Clinical trial NCT03559699
Conditions: Pyruvate Kinase Deficiency Anemia, Hemolytic
Sponsor: Agios Pharmaceuticals, Inc.
Start date: 2018-06-26; Completion date: 2020-11-12
Enrollment: 27 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Study AG348-C-007 was a multicenter study designed to evaluate the efficacy and safety of treatment with AG-348 in a minimum of 20, with up to 40, participants with pyruvate kinase (PK) deficiency, who were regularly receiving blood transfusions. The study was composed of two parts. During Part 1, Dose Optimization Period, participants started on a dose of 5 mg AG-348 administered twice daily. Over the course of Part 1 each participant's dose of AG-348 was sequentially increased to 20 mg twice a day, followed by 50 mg twice a day depending on their tolerance. During Part 2, Fixed-Dose Period, participants received AG-348 at their optimized dose from Part 1.

Detailed description:

Arms: arm 1: Participants received AG-348 tablets, administered orally, at a starting dose of 5 milligrams (mg), twice daily (BID), followed by two sequential dose level increases to 20 mg and 50 mg BID, for a period of 16 weeks in Part 1. This was followed by optimized dose BID, as determined by the investigator in Part 1, for a period of 24 weeks in Part 2.
Interventions: intervention 1: AG-348
Intervention descriptions: intervention 1: Part 1 (Dose Optimization Period): Participants began by receiving 5 mg orally, BID. Each participant's dose of AG-348 was sequentially increased to 20 mg BID followed by 50 mg BID depending on their response to AG-348 and their tolerance.

Part 2 (Fixed Dose Period): Optimized dose determined in Part 1.

Protocol documents:
Prot_000.pdf: 
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Agios Pharmaceuticals, Inc. 
CLINICAL STUDY PROTOCOL AG348-C-007 
 
AN OPEN-LABEL STUDY TO EVALUATE THE 
EFFICACY AND SAFETY OF AG-348 IN REGULARLY 
TRANSFUSED ADULT SUBJECTS WITH PYRUVATE 
KINASE (PK) DEFICIENCY  
Study Sponsor 
Agios Pharmaceuticals, Inc. 
88 Sidney Street  
Cambridge, MA 02139-4169, 
USA 
Phone: +1-617-649-8600 
Fax: 617-649-8618 
Study Medical Director 
, MD 
, 
 
Agios Pharmaceuticals, Inc.  
Phone: 
 
Email: 
 
Medical Monitor 
, MD, PhD 
 
Chiltern 
Phone: 
 
Email: 
 
EudraCT Number 
2017‐003803‐22 
NCT Number 
NCT03559699 
Document Version (Date) 
Revised 
Original Protocol, Version 1.0 (28 September 2017) 
Amendment 1, Version 2.0 (15 October 2018) 
Amendment 2, Version 3.0 (19 March 2019) 
This study will be conducted according to the protocol and in compliance with Good Clinical Practices 
(GCP) as described in the International Council for Harmonisation (ICH) of Technical Requirements for 
Pharmaceuticals for Human Use guidelines, the ethical principles stated in the Declaration of Helsinki, 
and other applicable regulatory requirements. 
 
CONFIDENTIALITY NOTE: 
The information contained in this document is confidential and proprietary to Agios Pharmaceuticals, Inc.  
Any distribution, copying, or disclosure is strictly prohibited unless such disclosure is required by federal 
regulations or state law. Persons to whom the information is disclosed must know that it is confidential and that it 
may not be further disclosed by them. 




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INVESTIGATOR’S AGREEMENT 
 
I understand that all documentation provided to me by Agios Pharmaceuticals, Inc. (Agios/the 
Sponsor) or its designated representative(s) concerning this study, which has not been previously 
published, will be kept in strict confidence. This documentation includes the study protocol, 
Investigator’s Brochure (IB), case report forms (CRFs), and other scientific data. 
This study will not commence without the prior written approval of a properly constituted 
Institutional Review Board (IRB)/Independent Ethics Committee (IEC). No changes will be 
made to the study protocol without the prior written approval of Agios and the IRB/IEC, except 
where necessary to eliminate an immediate hazard to the subject. 
I have read, understood, and agree to conduct this study as outlined in the protocol and in 
accordance with the guidelines and all applicable government regulations. 
 
 
 
 
Investigator Name (printed) 
 
Investigator Signature 
 
Date 
(DD MMM YYYY) 
 
 
 
 
 
 
Investigational site or name of institution and location (printed) 
 
 
 


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2. 
SYNOPSIS 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Title of Study:  
An Open-Label Study to Evaluate the Efficacy and Safety of AG-348 in Regularly Transfused Adult 
Subjects With Pyruvate Kinase (PK) Deficiency 
Study Center(s):  
This is a multicenter study that will be conducted in multiple countries.  
Phase of Development: 3 
Objectives:  
Primary: The primary objective of the study is to evaluate the efficacy of treatment with AG-348, as 
assessed by the reduction in transfusion burden. 
Secondary: The secondary objective of this study is to evaluate the safety of treatment with AG-348. 
Exploratory: 
• 
To determine the effect of AG-348 on markers of hemolysis, erythropoietic activity, and other 
indicators of clinical activity 
• 
To determine the effect of AG-348 on markers of iron metabolism and indicators of iron 
overload 
• 
To determine the effect of AG-348 on health-related quality of life (HRQoL), as determined 
using patient-reported outcomes 
• 
To evaluate the pharmacokinetics of AG-348 after oral administration 
• 
To evaluate the relationship of AG-348 pharmacokinetics to indicators of clinical activity 
• 
To evaluate the change in the levels of red blood cell (RBC)-specific form of pyruvate kinase 
(PKR) protein in whole blood 
Methodology: 
Study AG348-C-007 is a 2-part, multicenter, open-label, Phase 3 study consisting of a Dose 
Optimization Period (Part 1) followed by a Fixed-Dose Period (Part 2). This study will evaluate the 
efficacy and safety of treatment with AG-348 in a minimum of 20, with up to 40, adult subjects with 
pyruvate kinase deficiency (PK deficiency) who are regularly receiving blood transfusions. 
Prior to Part 1 of the study, there will be an 8-week Screening Period in which a subject’s complete 
transfusion history from 52 weeks prior to signing the Informed Consent Form (ICF) will be collected 
and recorded on the source documentation and electronic case report form (eCRF).  
The transfusion history information will be used as follows: 
• 
To determine whether the subject meets the study criteria for regular transfusion frequency  
• 
To calculate the Mean Transfusion Frequency (MTF) 
• 
To calculate the Individual Transfusion Trigger (TT), which is the mean (±0.5 g/dL or 
±0.31 mmol/L) of a subject’s collected historical pretransfusion hemoglobin (Hb) 
concentrations. For each transfusion, only the Hb concentration within 1 week prior to and 
closest to a transfusion will be included in this calculation. 


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• 
To calculate the Mean Number of Blood Units (MNU) transfused per transfusion 
• 
To function as historical control data to be used in the analysis 
All subjects will receive AG-348 in Parts 1 and 2 of the study. During Part 1 of the study, all subjects 
will start on a dose of 5 mg AG-348 administered twice daily (BID). Over the course of Part 1, each 
subject will undergo intrasubject dose optimization. Each subject’s dose level of AG-348 may be 
increased 2 times beyond the starting dose of 5 mg BID (ie, from 5 to 20 mg BID and from 20 to 
50 mg BID). In the Fixed-Dose Period of the study (Part 2), a subject will receive AG-348 at his/her 
optimized dose with no planned adjustments (ie, as a fixed dose) for a fixed period of 24 weeks. 
All subjects who remain on study during Part 2 through the Week 24 Visit may be eligible for an 
open-label extension study with AG-348. Subjects who continue on into an extension study will not be 
required to attend the Follow-up Visit. Subjects who continue the study through the Part 2 Week 24 
Visit on study drug but do not continue on into an extension study will attend the Follow-up Visit 
28±4 days after the last dose of study drug. Subjects who discontinue the study prior to the Part 2 
Week 24 Visit should attend the End of Study Visit 28±4 days after the last study visit that the subject 
attended or 28±4 days after the last dose of study drug, whichever is later.  
All subjects who discontinue study drug at any time during the study should undergo a dose taper as 
described in this protocol, unless an emergency situation justifies discontinuing the study drug 
abruptly. Whether the dose taper is performed or not, subjects discontinuing AG-348 should be 
monitored for signs of hemolysis and worsening of anemia. 
An overview of the study design is shown below. 
Figure: Overview of Design for Study AG348-C-007 
 
Abbreviation: BID = twice daily. 
1 Screening may be extended beyond 8 weeks if there is a delay in obtaining a subject’s complete transfusion 
history or to ensure that the first dose of study drug can be administered 2-7 days after the most recent 
transfusion, upon approval by the Medical Monitor (or designee).  
Number of Subjects (Planned):  
A minimum of 20, with up to 40, subjects  


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Diagnosis and Main Criteria for Inclusion: 
Inclusion Criteria: 
For enrollment into this study, subjects must meet all of the following criteria during the Screening 
Period: 
1. Have provided signed written informed consent prior to performing any study procedure, 
including screening procedures. 
2. Be aged 18 years or older. 
3. Have documented clinical laboratory confirmation of PK deficiency, defined as documented 
presence of at least 2 mutant alleles in the PKLR gene, of which at least 1 is a missense 
mutation, as determined per the genotyping performed by the study central genotyping 
laboratory.  
4. Have a history of a minimum of 6 transfusion episodes in the 52-week period prior to date of 
informed consent as documented in the transfusion history of the subject, which reflects the 
subject’s typical transfusion burden.  
5. Have complete records of transfusion history, defined as having the following available for the 
52 weeks prior to the date of informed consent: (1) all the transfusion dates, (2) the number of 
blood units transfused for all the transfusions, and (3) Hb concentrations within 1 week prior to 
transfusion for at least 80% of the transfusions. 
6. Have received at least 0.8 mg oral folic acid daily for at least 21 days prior to the first dose of 
study drug, to be continued daily during study participation.  
7. Have adequate organ function, as defined by: 
a. Serum aspartate aminotransferase (AST) ≤2.5 × upper limit of normal (ULN) (unless the 
increased AST is assessed by the Investigator as due to hemolysis and/or hepatic iron 
deposition) and alanine aminotransferase (ALT) ≤2.5 × ULN (unless the increased ALT is 
assessed by the Investigator as due to hepatic iron deposition). 
b. Normal or elevated levels of serum bilirubin. In subjects with serum bilirubin >ULN, the 
elevation must not be associated with choledocholithiasis, cholecystitis, biliary obstruction, 
or hepatocellular disease. Elevated bilirubin attributed to hemolysis with or without 
Gilbert’s syndrome is not exclusionary. 
c. Estimated glomerular filtration rate (GFR) ≥60 mL/min/1.73 m2, measured GFR 
≥60 mL/min, or calculated creatinine clearance (CrCL; Cockcroft-Gault) ≥60 mL/min. 
d. Absolute neutrophil count (ANC) ≥1.0 × 109/L. 
e. Platelet count ≥100 × 109/L, in the absence of a spleen, or platelet count ≥50 × 109/L, in 
the presence of a spleen and in the absence of any other cause of thrombocytopenia. 
f. Activated partial thromboplastin time (aPTT) and international normalized ratio (INR) 
≤1.25 × ULN, unless the subject is receiving therapeutic anticoagulants. 
8. For women of reproductive potential, have a negative serum pregnancy test during the 
Screening Period. Women of reproductive potential are defined as sexually mature women 
who have not undergone a hysterectomy, bilateral oophorectomy, or tubal occlusion or who 
have not been naturally postmenopausal (ie, who have not menstruated at all for at least the 
preceding 12 months prior to signing informed consent and have an elevated 
follicle-stimulating hormone level indicative of menopause during the Screening Period). 
9. For women of reproductive potential as well as men with partners who are women of 
reproductive potential, be abstinent as part of their usual lifestyle, or agree to use 2 forms of 
contraception, 1 of which must be considered highly effective, from the time of giving 
informed consent, during the study, and for 28 days following the last dose of study drug for 


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women and 90 days following the last dose of study drug for men. A highly effective form of 
contraception is defined as combined (estrogen and progestin containing) hormonal 
contraceptives (oral, intravaginal, or transdermal) known to be associated with inhibition of 
ovulation; progestin-only hormonal contraceptives (oral, injectable, or implantable) known to 
be associated with inhibition of ovulation; intrauterine device; intrauterine hormone releasing 
system; bilateral tube occlusion; or vasectomized partner. The second form of contraception 
can include an acceptable barrier method, which includes male or female condoms with or 
without spermicide, and cervical cap, diaphragm, or sponge with spermicide. Women of 
reproductive potential using hormonal contraception as a highly effective form of 
contraception must also utilize an acceptable barrier method while enrolled in the study and for 
at least 28 days after their last dose of study drug. 
10. Be willing to comply with all study procedures, in particular the Individual TT (calculated 
based on 52 weeks of transfusion history), for the duration of the study. 
Exclusion Criteria: 
Subjects who meet any of the following criteria during Screening will not be enrolled in the study: 
1. Are homozygous for the R479H mutation or have 2 non-missense mutations without the 
presence of another missense mutation in the PKLR gene, as determined per the genotyping 
performed by the study central genotyping laboratory.  
2. Have a significant medical condition that confers an unacceptable risk to participating in the 
study and/or that could confound the interpretation of the study data. Such significant medical 
conditions include, but are not limited to, the following: 
a. Poorly controlled hypertension (defined as systolic blood pressure [BP] >150 mmHg or 
diastolic BP >90 mmHg) refractory to medical management.  
b. History of recent (within 6 months prior to signing informed consent) congestive heart 
failure; myocardial infarction or unstable angina pectoris; hemorrhagic, embolic, or 
thrombotic stroke; deep venous thrombosis; or pulmonary or arterial embolism. 
c. Cardiac dysrhythmias judged as clinically significant by the Investigator. 
d. Heart-rate corrected QT interval-Fridericia’s method (QTcF) >450 msec with the 
exception of subjects with right or left bundle branch block. 
e. Clinically symptomatic cholelithiasis or cholecystitis. Prior cholecystectomy is not 
exclusionary. Subjects with symptomatic cholelithiasis or cholecystitis may be rescreened 
once the disorder has been treated and clinical symptoms have resolved. 
f. History of drug-induced cholestatic hepatitis. 
g. Iron overload sufficiently severe to result in a clinical diagnosis by the Investigator of 
cardiac (eg, clinically significant impaired left ventricular ejection fraction), hepatic (eg, 
fibrosis, cirrhosis), or pancreatic (eg, diabetes) dysfunction. 
h. Diagnosis of any other congenital or acquired blood disorder or any other hemolytic 
process, except mild allo-immunization, as a consequence of transfusion therapy. Genetic 
findings that in isolation are predicted to be insufficient to explain the observed clinical 
phenotype may be allowed (eg, heterozygous status for certain recessive RBC disorders). 
i. 
Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) antibody 
(Ab) with signs of active hepatitis B or C virus infection. If the subject is positive for 
HCVAb, a reverse transcriptase-polymerase chain reaction test will be conducted. Subjects 
with hepatitis C may be rescreened after receiving appropriate hepatitis C treatment. 
j. 
Positive test for human immunodeficiency virus-1 or -2 Ab. 


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k. Active infection requiring the use of parenteral antimicrobial agents or Grade ≥3 in 
severity (per National Cancer Institute Common Terminology Criteria for Adverse Events 
[NCI CTCAE]) within 2 months prior to the first dose of study drug. 
l. 
Diabetes mellitus judged to be under poor control by the Investigator or requiring 
>3 antidiabetic agents, including insulin (all insulins are considered 1 agent); use of insulin 
per se is not exclusionary.  
m. History of any primary malignancy, with the exception of curatively treated 
nonmelanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; or 
other primary tumor treated with curative intent, no known active disease present, and no 
treatment administered during the last 3 years. 
n. Unstable extramedullary hematopoiesis that could pose a risk of imminent neurologic 
compromise. 
o. Current or recent history of psychiatric disorder that, in the opinion of the Investigator or 
Medical Monitor (or designee), could compromise the ability of the subject to cooperate 
with study visits and procedures. 
3. Have a history of transfusions occurring on average more frequently than once every 3 weeks 
during the 52 weeks prior to signing informed consent.  
4. Have a splenectomy scheduled during the study drug period or have undergone splenectomy 
within 12 months prior to signing informed consent. 
5. Are currently enrolled in another therapeutic clinical trial involving ongoing therapy with any 
investigational or marketed product or placebo. Prior and subsequent participation in the PK 
Deficiency Natural History Study (NHS) (NCT02053480) or PK Deficiency Registry is 
permitted, however, concurrent participation is not. Therefore, subjects enrolling in this current 
study will be expected to temporarily suspend participation in the NHS or Registry. 
6. Have exposure to any investigational drug, device, or procedure within 3 months prior to the 
first dose of study drug.  
7. Have a prior bone marrow or stem cell transplant. 
8. Are currently pregnant or breastfeeding. 
9. Have a history of major surgery within 6 months of signing informed consent. Note that 
procedures such as laparoscopic gallbladder surgery are not considered major in this context. 
10. Are currently receiving medications that are strong inhibitors of cytochrome P450 (CYP) 3A4, 
strong inducers of CYP3A4, strong inhibitors of P-glycoprotein (P-gp), or digoxin (a P-gp 
sensitive substrate medication) that have not been stopped for a duration of at least 5 days or a 
timeframe equivalent to 5 half-lives (whichever is longer) prior to the first dose of study drug. 
11. Are currently receiving hematopoietic stimulating agents (eg, erythropoietins [EPOs], 
granulocyte colony stimulating factors, thrombopoietins) that have not been stopped for a 
duration of at least 28 days prior to the first dose of study drug. 
12. Have a history of allergy to sulfonamides if characterized by acute hemolytic anemia, 
drug-induced liver injury, anaphylaxis, rash of erythema multiforme type or Stevens-Johnson 
syndrome, cholestatic hepatitis, or other serious clinical manifestations. 
13. Have a history of allergy to AG-348 or its excipients (microcrystalline cellulose, 
croscarmellose sodium, sodium stearyl fumarate, and mannitol). 
14. Are currently receiving anabolic steroids, including testosterone preparations, within 28 days 
prior to the first dose of study drug. 


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Investigational Product, Dosage and Mode of Administration:  
AG-348 will be supplied as 5, 20, and 50 mg strength tablets to be administered orally. 
Duration of Treatment:  
The maximum total duration that a subject could receive AG-348 in this study is 40 weeks (not 
including the time required to taper study drug).  
The duration of AG-348 treatment at each dose level and the overall duration of the Dose Optimization 
Period (Part 1) of the study will be 16 weeks. The subject’s last visit in Part 1 will be the first visit in 
the Fixed-Dose Period (Part 2). In Part 2 of the study, subjects will receive their optimized dose of 
AG-348 for 24 weeks. 
Reference Therapy, Dosage and Mode of Administration:  
Not applicable. 
Criteria for Evaluation: 
Study Endpoints: 
Primary Endpoint:  
The primary endpoint of this study is the proportion of subjects who achieve a reduction in transfusion 
burden, defined as a ≥33% reduction in the number of RBC units transfused during the 24 weeks of 
Part 2 compared with the historical transfusion burden standardized to 24 weeks (Standardized Control 
Period). 
Secondary Endpoints: 
The secondary endpoints of the study are the following: 
• 
Annualized total number of RBC units transfused during the study (both Part 1 and Part 2) 
compared with the historical transfusion burden 
• 
Number of transfusion episodes during Part 2 compared with the Standardized Control Period 
• 
Proportion of subjects who become transfusion-free, defined as 0 transfusions administered 
during Part 2 
• 
Proportion of subjects who achieve Hb concentrations in the normal range at least once, 
8 weeks or more after a transfusion in Part 2 
• 
Safety endpoints, including the type, incidence, severity, and relationship to treatment of 
adverse events (AEs) and serious adverse events (SAEs), and AEs leading to treatment dose 
reduction, treatment interruption, and treatment discontinuation 
• 
Laboratory tests over time (eg, serum chemistry, liver function tests [LFTs], hematology, 
coagulation, lipids, sex steroids, urinalysis), physical examination findings, dual-energy X-ray 
absorptiometry (DXA) scans (hip and lumbar spine), vital signs, and 12-lead electrocardiogram 
(ECG) data 
Exploratory Endpoints: 
The exploratory endpoints of the study are the following: 
• 
Change from baseline in the following markers of hemolysis: bilirubin, lactate dehydrogenase 
(LDH), and haptoglobin levels 
• 
Change from baseline in markers of erythropoietic activity 
• 
Change from baseline in markers of iron metabolism and indicators of iron overload 
• 
Change from baseline over time in HRQoL scores (ie, Pyruvate Kinase Deficiency Impact 
Assessment [PKDIA], Pyruvate Kinase Deficiency Diary [PKDD], EuroQol-5D-5L 
[EQ-5D-5L]) 


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• 
Characterization of pharmacokinetic profile (drug concentrations over time) and determination 
of pharmacokinetic parameters of AG-348 (eg, area under the plasma concentration × time 
curve [AUC], maximum [peak] concentration [Cmax], and others as applicable) in Part 2 
• 
Exposure-response (or pharmacokinetic-pharmacodynamic) relationship between relevant 
pharmacokinetic parameters and endpoints that are indicators of clinical activity 
• 
Changes in total PKR protein levels 
Statistical Methods: 
Sample Size:  
Due to the rarity of the disease and the small patient population, the sample size is largely driven by 
feasibility. Given the feasibility results and uncertainty of the screen failure rate, the study will enroll a 
minimum of 20, with up to 40, subjects. With a sample size of 20, the power of the study is limited, 
that is, the power will be 58% to detect a response rate of 30% compared to a null rate of 10% with an 
exact test at 2-sided 0.05 significance level. There will be 75% power to detect a large response rate of 
35%, compared to the null of 10%. When the sample size is 40, the power will be at least 90%, even if 
the target response rate is 30%. 
Analysis Sets:  
Two analysis sets will be defined for evaluation of the study endpoints:  
• 
The Full Analysis Set (FAS) is defined as all subjects who received at least 1 dose of study 
drug. The FAS will be used for primary efficacy analyses and safety analyses, unless specified 
otherwise. 
• 
The Per Protocol Set (PPS) is defined as a subset of the FAS including subjects who have 
completed Part 2. The PPS will be used for sensitivity analysis for the primary endpoint. 
Statistical Methods: 
Statistical analysis details will be provided in the statistical analysis plan (SAP), which will be 
finalized before the database lock. 
Subject disposition, demographic and baseline characteristics, prior and concomitant medications, 
study drug exposure, study drug compliance, and other background characteristics will be summarized. 
All summaries will be based on the FAS unless otherwise specified in the SAP. 
Efficacy Analysis 
For the primary analysis of the primary endpoint, the number and proportion of subjects who achieve a 
transfusion reduction response, defined as ≥33% reduction in the number of RBC units transfused 
during the 24 weeks in Part 2 compared to the historical transfusion burden standardized to 24 weeks 
(Standardized Control Period), along with its 95% CI based on the exact binomial distribution, will be 
provided based on the FAS. Subjects who discontinue the study before completing 12 weeks of 
treatment in Part 2 will be considered nonresponders. No formal hypothesis test will be conducted. The 
focus will be given to descriptive statistics, including both the point estimate and the CIs.  
The similar set of analyses will be repeated based on PPS, to further evaluate the impact of early 
discontinuation on the efficacy results.  
For the analysis of the secondary efficacy endpoints of annualized total number of RBC units 
transfused during the study and number of transfusion episodes during Part 2, their change and 
percentage change from the corresponding normalized historical transfusion burden will be 
summarized using descriptive statistics.  
Additional analyses include: 
1. Number and percentage of subjects who are transfusion-free in the Fixed-Dose Period (Part 2), 
along with its 95% CI based on the exact binomial distribution, will be provided.  


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2. Number and percentage of subjects who achieve Hb concentrations in the normal range at least 
once, 8 weeks or more after a transfusion in Part 2, along with its 95% CI based on the exact 
binomial distribution, will be provided.  
Additional sensitivity analysis, supportive analysis, and analyses for exploratory endpoints and 
possibly additional endpoints will be described in the SAP. 
The overall safety profile of study drug will be assessed based on summary of treatment-emergent AEs, 
clinical laboratory values, ECGs, vital signs, physical examination findings, and DXA scans, based on 
the FAS. Only descriptive analysis will be performed (ie, no formal testing will be performed). 
Interim Analysis and Independent Data Monitoring Committee: 
No interim analysis is planned, but interim analyses may take place at any time during the study, if 
warranted, by the ongoing data and/or deemed necessary by the internal Agios team. No independent 
data monitoring committee is planned for this study. 
 


