necessary to eliminate an immediate hazard 
to trial patients. 
 
The Investigator will be responsible for the following: 
• 
Providing written summaries of the progress and status of the trial to the IRB/IEC annually or more 
frequently in accordance with the requirements, policies, and procedures established by the IRB/IEC. 
• 
Overall conduct of the trial at the site and adherence to requirements of ICH guideline for good 
clinical practice (E6(R2)) and applicable laws including but not limited to GDPR and applicable German 
privacy laws. 
• 
Submitting reports of SAEs according to the timeline and method outlined in the protocol. 
• 
Obtaining all required patient consents in accordance with applicable laws. 
 
The Sponsor will cover this trial by means of an adequate insurance of the participating patients which 
will be in place prior to the start of the trial. As per local regulations, details about the insurance are 
described in the patient information sheet and informed consent form. A copy of the insurance certificate 
is filed in the Investigator Site File and the patient can request a copy. 
4.2 
Patient Information and Consent 
All parties will ensure protection of patient personal data according to GDPR (and applicable German 
privacy laws) and will not include patient names on any forms, reports, publications, or in any other 
disclosures, except where required by laws. 
The informed consent document used in this trial, and any changes made during the course of the trial, 
must be prospectively approved by the IRB/EC. 
The investigator, or a person designated by the investigator, will obtain written informed consent from 
each patient before any trial specific activity is performed. The investigator and patient will each retain 
an original of the duly (by investigator and patient) signed ICF. 
Prior to execution of the clinical trial, the Sponsor works together with the investigator to prepare the 
written ICF and other written information investigator. The written ICF and any other written information 
will be submitted to the competent authority according to the local requirements. 
• 
The investigator is responsible for explaining the nature and purpose of the trial as well as other trial 
related matters to patients, using the written information. Also, the investigator is responsible for 
obtaining the patient’s full understanding and written consent to participate in the trial of their own 
free will. 
• 
The investigator or other responsible personnel who provided explanations (including collaborators 
who gave supportive information, if applicable) and the patient should sign and date the written 
information or write down his/her name and date the form. 
• 
ICF must be obtained by the time that the first observations/examinations of the pre-investigational 
period are performed. 
• 
The investigator or other responsible personnel should note the following when obtaining ICF from 
patients: 
o 
No patient may be subjected to undue influence, such as compulsory enrolment into a trial. 

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o 
The language and expressions used in the ICF should be as plain and understandable as possible.  
Patients should be given the opportunity to ask questions and receive satisfactory answers to the 
inquiry and should have adequate time to decide whether to participate in the trial. Written 
information should not contain any language or contents that causes the patients to waive or 
appears to waive any legal rights or that releases/mitigates or appears to release/mitigate the 
trial site, the investigator, collaborators, or the Sponsor from liability for negligence. 
4.3 
Data Protection 
• 
Trial patients will be assigned a unique trial patients identification number. Any trial patients’ records 
or datasets that are transferred to the Sponsor will contain this identifier only; trial patients names 
and any information which would make the trial patients identifiable will not be transferred. All 
laboratory specimens, evaluation forms, reports, and other records will be identified in a manner 
designed to maintain trial patients’ confidentiality. 
• 
The trial patients must be informed that the patients’ personal trial-related data will be used by the 
Sponsor in accordance with applicable data protection laws as stated in the ICF. The level of 
disclosure must also be explained to the trial patients as well as the patients’ rights including the right 
to access or erase his/her personal data. 
• 
The trial patients must be informed that the patients’ medical records may be reviewed by Clinical 
Quality Assurance auditors or other authorized personnel appointed by the Sponsor, by appropriate 
IRB/IEC members, and by inspectors from regulatory authorities. 
• 
All records will be kept in a secure storage area with limited access. 
4.4 
Patient Confidentiality 
Individual patient medical information obtained because of this trial is considered confidential and 
disclosure to third parties is prohibited. Such medical information may be given only after approval of the 
patient to the patient’s physician or to other appropriate medical personnel responsible for the patient’s 
well-being. 
The Sponsor, its board members, and its personnel shall not disclose any confidential information on 
patients obtained during the performance of their duties in the clinical trial without justifiable reasons. 
All individuals and organizations involved in the trial must pay very careful attention to protect patients’ 
privacy with appropriate measures, for example, by prohibiting the use of any private information that 
may identify a patient (e.g., name or address). These details shall be processed in accordance with GDPR 
and the applicable local and regional laws. 
Even though any individuals involved in the trial, including the trial monitors and auditors, may get to 
know matters related to patients’ privacy due to direct access to source documents, or from other 
sources, they may not disclose the content to third parties other than those designated by the Sponsor.  
 