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3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF 
FIGURES  
TABLE OF CONTENTS 
2. 
SYNOPSIS ...................................................................................................................4 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES ...............12 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS.............................18 
5. 
INTRODUCTION ......................................................................................................22 
5.1. 
Pyruvate Kinase Deficiency .......................................................................................22 
5.1.1. 
Epidemiology and Prevalence ....................................................................................22 
5.1.2. 
Biochemistry and Genetics .........................................................................................22 
5.1.3. 
Clinical Characteristics ...............................................................................................23 
5.1.3.1. 
Transfusion Frequency ...............................................................................................24 
5.2. 
Investigational Product (AG-348) ..............................................................................24 
5.2.1. 
Proposed Mechanism of Action of AG-348 ...............................................................24 
5.2.2. 
Summary of AG-348 Nonclinical Data With Potential Clinical Interest ...................25 
5.2.3. 
Summary of AG-348 Clinical Data ............................................................................25 
5.2.3.1. 
Summary of AG-348 Pharmacokinetics .....................................................................26 
5.2.3.2. 
Summary of AG-348 Pharmacodynamics ..................................................................26 
5.2.3.3. 
Summary of AG-348 Clinical Safety Data .................................................................27 
5.2.3.4. 
Summary of AG-348 Efficacy Data ...........................................................................27 
5.3. 
Study Rationale ...........................................................................................................27 
5.3.1. 
Justification of Study Design ......................................................................................28 
5.3.1.1. 
Justification Related to Sample Size...........................................................................28 
5.3.1.2. 
Justification of Using Individual Transfusion History Data as Comparator to 
Assess Efficacy of AG-348 ........................................................................................28 
5.3.1.3. 
Justification of Duration for Collection of Historical Data and Duration on 
Optimized Dose ..........................................................................................................29 
5.3.2. 
Rationale for Dose ......................................................................................................29 
5.3.2.1. 
Justification of Individual Dose Optimization ............................................................30 
6. 
TRIAL OBJECTIVES AND ENDPOINTS ...............................................................31 
6.1. 
Objectives ...................................................................................................................31 
6.1.1. 
Primary Objective .......................................................................................................31 
6.1.2. 
Secondary Objectives .................................................................................................31 


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6.1.3. 
Exploratory Objectives ...............................................................................................31 
6.2. 
Endpoints ....................................................................................................................31 
6.2.1. 
Primary Endpoint ........................................................................................................31 
6.2.2. 
Secondary Endpoints ..................................................................................................31 
6.2.3. 
Exploratory Endpoints ................................................................................................32 
7. 
INVESTIGATIONAL PLAN .....................................................................................33 
7.1. 
Study Design ...............................................................................................................33 
7.1.1. 
Overview of Study Design ..........................................................................................33 
7.1.2. 
Screening Period .........................................................................................................34 
7.1.2.1. 
Transfusion History ....................................................................................................34 
7.1.3. 
Part 1: Dose Optimization Period ...............................................................................35 
7.1.3.1. 
Transfusions During Part 1 .........................................................................................36 
7.1.4. 
Part 2: Fixed-Dose Period ...........................................................................................37 
7.1.4.1. 
Transfusions During Part 2 .........................................................................................37 
7.1.5. 
Discontinuation and Follow-up ..................................................................................37 
7.2. 
Number of Subjects ....................................................................................................38 
7.3. 
Criteria for Study Termination ...................................................................................38 
8. 
SELECTION AND WITHDRAWAL OF SUBJECTS ..............................................39 
8.1. 
Inclusion Criteria ........................................................................................................39 
8.2. 
Exclusion Criteria .......................................................................................................40 
8.3. 
Subject Withdrawal Criteria .......................................................................................42 
8.4. 
Subject Replacement ..................................................................................................43 
8.5. 
End of Study ...............................................................................................................43 
9. 
TREATMENT OF SUBJECTS ..................................................................................44 
9.1. 
Description of Study Drug ..........................................................................................44 
9.1.1. 
Study Drug ..................................................................................................................44 
9.1.2. 
Study Drug Packaging and Labeling ..........................................................................44 
9.1.3. 
Study Drug Storage .....................................................................................................44 
9.1.4. 
Study Drug Administration.........................................................................................44 
9.1.5. 
Study Drug Accountability .........................................................................................44 
9.1.6. 
Study Drug Handling and Disposal ............................................................................45 
9.2. 
Treatment Assignment ................................................................................................45 


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9.3. 
Criteria for Dose Reduction and Interruption of Study Drug for Safety 
Reasons .......................................................................................................................45 
9.3.1. 
Rapid Dose Taper .......................................................................................................47 
9.3.2. 
Gradual Dose Taper ....................................................................................................48 
9.4. 
Transfusions ................................................................................................................48 
9.5. 
Prior and Concomitant Medications ...........................................................................48 
9.5.1. 
Prohibited Medications ...............................................................................................49 
9.5.2. 
Allowed Concomitant Medications ............................................................................50 
9.6. 
Prior and Concomitant Non-Drug Therapies ..............................................................50 
9.7. 
Treatment Compliance ................................................................................................50 
9.8. 
Randomization and Blinding ......................................................................................50 
10. 
STUDY ASSESSMENTS ..........................................................................................51 
10.1. 
Informed Consent .......................................................................................................51 
10.2. 
Study Eligibility ..........................................................................................................51 
10.3. 
Medical and Surgical History .....................................................................................51 
10.3.1. 
Iron Overload-Related History ...................................................................................51 
10.4. 
Prior and Concomitant Medications ...........................................................................52 
10.5. 
PKLR Genotyping .......................................................................................................52 
10.6. 
Demographics .............................................................................................................52 
10.7. 
Assessment of Efficacy ...............................................................................................52 
10.7.1. 
Transfusion Burden ....................................................................................................52 
10.8. 
Assessments of Safety ................................................................................................52 
10.8.1. 
Vital Signs ..................................................................................................................52 
10.8.2. 
Physical Examination .................................................................................................52 
10.8.3. 
Laboratory Assessments .............................................................................................52 
10.8.4. 
Electrocardiogram .......................................................................................................52 
10.8.5. 
Dual-Energy X-ray Absorptiometry Scans .................................................................53 
10.8.6. 
Menstrual Cycle Diary ................................................................................................53 
10.9. 
Laboratory Assessments .............................................................................................53 
10.9.1. 
Safety Laboratory Assessments ..................................................................................54 
10.9.2. 
Other Laboratory Assessments ...................................................................................55 
10.10. 
Other Assessments ......................................................................................................55 
10.11. 
Pharmacokinetic Assessments ....................................................................................55 


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10.11.1. 
Blood Sample Collection ............................................................................................55 
10.11.2. 
Sample Analysis .........................................................................................................56 
10.12. 
Pharmacodynamic Assessments .................................................................................56 
10.13. 
Health-Related Quality of Life Assessments ..............................................................57 
10.13.1. 
Pyruvate Kinase Deficiency-Specific Health-Related Quality of Life 
Assessments ................................................................................................................57 
10.13.2. 
EuroQol-5D-5L ...........................................................................................................57 
10.13.3. 
Patient Global Impression of Severity ........................................................................57 
10.14. 
Study Conduct ............................................................................................................57 
10.14.1. 
Schedule of Assessments ............................................................................................57 
11. 
ADVERSE EVENTS..................................................................................................67 
11.1. 
Reporting Period for Adverse Events and Serious Adverse Events ...........................67 
11.2. 
Definition of Adverse Events .....................................................................................67 
11.2.1. 
Adverse Event .............................................................................................................67 
11.2.2. 
Serious Adverse Event ................................................................................................67 
11.2.3. 
Adverse Events Occurring Secondary to Other Events ..............................................68 
11.2.4. 
Pre-existing Medical Conditions ................................................................................68 
11.2.5. 
Abnormal Laboratory Values .....................................................................................69 
11.2.6. 
Adverse Events of Special Interest .............................................................................69 
11.2.6.1. 
Transaminase Increase ................................................................................................69 
11.3. 
Procedures for Reporting Adverse Events, Adverse Events of Special 
Interest, and Serious Adverse Events .........................................................................70 
11.3.1. 
Intensity ......................................................................................................................70 
11.3.2. 
Relationship to Study Drug ........................................................................................71 
11.4. 
Pregnancy Reporting ..................................................................................................71 
12. 
STATISTICAL METHODS .......................................................................................73 
12.1. 
Sample Size and Power ..............................................................................................73 
12.2. 
Analysis Sets ...............................................................................................................73 
12.2.1. 
Full Analysis Set .........................................................................................................73 
12.2.2. 
Per Protocol Set ..........................................................................................................74 
12.3. 
Statistical Analysis ......................................................................................................74 
12.3.1. 
General Methods .........................................................................................................74 
12.3.2. 
Background Characteristics ........................................................................................74 


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12.3.2.1. 
Subject Disposition .....................................................................................................75 
12.3.2.2. 
Demographics and Baseline Characteristics ...............................................................75 
12.3.2.3. 
Prior and Concomitant Medications ...........................................................................75 
12.3.3. 
Study Drug Exposure and Compliance .......................................................................76 
12.3.3.1. 
Study Drug Exposure ..................................................................................................76 
12.3.3.2. 
Study Drug Compliance .............................................................................................76 
12.3.4. 
Efficacy Analysis ........................................................................................................76 
12.3.4.1. 
Analysis of Transfusion Reduction ............................................................................76 
12.3.4.2. 
Analysis of Other Secondary Efficacy Endpoints ......................................................77 
12.3.5. 
Safety Analysis ...........................................................................................................78 
12.3.5.1. 
Adverse Events ...........................................................................................................78 
12.3.5.2. 
Clinical Laboratory Assessments ...............................................................................79 
12.3.5.3. 
Electrocardiogram .......................................................................................................79 
12.3.5.4. 
Vital Signs ..................................................................................................................79 
12.3.5.5. 
Physical Examination .................................................................................................79 
12.3.5.6. 
Dual-Energy X-ray Absorptiometry Scans .................................................................80 
12.4. 
Interim and Independent Data Monitoring Committee Analyses ...............................80 
12.4.1. 
Interim Analysis ..........................................................................................................80 
12.4.2. 
Independent Data Monitoring Committee Analysis ...................................................80 
13. 
ADMINISTRATIVE REQUIREMENTS ..................................................................81 
13.1. 
Good Clinical Practices ..............................................................................................81 
13.2. 
Ethical Considerations ................................................................................................81 
13.3. 
Subject Information and Informed Consent ...............................................................81 
13.4. 
Subject Confidentiality ...............................................................................................81 
13.5. 
Protocol Compliance ..................................................................................................82 
13.6. 
Data Management .......................................................................................................82 
13.7. 
Source Documentation and Electronic Case Report Form Completion .....................82 
13.8. 
Direct Access to Source Data .....................................................................................83 
13.9. 
Record Retention ........................................................................................................83 
13.10. 
Liability and Insurance ...............................................................................................83 
13.11. 
Publication of Study Findings and Use of Information ..............................................83 
14. 
REFERENCES ...........................................................................................................84 
 


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LIST OF TABLES 
Table 1: 
Dose Modification for Adverse Events Considered Related to Study Drug 
(Except for Events of Excessive Hemoglobin Response), Study AG348-C-
007 ..............................................................................................................................46 
Table 2: 
Rapid Dose Taper Regimen, Study AG348-C-007 ....................................................47 
Table 3: 
Gradual Dose Taper Regimen, Study AG348-C-007 .................................................48 
Table 4: 
Schedule of Assessments – Screening and Part 1 of Study AG348-C-007 ................58 
Table 5: 
Schedule of Assessments – Part 2 of Study AG348-C-007 ........................................63 
Table 6: 
Power Calculation .......................................................................................................73 
Table 7: 
Probability of Observing ≥1 Adverse Event Based on True Incidence Rate 
and Sample Size ..........................................................................................................73 
 
LIST OF FIGURES 
Figure 1: 
Glycolysis in Red Blood Cells of Patients With Pyruvate Kinase Deficiency ...........23 
Figure 2: 
Overview of Design for Study AG348-C-007 ............................................................34 
 


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4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation 
Definition 
Ab 
Antibody 
ADP 
Adenosine diphosphate 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALC 
Absolute lymphocyte count 
ALP 
Alkaline phosphatase  
ALT 
Alanine aminotransferase 
ANC 
Absolute neutrophil count 
aPTT 
Activated partial thromboplastin time 
AST 
Aspartate aminotransferase 
ATP 
Adenosine triphosphate 
AUC 
Area under the plasma concentration × time curve 
AUC0-12 
Area under the plasma concentration × time curve from 0 to 12 hours 
AUC0-last 
Area under the plasma concentration × time curve from time 0 to the time of the 
last measurable concentration 
BID 
Twice daily 
BMD 
Bone mineral density 
BP 
Blood pressure 
BUN 
Blood urea nitrogen 
CL/F 
Apparent total plasma clearance following oral (extravascular) dosing 
Cmax 
Maximum (peak) concentration 
CLp 
Plasma clearance 
CO2 
Carbon dioxide 
CrCL 
Calculated creatinine clearance 
CTCAE 
Common Terminology Criteria for Adverse Events 
CYP 
Cytochrome P450 enzymes 
2,3-DPG 
2,3-Diphosphoglycerate 
DXA 
Dual-energy X-ray absorptiometry 
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EDC 
Electronic data capture 


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Abbreviation 
Definition 
EPO 
Erythropoietin 
EQ-5D-5L 
EuroQol-5D-5L 
FAS 
Full Analysis Set 
FSH 
Follicle-stimulating hormone 
GCP(s) 
Good Clinical Practice(s) 
GDT 
Gradual Dose Taper 
GFR 
Glomerular filtration rate 
Hb 
Hemoglobin 
HBsAg 
Hepatitis B surface antigen 
hCG 
Human chorionic gonadotropin 
HCT 
Hematocrit 
HCV 
Hepatitis C virus 
HDL-C 
High-density lipoprotein cholesterol 
HIV 
Human immunodeficiency virus 
HR 
Hemoglobin response 
HRQoL 
Health-related quality of life 
IB 
Investigator’s Brochure 
IC50 
Half-maximal inhibitory concentration 
ICF 
Informed consent form 
ICH 
International Council for Harmonisation 
IEC 
Independent Ethics Committee 
IMP 
Investigational medicinal product 
INR 
International normalized ratio 
IRB 
Institutional Review Board 
LDH 
Lactate dehydrogenase 
LDL-C 
Low-density lipoprotein cholesterol 
LIC 
Liver iron concentration 
MAD 
Multiple-ascending dose 
max 
Maximum 
MCH 
Mean corpuscular hemoglobin 
MCHC 
Mean corpuscular hemoglobin concentration 
MCV 
Mean corpuscular volume 


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Abbreviation 
Definition 
min 
Minimum 
MNU 
Mean Number of Blood Units 
MRI 
Magnetic resonance imaging 
MTF 
Mean Transfusion Frequency 
n 
Number of subjects 
NCI 
National Cancer Institute 
NHS 
Natural History Study 
NRBC 
Nucleated red blood cell count  
NTBI 
Non-transferrin bound iron 
PD 
Pharmacodynamic(s) 
PEP 
Phosphoenolpyruvate 
PGIS 
Patient Global Impression of Severity 
P-gp 
P-glycoprotein 
PK deficient/cy 
Pyruvate kinase deficient/cy 
PKDD 
Pyruvate Kinase Deficiency Diary 
PKDIA 
Pyruvate Kinase Deficiency Impact Assessment 
PKL 
Liver-specific form of pyruvate kinase 
PKM 
Pyruvate kinase muscle isozyme 
PKR 
Red blood cell-specific form of pyruvate kinase 
PPS 
Per Protocol Set 
PRO 
Patient-reported outcome 
PTH 
Parathormone 
QD 
Once daily 
QOD 
Every other day 
QTcB 
Heart rate-corrected QT interval by Bazett’s method 
QTcF 
Heart rate-corrected QT interval by Fridericia’s method 
QnW 
Every n weeks 
RBC 
Red blood cell 
RDW 
Red cell distribution width  
RT-PCR 
Reverse transcriptase-polymerase chain reaction 
SAD 
Single-ascending dose 
SAE 
Serious adverse event 


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Abbreviation 
Definition 
SAP 
Statistical analysis plan 
SD 
Standard deviation 
t½ 
Terminal half-life 
T4 
Thyroxine 
TEAE 
Treatment-emergent adverse event 
TIBC 
Total iron-binding capacity  
Tmax 
Time to maximum (peak) concentration 
Tlast 
Time of last measurable concentration 
TSH 
Thyroid-stimulating hormone  
TRR 
Transfusion reduction response 
TT 
Transfusion Trigger 
ULN 
Upper limit of normal 
WBC 
White blood cell  
WT 
Wild-type 
Vss 
Volume of distribution at steady state 
Vz/F 
Volume of distribution during the terminal elimination phase following oral 
(extravascular) dosing 
 


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5. 
INTRODUCTION 
5.1. 
Pyruvate Kinase Deficiency 
Pyruvate kinase deficiency (PK deficiency) is a glycolytic enzymopathy that results in life-long, 
nonspherocytic hemolytic anemia. It is an autosomal recessive disease with a variable clinical 
presentation, ranging from mild to life threatening, which can be associated with severe, 
debilitating comorbidities.  
5.1.1. 
Epidemiology and Prevalence 
Epidemiological data for PK deficiency are scarce; however, the current estimated diagnosed 
prevalence of patients with PK deficiency in the United States (US) and European Union 5 
(EU5) (ie, France, Germany, Italy, Spain, and the United Kingdom) is approximately 2,400 cases 
(Carey et al, 2000; de Medicis et al, 1992). As with many rare genetic diseases, true prevalence 
of PK deficiency is not well understood (Beutler and Gelbart, 2000). Most recent estimates have 
been cited at approximately 1:20,000 to 1:485,000 (Beutler and Gelbart, 2000; Carey et al, 2000; 
Hirono et al, 2014; Zanella et al, 2007).  
5.1.2. 
Biochemistry and Genetics 
In normal cells, pyruvate kinase enzymatically catalyzes the metabolic conversion of 
phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) into pyruvate and adenosine 
triphosphate (ATP) as the final step in glycolysis. It is believed that PK deficiency leads to 
insufficient ATP production, resulting in red blood cell (RBC) hemolysis due to an impaired 
ability to maintain cellular membrane homeostasis (van Wijk and van Solinge, 2005). Indeed, 
PK deficiency has been reported to be associated with reduced RBC survival, as well as with 
impaired RBC maturation (Aizawa et al, 2003). 
Pyruvate kinase deficiency is the second most common of the glycolytic enzymopathies after 
glucose-6-phosphate dehydrogenase deficiency. Red blood cells from patients with 
PK deficiency are characterized by changes in metabolism associated with defective glycolysis, 
including a deficiency in ATP levels. Levels of 2,3-diphosphoglycerate (2,3-DPG), PEP, and 
other glycolytic intermediates upstream of the reaction catalyzed by the RBC-specific form of 
pyruvate kinase (PKR) have been reported to be elevated in patients with PK deficiency, 
reflecting the inhibition of glycolysis at the PKR step (Oski and Bowman, 1969) (see Figure 1). 
Red blood cells from patients with PK deficiency show less efficient utilization of glucose than 
the RBCs of healthy individuals (Tanaka et al, 1962). 


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Figure 1: 
Glycolysis in Red Blood Cells of Patients with Pyruvate Kinase Deficiency 
 
Abbreviations: 1,3-DPG = 1,3-diphosphoglycerate; 2,3-DPG = 2,3-diphosphoglycerate; 3-PG = 3-phosphoglycerate; 
ADP = adenosine diphosphate; ATP = adenosine triphosphate; FBP = fructose 1,6-biphosphate; 
PEP = phosphoenolpyruvate; PKR = red blood cell-specific form of pyruvate kinase.  
Note: Not all steps in glycolysis are shown. 
Pyruvate kinase deficiency is an autosomal recessive disease (patients must have 2 mutated 
alleles, usually as compound heterozygotes), and most patients with PK deficiency present with 
unique combinations of poorly characterized or private mutations. In addition to genetic 
heterogeneity, PK deficiency exhibits considerable phenotypic variability. For example, the US 
Pennsylvanian Amish community represents a subgroup of PK deficient patients with a relatively 
homogeneous genetic background (eg, homozygous R479H mutation, restricted marriage pool) 
and uniform lifestyle. Yet, despite these similarities, there is still considerable phenotypic 
variation within this community in terms of disease severity (Grace et al, 2015). 
A nondrug study protocol to obtain critical information regarding the natural history of 
PK deficiency and the range and incidence of related symptoms, treatments, and complications—
known as the PK Deficiency Natural History Study (NHS) (ClinicalTrials.gov, 2017)—has been 
developed by Boston Children’s Hospital (Boston, Massachusetts, US) and is funded and 
supported by Agios. This multicenter, global NHS is designed as a longitudinal cohort study with 
retrospective, baseline, and annual collection of data over a 2-year period. The study has thus far 
identified 123 mutations in 255 subjects (Bianchi et al, 2017). Fifty mutations or approximately 
40% of the identified mutations had not been previously described and were identified only as a 
consequence of the systematic genotyping effort for participants in this NHS. This highlights the 
evolving understanding of the genetic basis of PK deficiency. 
An attempt has been made to impose a system of classification onto this large collection of 
potential genotypes by dividing the genotypes into 2 classifications and 3 groups (Bianchi et al, 
2015). This classification showed that 79 of the 123 mutations (64.2%) were missense mutations, 
which are single nucleotide changes that result in amino acid substitutions in the PKR enzyme. 
The effects of these missense mutations can include a loss of catalytic efficiency and/or a loss of 
protein stability of the enzyme. Non-missense mutations include those that cause premature 
truncations of the enzyme, deletions or frameshifts, or mutations that affect splicing of the 
enzyme. Many of these non-missense mutations are predicted to be null alleles of PKR, resulting 
in a lack of functional protein expression (Bianchi et al, 2017). 
5.1.3. 
Clinical Characteristics 
The natural history of untreated PK deficiency is characterized by life-long hemolytic anemia 
and subsequent associated comorbidities, which can include a need for transfusions, 
susceptibility to infections after splenectomy, worsening anemia during pregnancy, and 
symptoms associated with chronic hemolytic anemia (Rider et al, 2011). Some patients with 
PK deficiency may present with severe hemolytic anemia in early infancy that requires 
immediate care. Unconjugated bilirubin is also often chronically elevated in patients with 


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PK deficiency; thus, pigmented gallstones are common in both children and adults with the 
disease. Additionally, iron overload is progressive and can ultimately lead to life-threatening 
symptoms.  
There are no generally agreed-upon definitions of “mild”, “moderate”, and “severe” disease 
because multiple factors—such as the degree of anemia, the level of bilirubin and severity of 
jaundice, and complications (including iron overload, transfusion need, and subjective feelings of 
fatigue and low energy level)—must be taken into account before an evaluation can be made.  
5.1.3.1. 
Transfusion Frequency 
There are no guidelines for transfusion management in patients with PK deficiency. Most adults 
with the disease have undergone splenectomy and require only sporadic or ad hoc transfusions, 
which are usually administered in the context of an acute hemolytic episode triggered by 
infection, trauma, or stress (Grace et al, 2018; Grace et al, 2016; Zanella et al, 2007; Zanella et 
al, 2005). These patients are considered “not regularly transfused.” 
A minority of adult patients with PK deficiency still require regular transfusions after 
splenectomy. Some adult patients who have not undergone splenectomy also require regular 
transfusions. There is no clear definition of what constitutes a regularly transfused patient with 
PK deficiency; data from the PK Deficiency NHS point to a wide range of transfusion 
frequencies in adults with the disease. For patients transfused infrequently, it can be difficult to 
determine retrospectively whether a patient is transfused mainly for cause or requires infrequent, 
but still regular transfusions. Experts have indicated patients receiving fewer than 6 transfusions 
per year (every 2 months on average over a 1-year period) are unlikely to require the transfusions 
to maintain hemoglobin (Hb) at an acceptable level. 
Analysis of the NHS study, which has data for a total of 255 subjects as of 28 April 2017, 
identified a maximum of 16 adult subjects (out of 98 non-Amish subjects) who reported 
receiving at least 6 transfusions in the year prior to enrollment (Grace et al, 2018). This severely 
limits the type of clinical trials that could be performed in this population. 
5.2. 
Investigational Product (AG-348) 
5.2.1. 
Proposed Mechanism of Action of AG-348 
AG-348 is a potent, broad-spectrum activator of PKR, 1 of 4 pyruvate kinase isoenzymes 
expressed in human tissues from 2 separate genes. Both PKR and the liver-specific form of 
pyruvate kinase (PKL) are splice isoforms of the PKLR gene, while pyruvate kinase muscle 
isozyme (PKM)1 and PKM2 are both expressed from the PKM gene. AG-348 is an allosteric 
activator of the PKR, PKL, and PKM2 isoenzymes, with similar activity for each. AG-348 acts 
by directly binding to the PKR tetramer and allosterically enhancing its affinity for PEP. 
As described in Section 5.1.2, the activity of the glycolytic pathway is disrupted in patients with 
PK deficiency. This disruption results in significantly reduced RBC lifespan and manifests 
clinically as nonspherocytic hemolytic anemia. In patients with PK deficiency, RBCs and their 
progenitors are characterized by changes in metabolism associated with defective glycolysis, 
including a buildup of PEP and the intermediate 2,3-DPG, and lowered levels of ATP. It is 
hypothesized that AG-348 restores the ability of RBCs to convert PEP + ADP to pyruvate + ATP 
and thereby normalizes RBC metabolism in patients with PK deficiency. 


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5.2.2. 
Summary of AG-348 Nonclinical Data with Potential Clinical Interest 
A series of exploratory pharmacology studies were conducted to characterize the ability of 
AG-348 to activate wild-type (WT) PKR and anemia-associated PKR mutants in vitro, ex vivo, 
and in vivo. 
Biochemical studies showed that AG-348 is a potent, broad-spectrum activator of recombinant 
PKR with low nanomolar potency against both WT and mutant enzymes. The effect of AG-348 
on PKR activity and a number of downstream pathway markers was evaluated in both human 
and murine RBCs and whole blood. AG-348 dose-response curves in human and murine RBCs 
showed increased PKR activity. AG-348 dose-response curves also showed increased ATP 
levels. 
The effects of AG-348 on PKR activity and RBC metabolism were also assessed in blood 
samples from subjects with PK deficiency. AG-348 activated PKR and induced metabolic 
changes (increased ATP levels and decreased 2,3-DPG levels) consistent with increased 
glycolytic pathway activity in RBCs from PK-deficient patients with different mutations in the 
PKR enzyme. Finally, a series of 3 in vivo pharmacology studies conducted in C57BL/6 mice 
confirmed the in vitro potency of AG-348 in increasing WT PKR enzyme activity and in 
modulating the levels of the downstream markers, ATP, and 2,3-DPG. Based on the data from 
these studies, a strong pharmacokinetic/pharmacodynamic (PD) relationship was established 
between AG-348 area under the plasma concentration × time curve from 0 to 12 hours (AUC0-12) 
and ATP/2,3-DPG AUC0-12 ratio.  
AG-348 was evaluated for its potential to inhibit binding and enzymatic activity in a panel of 
89 receptors, ion channels, and enzymes. AG-348 is a histamine H3 antagonist/inverse agonist 
and an aromatase inhibitor. Findings consistent with aromatase inhibition have been observed in 
the reproductive organs of male and female rats in toxicology studies of up to 6 months’ duration 
at exposures as low as 10,900 ng•hr/mL. No findings consistent with aromatase inhibition have 
been observed in monkeys in toxicology studies of up to 9 months’ duration. Effects consistent 
with aromatase inhibition and antagonism/inverse agonism at the H3 receptor have been 
observed in clinical studies (Section 5.2.3.3). 
Emesis was observed in monkey toxicology studies, and dose-dependent emesis was observed in 
a dedicated safety pharmacology study in ferrets. Based on animal studies, AG-348 may affect 
fertility in males and females. In animals, these effects were reversible after discontinuation of 
AG-348. AG-348 may also affect the ability to maintain pregnancy. 
Further details on these and other nonclinical studies, including nonclinical pharmacokinetics, 
are in the AG-348 Investigator’s Brochure (IB). 
5.2.3. 
Summary of AG-348 Clinical Data 
AG-348 has been evaluated in 4 clinical pharmacology studies in healthy subjects (3 completed 
and 1 ongoing) and 1 ongoing Phase 2, open-label, efficacy, and safety study (AG348-C-003, 
referred to as DRIVE-PK) in adult subjects with PK deficiency. 
The ongoing DRIVE-PK study is an open-label study intended for adult subjects with 
PK deficiency who are considered transfusion independent (per protocol definition) with 
screening Hb concentration ≤12 g/dL or ≤11 g/dL for males and females, respectively. The study 
is designed to evaluate the safety, tolerability, and potential indicators of clinical activity of 


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2 dose levels of AG-348 (50 and 300 mg twice daily [BID]) administered for up to 24 weeks in 
the Core Period and beyond 24 weeks in the Extension Period. The DRIVE-PK study is also 
intended to evaluate the pharmacokinetics of AG-348, the PD response (ATP and 2,3-DPG 
levels) after administration of AG-348, and additional PD biomarkers. 
A brief overview of AG-348 pharmacokinetic and PD data is provided in Section 5.2.3.1 and 
Section 5.2.3.2, respectively. An overview of the available safety data from these studies as well 
as preliminary efficacy results from the DRIVE-PK study is included in 
Section 5.2.3.3 and Section 5.2.3.4, respectively.  
Please refer to the AG-348 IB for additional details on all clinical studies and results. 
5.2.3.1. 
Summary of AG-348 Pharmacokinetics 
The pharmacokinetic profile of AG-348 has been well characterized in the Phase 1 
single-ascending dose (SAD) study (AG348-C-001) and Phase 1 multiple-ascending dose 
(MAD) study (AG348-C-002), conducted in healthy adult subjects. The pharmacokinetics of 
AG-348 increased in a dose-proportional manner across tested doses in the SAD study and at 
lower doses in the MAD study. At higher dose levels in the MAD study, a less than 
dose-proportional increase was observed, attributed to the cytochrome P450 (CYP) 3A4 
induction effect of AG-348. The effective half-life of AG-348 has been estimated to be 
approximately 3 to 6 hours.  
A capsule formulation was used in the SAD and MAD studies and the DRIVE-PK study. A 
tablet formulation is being used in several of the ongoing studies, was introduced into 
DRIVE-PK, and will also be used in this study. Prior to introducing the tablet formulation in 
clinical studies, a relative bioavailability study (AG348-C-005) was conducted in healthy 
subjects to compare the pharmacokinetics of the 2 formulations (ie, capsules and tablets). 
Systemic exposure to AG-348 appeared similar between formulations with an area under the 
plasma concentration × time curve (AUC) ratio of 1.05 and a maximum (peak) concentration 
(Cmax) ratio of 1.19 for the tablet formulation compared with the capsule formulation. These 
results suggest that no dose adjustments are required with the tablet formulation. Therefore, 
when the tablet formulation is used, the same dose as that of the capsule can be used in ongoing 
and future clinical studies. 
Additionally, in the ongoing Phase 2 DRIVE-PK study, conducted in adult subjects with 
PK deficiency, the pharmacokinetics of AG-348 in plasma have been evaluated. To date, 
pharmacokinetic data of AG-348 in adult subjects with PK deficiency were found to be similar to 
that observed in healthy adult subjects. 
Please refer to the AG-348 IB for detailed information regarding the pharmacokinetics of 
AG-348. 
5.2.3.2. 
Summary of AG-348 Pharmacodynamics 
In the SAD and MAD studies, the concentration of 2,3-DPG decreased in a dose-dependent 
manner and returned to levels close to baseline by 72 hours following the final dose of AG-348. 
In the SAD study, after a single dose of AG-348, a minimal increase in the concentration of ATP 
was observed at 24 to 120 hours postdose. In contrast to the SAD study, significant increases in 


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ATP were observed in the MAD study, and the concentration of ATP remained elevated through 
120 hours after the final dose of AG-348. 
In the DRIVE-PK study, conducted in adult subjects with PK deficiency, the PD responses of 
ATP and 2,3-DPG in whole blood have also been evaluated. In this study, no consistent pattern 
of decrease in concentration of 2,3-DPG or increase in ATP has been observed. The reason for 
this is not completely clear. 
Please refer to the AG-348 IB for detailed information regarding the PD properties of AG-348. 
5.2.3.3. 
Summary of AG-348 Clinical Safety Data 
Overall, AG-348 has been generally well tolerated among healthy adult subjects and adult 
subjects with PK deficiency. Important identified risks associated with administration of AG-348 
in clinical studies include bone mineral density (BMD) decrease (including osteoporosis and 
osteopenia due to aromatase inhibition), withdrawal hemolysis, and insomnia (not clinically 
serious, ie, not Grade 3 or Grade 4).  
Potential risks of AG-348 administration include anaphylactoid reaction, aromatase inhibition, 
gastrointestinal disturbances, transaminase increases, and triglyceride increase. Transaminase 
increases are adverse events of special interest (AESIs) for AG-348. Please refer to 
Section 11.2.6 for additional information on AESIs and the current AG-348 IB for a more 
detailed overview of available safety data. 
5.2.3.4. 
Summary of AG-348 Efficacy Data 
In the ongoing Phase 2 DRIVE-PK study in adult subjects with PK deficiency, the efficacy of 
AG-348 is primarily analyzed via evaluation of changes in Hb concentrations.  
As of a data cutoff of 14 July 2017, a preliminary analysis indicates that of the 52 subjects who 
received AG-348 during the Core Period, 26 subjects (50.0%) achieved maximum increases in 
Hb >1 g/dL (Grace et al, 2017). Of the 42 subjects with ≥1 missense mutation, 25 subjects 
(59.5%) had an Hb increase >1.0 g/dL. The majority of Hb increases were rapid and sustained. 
The median time to the first observation of an Hb increase >1 g/dL above baseline was 10 days 
(range, 7 to 187 days). 
Overall, treatment with AG-348 has resulted in Hb responses that are rapid in onset, robust, and 
sustained with prolonged treatment with AG-348. In summary, this clinical evidence from 
Study AG348-C-003 suggests that treatment with AG-348 has the ability to provide a significant 
clinical benefit to patients.  
5.3. 
Study Rationale 
The totality of the preliminary data from the Phase 2 DRIVE-PK study, combined with the data 
from the 2 completed Phase 1 SAD and MAD studies, support continued clinical development of 
AG-348 for the treatment of patients with PK deficiency. In order to evaluate the efficacy and 
safety of AG-348 across the disease spectrum of PK deficiency, 2 studies investigating the 
treatment of AG-348 in subjects with PK deficiency will be conducted. This protocol, 
AG348-C-007, will be conducted in adult subjects with PK deficiency who are regularly 
receiving transfusions, while a separate study, AG348-C-006, will be conducted in adult subjects 
with PK deficiency who are not regularly receiving transfusions.  


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A clear and serious unmet medical need exists for patients with PK deficiency. At present, there 
are no approved, disease-specific therapeutic agents for the treatment of patients with 
PK deficiency; rather, available treatment options are supportive only. Many adult patients 
underwent splenectomy in childhood as a means to reduce hemolysis and eliminate the need for 
regular transfusions. However, some patients, despite undergoing splenectomy, still require 
regular transfusions to maintain their Hb concentration at a level considered acceptable. Iron 
overload, an unavoidable complication of chronic hemolytic anemia, which is usually 
incompletely controlled by iron chelation therapy, is made worse by splenectomy and frequent 
transfusions. The consequences of transfusion-related iron overload if inadequately treated 
include liver dysfunction, cardiomyopathy (which may be fatal), and endocrinopathy, including 
diabetes, hypogonadism, and infertility. All this contributes to a clear unmet medical need for the 
regularly transfused population of patients with PK deficiency.  
Treatment with AG-348 has the potential to correct the underlying pathology of PK deficiency 
by activating PKR and increasing glycolytic pathway activity in RBCs to reduce hemolysis and 
provide clinical benefit to subjects with PK deficiency. This clinical study will determine 
whether AG-348 provides benefit to the regularly transfused patient population by decreasing 
their need for regular transfusions. 
5.3.1. 
Justification of Study Design 
Given the rarity of patients with PK deficiency in general and the extreme rarity of regularly 
transfused patients with PK deficiency (less than 20% of the non-Amish adult population with 
PK deficiency according to the NHS), it is not possible to conduct an adequately powered 
randomized controlled study. Therefore, Study AG348-C-007 has a single-arm design in which 
each subject’s own historical transfusion data will be used as their individual control. Efficacy 
will be measured by the change in transfusion burden when subjects are on an individually 
optimized dose of AG-348 compared with each subject’s historical transfusion burden. Subjects 
will be treated on study for up to 40 weeks, including up to 16 weeks for dose optimization and 
24 weeks on their individual optimized dose. This length of time was selected in order to have a 
reasonable estimate of the transfusion frequency on a stable dose of AG-348 in subjects whose 
historical transfusion frequency could range from every 3 weeks to every 8 weeks. Additional 
details on the study design are provided in Section 7.1. See Section 5.3.1.1, Section 5.3.1.2, and 
Section 5.3.1.3 for further justification regarding specific aspects of the study design. 
5.3.1.1. 
Justification Related to Sample Size 
As previously mentioned (Section 5.3.1), it is estimated that less than 20% of the non-Amish 
adult population with PK deficiency is regularly transfused. A feasibility assessment and 
outreach effort to evaluate the accessibility of a subject population meeting the criteria for this 
proposed Phase 3 study have thus far identified 36 potential subjects in 22 sites within 
13 countries. Although there is uncertainty with the screen failure rate, a number of these 
patients may be ineligible for the trial, for a variety of reasons. 
5.3.1.2. 
Justification of Using Individual Transfusion History Data as Comparator to 
Assess Efficacy of AG-348 
Each subject’s own historical transfusion data will be used as his/her individual control to assess 
efficacy (ie, reduction in transfusion need while the subject is receiving AG-348). Using 


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historical controls rather than a placebo control group is an established method in rare diseases 
(Moscicki and Tandon, 2017). More specifically, individual patients’ transfusion histories have 
been used as a comparator in multiple studies conducted in frequently transfused patients with 
various chronic anemias (eg, beta-thalassemia, paroxysmal nocturnal hemoglobinuria). It is 
especially valuable in heterogeneous patient populations, such as regularly transfused patients 
with PK deficiency where the absence of transfusion guidelines translates to patients with 
apparently similar degrees of anemia potentially being treated differently. 
5.3.1.3. 
Justification of Duration for Collection of Historical Data and Duration on 
Optimized Dose 
This study includes a collection of 52 weeks of historical transfusion data prior to screening. The 
duration of 52 weeks was selected in order to limit the impact of any ad hoc transfusion 
administered for an acute hemolytic episode on the estimation of each subject’s basal transfusion 
need.  
The subjects enrolled into this study will have a transfusion frequency at a minimum of around 
every 8 weeks. Therefore, a 16-week duration for dose optimization followed by a 24-week 
duration on a stable dose should ensure an appropriate estimate of the transfusion rate while 
subjects are receiving AG-348. 
5.3.2. 
Rationale for Dose 
To assist with dose selection, an exposure-response analysis was conducted using 
pharmacokinetic, efficacy, and safety data from the DRIVE-PK study conducted in subjects who 
do not require regular transfusions. Briefly, a sequential population pharmacokinetic-efficacy 
model was developed using increase in Hb as the efficacy endpoint, while a binary logistic 
regression approach incorporating the safety endpoints of alanine aminotransferase (ALT), 
aspartate aminotransferase (AST), total and free testosterone, estrone, estradiol, insomnia, and 
hot flush was used for the analysis of exposure-safety relationship. Insomnia was the only safety 
event that was found to be significant in the logistic regression analysis.  
Following model development, simulations were conducted to select 3 dose levels for evaluation 
in Phase 3 studies:  
1. The low dose level (5 mg BID) was identified as a dose at which patients were likely to 
have an Hb increase of ≥1.5 g/dL (0.93 mmol/L) from baseline without exceeding the 
upper limit of normal (ULN) Hb (efficacy criterion), with a minimal probability of 
occurrence of Grade ≥1 insomnia (safety criterion).  
2. Since the mid dose (20 mg BID) involves an intra-patient dose level increase, this dose 
level was selected such that it would result in a reasonable increase (approximately 2- to 
2.5-fold) in exposure compared to the predicted exposure at the low dose level. 
3. Since the high dose (50 mg BID) also involves an intra-patient dose level increase, this 
dose level was selected such that it would result in a reasonable increase (approximately 
2- to 2.5-fold) in exposure compared to the predicted exposure at the mid dose level. 
Using these criteria and simulations from the population pharmacokinetic-efficacy-safety 
analyses, doses of 5 mg BID, 20 mg BID, and 50 mg BID were selected for the Dose 
Optimization Period in Study AG348-C-006, which will enroll subjects with PK deficiency who 


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are not regularly transfused. Patients who are regularly transfused and those who do not need 
regular transfusions represent the same disease caused by the same genetic deficiency in the 
PKLR gene; therefore, the same doses have been selected for this current study. 
5.3.2.1. 
Justification of Individual Dose Optimization 
Preliminary results of the DRIVE-PK study indicate that multiple subjects had to have their 
randomized dose level reduced because of excess increases in Hb or the occurrence of adverse 
events (AEs) such as insomnia, headache, or nausea. Dose reductions led to the resolutions of 
most of these AEs and the maintenance of a satisfactory but not excessive level of Hb where 
applicable.  
Individual dose optimization is incorporated in this study to allow each subject to gradually 
increase his/her dose of AG-348 in order to identify a dose that confers maximum benefit with 
minimum risk to that subject. For this reason, all subjects will start at a low dose level (5 mg 
BID) with 2 sequential steps for dose level increases (ie, from 5 to 20 mg BID and from 20 to 
50 mg BID), depending on safety (see Section 7.1.3 for dose optimization guidelines). 


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6. 
TRIAL OBJECTIVES AND ENDPOINTS 
6.1. 
Objectives 
6.1.1. 
Primary Objective 
The primary objective of the study is to evaluate the efficacy of treatment with AG-348, as 
assessed by the reduction in transfusion burden. 
6.1.2. 
Secondary Objectives 
The secondary objective of this study is to evaluate the safety of treatment with AG-348.  
6.1.3. 
Exploratory Objectives 
The exploratory objectives of the study are the following: 
• To determine the effect of AG-348 on markers of hemolysis, erythropoietic activity, 
and other indicators of clinical activity 
• To determine the effect of AG-348 on markers of iron metabolism and indicators of 
iron overload 
• To determine the effect of AG-348 on health-related quality of life (HRQoL), as 
determined using patient-reported outcomes (PROs) 
• To evaluate the pharmacokinetics of AG-348 after oral administration 
• To evaluate the relationship of AG-348 pharmacokinetics to indicators of clinical 
activity 
• To evaluate the change in the levels of PKR protein in whole blood 
6.2. 
Endpoints 
6.2.1. 
Primary Endpoint 
The primary endpoint of this study is the proportion of subjects who achieve a reduction in 
transfusion burden, defined as a ≥33% reduction in the number of RBC units transfused during 
the 24 weeks of Part 2 compared with the historical transfusion burden standardized to 24 weeks 
(Standardized Control Period). 
6.2.2. 
Secondary Endpoints 
The secondary endpoints of the study are the following:  
• Annualized total number of RBC units transfused during the study (both Part 1 and 
Part 2) compared with the historical transfusion burden 
• Number of transfusion episodes during Part 2 compared with the Standardized 
Control Period  
• Proportion of subjects who become transfusion-free, defined as 0 transfusions 
administered during Part 2 


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• Proportion of subjects who achieve Hb concentrations in the normal range at least 
once, 8 weeks or more after a transfusion in Part 2 
• Safety endpoints, including the type, incidence, severity, and relationship to treatment 
of AEs and serious adverse events (SAEs), and AEs leading to treatment dose 
reduction, treatment interruption, and treatment discontinuation 
• Laboratory tests over time (eg, serum chemistry, liver function tests [LFTs], 
hematology, coagulation, lipids, sex steroids, urinalysis), physical examination (PE) 
findings, dual-energy X-ray absorptiometry (DXA) scans (hip and lumbar spine), 
vital signs, and 12-lead electrocardiogram (ECG) data  
6.2.3. 
Exploratory Endpoints 
The exploratory endpoints of the study are the following: 
• Change from baseline in the following markers of hemolysis: bilirubin, lactate 
dehydrogenase (LDH), and haptoglobin levels 
• Change from baseline in markers of erythropoietic activity 
• Change from baseline in markers of iron metabolism and indicators of iron overload 
• Change from baseline over time in HRQoL scores (ie, Pyruvate Kinase Deficiency 
Impact Assessment [PKDIA], Pyruvate Kinase Deficiency Diary [PKDD], 
EuroQol-5D-5L [EQ-5D-5L]) 
• Characterization of pharmacokinetic profile (drug concentrations over time) and 
determination of pharmacokinetic parameters of AG-348 (eg, AUC, Cmax, and others 
as applicable) in Part 2  
• Exposure-response (or pharmacokinetic-PD) relationship between relevant 
pharmacokinetic parameters and endpoints that are indicators of clinical activity 
• Change from baseline in PKR protein levels 


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7. 
INVESTIGATIONAL PLAN 
7.1. 
Study Design 
7.1.1. 
Overview of Study Design 
This is a 2-part, multicenter, open-label, Phase 3 study consisting of a Dose Optimization Period 
(Part 1) followed by a Fixed-Dose Period (Part 2), as shown in Figure 2. This study will evaluate 
the efficacy and safety of treatment with AG-348 in a minimum of 20, with up to 40, adult 
subjects with PK deficiency who are regularly receiving blood transfusions.  
Prior to Part 1 of the study, there will be an 8-week Screening Period in which a subject’s 
complete transfusion history from 52 weeks prior to signing the Informed Consent Form (ICF) 
will be documented. This information, taken from the subject’s medical record and entered into 
the electronic case report form (eCRF), will be used to inform study eligibility and analyze data. 
All subjects will receive AG-348 in Parts 1 and 2 of the study. During Part 1 of the study, all 
subjects will start on a dose of 5 mg AG-348 administered BID. Over the course of Part 1 of the 
study each subject will undergo intrasubject dose optimization per Section 7.1.3. Each subject’s 
dose level of AG-348 may be increased 2 times beyond the starting dose of 5 mg BID (ie, from 5 
to 20 mg BID and from 20 to 50 mg BID). In the Fixed-Dose Period of the study (Part 2), a 
subject will receive AG-348 at his/her optimized dose with no planned adjustments (ie, as a fixed 
dose) for a fixed period of 24 weeks. 
All subjects who remain on study during Part 2 through the Week 24 Visit may be eligible for an 
open-label extension study with AG-348. Subjects who continue on into an extension study will 
not be required to attend the Follow-up Visit. Subjects who continue the study through the 
Part 2 Week 24 Visit on study drug but do not continue on into an extension study will attend the 
Follow-up Visit 28±4 days after the last dose of study drug. 
Subjects who discontinue the study prior to the Part 2 Week 24 Visit should attend the End of 
Study Visit 28±4 days after the last study visit that the subject attended or 28±4 days after the 
last dose of study drug, whichever is later.  
All subjects who discontinue study drug at any time during the study should undergo a dose taper 
as described in Section 9.3, unless an emergency situation justifies discontinuing the study drug 
abruptly. Whether the dose taper is performed or not, subjects discontinuing AG-348 should be 
monitored for signs of hemolysis and worsening of anemia. 
The maximum total duration that a subject can receive AG-348 in this study is 40 weeks (not 
including the dose taper). 
The Screening Period, Part 1, Part 2, and follow-up of the study are described in more detail in 
Section 7.1.2, Section 7.1.3, Section 7.1.4, and Section 7.1.5, respectively. Details for dose 
reductions or interruptions required for reasons of safety are in Section 9.3. 