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5. 
DOCUMENTATION 
5.1 
Archiving 
Unless other Union law requires archiving for a longer period, the Sponsor and the Principal Investigator 
shall archive the content of the clinical trial master file for at least 25 years after the end of the clinical 
trial. However, the medical files of subjects shall be archived in accordance with national law.  
5.2 
Recording of Data in Source Documents and eCRFs 
Wherever possible, all data will be entered directly into the eCRFs. In some cases, source documents will 
be used. 
 
A Data Management Plan will be written by the Sponsor or delegate which will be finalized prior to the 
first dosing.  

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6. 
CONFIDENTIALITY AND PUBLICATION POLICY 
By signing this protocol, the Principal Investigator reaffirms to the Sponsor that the Investigator will 
maintain in confidence all information furnished to the Investigator or resulting from this trial. The 
Investigator will only divulge such information as may be necessary to the IRB/IEC and the members of 
the staff and the patients who are involved in this trial. 
The Sponsor supports publication of the trial results, regardless of the outcome. It is understood that all 
data related to the trial remains the sole and exclusive property of the Sponsor. The Trial Center and/or 
Investigator may publish the results from the Trial; provided, however, that the Trial Center and/or 
Investigator submits the proposed publication to the Sponsor for review at least sixty (60) days prior to 
the date of the proposed publication. The Sponsor shall have the right to request modifications or removal 
from the proposed publication any Sponsor Information. The Trial Center and/or Investigator shall 
remove such Sponsor Information from the publication but only to the extent that doing so does not 
interfere with the complete and accurate presentation and interpretation of the Trial results. The Trial 
Center and/or Investigator shall consider such Sponsor’s other comments in good faith but has no 
obligation to incorporate them. If such publication may affect the patentability of any invention to which 
the Sponsor has rights, the Sponsor shall have the right to request an additional delay to the proposed 
disclosure of no more than hundred-twenty (120) days so as to allow Sponsor to preserve its intellectual 
property. 
 
 

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8. 
REFERENCES 
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Bowden, S. A. & Foster, B. L. Profile of asfotase alfa in the treatment of hypophosphatasia: design, 
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Kolbus, N., Beuche, W., Felgenhauer, K. & Mäder, M. Definition of a Discontinuous Immunodominant Epitope 
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Whyte, M. P. et al. Asfotase alfa therapy for children with hypophosphatasia. JCI Insight 1, (2016). 
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Whyte, M. P. et al. Asfotase alfa for infants and young children with hypophosphatasia: 7 year outcomes of a 
single-arm, open-label, phase 2 extension trial. Lancet Diabetes Endocrinol. 7, 93–105 (2019). 
7. 
Shapiro, J. R. & Lewiecki, E. M. Hypophosphatasia in Adults: Clinical Assessment and Treatment 
Considerations: HYPOPHOSPHATASIA IN ADULTS. J. Bone Miner. Res. 32, 1977–1980 (2017). 
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Gasque, K. C. S. et al. Improvement of the skeletal and dental hypophosphatasia phenotype in Alpl−/− mice 
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Pickkers, P. et al. Effect of Human Recombinant Alkaline Phosphatase on 7-Day Creatinine Clearance in 
Patients with Sepsis-Associated Acute Kidney Injury. JAMA 320, 1998–2009 (2018). 
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CTFG - Clinical Trials Facilitation and Coordination Group. Recommendations related to contraception and 
pregnancy testing in clinical trials Version 1.1 (21Sep2020)