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Figure 2: 
Overview of Design for Study AG348-C-007 
 
Abbreviation: BID = twice daily. 
1 Screening may be extended beyond 8 weeks if there is a delay in obtaining a subject’s complete transfusion history 
or to ensure that the first dose of study drug can be administered 2-7 days after the most recent transfusion, upon 
approval by the Medical Monitor (or designee).  
7.1.2. 
Screening Period 
Screening will occur over an 8-week period after a subject provides written informed consent. 
During the Screening Period, each subject’s transfusion history in the past 52 weeks will be 
collected. The Investigator will determine whether each subject meets all of the inclusion criteria 
and none of the exclusion criteria. Eligibility of each subject will be confirmed by the Medical 
Monitor (or designee). A subject will not be considered enrolled (and cannot receive his/her first 
dose of study drug) until eligibility is confirmed. 
7.1.2.1. 
Transfusion History 
As described in Section 5.3.1.2, an established method of obtaining historical control data will be 
used to collect subjects’ transfusion history at Screening. For each subject, the following 
transfusion history information for the 52 weeks prior to signing the ICF will be collected and 
recorded on the source documentation and eCRF: 
• Dates of all transfusions  
• Pre-transfusion Hb concentrations (Hb concentrations within 1 week prior to each 
transfusion episode) for at least 80% of the transfusions  
• For each transfusion, number of blood units transfused  
• When available, hematocrit (HCT) and volume of transfused blood units 
The transfusion history information will be used as follows: 
• To determine whether the subject meets the study criteria for regular transfusion 
frequency (see Inclusion Criterion #4 [Section 8.1] and Exclusion Criterion #3 
[Section 8.2])  
• To calculate the Mean Transfusion Frequency (MTF)  


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• To calculate the Individual Transfusion Trigger (TT), which is the mean (±0.5 g/dL 
or ±0.31 mmol/L) of a subject’s collected historical pretransfusion Hb concentrations. 
For each transfusion, only the Hb concentration within 1 week prior to and closest to 
a transfusion will be included in this calculation.  
• To calculate the Mean Number of Blood Units (MNU) transfused per transfusion 
• To function as historical control data to be used in the analysis 
The MTF (transfusion every X week[s]) will be calculated by dividing 52 weeks by the total 
number of transfusions received in the 52-week period prior to informed consent. For example, a 
subject who received 13 transfusions in the 52-week period would have an MTF of every 
4 weeks (52 weeks/13 transfusions = transfusion every 4 weeks). Fractional numbers will be 
rounded to the closest integer (eg, 52 weeks/17 transfusions = 3.06 weeks rounded to every 
3 weeks). All transfusions will be included in this calculation, and no attempt will be made to 
distinguish transfusions administered for cause (eg, acute infection) and transfusions 
administered systematically. 
The Individual TT will be the sum of the Hb concentrations within 1 week prior to and closest to 
each transfusion (when available) for the transfusions in the 52-week period prior to informed 
consent divided by the number of such available Hb concentrations during that period, calculated 
to the first decimal point, if expressed in g/dL, or to the second decimal point, if expressed in 
mmol/L. A margin of ±0.5 g/dL (or ±0.31 mmol/L) will be attached to this value. For example, a 
subject who had 7 pre-transfusion Hb concentrations in the 52-week period prior to informed 
consent would have an Individual TT of 7.0±0.5 g/dL based on the following pre-transfusion Hb 
concentrations (g/dL): 7.0, 7.2, 6.8, 6.8, 6.7, 7.2, and 7.1. 
The MNU will be the sum of the number of units transfused at each transfusion during the 
52-week period prior to informed consent divided by the number of transfusions during this 
period. Fractional numbers will be rounded to the closest integer 
(eg, 13 units/6 transfusions = 2.2 units rounded to 2 units; 25 units/10 transfusions = 2.5 units 
rounded to 3 units).  
A subject’s Screening Period duration may be extended beyond 8 weeks upon the Medical 
Monitor’s (or designee’s) approval if there is a delay in obtaining a subject’s complete 
transfusion history, or the subject is projected to need a transfusion within 1 week of his/her 
scheduled first dose to ensure that treatment with AG-348 is always started 2-7 days after a 
subject's most recent transfusion in the Screening Period (referred to as Transfusion 0). 
7.1.3. 
Part 1: Dose Optimization Period 
The goal of the Dose Optimization Period is to maximize a subject’s increase in Hb while 
maintaining an acceptable safety profile. All subjects will receive an initial dose of 5 mg BID of 
AG-348, with 2 sequential steps for dose level increase (ie, from 5 to 20 mg BID and from 20 to 
50 mg BID; no increases beyond 50 mg BID will be allowed).  
The first dose of study drug on Day 1 should be taken at the study site following all Day 1 
assessments (with the exception of eDiary assessments) as depicted in the Schedule of 
Assessments (Section 10.14.1, Table 4).  


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Subjects will be assessed for safety and efficacy during Part 1 to determine if their dose should 
be increased, maintained at the current level, or decreased. The Investigator will be expected to 
be vigilant about monitoring each subject’s Hb concentrations throughout the study in order to 
evaluate the need for a transfusion (by comparing the subject’s Hb concentration with his/her 
Individual TT). Investigators will assess the overall status of the subject and the subject’s 
tolerability to make a decision on study drug dose. The following rules apply: 
• At the Week 4 Visit, study drug dose should be increased to the next dose level (ie, 
from 5 mg BID to 20 mg BID) if the subject has not experienced any study drug-
related safety issues.  
• The dose should be increased either at the Week 10 Visit if the subject has reached 
his/her Individual TT (and has been transfused) between Week 8 and Week 10, or 
when the subject reaches the Week 12 Visit (without having reached his/her 
Individual TT between Week 8 and Week 12). In either case, the study drug dose 
should be increased to the next dose level if the subject has not experienced any study 
drug-related safety issues. 
If the dose was not increased at the Week 4 Visit, and the Investigator is considering increasing 
the dose at a subsequent visit, the Investigator should contact the Medical Monitor (or designee) 
for guidance. 
The study drug dose should not be increased at any of these visits if Hb concentration from the 
previous visits (per the central laboratory analysis) is ≥14.5 g/dL (9.0 mmol/L) in males or 
≥13.0 g/dL (8.07 mmol/L) in females. 
If the Investigator deems it necessary to reduce the study drug dose for safety reasons, the 
subject’s dose may be reduced to 1 of the 2 available lower dose levels (ie, 5 mg BID or 20 mg 
BID). If the subject is already receiving 5 mg BID and/or cannot tolerate BID dosing, another 
regimen may be allowed after discussion with, and approval by, the Medical Monitor (or 
designee). 
Dose escalation or re-introduction should be avoided after the Week 12 Visit but may be 
permitted after discussion with the Medical Monitor (or designee) for subjects who had their 
dose reduced or suspended for reasons related to safety.  
If questions arise about whether the dose level of a given subject should be increased, 
maintained, or decreased, the site is advised to contact the Medical Monitor (or designee) for 
discussion. At any time during the study, the Investigator can reduce the subject’s dose or 
interrupt study drug for reasons related to safety (as described in Section 9.3). 
Assessments and timing details for Part 1 are outlined in the Schedule of Assessments (Table 4).  
7.1.3.1. 
Transfusions During Part 1 
The scheduled hematology assessments in Part 1 will be used to determine whether a subject has 
reached his/her Individual TT, in which case the subject should be transfused with his/her MNU 
of packed RBCs.  
Subjects, in particular those with a historical MTF of every 3 or 4 weeks, may need additional 
hematology assessments (Hb concentration) if the subject’s Individual TT has not been reached 
at a scheduled visit. Blood sampling for these additional assessments (unscheduled visits) should 


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be analyzed by the central laboratory but, in extenuating situations, may be conducted and 
analyzed by a local laboratory or physician’s office local to the subject, as feasible and allowed 
by Investigators and local regulations. 
All on-study transfusion data will be recorded as noted in Section 9.4. 
7.1.4. 
Part 2: Fixed-Dose Period 
In Part 2 of the study, subjects will receive their optimized dose of AG-348 for 24 weeks. Dose 
escalation or re-introduction should be avoided during Part 2 but may be permitted after 
discussion with the Medical Monitor (or designee) for subjects who had their dose reduced or 
suspended for reasons related to safety.  
Assessments and timing details for Part 2 are outlined in the Schedule of Assessments (Table 5). 
7.1.4.1. 
Transfusions During Part 2 
Subjects should continue to be transfused when their Hb concentration decreases to their 
Individual TT. Some subjects may need additional hematology assessments (Hb concentration), 
for example, if the subject’s Individual TT has not been reached at a scheduled visit. Blood 
sampling for these additional assessments (unscheduled visits) should be analyzed by the central 
laboratory but, in extenuating situations, may be conducted and analyzed by a local laboratory or 
physician’s office local to the subject, as feasible and allowed by Investigators and local 
regulations. 
All on-study transfusion data will be recorded as noted in Section 9.4. 
7.1.5. 
Discontinuation and Follow-up 
Subjects who discontinue the study prior to the Part 2 Week 24 Visit should attend the End of 
Study Visit 28±4 days after the last study visit that the subject attended or 28±4 days after the 
last dose of study drug, whichever is later. This visit will be identical to the Part 2 Week 24 Visit 
with these exceptions: no study drug will be dispensed at the End of Study Visit, blood sampling 
for pharmacokinetic profile will not be drawn, PKR protein will not be assessed, and if the 
subject has not already done so, they should return their eDiary and study drug. 
Subjects who continue study drug through Part 2 Week 24 should continue taking study drug at 
least through the morning dose of the Part 2 Week 24 Visit. All subjects who remain on study 
during Part 2 through the Week 24 Visit may be eligible for an open-label extension study with 
AG-348. 
• Subjects who continue study drug through the Part 2 Week 24 Visit but do not 
continue on into an extension study should undergo a dose taper and then attend the 
Follow-up Visit 28±4 days after the last dose of study drug. 
• Subjects who continue on into an extension study will not be required to attend the 
Follow-up Visit.  
• Subjects who initiate or are undergoing a dose taper for a reason related to safety and 
for whom participation in an extension study remains undetermined should perform 
the taper as detailed in Section 9.3. Transition to an extension study should then be 
discussed with the Medical Monitor (or designee). 


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All subjects who discontinue study drug at any time during the study should undergo a dose taper 
(see Section 9.3), unless an emergency situation justifies discontinuing the study drug abruptly. 
Guidelines for the follow-up of subjects with any AE at the subject’s completion of the study are 
described in Section 11. 
7.2. 
Number of Subjects 
A minimum of 20, with up to 40, subjects are planned for enrollment.  
7.3. 
Criteria for Study Termination 
This study may be prematurely terminated if, in the opinion of the Sponsor, there is sufficient 
reasonable cause. In the event of such action, written notification documenting the reason for 
study termination will be provided to each Investigator. 
Circumstances that may warrant termination include, but are not limited to, the following: 
• Determination of unexpected, significant, or unacceptable risk to subjects 
• Plans to modify, suspend, or discontinue the development of the study drug 
• Decisions of competent authorities or Institutional Review Board (IRB)/Independent 
Ethics Committee (IEC) 
• Other administrative reasons 
Should the study be closed prematurely, all study materials must be returned to the Sponsor or 
the Sponsor’s designee.  


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8. 
SELECTION AND WITHDRAWAL OF SUBJECTS 
8.1. 
Inclusion Criteria 
For enrollment into this study, subjects must meet all of the following criteria during the 
Screening Period: 
1. Have provided signed written informed consent prior to performing any study procedure, 
including screening procedures. 
2. Be aged 18 years or older. 
3. Have documented clinical laboratory confirmation of PK deficiency, defined as 
documented presence of at least 2 mutant alleles in the PKLR gene, of which at least 1 is 
a missense mutation, as determined per the genotyping performed by the study central 
genotyping laboratory.  
4. Have a history of a minimum of 6 transfusion episodes in the 52-week period prior to 
date of informed consent as documented in the transfusion history of the subject, which 
reflects the subject’s typical transfusion burden. 
5. Have complete records of transfusion history, defined as having the following available 
for the 52 weeks prior to the date of informed consent: (1) all the transfusion dates, (2) 
the number of blood units transfused for all the transfusions, and (3) Hb concentrations 
within 1 week prior to transfusion for at least 80% of the transfusions. 
6. Have received at least 0.8 mg oral folic acid daily for at least 21 days prior to the first 
dose of study drug, to be continued daily during study participation.  
7. Have adequate organ function, as defined by: 
a. Serum AST ≤2.5 × ULN (unless the increased AST is assessed by the Investigator as 
due to hemolysis and/or hepatic iron deposition) and ALT ≤2.5 × ULN (unless the 
increased ALT is assessed by the Investigator as due to hepatic iron deposition). 
b. Normal or elevated levels of serum bilirubin. In subjects with serum bilirubin >ULN, 
the elevation must not be associated with choledocholithiasis, cholecystitis, biliary 
obstruction, or hepatocellular disease. Elevated bilirubin attributed to hemolysis with 
or without Gilbert’s syndrome is not exclusionary. 
c. Estimated glomerular filtration rate (GFR) ≥60 mL/min/1.73 m2, measured GFR 
≥60 mL/min, or calculated creatinine clearance (CrCL; Cockcroft-Gault) 
≥60 mL/min. 
d. Absolute neutrophil count (ANC) ≥1.0 × 109/L. 
e. Platelet count ≥100 × 109/L, in the absence of a spleen, or platelet count ≥50 × 109/L, 
in the presence of a spleen and in the absence of any other cause of 
thrombocytopenia. 
f. Activated partial thromboplastin time (aPTT) and international normalized ratio 
(INR) ≤1.25 × ULN, unless the subject is receiving therapeutic anticoagulants. 
8. For women of reproductive potential, have a negative serum pregnancy test during the 
Screening Period. Women of reproductive potential are defined as sexually mature 
women who have not undergone a hysterectomy, bilateral oophorectomy, or tubal 
occlusion or who have not been naturally postmenopausal (ie, who have not menstruated 


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at all for at least the preceding 12 months prior to signing informed consent and have an 
elevated follicle-stimulating hormone (FSH) level indicative of menopause during the 
Screening Period). 
9. For women of reproductive potential as well as men with partners who are women of 
reproductive potential, be abstinent as part of their usual lifestyle, or agree to use 2 forms 
of contraception, 1 of which must be considered highly effective, from the time of giving 
informed consent, during the study, and for 28 days following the last dose of study drug 
for women and 90 days following the last dose of study drug for men. A highly effective 
form of contraception is defined as combined (estrogen and progestin containing) 
hormonal contraceptives (oral, intravaginal, or transdermal) known to be associated with 
inhibition of ovulation; progestin-only hormonal contraceptives (oral, injectable, or 
implantable) known to be associated with inhibition of ovulation; intrauterine device; 
intrauterine hormone releasing system; bilateral tube occlusion; or vasectomized partner. 
The second form of contraception can include an acceptable barrier method, which 
includes male or female condoms with or without spermicide, and cervical cap, 
diaphragm, or sponge with spermicide. Women of reproductive potential using hormonal 
contraception as a highly effective form of contraception must also utilize an acceptable 
barrier method while enrolled in the study and for at least 28 days after their last dose of 
study drug. 
10. Be willing to comply with all study procedures, in particular the Individual TT 
(calculated based on 52 weeks of transfusion history), for the duration of the study. 
8.2. 
Exclusion Criteria 
Subjects who meet any of the following criteria during Screening will not be enrolled in the 
study: 
1. Are homozygous for the R479H mutation or have 2 non-missense mutations, without the 
presence of another missense mutation, in the PKLR gene as determined per the 
genotyping performed by the study central genotyping laboratory.  
2. Have a significant medical condition that confers an unacceptable risk to participating in 
the study and/or that could confound the interpretation of the study data. Such significant 
medical conditions include, but are not limited to, the following: 
a. Poorly controlled hypertension (defined as systolic blood pressure [BP] >150 mmHg 
or diastolic BP >90 mmHg) refractory to medical management.  
b. History of recent (within 6 months prior to signing informed consent) congestive 
heart failure; myocardial infarction or unstable angina pectoris; hemorrhagic, 
embolic, or thrombotic stroke; deep venous thrombosis; or pulmonary or arterial 
embolism. 
c. Cardiac dysrhythmias judged as clinically significant by the Investigator. 
d. Heart-rate corrected QT interval-Fridericia’s method (QTcF) >450 msec with the 
exception of subjects with right or left bundle branch block. 
e. Clinically symptomatic cholelithiasis or cholecystitis. Prior cholecystectomy is not 
exclusionary. Subjects with symptomatic cholelithiasis or cholecystitis may be 
rescreened once the disorder has been treated and clinical symptoms have resolved. 


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f. History of drug-induced cholestatic hepatitis. 
g. Iron overload sufficiently severe to result in a clinical diagnosis by the Investigator of 
cardiac (eg, clinically significant impaired left ventricular ejection fraction), hepatic 
(eg, fibrosis, cirrhosis), or pancreatic (eg, diabetes) dysfunction. 
h. Diagnosis of any other congenital or acquired blood disorder or any other hemolytic 
process, except mild allo-immunization, as a consequence of transfusion therapy. 
Genetic findings that in isolation are predicted to be insufficient to explain the 
observed clinical phenotype may be allowed (eg, heterozygous status for certain 
recessive RBC disorders). 
i. Positive test for HBsAg or hepatitis C virus (HCV) antibody (Ab) with signs of active 
hepatitis B or C virus infection. If the subject is positive for HCVAb, a reverse 
transcriptase-polymerase chain reaction test will be conducted. Subjects with 
hepatitis C may be rescreened after receiving appropriate hepatitis C treatment. 
j. Positive test for HIV-1 or -2 Ab. 
k. Active infection requiring the use of parenteral antimicrobial agents or Grade ≥3 in 
severity (per NCI CTCAE [National Cancer Institute Common Terminology Criteria 
for Adverse Events]) within 2 months prior to the first dose of study drug. 
l. Diabetes mellitus judged to be under poor control by the Investigator or requiring 
>3 antidiabetic agents, including insulin (all insulins are considered 1 agent); use of 
insulin per se is not exclusionary.  
m. History of any primary malignancy, with the exception of curatively treated 
nonmelanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; 
or other primary tumor treated with curative intent, no known active disease present, 
and no treatment administered during the last 3 years. 
n. Unstable extramedullary hematopoiesis that could pose a risk of imminent neurologic 
compromise. 
o. Current or recent history of psychiatric disorder that, in the opinion of the Investigator 
or Medical Monitor (or designee), could compromise the ability of the subject to 
cooperate with study visits and procedures. 
3. Have a history of transfusions occurring on average more frequently than once every 
3 weeks during the 52 weeks prior to signing informed consent.  
4. Have a splenectomy scheduled during the study drug period or have undergone 
splenectomy within 12 months prior to signing informed consent. 
5. Are currently enrolled in another therapeutic clinical trial involving ongoing therapy with 
any investigational or marketed product or placebo. Prior and subsequent participation in 
the PK Deficiency NHS (NCT02053480) or PK Deficiency Registry is permitted, 
however, concurrent participation is not. Therefore, subjects enrolling in this current 
study will be expected to temporarily suspend participation in the NHS or Registry. 
6. Have exposure to any investigational drug, device, or procedure within 3 months prior to 
the first dose of study drug.  
7. Have a prior bone marrow or stem cell transplant. 
8. Are currently pregnant or breastfeeding. 


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9. Have a history of major surgery within 6 months of signing informed consent. Note that 
procedures such as laparoscopic gallbladder surgery are not considered major in this 
context. 
10. Are currently receiving medications that are strong inhibitors of CYP3A4, strong 
inducers of CYP3A4, strong inhibitors of P-glycoprotein (P-gp), or digoxin (a P-gp 
sensitive substrate medication) that have not been stopped for a duration of at least 5 days 
or a timeframe equivalent to 5 half-lives (whichever is longer) prior to the first dose of 
study drug. 
11. Are currently receiving hematopoietic stimulating agents (eg, erythropoietins [EPOs], 
granulocyte colony stimulating factors, thrombopoietins) that have not been stopped for a 
duration of at least 28 days prior to the first dose of study drug. 
12. Have a history of allergy to sulfonamides if characterized by acute hemolytic anemia, 
drug-induced liver injury, anaphylaxis, rash of erythema multiforme type or 
Stevens-Johnson syndrome, cholestatic hepatitis, or other serious clinical manifestations. 
13. Have a history of allergy to AG-348 or its excipients (microcrystalline cellulose, 
croscarmellose sodium, sodium stearyl fumarate, and mannitol). 
14. Are currently receiving anabolic steroids, including testosterone preparations, within 
28 days prior to the first dose of study drug. 
8.3. 
Subject Withdrawal Criteria 
Subjects may withdraw from the study at any time for any reason. Subjects will be withdrawn 
from study drug and study-related procedures under the following conditions: 
• Withdrawal of consent 
• Development of an intercurrent medical condition that precludes further participation 
in the study 
• Subject requires use of a prohibited concomitant medication (Section 9.5.1) 
• Investigator decision 
• Persistent nonadherence to protocol requirements 
• Pregnancy 
• Lost to follow-up 
Subjects who discontinue treatment will be encouraged to return for subsequent scheduled visits 
(or at a minimum the End of Study Visit) unless they withdraw consent. Should a subject decide 
to withdraw consent, all efforts will be made to complete and report the protocol-defined study 
observations up to the time of the subject’s withdrawal as completely as possible and to 
determine the reason for withdrawal. 
In the event a subject is withdrawn from the study drug or the study, the Medical Monitor (or 
designee) must be informed.  


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When a subject withdraws from the study drug or withdraws from the study, the primary reason 
for treatment discontinuation or study discontinuation must be recorded in the appropriate 
section of the eCRF. 
Refer to Section 11 for details regarding the follow-up of AEs ongoing at the time a subject 
discontinues treatment.  
8.4. 
Subject Replacement 
Subjects who discontinue the study for reasons other than AEs may be replaced at the Sponsor’s 
discretion. 
8.5. 
End of Study 
End of Study is defined as the time at which all subjects have completed the study or are lost to 
follow-up. 


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9. 
TREATMENT OF SUBJECTS 
9.1. 
Description of Study Drug 
9.1.1. 
Study Drug 
AG-348 will be supplied as 5, 20, and 50 mg strength tablets to be administered orally. Please 
see the IB for further details regarding AG-348. AG-348 is provided for investigational use only 
(considered an investigational medicinal product [IMP]) and is to be used only within the context 
of this study. All study drug product will be supplied by the Sponsor. 
9.1.2. 
Study Drug Packaging and Labeling 
AG-348 will be supplied in appropriate containers with child-resistant closures and will be 
labeled appropriately as IMP for this study. Packaging and labeling will be prepared to meet all 
regulatory requirements. 
This is an open-label study; there are no additional requirements regarding the physical aspect of 
blinding related to packaging and labeling. 
9.1.3. 
Study Drug Storage 
AG-348 tablets must be stored according to the respective package label. All study drug products 
must be stored in a secure, limited-access location and may be dispensed only by the Investigator 
or by a member of the staff specifically authorized by the Investigator.  
9.1.4. 
Study Drug Administration 
AG-348 tablets are to be taken orally and swallowed whole with water. The tablets are not to be 
crushed, chewed, or dissolved in water. Doses of study drug may be taken with or without food. 
Subjects will take 1 tablet BID, approximately 12 hours apart (ie, 12 hours±2 hours). Subjects 
will be instructed to complete a dosing diary in the eDiary dispensed during Screening. 
If a subject misses a scheduled dose by 4 hours or less, he or she should still take that dose. If a 
subject misses a scheduled dose by more than 4 hours, he or she should skip that dose. If a dose 
is skipped, the next dose should then be taken approximately 24 hours from the previous dose. If 
a subject experiences a dose interruption for non-safety reasons, they should be re-started on the 
prescribed dose as soon as possible. 
Subjects should be advised not to abruptly interrupt or discontinue dosing without first 
speaking with the treating Investigator except in case of medical emergency; abrupt 
interruption or discontinuation of AG-348 may result in withdrawal hemolysis. If a subject 
needs to interrupt or permanently discontinue study drug at any time during the study, 
guidance is provided in Section 9.3 and Section 7.1.5. 
9.1.5. 
Study Drug Accountability 
Accountability for the study drug at the clinical facility is the responsibility of the Investigator. 
The Investigator will ensure that the study drug is used only in accordance with this protocol. 
Where allowed, the Investigator may choose to assign drug accountability responsibilities to a 
pharmacist or other appropriate individual. 


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The Investigator or delegate will maintain accurate drug accountability records indicating the 
drug’s delivery date to the site, inventory at the site, use by each subject, and return to the 
Sponsor or the Sponsor’s designee (or disposal of the drug, if approved by the Sponsor). These 
records will adequately document that the subjects were provided the doses as specified in the 
protocol and should reconcile all study drug received from the Sponsor. Accountability records 
will include dates, quantities, batch/serial numbers, expiration dates (if applicable), and subject 
numbers. The Site Monitor will review drug accountability at the site on a schedule agreed to by 
the Sponsor. 
Study drug must not be used for any purpose other than the present study.  
All unused and used study drug will be retained at the site until it is inventoried by the Site 
Monitor. All used, unused, or expired study drug will be returned to the Sponsor or the Sponsor’s 
designee or, if authorized, disposed of at the study site per the site’s Standard Operating 
Procedures and documented. 
Study drug is expected to be dispensed to the subject at the study site. If necessary, in 
exceptional circumstances and with agreement of the Sponsor (or representative), study drug can 
be provided to the subject’s home, if acceptable by practice and allowed by local regulations. 
9.1.6. 
Study Drug Handling and Disposal 
All unused study drug must be properly disposed of in compliance with local procedures and 
governing regulations. Documentation of the method of destruction should be maintained in the 
Investigator’s files. 
9.2. 
Treatment Assignment 
All subjects enrolled in this study will receive AG-348. 
9.3. 
Criteria for Dose Reduction and Interruption of Study Drug for 
Safety Reasons 
It is important that a subject does not abruptly interrupt study drug due to the risk of 
withdrawal hemolysis, except in case of medical emergency. 
The Investigator will monitor all subjects for safety. A subject’s dose may be reduced 
(to 5 mg BID or 20 mg BID) or interrupted for safety reasons (ie, excessive Hb response or AEs 
considered related to study drug) as described below. Guidelines for dose modification for AEs 
considered related to study drug (except for events of excessive Hb response) are provided in 
Table 1. After careful consideration of the relative risk of maintaining the subject on the study 
drug versus withdrawal hemolysis when stopping study drug abruptly versus reducing the dose 
rapidly, the Investigator should determine if a gradual dose taper (GDT; Table 3) usually 
indicated for the management of nonacute AEs, a rapid dose taper (RDT; Table 2) usually 
indicated for the management of acute AEs or SAEs, or abrupt discontinuation of study drug 
usually indicated to receive emergency medical treatment is necessary. In all cases, study drug 
dose reductions, interruptions, or discontinuations should be discussed with the Medical Monitor 
(or designee). 


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For a subject with an excessive Hb response higher than 2 g/dL (1.24 mmol/L) below the ULN 
(ie, higher than 15.0 g/dL [9.31 mmol/L] in men and 13.5 g/dL [8.38 mmol/L] in women) up to 
the ULN (ie, 17.0 g/dL [10.55 mmol/L] in men and 15.5 g/dL [9.62 mmol/L] in women), a dose 
decrease should be considered for the subject to the next lower dose level, without a need for 
taper (ie, if the subject experienced an excessive Hb response at 50 mg BID, his/her dose will be 
decreased to 20 mg BID). The same rule applies to the decrease from 20 mg BID to 5 mg BID. 
For a subject with an excessive Hb response higher than the ULN, the event should be reported 
as an AE and the subject’s dose will be decreased to the next lower dose level without a need for 
taper (ie, if the subject experienced an excessive Hb response at 50 mg BID, his/her dose will be 
decreased to 20 mg BID). The same rule applies to the decrease from 20 mg BID to 5 mg BID. 
Table 1: 
Dose Modification for Adverse Events Considered Related to Study Drug 
(Except for Events of Excessive Hemoglobin Response), Study AG348-C-007  
Related Adverse 
Event(s) Severity 
Dose Modification 
Grade 1 
None required. 
Grade 2 
None required. Contact the Medical Monitor (or designee) to discuss specific 
cases that may need to be managed as Grade 3 events (see below). 
Grade 3 
After careful consideration of the relative risk of maintaining the subject on the 
study drug versus the risk of withdrawal hemolysis when stopping AG-348 
abruptly or reducing the dose rapidly, the Investigator should determine which 
of the following options is appropriate: 
• 
Maintaining the current dose, or 
• 
Performing a dose taper, or 
• 
Stopping the study drug abruptly 
If the decision is made to maintain the current dose of study drug, then no dose 
changes are required. At least once weekly monitoring should be performed 
until the event resolves to baseline or Grade 1 (whichever is lower). If the 
event persists, stopping the study drug abruptly or performing a dose taper 
should be considered. 
If the decision is made to stop the study drug abruptly or perform a dose taper, 
the below instructions should be followed for re-introduction or escalation of 
the study drug, respectively.  
In all cases, re-introduction and escalation of study drug should be 
performed only after discussion with the Medical Monitor (or designee).1 
• 
Restarting treatment after dosing was stopped:  
Once the event resolves to baseline or Grade 1 (whichever is lower) 
and the decision is made to restart AG-348, the study drug should be 
re-introduced at the 5 mg BID dose level. If the event does not reoccur 
after at least 4 weeks on 5 mg BID (with at least once weekly 
monitoring), the dose may be increased from 5 mg BID to 20 mg BID. 
If the event does not reoccur after at least 4 weeks on 20 mg BID (with 
at least once weekly monitoring), the dose may be increased from 
20 mg BID to 50 mg BID. 
• 
Events resolving during the dose taper (ie, the subject is still on study 


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Related Adverse 
Event(s) Severity 
Dose Modification 
drug):  
If during the dose taper, the event resolves to baseline or Grade 1 
(whichever is lower), the study drug should be maintained at the dose 
at which the event resolved for at least 4 weeks (with at least once 
weekly monitoring). If the event does not reoccur after at least 
4 weeks, then it should be considered to increase the dose to the next 
highest BID dose (5 mg BID, 20 mg BID, or 50 mg BID) with at least 
once weekly monitoring. If the event does not reoccur after at least 4 
weeks (with at least once weekly monitoring), and the subject is not 
already receiving 50 mg BID, then it should be considered to increase 
the dose to the next BID dose level. 
If the AE reoccurs at any point during the above scenarios, the subject should 
undergo a dose taper or stop AG-348 abruptly if necessitated by the risk of the 
AE. If the subject undergoes a dose taper and the AE resolves during the taper, 
study drug should be maintained at the next lowest BID dose below the dose at 
which the AE resolved. If the subject cannot tolerate BID dosing, another 
regimen may be allowed after discussion with, and approval by, the Medical 
Monitor (or designee). If the AE does not resolve to baseline or Grade 1 
(whichever is lower) after the dose is decreased, it should be considered to 
decrease the dose further. If the AE still does not resolve, dose should be 
permanently discontinued. 
Grade 4 
After careful consideration of the relative risk of withdrawal hemolysis when 
stopping AG-348 abruptly versus reducing the dose rapidly, the Investigator 
should determine which of the following options is appropriate: 
• 
Performing a rapid dose taper or 
• 
Stopping the study drug abruptly 
If the event resolves and the Investigator believes that restarting study drug is 
justified, the Medical Monitor (or designee) should be consulted before any 
further study drug is administered. 
Abbreviations: AE = adverse event; BID = twice daily; Hb = hemoglobin; RDT = rapid dose taper. 
1 The study drug dose should not be increased if Hb concentration (per the central laboratory analysis) is ≥14.5 g/dL 
(9.0 mmol/L) in males or ≥13.0 g/dL (8.07 mmol/L) in women. 
9.3.1. 
Rapid Dose Taper 
In subjects undergoing an RDT, the regimen as detailed in Table 2 will be followed. Subjects 
going through the RDT should be monitored for signs of hemolysis and worsening of anemia. 
Table 2: 
Rapid Dose Taper Regimen, Study AG348-C-007 
Starting Dose 
First Step 
×3 days 
Second Step 
×3 days 
Third Step 
×3 days 
5 mg BID 
5 mg QD 
5 mg QOD 
n/a 
20 mg BID 
20 mg QD 
20 mg QOD 
n/a 
50 mg BID 
50 mg QD 
20 mg QD 
20 mg QOD 


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Abbreviations: BID = twice daily; n/a = not applicable; QD = once daily; QOD = every other day. 
9.3.2. 
Gradual Dose Taper 
In subjects undergoing a GDT, the regimen detailed below and in Table 3 will be followed.  
For subjects on either 5 mg BID or 20 mg BID of study drug, the GDT will start with subjects 
reducing the frequency of dosing from their usual regimen of 1 tablet BID to 1 tablet QD for 
7 days, followed by 1 tablet QOD for 7 days prior to stopping study drug altogether (see Table 
3).  
For subjects on 50 mg BID, the GDT will start with subjects reducing the frequency of dosing 
from their usual regimen of 1 tablet BID to 1 tablet QD for 7 days; then they will switch to 
1 tablet of 20 mg QD for 7 days, followed by 1 tablet 20 mg QOD for 7 days prior to stopping 
study drug altogether (see Table 3). Dose tapers may be discussed with the Medical Monitor (or 
designee). 
Subjects going through a GDT should be monitored for signs of hemolysis and worsening of 
anemia. If a GDT is performed to discontinue dosing permanently, subjects may stop taking the 
study drug after the taper has been completed. 
Table 3: 
Gradual Dose Taper Regimen, Study AG348-C-007 
Starting Dose 
First Step 
×7 days 
Second Step 
×7 days 
Third Step 
×7 days 
5 mg BID 
5 mg QD 
5 mg QOD 
n/a 
20 mg BID 
20 mg QD 
20 mg QOD 
n/a 
50 mg BID 
50 mg QD 
20 mg QD  
20 mg QOD 
Abbreviations: BID = twice daily; n/a = not applicable; QD = once daily; QOD = every other day. 
9.4. 
Transfusions 
Transfusion history should be recorded as noted in Section 7.1.2.1.  
Transfusion(s) during the Screening Period will be administered according to the usual care of 
the subject (ie, without regard to the subject’s Individual TT) and will be recorded but not 
included in the subject’s transfusion history. During the Screening Period, the last transfusion 
should be administered 2 to 7 days before Day 1. 
For each transfusion administered at any time during the study, date of transfusion, number of 
RBC units transfused, HCT, and volume of RBC units will be recorded. 
9.5. 
Prior and Concomitant Medications 
Prior medications are defined as those administered anytime with the 28 days prior to signing of 
the ICF until the first dose of study drug. Concomitant medications are defined as those 
administered from the point of first dose of study drug through the subject’s completion of the 
study. All prior and concomitant medications must be recorded in the appropriate section of the 
source documentation and eCRF along with any dosage information, dates of administration, 
mode of administration, and reason for use. For non-drug therapies, please reference Section 9.6. 


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9.5.1. 
Prohibited Medications 
Concomitant use of investigational drugs is not allowed while subjects are participating in this 
study. All subjects must discontinue any investigational drug no less than 3 months prior to the 
first dose of study drug. 
In vitro studies using human liver microsomes and recombinant CYP enzymes have shown that 
AG-348 is primarily metabolized by CYP3A4 (>70%), with minor contributions from CYP2C9, 
CYP2C8, and CYP1A2. In addition, AG-348 has been shown to be a weak time-dependent 
CYP3A4 inhibitor and a potential inducer of CYP3A4 and CYP2B6 in vitro. In vitro transporter 
studies have shown that AG-348 is a substrate and inhibitor of P-gp.  
Also, AG-348 exhibits pH-dependent solubility. Therefore, proton-pump inhibitors and 
H2-receptor antagonists may decrease the absorption of AG-348.  
Based on these results, below is a list of concomitant therapy to be avoided and concomitant 
therapy requiring careful monitoring. 
The following are prohibited at all times during participation in this study: 
• Strong inhibitors of CYP3A4 (listed in Appendix 5 of the AG-348 IB) 
• Products known to inhibit CYP3A4, such as grapefruit or grapefruit juice 
• Strong inducers of CYP3A4 (listed in Appendix 5 of the AG-348 IB) 
• Strong inhibitors of P-gp (listed in Appendix 5 of the AG-348 IB) 
• Digoxin, a P-gp transporter-sensitive substrate 
If a subject is taking any medication listed in the AG-348 IB and/or digoxin prior to enrolling in 
the study, the medication must be discontinued at least 5 days or a time frame equivalent to 
5 half-lives (whichever is longer) prior to Day 1 dosing. 
• Hematopoietic stimulating agents (eg, EPOs, granulocyte colony stimulating factors, 
thrombopoietins) must be discontinued no less than 28 days prior to the first dose of 
study drug. B12 injections are permitted for subjects with a prior diagnosis of B12 
deficiency syndromes. Subjects must be repleted to stability of the Hb and mean 
corpuscular volume (MCV) prior to enrollment in the study. 
• Anabolic steroids, including testosterone preparations, administered for anemia must 
be discontinued no less than 28 days prior to the first dose of study drug. 
The medications that fall under the categories mentioned below should be avoided and replaced 
with alternative treatments. If this is not possible, subjects receiving these medications should be 
carefully monitored. A general monitoring guideline for Investigators whose subjects take 
medications that fall under the categories mentioned below is as follows: Investigators must 
monitor subjects for lack of efficacy of the prescribed medication or for side effects arising from 
the medication. If either a lack of efficacy of the prescribed medication or side effects suspected 
to be related to the prescribed medication are noticed, then the Investigator should make 
appropriate modifications to the dose of the prescribed medication or find alternatives to the 
prescribed medication. 
• Corticosteroids (sensitive substrates of CYP3A4 and weak CYP3A4 inducers) 


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• Sensitive substrates of CYP3A4 (listed in Appendix 5 of the AG-348 IB) 
• Moderate inhibitors of CYP3A4 (listed in Appendix 5 of the AG-348 IB) 
• Sensitive substrates of CYP2B6 (listed in Appendix 5 of the AG-348 IB) 
• Proton-pump inhibitors and H2-receptor antagonists (listed in Appendix 5 of the 
AG-348 IB). Antacids, such as magnesium hydroxide and aluminum hydroxide, can 
be used with AG-348. 
AG-348, being a potential CYP3A4 inducer, has the potential to reduce the effectiveness of oral 
contraceptives. Therefore, women using oral contraceptives must also utilize a barrier method 
contraceptive while enrolled in the study and until at least 28 days after their last dose of study 
drug, as specified in Inclusion Criterion #9 (see Section 8.1). 
9.5.2. 
Allowed Concomitant Medications 
Medications other than those specified above (Section 9.5.1) are permitted during the study. All 
intercurrent medical conditions will be treated at the discretion of the Investigator according to 
acceptable local standards of medical care. Subjects may receive analgesics, antiemetics, 
anti-infectives, and antipyretics as medically indicated and consistent with the guidance in 
Section 9.5.1.  
Subjects may continue iron chelation therapy with deferoxamine, deferasirox, or deferiprone. As 
iron overload is a long-term complication of PK deficiency, any initiation, completion, or change 
of iron chelation therapy will be of particular interest. Data about chelation therapy use will be 
carefully collected. 
Subjects must continue taking at least 0.8 mg oral folic acid daily for the duration of the study.  
9.6. 
Prior and Concomitant Non-Drug Therapies 
Prior non-drug therapies determined to be relevant for medical/surgical history and/or eligibility 
criteria, such as major surgeries within 6 months prior to signing the ICF (see Section 8.2, 
Exclusion Criterion #9) and splenectomy within 12 months prior to signing the ICF (see 
Section 8.2, Exclusion Criterion #4) should be recorded. 
Relevant concomitant non-drug therapies used to treat an AE should be collected from the 
signing of ICF until the subject completes the study. 
9.7. 
Treatment Compliance 
Treatment compliance will be assessed by drug accountability (ie, number of tablets dispensed). 
9.8. 
Randomization and Blinding 
This is an open-label study, and no blinding is involved. All subjects will receive AG-348 as 
described in Section 9.2. There is no randomization. 


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10. 
STUDY ASSESSMENTS 
The timing of all study assessments is indicated in the Schedule of Assessments for Part 1 
(Table 4) and Part 2 (Table 5) of the study. 
10.1. 
Informed Consent 
A description of the study is to be presented to each potential subject and a signed and dated ICF 
is to be obtained before any study-specific procedures are performed. The ICF will contain a 
separate section regarding the option to use leftover biological samples for analysis of additional 
biomarkers; subjects may opt in or decline; this will not affect their eligibility for the study. 
10.2. 
Study Eligibility 
A subject’s eligibility will be assessed and confirmed during the Screening Period. The 
Investigator will determine whether each subject meets all the inclusion criteria and none of the 
exclusion criteria. Eligibility of each subject will be confirmed by the Medical Monitor (or 
designee). A subject will not be considered enrolled (and cannot receive his/her first dose of 
study drug) until eligibility is confirmed. 
If a subject is determined to be ineligible for the study, the subject will be excluded from 
participation.  
If study ineligibility is due to a transient condition (eg, prohibited concomitant medication), the 
subject could be rescreened after the criterion that the subject failed to meet has resolved. In the 
case of a subject declared ineligible according to a previous version of the protocol, the subject 
may be rescreened if the subject could be eligible according to the current version of the 
protocol. 
The following assessments will not need to be repeated at rescreening if they were performed 
correctly at Screening: transfusion history unless additional transfusions have been administered, 
demographics, medical/surgical history unless new information needs to be added, liver MRI 
unless done more than 6 months prior, DXA scan unless done more than 6 months prior, and 
PKLR genotyping. 
10.3. 
Medical and Surgical History 
All medical and surgical history deemed to be relevant per the Investigator (in particular, 
pertaining, but not limited to, the diagnosis of PK deficiency) and current medical conditions are 
to be recorded on the source documentation and included in the eCRF during Screening.  
Prior history of splenectomy and/or cholecystectomy must be documented and included in the 
eCRF during Screening for all subjects. 
10.3.1. 
Iron Overload-Related History 
Historical use of chelation therapy for the 12 months prior to signing the ICF should be recorded, 
including type of iron chelation therapy, start date, stop date, and dose. Additionally, serum iron, 
ferritin, transferrin saturation, and LIC data should be recorded for the 12 months prior to signing 
the ICF. 


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10.4. 
Prior and Concomitant Medications 
Prior and concomitant medications will be captured as described in Section 9.5 and per the 
Schedule of Assessments (Section 10.14.1).  
10.5. 
PKLR Genotyping 
The PKLR genotyping and genotype classification (ie, missense or non-missense) will be 
performed during Screening by a central genotyping laboratory for confirmation of study 
eligibility.  
10.6. 
Demographics 
Subject demographic data will include gender, year of birth, race, and ethnicity. 
Race and ethnicity data will be collected to ensure that any race- and ethnicity-related 
specificities in the safety, pharmacokinetics, and/or efficacy of AG-348 can be captured and 
interpreted accurately.  
Collection of demographic data will be modified by country regulatory requirements, as 
appropriate.  
10.7. 
Assessment of Efficacy 
10.7.1. 
Transfusion Burden 
Transfusion burden (number of RBC units received over a 24-week period) will be assessed 
based on the transfusion information collected in Part 2 of the study. Information on transfusions 
administered during the study will be recorded as specified in Section 9.4. 
10.8. 
Assessments of Safety 
10.8.1. 
Vital Signs 
Vital signs will be recorded and will include systolic and diastolic BP, heart rate, and body 
temperature.  
10.8.2. 
Physical Examination 
Complete physical examinations will be performed according to the Schedules of Assessments 
(Section 10.14.1) and additionally when clinically indicated, at the discretion of the Investigator.  
Height will be documented during Screening only. 
10.8.3. 
Laboratory Assessments 
Safety laboratory assessments are described in Section 10.9.1. 
10.8.4. 
Electrocardiogram 
The 12-lead ECGs should be performed following 5 minutes of recumbency and in triplicate 
using the ECG machine provided by the central vendor and according to the vendor manual. The 
ECG at Part 2 Week 24 will be performed prior to, but within the same window as, the 


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pharmacokinetic sample collected 1 hour (±5 minutes) after the study drug dose administration 
to align with the approximate Tmax (time to maximum [peak] concentration) of the study drug. 
The ECGs will be read promptly by a qualified physician at the study site to detect any eligibility 
or safety issue. Only QTcF (not heart rate-corrected QT interval by Bazett’s method [QTcB]) 
will be used for determination of eligibility. In addition to the local read, the ECGs will be sent 
promptly to the central vendor for a data analysis read. 
An ECG will be repeated if clinically significant abnormalities are observed, if artifacts are 
present, or if machine/equipment errors occur.  
10.8.5. 
Dual-Energy X-ray Absorptiometry Scans 
The DXA scans of the lumbar spine and proximal femur (trochanter and inter-trochanter, which 
comprise the total hip and femoral neck) will be performed according to the instructions 
provided by the central vendor (see vendor’s manual for details). The DXA scans will be 
transmitted promptly to the central vendor for assessment of technical adequacy and may have to 
be repeated (before first dose for the screening DXA scan) if not technically adequate. The DXA 
scans will be read and interpreted by the central vendor. 
10.8.6. 
Menstrual Cycle Diary 
Menstruating female subjects will be required to fill out an electronic menstrual cycle diary for 
each menstrual period in order to detect any change in menstrual cycles. Subjects will record the 
start date, stop date, and any notable characteristics of each menstrual cycle in the eDiary 
provided at Screening. 
10.9. 
Laboratory Assessments 
All clinical laboratory evaluations are to be performed by a central laboratory. If Investigators 
believe that it is clinically indicated to obtain urgent safety laboratory results from their own 
local laboratories on the day of the subject’s visit and before the results are returned from the 
designated central laboratory, they are free to exercise their discretion to do so. In this case, a 
sample should be sent, in parallel, to the central laboratory. Decisions about subject eligibility 
and subjects’ management are to be based on the results obtained from the central laboratory, 
unless an urgent safety issue requires performing a local test, and except for pregnancy testing 
from Day 1 until the end of the study, which should be done locally. 
Blood samples for clinical evaluations will be collected according to the Schedules of 
Assessments (Section 10.14.1). All blood sample draws for screening tests should be performed 
as long after a previous transfusion as possible. In addition, all clinically significant laboratory 
abnormalities noted on testing will be followed by repeat testing and further investigated 
according to the judgment of the Investigator.  
Safety laboratory assessments and other laboratory assessments are listed in Section 10.9.1 and 
Section 10.9.2, respectively.  


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10.9.1. 
Safety Laboratory Assessments 
The following safety laboratory parameters will be measured: 
Hematology: 
Complete blood count with differential (HCT, Hb, RBC count, 
percent reticulocyte, absolute reticulocyte count [if available], 
immature reticulocyte fraction [IRF-H and IRF-M+H] [if 
available], MCV, MCH, MCHC, red cell distribution width, 
nucleated RBC count, white blood cell count, ANC, absolute 
lymphocyte count, absolute monocyte count, eosinophil count, 
basophil count, and platelet count) 
Serum Chemistry: 
Sodium, potassium, chloride, calcium, magnesium, phosphorus, 
carbon dioxide or bicarbonate, albumin, total protein, glucose, 
blood urea nitrogen or urea, creatinine, and uric acid. At Screening, 
estimated GFR, measured GFR, or calculated CrCL 
(Cockcroft-Gault) will be assessed. 
Lactate Dehydrogenase 
and Haptoglobin: 
LDH and haptoglobin 
Liver Function Tests: 
Alkaline phosphatase (ALP), ALT, AST, total bilirubin, direct 
bilirubin, and indirect bilirubin 
Sex Steroid Testing: 
Testosterone (total and free), estrone, and estradiol  
Lipids: 
Total cholesterol, high-density lipoprotein cholesterol, low-density 
lipoprotein cholesterol, and triglycerides measured by standard 
method and nuclear magnetic resonance 
Coagulation Studies: 
Fibrinogen, aPTT, and INR 
Dipstick Urinalysis: 
Protein, glucose, leukocytes, and blood 
Pregnancy (Human 
Chorionic 
Gonadotropin [hCG]) 
Test: 
Serum or urine pregnancy tests (with the exception of the 
screening pregnancy test, which must be serum) 
UGT1A1 Genotyping: 
To detect subjects with Gilbert’s syndrome 
For hematology parameters, the most recent nonmissing measurements prior to Transfusion 0 
(the most recent transfusion occurring in the Screening Period prior to the start of study drug on 
Day 1) will be defined as the baseline. This is to limit the impact of transfusion on these 
hematology laboratory parameters. For transaminase (AST and ALT), the baseline will be 
defined as the average of the Screening and Day 1 values. 


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10.9.2. 
Other Laboratory Assessments 
A blood sample for serology, including HBsAg, HCVAb screen, and HIV-1 and HIV-2 Ab, will 
be collected from all subjects for eligibility criteria. 
The following other laboratory parameters will be assessed: 
• FSH (to confirm postmenopausal status) 
• Iron panel and related markers (iron, serum ferritin, total iron-binding capacity 
[TIBC], transferrin saturation, non-transferrin bound iron [NTBI], hepcidin, 
C-reactive protein [CRP], and other markers of iron metabolism). Remaining sample 
may be used for analyses of lipoproteins (only in subjects who have agreed to this 
optional analysis in the ICF). 
• Markers of erythropoietic activity: EPO, erythroferrone, soluble transferrin receptor, 
and other makers of erythropoiesis 
• Assessments of complications of iron overload: thyroxine, thyroid-stimulating 
hormone, parathormone, fructosamine, and vitamin D 
All blood sample draws for screening tests should be performed as long after a previous 
transfusion as possible. 
10.10. 
Other Assessments 
Liver iron concentration will be measured by MRI based on the measurement and imaging of 
proton transverse relaxation rates (R2). The MRI data collected at the site will be transferred 
immediately to a central vendor for technical assessment and analysis. For further details, please 
refer to the vendor’s manual. 
10.11. 
Pharmacokinetic Assessments 
10.11.1. 
Blood Sample Collection 
On days of pharmacokinetic blood sample collection, the morning dose of study drug must be 
administered at the study site. On days where a predose sample is required, the study drug must 
be administered after the predose sample is taken. Plasma samples for pharmacokinetic analysis 
of AG-348 will be collected at the following time points (note: time points are listed in relation 
to the morning dose of study drug): 
• 
Week 24 Visit in Part 2 (full profile blood sampling)  
o Predose (within 1 hour prior to study drug administration) 
o 0.5 hours (±5 minutes) post study drug administration 
o 1 hour (±5 minutes) post study drug administration 
o 2 hours (±5 minutes) post study drug administration 
o 4 hours (±30 minutes) post study drug administration 
o 8 hours (±30 minutes) post study drug administration 


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The actual date and time of sample collection will be recorded in the source documents and 
eCRF. An explanation should be provided in the source documents for any missed or mishandled 
pharmacokinetic samples as well as for any samples collected outside the time windows.  
10.11.2. 
Sample Analysis 
Pharmacokinetic samples will be analyzed for AG-348 using a validated liquid 
chromatography-tandem mass spectrometry method. Remaining samples may be used for 
analyses of AG-348 metabolism (only in subjects who have agreed to this optional analysis in 
the ICF). 
Plasma pharmacokinetic parameters will be computed, when data allow, using standard 
noncompartmental methods based on observed plasma AG-348 concentrations and on actual 
sample collection times. These parameters will include, but may not be limited to, the following: 
• AUC0-last: The area under the plasma concentration × time curve from time 0 to the 
time of the last measurable concentration 
• Tlast: Time of last measurable concentration 
• Cmax: Maximum (peak) concentration  
• Tmax: Time to maximum (peak) concentration  
• λz: Apparent terminal elimination rate constant, calculated from a semi-log plot of the 
plasma concentration versus time curve  
• t½: Terminal half-life  
• CL/F: The apparent total plasma clearance (CLp) following oral (extravascular) 
dosing 
• Vz/F: The apparent volume of distribution during the terminal elimination phase 
following oral (extravascular) dosing 
10.12. 
Pharmacodynamic Assessments 
Pharmacodynamic samples to measure PKR protein levels in whole blood will be taken during 
Screening between Day -7 and Day -2 before Transfusion 0 is administered, at the Day 1 Visit in 
Part 1 before administration of the first dose, and during Part 2 at the Week 24 Visit. Additional 
analysis of exploratory biomarkers (PKM protein levels and levels of intermediates in the 
metabolic pathways affected by PKR) to further the understanding of the mechanism of action of 
AG-348 may be performed on leftover samples in subjects who have agreed to this optional 
analysis in the ICF. 
On days of sample collection (except Transfusion 0), the morning dose of study drug must be 
administered at the study site following the predose collections. The predose sample for 
PD assessments during Part 2 at the Week 24 Visit should be collected within 60 minutes prior to 
study drug administration. Procedures for sample collection and processing will be provided in a 
separate study manual. 


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10.13. 
Health-Related Quality of Life Assessments 
Subjects will use the eDiary (provided during Screening) to record responses to each of the 
HRQoL assessments (Section 10.13.1, Section 10.13.2, and Section 10.13.3). The HRQoL 
assessments should be completed in the evening (between 5:00-11:00 PM) within the window 
for the relevant study visit as indicated in the Schedule of Assessments (Section 10.14.1). 
10.13.1. 
Pyruvate Kinase Deficiency-Specific Health-Related Quality of Life Assessments 
The PKDD is a 7-item PRO measure of the core signs and symptoms associated with 
PK deficiency in adults. Subjects rate their experience with symptoms of PK deficiency on the 
present day. The symptoms include those associated with tiredness, jaundice, bone pain, 
shortness of breath, and energy level.  
The PKDIA is a 12-item PRO measure of the common impacts of PK deficiency on activities of 
daily living. Subjects rate how PK deficiency has impacted aspects of daily living in the past 
7 days, including impacts on relationships; perceived appearance; work performance; and 
leisure, social, mental, and physical activities.  
These 2 tools were developed by the Sponsor to systematically assess and capture changes in 
symptom burden and impact on HRQoL.  
10.13.2. 
EuroQol-5D-5L 
The EQ-5D-5L is a standardized instrument for evaluating quality of life over 5 dimensions: 
mobility, self-care, usual activities, pain, and mood. It is applicable over a broad range of health 
conditions and is used widely in clinical trials. 
10.13.3. 
Patient Global Impression of Severity 
The Patient Global Impression of Severity (PGIS) is a single-item questionnaire used to rate the 
subject’s impression of the severity of his/her condition. 
10.14. 
Study Conduct 
10.14.1. 
Schedule of Assessments 
The Schedule of Assessments for Part 1 of the study is in Table 4 and the Schedule of 
Assessments for Part 2 of the study is in Table 5. 


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Table 4: 
Schedule of Assessments – Screening and Part 1 of Study AG348-C-007 
Visit: 
Screening1 
P1D12 
P1W2 
P1W4 
P1W6 
P1W8 
P1W10 
P1W12 
P1W14 
P1W163 
Study Day: 
(Window [days]) 
D -56 to 
D -7 
Transfusion 
04 
D -7 to D -2 
D1 
D15 
(±3 d) 
D29 
(±3 d) 
D43 
(±3 d) 
D57 
(±3 d) 
D71 
(±3 d) 
D85 
(±3 d) 
D99 
(±3 d) 
D113 
(±3 d) 
(P1W16 is the 
same day as 
P2D1)3 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
Transfusion history5 
X 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
Medical/surgical history6 
X 
 
 
 
 
 
 
 
 
 
 
Prior medications  
X 
 
 
 
 
 
 
 
 
 
 
Physical examination7 
X 
 
X 
 
 
 
 
 
X 
 
X 
Vital signs8 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG9 
X 
 
X 
 
 
 
 
 
 
 
X 
Liver MRI to assess LIC10 
X 
 
 
 
 
 
 
 
 
 
X 
DXA scan10 
X 
 
 
 
 
 
 
 
 
 
X 
Dispense eDiary11 
X 
 
 
 
 
 
 
 
 
 
 
HRQoL assessments11,12 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Dosing diary11 
 
 
X 
Menstrual cycle diary11 
X 
PKLR genotyping13 
X 
 
 
 
 
 
 
 
 
 
 
Clinical laboratory evaluations14 
HBsAg, HCVAb, HIV-1, 
and -2 Ab15 
X 
 
 
 
 
 
 
 
 
 
 
FSH16 
X 
 
 
 
 
 
 
 
 
 
 


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Visit: 
Screening1 
P1D12 
P1W2 
P1W4 
P1W6 
P1W8 
P1W10 
P1W12 
P1W14 
P1W163 
Study Day: 
(Window [days]) 
D -56 to 
D -7 
Transfusion 
04 
D -7 to D -2 
D1 
D15 
(±3 d) 
D29 
(±3 d) 
D43 
(±3 d) 
D57 
(±3 d) 
D71 
(±3 d) 
D85 
(±3 d) 
D99 
(±3 d) 
D113 
(±3 d) 
(P1W16 is the 
same day as 
P2D1)3 
Hematology17 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum chemistry18 
X 
 
X 
 
 
 
 
 
X 
 
X 
Liver function tests19 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
LDH and haptoglobin 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
T4, TSH, PTH, 
fructosamine, and 
vitamin D 
 
 
X 
 
 
 
 
 
 
 
 
Coagulation studies20 
X 
 
X 
 
 
 
 
 
X 
 
 
Urinalysis21 
X 
 
X 
 
 
 
 
 
X 
 
 
Pregnancy test22 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Iron panel and related 
markers23 
X 
 
X 
 
 
 
 
 
X 
 
 
Markers of erythropoietic 
activity23 
X 
 
X 
 
 
 
 
 
X 
 
 
Lipids24 
 
 
X 
 
 
X 
 
 
X 
 
X 
Sex steroids25 
 
 
X 
 
X 
 
X 
 
X 
 
X 
PKR protein26 
 
X 
X 
 
 
 
 
 
 
 
 
Eligibility confirmation27 
 
X 
 
 
 
 
 
 
 
 
UGT1A1 genotyping28 
 
 
X 
 
 
 
 
 
 
 
 
Dispense study drug29 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Study drug dosing 
 
 
X 
Return study drug  
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 


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Visit: 
Screening1 
P1D12 
P1W2 
P1W4 
P1W6 
P1W8 
P1W10 
P1W12 
P1W14 
P1W163 
Study Day: 
(Window [days]) 
D -56 to 
D -7 
Transfusion 
04 
D -7 to D -2 
D1 
D15 
(±3 d) 
D29 
(±3 d) 
D43 
(±3 d) 
D57 
(±3 d) 
D71 
(±3 d) 
D85 
(±3 d) 
D99 
(±3 d) 
D113 
(±3 d) 
(P1W16 is the 
same day as 
P2D1)3 
Concomitant medications/ 
transfusions30 
 
X 
SAEs/AEs/AESIs31 
X 
Abbreviations: Ab = antibody; AE = adverse event; AESI = adverse event of special interest; ALC = absolute lymphocyte count; ALP = alkaline phosphatase; ALT = alanine 
aminotransferase; ANC = absolute neutrophil count; aPTT = activated partial thromboplastin time; AST = aspartate aminotransferase; BP = blood pressure; BUN = blood urea 
nitrogen; CBC = complete blood count; CO2 = carbon dioxide; CrCL = calculated creatinine clearance; CRP = C-reactive protein; D = Study Day; d = days; DXA = dual-
energy X-ray absorptiometry; ECG = electrocardiogram; EQ-5D-5L = EuroQol-5D-5L; FSH = follicle-stimulating hormone; GFR = glomerular filtration rate; 
Hb = hemoglobin; HBsAg = hepatitis B surface antigen; hCG = human chorionic gonadotropin; HCV = hepatitis C virus; HCT = hematocrit; HDL-C = high-protein lipoprotein 
cholesterol; HIV = human immunodeficiency virus; HRQoL = health-related quality of life; ICF = Informed Consent Form; INR = international normalized ratio; LDH = 
lactate dehydrogenase; LDL-C = low-density lipoprotein cholesterol; LIC = liver iron concentration; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular 
hemoglobin concentration; MCV = mean corpuscular volume; MRI = magnetic resonance imaging; NRBC = nucleated red blood cell count; NTBI = non-transferrin bound 
iron; P = Part; PGIS = Patient Global Impression of Severity; PKDD = Pyruvate Kinase Deficiency Diary; PKDIA = Pyruvate Kinase Deficiency Impact Assessment; PKR = 
red blood cell-specific form of pyruvate kinase; PTH = parathormone; RBC = red blood cell; RDW = red cell distribution width; RT-PCR = reverse transcriptase-polymerase 
chain reaction; SAE = serious adverse event; TIBC = total iron-binding capacity; TSH = thyroid-stimulating hormone; T4 = thyroxine; UGT1A1 = uridine diphosphate 
glucuronosyltransferase 1A1 gene; W = Week; WBC = white blood cell. 
Note: Whenever more than 1 assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive 
assessment (eg, vital signs, ECG, blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time (if applicable) 
1 A subject’s Screening Period duration may be extended beyond 8 weeks upon the Medical Monitor’s (or designee’s) approval if there is a delay in obtaining a subject’s 
complete transfusion history or the subject is projected to need a transfusion within 1 week of his/her scheduled first dose to ensure that treatment with AG-348 is always 
started 2-7 days after a subject’s most recent transfusion in the Screening Period (referred to as Transfusion 0). All blood sample draws for screening tests should be 
performed as long after a previous transfusion as possible. 
2 The Day 1 visit should take place 2-7 days after the last transfusion in the Screening Period (ie, Transfusion 0). The first dose of study drug on Day 1 should be taken at the 
study site following all Day 1 assessments (with the exception of eDiary assessments; these assessments should be performed in the evening of Day 1). 
3 The Part 1 Week 16 Visit will mark the transition from Part 1 into Part 2 (ie, Part 1 Week 16 is the same as Part 2 Day 1, marking the end of Part 1 and the beginning of 
Part 2). 
4 Some subjects may require more than 1 transfusion during the Screening Period. Transfusion 0 is defined as the most recent transfusion occurring in the Screening Period 
prior to the start of study drug on Day 1. This transfusion does not need to be conducted at the study site, however blood samples indicated in the table will be collected and 
sent to the central laboratory for analysis. These samples must be drawn before Transfusion 0 is administered and within the Day -7 to Day -2 window.  
5 The complete transfusion history (as defined in Section 7.1.2.1) for the entire 52-week period prior to informed consent must be collected and will be used to inform study 
eligibility and analyze data.  
6 All medical and surgical history deemed to be relevant per the Investigator (in particular, pertaining, but not limited to, the diagnosis of PK deficiency), current medical 
conditions, and prior history of splenectomy and/or cholecystectomy are to be recorded. Additionally, serum iron, ferritin, transferrin saturation, and LIC data should be 
recorded for the 12 months prior to signing the ICF. Historical use of chelation therapy for the 12 months prior to signing the ICF should be recorded, including type of iron 
chelation therapy, start date, stop date, and dose. 


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7 A complete physical examination will be performed. Height will be documented during Screening only. Additional physical examinations may be performed when clinically 
indicated, at the discretion of the Investigator. 
8 Vital signs of systolic and diastolic BP, heart rate, and body temperature will be collected. 
9 The 12-lead ECGs will be conducted using the equipment provided by the vendor and according to the vendor manual after 5 minutes of recumbency and in triplicate. The 
ECGs will be read promptly by a qualified physician at the study site to detect any eligibility or safety issue. In addition to the local read, the ECGs will be sent promptly to 
the central vendor for a data analysis read. 
10 The DXA and liver MRI scan(s) should be performed per the vendor manuals. If the Screening DXA and/or MRI scan(s) are deemed to be of low quality by the central 
vendors, the scan(s) will be repeated before the first dose of study drug is administered. 
11 All subjects will be given an eDiary to record responses to HRQoL assessments and dosing. In addition, menstruating female subjects will record their menstrual cycles (start 
date, stop date, and change in characteristics) in the eDiary; the menstrual diary can be completed at any time of day. The dosing diary should be completed every day in the 
evening (between 5:00-11:00 PM). 
12 Health-related quality of life will be assessed using PKDD, PKDIA, EQ-5D-5L, and PGIS. Note that PGIS is collected at Day 1 only. The HRQoL assessments should be 
completed in the evening (between 5:00-11:00 PM) within the window for the relevant study visit. 
13 For eligibility confirmation, the PKLR genotyping must be analyzed by the central laboratory; sample must be drawn early in the Screening Period to ensure results are back 
in time to allow the Investigator to assess eligibility. 
14 On Day 1 of Part 1, all clinical laboratory evaluations should be collected predose. All clinical laboratory evaluations should be analyzed centrally, except the urine or serum 
pregnancy tests performed after Screening. For an urgent safety issue that may require local laboratory evaluations, refer to Section 10.9 of the protocol. 
15 If the subject is positive for HCVAb, an RT-PCR test will be conducted. The subject is ineligible to enroll if active hepatitis C is present. The subject may be rescreened after 
receiving appropriate hepatitis C treatment. 
16 The FSH assessment will be performed only at Screening for female subjects for confirmation of postmenopausal status (ie, female subjects who have not menstruated at all 
for at least the preceding 12 months prior to signing the ICF). Samples should be drawn in the morning (does not need to be fasting). 
17 Hematology parameters (ie, CBC with differential) will include HCT, Hb, RBC count, percent reticulocyte, absolute reticulocyte count (if available), immature reticulocyte 
fraction (IRF-H and IRF-M+H) (if available), MCV, MCH, MCHC, RDW, NRBC, WBC count, ANC, ALC, absolute monocyte count, eosinophil count, basophil count, and 
platelet count. 
18 Serum chemistry parameters will be collected after an overnight fast (with the exception of the screening assessment, which can be done without fasting) and will include 
sodium, potassium, chloride, calcium, magnesium, phosphorus, CO2 or bicarbonate, albumin, total protein, glucose, BUN or urea, creatinine, and uric acid. At Screening, 
estimated GFR, measured GFR, or calculated CrCL (Cockcroft-Gault) will be assessed. If the subject reports that he/she did not adhere to an overnight fast, these samples 
should not be collected at the scheduled visit (with the exception of the screening assessment, which can be done without fasting). Instead, these samples should be collected 
within 2 weeks of the original time point at the next scheduled visit or at an unscheduled visit, after the subject has adhered to an overnight fast. 
19 Liver function tests are ALP; ALT; AST; and total, direct, and indirect bilirubin. 
20 Coagulation parameters will include fibrinogen, aPTT, and INR. 
21 Urinalysis will be performed by a dipstick method and will include assessments of protein, glucose, leukocytes, and blood. 
22 A serum pregnancy (hCG) test must be done at Screening and sent to the central laboratory. A urine or serum pregnancy (hCG) test must be done and documented to be 
negative on Day 1 before administration of the first dose of AG-348. A urine or serum pregnancy (hCG) test must be repeated every 4 weeks at the indicated visits and must 
also be done at any point throughout the study if pregnancy is clinically suspected. All pregnancy tests, other than the test performed at Screening, should be performed 
locally.  
23 The iron panel and related markers will include iron, serum ferritin, TIBC, transferrin saturation, NTBI, hepcidin, CRP, and other markers of iron metabolism. 
Erythropoietin, erythroferrone, soluble transferrin receptor, and other markers of erythropoiesis will be collected as markers of erythropoietic activity. Remaining sample may 
be used for analyses of lipoproteins (only in subjects who have agreed to this optional analysis in the ICF). 
24 For lipid testing samples for total cholesterol, HDL-C, LDL-C, and triglyceride will be collected after an overnight fast (see central laboratory vendor manual for detail). If 
the subject reports that they did not adhere to an overnight fast, these samples should not be collected at the scheduled visit. Instead, these samples should be collected within 
2 weeks of the original time point at the next scheduled visit or at an unscheduled visit, after the subject has adhered to an overnight fast. 
25 Sex steroid testing will assess testosterone (total and free), estrone, and estradiol. Samples should be drawn in the morning (does not need to be fasting). 


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26 Samples for PKR protein are to be collected prior to Transfusion 0 and predose on Day 1. On days of sample collection (except for Transfusion 0), the morning dose of study 
drug must be administered at the study site following the predose collections. 
27 The Investigator will determine whether each subject meets all the inclusion criteria and none of the exclusion criteria. Eligibility of each subject will be confirmed by the 
Medical Monitor (or designee) during the Screening Period. In addition, on Day 1, the site must verify that a subject who has been considered eligible after completing 
Screening has a negative urine or serum pregnancy test, if applicable, before the subject can be dosed. 
28 UGT1A1 genotyping results are not required prior to enrollment. 
29 Study drug is expected to be dispensed to the subject at the study site. If necessary, in exceptional circumstances and with agreement of the Sponsor (or representative), study 
drug can be provided to the subject’s home, if acceptable by practice and allowed by local regulations. 
30 Collection of transfusion information for transfusions administered during the study will include the number of transfusions, date of the transfusions, HCT, volume of RBC 
units, and the number of RBC units transfused.  
31 The Investigator should ask the subject for information regarding sleep patterns, signs, and symptoms associated with insomnia. 


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Table 5: 
Schedule of Assessments – Part 2 of Study AG348-C-007 
Visit: 
P2W4 
P2W8 
P2W12 
P2W18 
P2W24 or 
EOS1 
Follow-up2 
Study Day: 
(Window [days]) 
D29 
(±3 d) 
D57 
(±3 d) 
D85 
(±3 d) 
D127 
(±3 d) 
D1691 
(±3 d) 
28 d after the last dose2 
(±4 d) 
Physical examination3 
 
 
X 
 
X 
 
Vital signs4 
X 
X 
X 
X 
X 
X 
12-lead ECG5 
 
 
 
 
X 
X 
Liver MRI to assess LIC6 
 
 
 
 
X 
 
DXA scan6 
 
 
 
 
X6 
 
HRQoL assessments7,8 
X 
X 
X 
X 
X 
X2 
Dosing diary8 
X 
Menstrual diary8 
X 
Clinical laboratory evaluations9 
Hematology10 
X 
X 
X 
X 
X 
X 
Serum chemistry11 
 
 
X 
 
X 
X 
Liver function tests12 
X 
X 
X 
X 
X 
X 
LDH and haptoglobin 
X 
X 
X 
X 
X 
X 
T4, TSH, PTH, fructosamine, and vitamin D 
 
 
 
 
X 
 
Coagulation studies13 
 
 
X 
 
X 
 
Urinalysis14 
 
 
X 
 
X 
X 
Iron panel and related markers15 
 
 
X 
 
X 
 
Markers of erythropoietic activity15 
 
 
X 
 
X 
 
Lipids16 
X 
X 
X 
X 
X 
X 
Sex steroids17 
X 
X 
X 
X 
X 
X 
Pregnancy tests18 
X 
X 
X 
X 
X 
X 


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Visit: 
P2W4 
P2W8 
P2W12 
P2W18 
P2W24 or 
EOS1 
Follow-up2 
Study Day: 
(Window [days]) 
D29 
(±3 d) 
D57 
(±3 d) 
D85 
(±3 d) 
D127 
(±3 d) 
D1691 
(±3 d) 
28 d after the last dose2 
(±4 d) 
Blood sampling for pharmacokinetic profile19 
 
 
 
 
X1 
 
PKR protein20 
 
 
 
 
X1 
 
Dispense study drug21 
X 
X 
X 
X 
X1 
 
Study drug dosing 
X 
 
Return study drug 
X 
X 
X 
X 
X1 
X 
Return eDiary 
 
 
 
 
X1 
X 
Concomitant medications/transfusions22 
X 
SAEs/AEs/AESIs23 
X 
Abbreviations: AE = adverse event; AESI = adverse event of special interest; ALC = absolute lymphocyte count; ALP = alkaline phosphatase; ALT = alanine aminotransferase; 
ANC = absolute neutrophil count; aPTT = activated partial thromboplastin time; AST = aspartate aminotransferase; BP = blood pressure; BUN = blood urea nitrogen; 
CBC = complete blood count; CO2 = carbon dioxide; CRP = C-reactive protein; D = Study Day; d = days; DXA = dual-energy X-ray absorptiometry; ECG = electrocardiogram; 
EOS = end of study; EQ-5D-5L = EuroQol-5D-5L; Hb = hemoglobin; hCG = human chorionic gonadotropin; HCT = hematocrit; HDL-C = high-density lipoprotein cholesterol; 
HRQoL = health-related quality of life; ICF = Informed Consent Form; INR = international normalized ratio; LDH = lactate dehydrogenase; LDL-C = low-density lipoprotein 
cholesterol; LIC = liver iron concentration; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume; 
MRI = magnetic resonance imaging; NRBC = nucleated red blood cell count; NTBI = non-transferrin bound iron; P = Part; PGIS = Patient Global Impression of Severity; 
PKDD = Pyruvate Kinase Deficiency Diary; PKDIA = Pyruvate Kinase Deficiency Impact Assessment; PKR = red blood cell-specific form of pyruvate kinase; 
PTH = parathormone; RBC = red blood cell; RDW = red cell distribution width; SAE = serious adverse event; T4 = thyroxine; TIBC = total iron-binding capacity; Tmax = Time to 
maximum (peak) concentration; TSH = thyroid-stimulating hormone; W = Week; WBC = white blood cell. 
Note: Whenever more than 1 assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive assessment 
(eg, vital signs, ECG, blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time (if applicable). The order of procedures may be 
revised with prior discussion between Sponsor and site. 
1 Subjects who continue study drug through Part 2 Week 24 should continue taking study drug at least through the morning dose of the Part 2 Week 24 Visit. All subjects who 
remain on study during Part 2 through the Week 24 Visit may be eligible for an open-label extension study with AG-348. Subjects who discontinue the study prior to the 
Part 2 Week 24 Visit should attend the End of Study Visit 28±4 days after the last study visit that the subject attended or 28±4 days after the last dose of study drug, 
whichever is later. Note, this may occur at any time during the study (Part 1 and Part 2). The End of Study Visit will be identical to the Part 2 Week 24 Visit with these 
exceptions: no study drug will be dispensed, blood sampling for pharmacokinetic profile will not be drawn, PKR protein will not be assessed, and if the subject has not 
already done so, they should return their eDiary and study drug. 
2 Subjects who continue study drug through the Part 2 Week 24 Visit but do not continue on into an extension study should undergo a dose taper and then attend the Follow-up 
Visit 28 ±4 days after the last dose of study drug (with the exception of HRQoL assessments, which will occur 28±4 days after the subject’s last full dose of study drug). 
Subjects who continue on into an extension study will not be required to attend the Follow-up Visit. Subjects who initiate or are undergoing a dose taper for a reason related 
to safety and for whom participation in an extension study remains undetermined should perform the taper, and a transition to an extension study should be discussed with the 
Medical Monitor (or designee). 


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3 A complete physical examination will be performed. Additional physical examinations may be performed when clinically indicated, at the discretion of the Investigator. 
4 Vital signs of systolic and diastolic BP, heart rate, and body temperature will be collected. 
5 The 12-lead ECGs will be conducted using the equipment provided by the vendor and according to the vendor manual after 5 minutes of recumbency and in triplicate. The 
ECGs will be read promptly by a qualified physician at the study site to detect any eligibility or safety issue. In addition to the local read, the ECGs will be sent promptly to 
the central vendor for a data analysis read. The ECG at Part 2 Week 24 will be performed prior to, but within the same window as, the pharmacokinetic sample collected 
1 hour (±5 minutes) after the study drug dose administration to align with the approximate Tmax of the study drug. The ECG performed as part of the EOS Visit can be 
collected at any time during the visit. 
6 The DXA and liver MRI scan(s) should be performed per the vendor manuals. If the DXA and/or MRI scan(s) are deemed to be of low quality by the central vendor, the 
scan(s) will be repeated no later than the next visit. The DXA scan for the EOS Visit will be performed only for these subjects if they have been on AG-348 for at least 22 
weeks (since Part 1 Day 1). 
7 Health-related quality of life will be assessed using PKDD, PKDIA, EQ-5D-5L, and PGIS. Note that PGIS should be collected only at the Week 24 Visit or the EOS Visit (if 
applicable). The HRQoL assessments should be completed in the evening (between 5:00-11:00 PM) within the window for the relevant study visit. 
8 All subjects will record responses to HRQoL assessments and keep a dosing diary in an eDiary dispensed at Screening. In addition, menstruating female subjects will record 
their menstrual cycles (start date, stop date, and characteristics) in the eDiary; the menstrual diary can be completed at any time of day. The dosing diary should be completed 
every day in the evening (between 5:00-11:00 PM). 
9 All clinical laboratory evaluations should be analyzed centrally, except the urine or serum pregnancy tests. For an urgent safety issue that may require local laboratory 
evaluations, refer to Section 10.9 of the protocol. 
10 Hematology parameters (ie, CBC with differential) will include HCT, Hb, RBC count, percent reticulocyte, absolute reticulocyte count (if available), immature reticulocyte 
fraction (IRF-H and IRF-M+H) (if available), MCV, MCH, MCHC, RDW, NRBC, WBC count, ANC, ALC, absolute monocyte count, eosinophil count, basophil count, and 
platelet count. 
11 Serum chemistry parameters will be collected after an overnight fast and will include sodium, potassium, chloride, calcium, magnesium, phosphorus, CO2 or bicarbonate, 
albumin, total protein, glucose, BUN or urea, creatinine, and uric acid. If the subject reports that he/she did not adhere to an overnight fast, these samples should not be 
collected at the scheduled visit. Instead, these samples should be collected within 2 weeks of the original time point at the next scheduled visit or at an unscheduled visit, after 
the subject has adhered to an overnight fast. 
12 Liver function tests are ALP; ALT; AST; and total, direct, and indirect bilirubin. 
13 Coagulation parameters will include fibrinogen, aPTT, and INR. 
14 Urinalysis will be performed by a dipstick method and will include assessments of protein, glucose, leukocytes, and blood. 
15 The iron panel and related markers will include iron, serum ferritin, TIBC, transferrin saturation, NTBI, hepcidin, CRP, and other markers of iron metabolism. 
Erythropoietin, erythroferrone, soluble transferrin receptor, and other markers of erythropoiesis will be collected as markers of erythropoietic activity. Remaining sample 
may be used for analyses of lipoproteins (only in subjects who have agreed to this optional analysis in the ICF). 
16 For lipid testing samples for total cholesterol, HDL-C, LDL-C, and triglyceride will be collected after an overnight fast (see central laboratory vendor manual for detail). If 
the subject reports that they did not adhere to an overnight fast, these samples should not be collected at the scheduled visit. Instead, these samples should be collected within 
2 weeks of the original time point at the next scheduled visit or at an unscheduled visit, after the subject has adhered to an overnight fast. 
17 Sex steroid testing will assess testosterone (total and free), estrone, and estradiol. Samples should be drawn in the morning (does not need to be fasting). 
18 A urine or serum pregnancy (hCG) test must be repeated every 4-6 weeks at the indicated visits and must also be done at any point throughout the study if pregnancy is 
clinically suspected. All pregnancy tests, other than the test performed at Screening, should be performed locally. 
19 Plasma samples for pharmacokinetic analysis will be collected at the following time points: predose (within 1 hour prior to dosing) and 0.5 hour (±5 minutes), 1 hour 
(± 5 minutes), 2 hours (±5 minutes), 4 hours (±30 minutes), and 8 hours (±30 minutes) postdose. On days of pharmacokinetic blood sample collection, the morning dose of 
study drug must be administered at the study site; on days where a predose sample is required, the study drug must be administered after the predose sample is taken.  
20 Samples for PKR protein are to be collected predose (within 60 minutes prior to study drug administration). On days of sample collection, the morning dose of study drug 
must be administered at the study site following the predose collections. 


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21 Study drug is expected to be dispensed to the subject at the study site. If necessary, in exceptional circumstances and with agreement of the Sponsor (or representative), study 
drug can be provided to the subject’s home, if acceptable by practice and allowed by local regulations. 
22 Collection of transfusion information for transfusions administered during the study will include the number of transfusions, date of the transfusions, HCT, volume of RBC 
units, and the number of RBC units transfused. 
23 The Investigator should ask the subject for information regarding sleep patterns, signs, and symptoms associated with insomnia. 
 


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11. 
ADVERSE EVENTS 
11.1. 
Reporting Period for Adverse Events and Serious Adverse Events 
Monitoring of AEs, including frequency, severity, and characterization of SAEs, AESIs, and 
AEs leading to discontinuation will be conducted throughout the study. Adverse events and 
SAEs will be recorded in the source documentation and eCRF from the time of the signing of 
ICF through the subject’s completion of study or withdrawal of consent, whichever occurs first. 
All AEs will be monitored until resolution of the AE to baseline, the AE is considered stable 
within the context of the trial, the subject is lost to follow-up, or until 28 days after the last dose 
of study drug unless the subject initiates an extension study, in which case ongoing AEs will be 
reported in the extension study database following consenting of the subject.  
All SAEs will be followed until final outcome of the SAE is known, the subject is lost to 
follow-up, or the subject initiates an extension study, in which case the SAE follow-up 
information will be reported in the extension study database. Any SAEs that are assessed as 
related to study drug that occur ≥28 days post-treatment are to be reported to the Sponsor directly 
by the Investigator. 
Adverse events will be evaluated by the Investigator and recorded per Section 11.3. Any AEs 
already documented at a previous assessment and designated as ongoing will be reviewed at 
subsequent visits or assessment time points as necessary. If these AEs have resolved, this will be 
documented. 
All AEs will be graded using the NCI CTCAE v4.03 grading system. 
11.2. 
Definition of Adverse Events 
11.2.1. 
Adverse Event 
A clinical AE is any untoward medical occurrence in a patient or clinical investigation subject 
administered a pharmaceutical product and which does not necessarily have to have a causal 
relationship with the study drug. An AE can, therefore, be any unfavorable and unintended sign 
(including an abnormal laboratory finding, for example), symptom, or disease temporally 
associated with the use of a medicinal product, whether or not considered related to the 
medicinal product. 
Pre-existing conditions that worsen during a study are to be reported as AEs. Withdrawal 
hemolysis is to be reported as a study drug-related AE. 
11.2.2. 
Serious Adverse Event 
An AE or suspected adverse reaction is considered serious if, in the view of either the 
Investigator or the Sponsor, it results in any of the following outcomes: 
• Death 


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• Life threatening (meaning that the subject was at immediate risk of death from the 
reaction as it occurred, but it does not include a reaction that hypothetically might 
have caused death had it occurred in a more severe form) 
• Inpatient hospitalization or prolongation of existing hospitalization. Hospitalization 
admissions and/or surgical operations scheduled to occur during the study period but 
planned prior to study entry are not considered AEs if the illness or disease existed 
before the subject was enrolled in the study, provided that it did not deteriorate in an 
unexpected manner during the study (eg, surgery performed earlier than planned). 
• A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• Congenital anomaly/birth defect 
• Important medical event. An important medical event is an event that may not result 
in death, be life threatening, or require hospitalization but may be considered an SAE 
when, based upon appropriate medical judgment, it may jeopardize the subject and 
may require medical or surgical intervention to prevent 1 of the outcomes listed in the 
definitions for SAEs. Examples of such medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood 
dyscrasias or convulsions that do not result in inpatient hospitalization, or the 
development of drug dependency or drug abuse. 
11.2.3. 
Adverse Events Occurring Secondary to Other Events 
In general, AEs occurring secondary to other events (eg, cascade events or clinical sequelae) 
should be identified by their primary cause, with the exception of severe or serious secondary 
events. However, medically significant AEs occurring secondary to an initiating event that are 
separated in time should be recorded as independent events on the Adverse Event eCRF. Please 
refer to the eCRF completion guidance for examples of how to record events occurring 
secondary to other events. 
All AEs should be recorded separately on the Adverse Event eCRF if it is unclear as to whether 
the events are associated. 
11.2.4. 
Pre-existing Medical Conditions 
A pre-existing medical condition is one that is present during Screening for this study. Such 
conditions should be recorded on the Medical History eCRF. 
A pre-existing medical condition should be recorded as an AE only if the frequency, severity, or 
character of the condition worsens during the study. When recording such events on the Adverse 
Event eCRF, it is important to convey the concept that the pre-existing condition has changed by 
including applicable descriptors (eg, “more frequent headaches”). 


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11.2.5. 
Abnormal Laboratory Values 
Abnormal laboratory tests should be repeated as soon as possible for confirmation. Not every 
laboratory abnormality qualifies as an AE. A laboratory test result should be reported as an AE if 
it meets any of the following criteria: 
a. Accompanied by clinical symptoms 
b. Results in a change in study drug (eg, dosage modification, treatment interruption, or 
treatment discontinuation) 
c. Results in a medical intervention (eg, potassium supplementation for hypokalemia) or 
a change in concomitant therapy 
d. Clinically significant in the Investigator’s judgment 
It is the Investigator’s responsibility to review all laboratory findings. Medical and scientific 
judgment should be exercised in deciding whether an isolated laboratory abnormality should be 
classified as an AE. 
If a clinically significant laboratory abnormality is a sign of a disease or syndrome, only the 
diagnosis should be recorded on the Adverse Event eCRF. 
If a clinically significant laboratory abnormality is not a sign of a disease or syndrome, the 
abnormality itself should be recorded on the Adverse Event eCRF, along with a descriptor 
indicating if the test result is above or below the normal range. 
11.2.6. 
Adverse Events of Special Interest 
An AESI can be serious or nonserious. Ongoing monitoring and rapid communication (within 
24 hours) by the Investigator to the Sponsor is required to allow for further characterization and 
reporting to regulatory authorities. 
11.2.6.1. Transaminase Increase 
Transaminase increase is an AESI for AG-348. In the event of a transaminase increase of 
>2.5 × baseline (defined as the mean of the Screening and Day 1 values) or an increase in AST 
or ALT to Grade ≥2 in severity, whichever is lower. The study site should report this occurrence 
to the Sponsor, using the AESI page in the eCRF, within 24 hours of their first knowledge of the 
event.  
An LFT panel should then be performed weekly until the transaminases have decreased to 
<2.5 × baseline (defined as the average of the Screening and Day 1 values). Additionally, the 
following tests should be performed to gain further information on the possible cause of the 
transaminase increase: 
1. Rule out biliary obstruction by liver imaging (liver CT scan, liver MRI, liver ultrasound, 
or magnetic resonance cholangiopancreatography, as clinically indicated).  
2. Viral screen for Epstein-Barr virus (EBV) Abs, cytomegalovirus (CMV) Abs, Hepatitis A 
Ab, HBsAg, HCVAb (with an RT-PCR test performed if HCVAb is positive), HIV-1Ab, 
and HIV-2Ab using the central laboratory. 
3. Autoimmune hepatitis panel consisting of the following: serum antinuclear antibody, 
antismooth muscle antibody, liver-kidney microsomal type 1 antibody, antibody to 


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soluble liver antigen, and antimitochondrial antibody when transaminase increase meets 
the criteria of AESI and repeated 4 weeks later using the central laboratory if the results 
were negative in the first time.  
The Investigator should refer to Section 9.3 to determine if a dose adjustment is needed. If the 
Investigator is not sure whether or not a dose adjustment is needed, they should consult with the 
Medical Monitor (or designee). 
11.3. 
Procedures for Reporting Adverse Events, Adverse Events of Special 
Interest, and Serious Adverse Events 
Each subject must be carefully monitored for the development of any AEs. This information 
should be obtained in the form of nonleading questions (eg, “How are you feeling?”) and from 
signs and symptoms detected during each examination, observations of study personnel, and 
spontaneous reports from subjects. 
The Investigator should ask the subject for information regarding sleep patterns, signs, and 
symptoms associated with insomnia. 
All AEs (serious and nonserious) spontaneously reported by the subject and/or in response to an 
open question from study personnel or revealed by observation, physical examination, or other 
diagnostic procedures will be recorded on the appropriate page of the eCRF. Any clinically 
relevant deterioration in laboratory assessments or other clinical finding is considered an AE and 
must be recorded on the appropriate pages of the eCRF. When possible, signs and symptoms 
indicating a common underlying pathology should be noted as 1 comprehensive event. 
Any deaths and any AEs assessed as life threatening are to be reported immediately. All SAEs 
are to be reported within 24 hours from the point in time when the Investigator becomes aware of 
the SAE on the appropriate page of the eCRF. All SAEs must be reported whether or not they are 
considered causally related to the study drug. 
In the event that the electronic data capture (EDC) system is unavailable, a paper SAE and fax 
coversheet should be completed and faxed/emailed to the Sponsor within no more than 24 hours 
after learning of the event using the contact details provided to Investigators in the Serious 
Adverse Event Report Form Completion Guidelines. 
Excessive Hb responses should only be reported as an AE if they meet the criteria for Hb 
increased per CTCAE (ie, Hb concentration is higher than the subject’s gender-specific ULN). 
Any reports of excessive Hb response should be graded using the CTCAE grading system. 
If there are serious, unexpected adverse drug reactions associated with the use of AG-348, the 
Sponsor will notify the appropriate regulatory agency(ies) and all participating Investigators on 
an expedited basis. The local IRB/IEC will be promptly notified based on local regulations 
where required by the IRB/IEC of all serious, unexpected adverse drug reactions involving risk 
to human subjects. 
11.3.1. 
Intensity 
The intensity of all AEs will be graded according to the NCI CTCAE. It is important to 
distinguish between SAEs and AEs with a severe intensity. An AE of severe intensity may not be 
considered serious. Severity is a measure of intensity, whereas seriousness is defined by the 


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criteria in Section 11.2.2. For example, a severe headache without any further findings would not 
be considered an SAE. Alternatively, a mild presentation of a serious event such as a myocardial 
infarction assessed as mild by a cardiologist that leads to hospitalization would be considered an 
SAE. 
Severity of all AEs, including clinically significant treatment-emergent laboratory abnormalities, 
will be graded according to the NCI CTCAE v4.03. Adverse events not listed by the CTCAE 
will be graded as follows: 
• Mild (Grade 1): The event is noticeable to the subject but does not interfere with 
routine activity. 
• Moderate (Grade 2): The event interferes with routine activity but responds to 
symptomatic therapy or rest. 
• Severe (Grade 3): The event significantly limits the subject’s ability to perform 
routine activities despite symptomatic therapy. 
• Life threatening (Grade 4): An event in which the subject is at risk of death at the 
time of the event. 
• Fatal (Grade 5): An event that results in the death of the subject. 
11.3.2. 
Relationship to Study Drug 
Relationship to study drug administration will be determined by the Investigator according to the 
following criteria: 
• Not Related: AEs will be considered related unless they fulfill the criteria as specified 
below:  
o Evidence exists that the AE has an etiology other than the study drug 
(eg, pre-existing medical condition, underlying disease, intercurrent illness, 
concomitant medication); and/or  
o The AE has no plausible temporal relationship to the administration of the study 
drug (eg, cancer diagnosed 2 days after the first dose of study drug). 
• Related: AEs will be considered related if they fulfill the criteria as specified below: 
o There is a plausible temporal relationship between the onset of the AE and 
administration of the study drug, and the AE cannot be readily explained by the 
subject’s clinical state, intercurrent illness, or concomitant therapies; and/or 
o The AE follows a known pattern of response to the study drug; and/or 
o The AE abates or resolves upon discontinuation of the study drug or dose 
reduction and, if applicable, reappears upon re-challenge. 
11.4. 
Pregnancy Reporting 
Pregnancy is neither an AE nor an SAE, unless a complication relating to the pregnancy occurs 
(eg, spontaneous abortion, which may qualify as an SAE). However, any pregnancy in a 
participating female subject that occurs during this study or within 28 days following the last 


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dose of AG-348 must be reported to the Sponsor or Medical Monitor (or designee) within 
24 hours of being notified of the pregnancy. Any pregnancy in a female sexual partner of a 
participating male subject, that occurs during this study or within 90 days following the last dose 
of AG-348, must be reported to the Sponsor or Medical Monitor (or designee) within 24 hours of 
being notified of the pregnancy, if acceptable by practice and allowed by local regulations. If the 
pregnancy in a female sexual partner of a participating male subject occurs after the male subject 
has completed the study, the pregnancy must be reported to the Sponsor directly by the 
Investigator.  
The Investigator must follow up and document the course and outcome of all pregnancies even if 
the subject was discontinued from the study or if the study has finished. The female subject or 
female sexual partner of a male subject should receive any necessary counseling regarding the 
risks of continuing the pregnancy and the possible effects on the fetus. Monitoring should 
continue until conclusion of the pregnancy. 
All outcomes of pregnancy in a female study participant or consented female sexual partner of a 
male participant must be reported by the Investigator to the Sponsor or Sponsor’s designee on a 
Pregnancy Outcome Report form within 28 days after he/she has gained knowledge of the 
delivery or elective abortion. 
Any SAE that occurs during pregnancy in a female study participant or consented female sexual 
partner of a male participant must be recorded on the SAE report form (eg, maternal serious 
complications, spontaneous or therapeutic abortion, ectopic pregnancy, stillbirth, neonatal death, 
congenital anomaly, or birth defect) and reported within 24 hours in accordance with the 
procedure for reporting SAEs. 
Women of reproductive potential as well as men with partners who are women of reproductive 
potential must agree to be abstinent as part of their usual lifestyle or agree to use 2 forms of 
contraception, 1 of which must be considered highly effective, from the time of giving informed 
consent, during the study, and for 28 days following the last dose of study drug for women and 
90 days following the last dose of study drug for men. Periodic abstinence (eg, calendar, 
symptothermal, and post-ovulation methods) and withdrawal are not acceptable methods of 
contraception. 


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12. 
STATISTICAL METHODS 
12.1. 
Sample Size and Power 
Due to the rarity of the disease and the small patient population, the sample size is largely driven 
by feasibility. Given the feasibility results and uncertainty of the screen failure rate, the study 
will enroll a minimum of 20, with up to 40, subjects. With a sample size of 20, the power of the 
study is limited, that is, the power will be 58% to detect a response rate of 30% compared to a 
null rate of 10% with an exact test at 2-sided 0.05 significance level. There will be 75% power to 
detect a large response rate of 35%, compared to the null of 10%. When the sample size is 40, 
the power will be at least 90%, even if the target response rate is 30%. The power for different 
scenarios is listed in Table 6. 
Table 6: 
Power Calculation 
Sample Size 
Target Response Rate (null=10%) 
25% 
30% 
35% 
20 
0.38 
0.58 
0.75 
30 
0.48 
0.71 
0.87 
40 
0.71 
0.90 
0.97 
The probability of observing at least 1 AE based on different true incidence rate and sample sizes 
is displayed in Table 7. 
Table 7: 
Probability of Observing ≥1 Adverse Event Based on True Incidence Rate 
and Sample Size 
Sample Size 
AE Incidence 
5% 
10% 
20 
64.2% 
87.8% 
30 
78.5% 
95.8% 
40 
87.1% 
98.5% 
Abbreviation: AE = adverse event. 
12.2. 
Analysis Sets 
Assignment of subjects to analysis sets will be done before the clinical database lock for the 
study. All Subjects will refer to all subjects who were enrolled or dosed (ie, all subjects in the 
study). 
Two analysis sets will be defined for evaluation of the study endpoints: Full Analysis Set (FAS) 
and Per Protocol Set (PPS). 
12.2.1. 
Full Analysis Set 
The FAS is defined as all subjects who received at least 1 dose of study drug. 


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The FAS will be used for primary efficacy analyses and safety analyses unless specified 
otherwise. Additional information may be further provided by the optimized dose. 
12.2.2. 
Per Protocol Set 
The PPS is defined as a subset of the FAS including subjects who have completed Part 2.  
The PPS will be used for sensitivity analysis as specified in Section 12.3.4.1.  
12.3. 
Statistical Analysis 
This section presents a summary of the planned primary analyses of efficacy and safety for this 
study. Additional supportive and exploratory analyses will be specified in the final statistical 
analysis plan (SAP), which will be finalized before the database lock. 
12.3.1. 
General Methods 
All individual subject data for all subjects will be presented in data listings. 
Continuous variables will be summarized using the following descriptive summary statistics: 
the number of subjects (n), mean, standard deviation (SD), median, minimum value (min), and 
maximum value (max).  
Categorical variables will be summarized using counts and percentages.  
Baseline value: Unless otherwise specified, the baseline value will be defined as the most recent 
nonmissing measurement (scheduled or unscheduled) collected before the initial administration 
of study drug.  
• For hematology parameters, the most recent nonmissing measurements prior to 
Transfusion 0 (the most recent transfusion occurring in the Screening Period prior to 
the start of study drug on Day 1) will be defined as the baseline. This is to limit the 
impact of transfusion on these hematology laboratory parameters. 
• Historical transfusion frequency will be based on the historical data during the 
52 weeks prior to screening. Details are specified in Section 12.3.2.  
• For transaminases (AST and ALT), the baseline will be defined as the average of the 
assessment of the Screening and Day 1 values. 
Change (Absolute Change) from baseline will be calculated as postbaseline value - baseline 
value. 
Relative change from baseline will be calculated and presented in percentage as 
100 × (postbaseline value - baseline value) / baseline value. 
Treatment-emergent period is defined as from the first dose of study drug to 28 days after the 
last dose of study drug. 
12.3.2. 
Background Characteristics 
Subject disposition, demographic and baseline characteristics, prior and concomitant 
medications, and other background characteristics will be summarized. Additionally, all subject 


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data will be presented in subject data listings. All summaries will be based on the FAS unless 
otherwise specified in the SAP.  
12.3.2.1. Subject Disposition 
Number and percentage of subjects in the following categories will be summarized as 
appropriate: 
• Enrolled 
• Dosed (FAS) 
• Completed treatment in Part 1 
• Prematurely discontinued treatment in Part 1 and the reasons for discontinuation 
• Completed treatment in Part 2 (PPS) 
• Prematurely discontinued treatment in Part 2 and the reasons for discontinuation 
• Completed study 
• Prematurely discontinued the study and the reasons for discontinuation 
12.3.2.2. Demographics and Baseline Characteristics 
Demographic, background (eg, medical history), and baseline characteristics will be 
summarized. The demographics, baseline characteristics, and medical history summary will be 
presented for the FAS. 
12.3.2.3. Prior and Concomitant Medications 
Medications used in this study will be coded using the World Health Organization Drug 
Dictionary Enhanced and categorized as the following:  
• Prior medication: any medication that started before initial dosing of study drug, 
regardless of when it ended 
• Concomitant medication: medication continued or newly received during the 
treatment-emergent period 
• Post-treatment medication: medication continued or newly received after the 
treatment-emergent period, if the subject is still enrolled in the study 
A given medication can be classified as a prior medication, a concomitant medication, or a 
post-treatment medication; both prior and concomitant; both concomitant and post-treatment; or 
prior, concomitant, and post-treatment. If a medication has a missing or partially missing 
start/end date or time and it cannot be determined whether it was taken before, during, or after 
the treatment-emergent period, it will be considered as prior, concomitant, and post-treatment 
medications. 
Prior medications and concomitant medications will be summarized descriptively based on the 
FAS. Post-treatment medications will be listed for each subject.  


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12.3.3. 
Study Drug Exposure and Compliance 
12.3.3.1. Study Drug Exposure 
Duration of study drug exposure is defined as follows: last dose date − first dose date + 1 day, 
regardless of any interruptions in dosing. If the last dose date of study drug is missing, the 
subject’s treatment discontinuation or completion date will be used for analysis purposes. 
Duration of study drug exposure will be summarized descriptively as a quantitative variable (n, 
mean, SD, median, min, and max). 
Exposure summaries will be based on FAS. 
12.3.3.2. Study Drug Compliance 
Study drug compliance will be assessed by percentage of days on treatment and percentage of 
tablets taken. The percentage of days on treatment will be calculated as follows:  
100 × total number of single days on study drug / (duration of study drug exposure + total 
number of days study drug interrupted after last dose, if any) 
Single day on study drug is collected from the subject’s diary data. If it is unknown whether the 
subject received treatment on a specified day, then it will be assumed that no treatment was taken 
on that day. 
Percentage of tablets taken will be calculated as follows: 
100 × (total number of tablets administered) / (total number of tablets intended)  
Both percentage of days on treatment and percentage of tablets taken will be summarized 
descriptively as quantitative variables (n, mean, SD, median, min, and max). The number and 
percentage of subjects whose compliance is <80% or ≥80% will be summarized. 
Study drug compliance will be based on the FAS.  
12.3.4. 
Efficacy Analysis 
12.3.4.1. Analysis of Transfusion Reduction 
Historical transfusion RBC units standardized to 24 weeks will be calculated as the total number 
of transfusion RBC units over the 52 weeks prior to the informed consent × 24/52. The Part 2 
on-study 24-week transfusion RBC units will be calculated as the total number of transfused 
RBC units received from the first day in Part 2 until the last full dose before starting the dose 
taper (if applicable) in Part 2 divided by corresponding number of days × 168 (24 weeks). 
Both the historical transfusion RBC units standardized to 24 weeks and on-study 24-week 
transfusion RBC units will be summarized using descriptive statistics (n, mean, SD, median, 
min, and max). The change in transfusion RBC units (calculated as the on-study 24-week 
transfusion RBC units – historical transfusion RBC units standardized to 24 weeks) and the 
percentage change (calculated as [on-study 24-week transfusion RBC units – historical 
transfusion RBC units standardized to 24 weeks] / historical transfusion RBC units standardized 
to 24 weeks) will also be summarized based on the FAS. In addition, the number and percentage 


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of subjects with different categories (<0, ≥0 to <20%, ≥20% to <33%, ≥33% to 50%, ≥50%) of 
percentage change will also be provided. 
Primary Analysis 
The number and proportion of subjects who achieve a transfusion reduction response (TRR), 
defined as ≥33% reduction in the number of RBC units transfused during the 24 weeks in Part 2 
compared to the historical transfusion burden standardized to 24 weeks (Standardized Control 
Period), along with its 95% CI based on the exact binomial distribution, will be provided based 
on the FAS. Subjects who completed at least 12 weeks of treatment in Part 2 will be evaluated 
for their transfusion reduction status based on change in transfusion RBC units. Subjects who 
discontinue the study before completing 12 weeks of treatment in Part 2 will be considered 
nonresponders. Given the open-label, single-arm nature of the study and limited power of the 
study, no formal hypothesis test will be conducted. The focus will be given to descriptive 
statistics, including both point estimate and the CIs. 
The similar set of analyses will be repeated based on PPS to further evaluate the impact of early 
discontinuation on the efficacy results. Details will be provided in the final SAP. 
12.3.4.2. Analysis of Other Secondary Efficacy Endpoints 
Annualized total number of RBC units transfused during the study compared with the 
historical transfusion burden 
Annualized total number of RBC units transfused during the study (both Part 1 and Part 2) will 
be calculated as the total number of RBC units transfused during the study divided by the total 
number of days from the first dose date until the last day on study × 364. 
Both the historical transfusion RBC units and on-study annualized transfusion RBC units will be 
summarized using descriptive statistics (n, mean, SD, median, min, and max). The change in 
annualized transfusion RBC units and the percentage change will also be summarized based on 
the FAS. The number and percentage of subjects with different categories of percentage change 
will also be provided. 
Number of transfusion episodes 
Number of transfusion episodes during the Standardized Control Period (24 weeks), and number 
of transfusion episodes during Part 2 (standardized to 24 weeks), their change and percentage 
change will be summarized using descriptive statistics. Transfusions received over up to 
3 consecutive days will be considered as a single transfusion event. 
Proportion of subjects transfusion free 
Number and percentage of subjects who are transfusion free in the Fixed-Dose Period (Part 2), 
along with its 95% CI based on the exact binomial distribution, will be provided. 
Number of subjects with normal Hb  
Number and percentage of subjects who achieve Hb concentrations in the normal range at least 
once, 8 weeks or more after a transfusion in Part 2, along with its 95% CI based on the exact 
binomial distribution, will be provided. 


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Additional sensitivity analysis, supportive analysis (including additional modeling of the 
secondary endpoints), and analyses for exploratory endpoints and possibly additional endpoints 
will be described in the SAP. 
12.3.5. 
Safety Analysis 
The overall safety profile of study drug will be assessed in terms of the following safety and 
tolerability endpoints: 
• Incidence of treatment-emergent adverse events (TEAEs) 
• Clinical laboratory values 
• ECGs (standard 12-lead) 
• Vital signs 
• Physical examination findings 
• DXA scans 
Safety endpoints will be analyzed based on the FAS. Only descriptive analysis of safety will be 
performed (ie, no formal testing will be performed). 
12.3.5.1. Adverse Events 
For analysis purposes, AEs will be classified as pretreatment AEs, TEAEs, or 
post-treatment AEs.  
• Pretreatment AE: any AE that started before initial dosing of study drug. 
• TEAE: any AE that increased in severity or that was newly developed during the 
treatment-emergent period. 
• Post-treatment AE: any AE that increased in severity or that was newly developed 
after 28 days after the last dose of study drug. 
For AEs with missing or partial start dates, if there is no clear evidence that the AEs started 
before or after study drug, then the AEs will be classified as TEAEs. 
The AE analysis will be provided for Part 1 and Part 2 separately as well as combining Part 1 
and Part 2. Adverse events summary tables will be presented for TEAEs only and will include 
the following: 
• All TEAEs  
• TEAEs by relationship  
• TEAEs by maximal severity 
• TEAEs leading to treatment discontinuation 
• TEAEs leading to treatment interruption 
• Serious TEAEs 
• TEAEs leading to death 


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Summaries will be presented by Medical Dictionary for Regulatory Activities system organ class 
and preferred term using frequency counts and percentages (ie, number and percentage of 
subjects with an event as well as total number of events). When summarizing the number and 
percentage of subjects with an event, subjects with multiple occurrences of the same AE or a 
continuing AE will be counted once, only the maximum severity level will be presented in the 
severity summaries, and the worst/highest relationship level in the relationship summaries. A 
separate table will summarize all TEAEs when each of them is considered unique, hereafter 
referred to as an AE count table.  
In addition, listings containing individual subject data for all TEAEs leading to treatment 
discontinuation, TEAEs leading to treatment interruption, deaths, and serious AEs will be 
provided separately. All AEs, including pre- and post-treatment AEs, will be presented in 
individual subject data listings. 
12.3.5.2. Clinical Laboratory Assessments 
For laboratory measurements, the raw values and change from baseline values of the continuous 
laboratory results will be summarized at each scheduled time point.  
For selected laboratory parameters, the number and percentage of subjects with shift changes 
from baseline (normal/missing, high, and low according to the reference range) to 
the highest/lowest laboratory evaluation during the treatment-emergent period will be tabulated. 
The details will be provided in the SAP. 
The number and percentage of subjects with transaminase increases of >2.5 × baseline or those 
that have increased to ≥Grade 2 (AESI of elevated transaminase as defined in Section 11.2.6.1) 
will be summarized. 
Results of urinalysis and the serum and urine pregnancy tests will be listed in individual subject 
data listings only. In addition, a listing containing individual subject hematology, chemistry, and 
coagulation values outside the reference ranges will be provided. This listing will include data 
from scheduled and unscheduled time points. 
12.3.5.3. Electrocardiogram 
For ECG measurements, a summary of raw values and change from baseline values will be 
provided at each scheduled time point for the following standard digital ECG measurements: PR, 
QRS, QT, and QT corrected for heart rate intervals (QTcB and QTcF), and heart rate. 
The number and percentage of subjects with shift changes from baseline (normal/missing, 
clinically insignificant, and potentially clinically significant) to the worst ECG evaluation during 
the TEAE period will be tabulated.  
12.3.5.4. Vital Signs 
For vital signs measurements, the raw values and change from baseline values will be 
summarized at each scheduled time point: systolic and diastolic BP (mm Hg), body temperature 
(°C), and heart rate (beats per minute).  
12.3.5.5. Physical Examination 
Physical examination findings will be presented as a data listing only. 


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12.3.5.6. Dual-Energy X-ray Absorptiometry Scans 
For DXA scans, the raw values of BMD, T- and Z-scores, and change from baseline values will 
be summarized for each scanned area and at each scheduled time point.  
12.4. 
Interim and Independent Data Monitoring Committee Analyses 
12.4.1. 
Interim Analysis 
No interim analysis is planned, but interim analyses may take place at any time during the study 
if warranted by the ongoing data and/or deemed necessary by the internal Sponsor team. 
12.4.2. 
Independent Data Monitoring Committee Analysis 
No independent data monitoring committee is planned for this study. 
 


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13. 
ADMINISTRATIVE REQUIREMENTS 
13.1. 
Good Clinical Practices 
The study will be conducted in accordance with the International Council for Harmonisation 
(ICH) for Good Clinical Practice (GCP) and the appropriate regulatory requirement(s). The 
Investigator will be thoroughly familiar with the appropriate use of the study drug as described in 
the protocol and the IB. Essential clinical documents will be maintained to demonstrate the 
validity of the study and the integrity of the data collected. Master files should be established at 
the beginning of the study, maintained for the duration of the study, and retained according to the 
appropriate regulations.  
13.2. 
Ethical Considerations 
The study will be conducted in accordance with ethical principles founded in the Declaration of 
Helsinki. 
The Investigator must obtain IRB/IEC approval for the investigation and must submit written 
documentation of the approval to the Sponsor before he/she can enroll any subject into the study. 
The IRB/IEC will review all appropriate study documentation in order to safeguard the rights, 
safety, and well-being of the subjects. The study will be conducted only at sites where IRB/IEC 
approval has been obtained. The protocol, IB, ICF, advertisements (if applicable), written 
information given to the subjects, safety updates, annual progress reports, and any revisions to 
these documents will be provided to the IRB/IEC.  
The IRB/IEC is to be notified of any amendment to the protocol in accordance with local 
requirements. Progress reports and notifications of serious unexpected adverse drug reactions are 
to be provided to the IRB/IEC according to local regulations and guidelines. 
13.3. 
Subject Information and Informed Consent 
The Investigator or trained designee will ensure that the subject is given full and adequate oral 
and written information about the nature, purpose, possible risk, and benefit of the study. 
Subjects must also be notified that they are free to discontinue from the study at any time. The 
subject should be given the opportunity to ask questions and allowed time to consider the 
information provided. 
After the study has been fully explained, written informed consent will be obtained from the 
subject prior to study participation.  
The subject’s signed and dated informed consent must be obtained before conducting any 
study-related procedures. The Investigator must maintain the original, signed consent form. A 
copy of the signed form must be given to the subject. 
The method of obtaining and documenting the informed consent and the contents of the consent 
will comply with ICH-GCP and all applicable regulatory requirement(s). 
13.4. 
Subject Confidentiality 
In order to maintain subject privacy, all source documents, study drug accountability records, 
study reports, and communications will identify the subject by the assigned subject number. The 


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Investigator will grant monitor(s) and auditor(s) from the Sponsor or the Sponsor’s designee and 
regulatory authority(ies) access to the subject’s original medical records for verification of data 
gathered on the source documents and to audit the data collection process. The subject’s 
confidentiality will be maintained and will not be made publicly available to the extent permitted 
by the applicable laws and regulations. 
13.5. 
Protocol Compliance 
The Investigator will conduct the study in compliance with the protocol. Modifications to the 
protocol should not be made without agreement of both the Investigator and the Sponsor. 
Changes to the protocol will require written IRB/IEC approval/favorable opinion prior to 
implementation, except when the modification is needed to eliminate an immediate hazard(s) to 
subjects. The IRB/IEC may provide, if applicable, where regulatory authority(ies) permit, 
expedited review and approval/favorable opinion for minor change(s) in ongoing studies that 
have the approval/favorable opinion of the IRB/IEC. The Sponsor or designee will submit all 
protocol modifications to the regulatory authority(ies) in accordance with the governing 
regulations.  
When immediate deviation from the protocol is required to eliminate an immediate hazard(s) to 
subjects, the Investigator will contact the Medical Monitor (or Medical Director), if 
circumstances permit, to discuss the planned course of action. Any departures from the protocol 
must be fully documented in the source documents/database. 
13.6. 
Data Management 
All data for the subjects recruited for the trial will be entered onto the eCRFs via an EDC system 
provided by the Sponsor or designee. Only authorized staff may enter data onto the eCRFs. If an 
entry error is made, the corrections to the eCRFs will be made according to eCRF guidelines by 
an authorized member of the site staff. 
Electronic case report forms will be checked for correctness against source document data by the 
Sponsor’s Monitor. If any entries into the eCRF are incorrect or incomplete, the monitor will ask 
the Investigator or the study site staff to make appropriate corrections, and the corrected eCRF 
will again be reviewed for completeness and consistency. Any discrepancies will be noted in the 
eCRF system by means of electronic data queries. Authorized site staff will be asked to respond 
to all electronic queries according to the eCRF guidelines. 
13.7. 
Source Documentation and Electronic Case Report Form 
Completion 
Source documents will be completed for each study subject. It is the Investigator’s responsibility 
to ensure the accuracy, completeness, and timeliness of the data reported in the subject’s source 
document/eCRF. The source document should indicate the subject’s participation in the study 
and should document the dates and details of study procedures, AEs, and subject status. 
The Investigator or designated representative should complete the source document as soon as 
possible after information is collected for a subject’s examination, treatment, or any other study 
procedure. Any outstanding entries must be completed after the final examination. An 


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explanation should be given for all missing data. The Investigator will retain all completed 
source documents. 
13.8. 
Direct Access to Source Data 
The study will be monitored by the Sponsor or the Sponsor’s designee. Monitoring will be 
performed by personal visits from a representative of the Sponsor (Site Monitor) and will include 
on-site review of the source documents for completeness and clarity, cross-checking with source 
documents, and clarification of administrative matters will be performed. The review of medical 
records will be performed in a manner to ensure that subject confidentiality is maintained. 
The Site Monitor will ensure that the investigation is conducted according to protocol design and 
regulatory requirements by frequent communications (letter, telephone, email, and fax). 
All unused study drug and other study materials should be destroyed or returned to the Sponsor 
or designee after the study has been completed, as directed by the Sponsor. 
Regulatory authorities, the IRB/IEC, and/or the Sponsor’s clinical quality assurance group or 
designee may request access to all source documents, database, and any other applicable study 
documentation for an on-site audit or inspection. Direct access to these documents must be 
guaranteed by the Investigator, who must provide support at all times for these activities.  
13.9. 
Record Retention 
The Investigator will maintain all study records according to ICH-GCP and applicable regulatory 
requirement(s). Records will be retained for at least 2 years after the last marketing application 
approval or 2 years after formal discontinuation of the clinical development of the investigational 
product or according to applicable regulatory requirement(s). If the Investigator withdraws from 
the responsibility of keeping the study records, custody must be transferred to a person willing to 
accept the responsibility. The Sponsor must be notified in writing if a custodial change occurs. 
13.10. 
Liability and Insurance 
The Sponsor has subscribed to an insurance policy covering, in its terms and provisions, its legal 
liability for injuries caused to participating persons and arising out of this research performed 
strictly in accordance with the scientific protocol as well as with applicable law and professional 
standards. 
13.11. 
Publication of Study Findings and Use of Information 
All information regarding AG-348 supplied by the Sponsor or designee to the Investigator is 
privileged and confidential information. The Investigator agrees to use this information only to 
conduct the study and not to use it for any other purpose without explicit consent from the 
Sponsor. 
It is understood that there is an obligation on the Investigator’s part to provide the Sponsor with 
the complete data obtained during the study. Such information will be used in the clinical 
development of AG-348 and may be disclosed to regulatory authorities, other Investigators, 
corporate partners, or consultants, as required. 


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14. 
REFERENCES 
Aizawa S, Kohdera U, Hiramoto M, et al. Ineffective erythropoiesis in the spleen of a patient 
with pyruvate kinase deficiency. Am J Hematol. 2003;74(1):68-72. 
 
Beutler E, Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene 
frequency in the general white population. Blood. 2000;95(11):3585-3588. 
 
Bianchi P, Fermo E, Lezon-Geyda K, et al. Molecular characterization of 140 patients in the 
Pyruvate Kinase Deficiency (PKD) Natural History Study (NHS): Report of 20 new variants.  
American Society of Hematology (ASH) 57th Annual Meeting & Exposition; 05-08 December, 
2015, 2015; Orlando, FL. 
 
Bianchi P, Fermo E, Lezon-Geyda K, et al. Genotype-Phenotype Correlation and Molecular 
Heterogeneity in Pyruvate Kinase Deficiency: Data from the PKD Natural History Study.  59th 
American Society of Hematology Annual Meeting2017; San Diego, CA, USA. 
 
Carey PJ, Chandler J, Hendrick A, et al. Prevalence of pyruvate kinase deficiency in northern 
European population in the north of England. Northern Region Haematologists Group. Blood. 
2000;96(12):4005-4006. 
 
ClinicalTrials.gov [Internet]. Boston Children's Hospital; Pyruvate Kinase Deficiency Natural 
History Study (PK Deficiency NHS). Agios Pharmaceuticals, Inc; 
2017. https://clinicaltrials.gov/ct2/show/NCT02053480. Accessed 21 March, 2017. 
 
de Medicis E, Ross P, Friedman R, et al. Hereditary nonspherocytic hemolytic anemia due to 
pyruvate kinase deficiency: a prevalence study in Quebec (Canada). Hum Hered. 
1992;42(3):179-183. 
 
Grace R, Holmes M, Barcellini W, et al. The Phenotypic Spectrum of Pyruvate Kinase 
Deficiency (PK deficiency) from the PK deficiency Natural History Study: Description of Four 
Severity Groups by Anemia Status.  American Society for Hematology 57th Annual Meeting & 
Exposition2015; Orlando, FL. 
 
Grace RF, Bianchi P, van Beers EJ, et al. Clinical spectrum of pyruvate kinase deficiency: data 
from the Pyruvate Kinase Deficiency Natural History Study. Blood. 2018;131(20):2183-2192. 
 
Grace RF, Layton DM, Galacteros F, et al. Result Update from the DRIVE PK Study: Effects of 
AG-348, a pyruvate kinase activator, in patients with pyruvate kinase deficiency: Updated results 
from the DRIVE PK study [Abstract 2194]. American Society of Hematology (ASH) 59th Annual 
Meeting & Exposition; 9 December, 2017; Atlanta, GA. 
 
Grace RF, Rose C, Layton DM, et al. Effects of AG-348, a Pyruvate Kinase Activator, on Anemia 
and Hemolysis in Patients with Pyruvate Kinase Deficiency: Data from the DRIVE PK Study.  
21st European Hematology Association2016; Copenhagen, Denmark. 
 


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Hirono A, Kanno H, Miwa S, Beutler E. Chapter 182 Pyruvate Kinase Deficiency and Other 
Enzymopathies of the Erythrocyte. New York, NY: McGraw Hill; 2014. 
 
Moscicki RA, Tandon PK. Drug-Development Challenges for Small Biopharmaceutical 
Companies. N Engl J Med. 2017;376(5):469-474. 
 
Oski FA, Bowman H. A low Km phosphoenolpyruvate mutant in the Amish with red cell 
pyruvate kinase deficiency. Br J Haematol. 1969;17(3):289-297. 
 
Rider NL, Strauss KA, Brown K, et al. Erythrocyte pyruvate kinase deficiency in an old-order 
Amish cohort: longitudinal risk and disease management. Am J Hematol. 2011;86(10):827-834. 
 
Tanaka KR, Valentine WN, Miwa S. Pyruvate kinase (PK) deficiency hereditary nonspherocytic 
hemolytic anemia. Blood. 1962;19:267-295. 
 
van Wijk R, van Solinge WW. The energy-less red blood cell is lost: erythrocyte enzyme 
abnormalities of glycolysis. Blood. 2005;106(13):4034-4042. 
 
Zanella A, Fermo E, Bianchi P, Chiarelli LR, Valentini G. Pyruvate kinase deficiency: the 
genotype-phenotype association. Blood Rev. 2007;21(4):217-231. 
 
Zanella A, Fermo E, Bianchi P, Chiarelli LR, Valentini G. Pyruvate kinase deficiency: the 
genotype-phenotype association. Blood Reviews. 2007;21(4):217-231. 
 
Zanella A, Fermo E, Bianchi P, Valentini G. Red cell pyruvate kinase deficiency: molecular and 
clinical aspects. Br J Haematol. 2005;130(1):11-25. 
 

SAP_001.pdf: 
 
Official Title: An Open-Label Study To Evaluate the Efficacy and Safety of AG-348 
in Regularly Transfused Adult Subjects With Pyruvate Kinase (PK) 
Deficiency 
NCT Number: NCT03559699 
Document Date(s): SAP Version 3.0 – 25 September 2020 
 
 


Study AG348-C-007 
 
Statistical Analysis Plan (3.0) 
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STATISTICAL ANALYSIS PLAN 
An Open-Label Study to Evaluate the Efficacy and Safety of AG-348 in Regularly 
Transfused Adult Subjects with Pyruvate Kinase (PK) Deficiency 
 
 
AG348-C-007  
 
 
 
 
 
 
 
 
 
Version: 3.0 
Date: 25-Sep-2020  
 
 
 
 
 
CONFIDENTIALITY NOTE: 
The information contained in this document is confidential and proprietary to Agios Pharmaceuticals, Inc. 
Any distribution, copying, or disclosure is strictly prohibited unless such disclosure is required by federal 
regulations or state law. Persons to whom the information is disclosed must know that it is confidential and that 
it may not be further disclosed by them. 
 
DocuSign Envelope ID: 699AC215-548C-41A9-AAC2-332914A51AAF




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Statistical Analysis Plan (3.0) 
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TABLE OF CONTENTS 
TABLE OF CONTENTS ............................................................................................ 3 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ............................. 6 
1. 
VERSION HISTORY .............................................................................. 8 
2. 
INTRODUCTION ................................................................................... 9 
3. 
TRIAL OBJECTIVES AND ENDPOINTS ............................................ 9 
3.1. 
Objectives ................................................................................................ 9 
3.1.1. 
Primary Objective .................................................................................... 9 
3.1.2. 
Secondary Objectives .............................................................................. 9 
3.1.3. 
Exploratory Objectives ............................................................................ 9 
3.2. 
Endpoints ............................................................................................... 10 
3.2.1. 
Primary Endpoint ................................................................................... 10 
3.2.2. 
Secondary Endpoints ............................................................................. 10 
3.2.3. 
Exploratory Endpoints ........................................................................... 10 
4. 
STUDY DESIGN .................................................................................. 11 
5. 
ANALYSIS DATA SETS ..................................................................... 12 
6. 
GENERAL STATISTICAL CONSIDERATIONS ............................... 12 
6.1. 
Randomization, Blinding, Unblinding, and Crossover .......................... 12 
6.2. 
Sample Size Determination and Decision Rules ................................... 12 
6.2.1. 
Sample Size Determination ................................................................... 12 
6.2.2. 
Decision Rules ....................................................................................... 13 
6.3. 
Definitions ............................................................................................. 13 
6.3.1. 
Study Drug and Study Treatment .......................................................... 13 
6.3.2. 
Start and End Dates of Study Drug and Study Treatment ..................... 13 
6.3.3. 
Study Day .............................................................................................. 13 
6.3.4. 
Baseline .................................................................................................. 13 
6.3.5. 
On-Treatment Period and Optimized Dose ........................................... 14 
6.4. 
General Methods .................................................................................... 15 
6.4.1. 
Data Handling After Cutoff Date .......................................................... 15 
6.4.2. 
Standard Derivations and Reporting Conventions ................................ 15 
6.4.3. 
Pooling of Data Across Sites ................................................................. 16 
6.4.4. 
Continuous and Categorical Variables .................................................. 16 
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6.4.5. 
Unscheduled Visits ................................................................................ 16 
6.5. 
Methods for Handling Missing Data ..................................................... 18 
6.5.1. 
Adverse Event and Concomitant Medication Start Dates ..................... 18 
6.5.2. 
Adverse Event and Concomitant Medication End Dates ...................... 18 
6.5.3. 
Exposure ................................................................................................ 19 
7. 
STATISTICAL ANALYSES ................................................................ 19 
7.1. 
Subject Disposition ................................................................................ 19 
7.2. 
Protocol Deviations ............................................................................... 20 
7.3. 
Demographic and Other Baseline Characteristics ................................. 20 
7.3.1. 
Demographics and Physical Measurements .......................................... 20 
7.3.2. 
Disease Characteristics .......................................................................... 21 
7.3.3. 
Medical History ..................................................................................... 22 
7.3.4. 
Prior Therapies ....................................................................................... 22 
7.4. 
Exposure to Study Drug and Compliance ............................................. 22 
7.4.1. 
Treatment Duration and Exposure ......................................................... 22 
7.4.2. 
Dose Modifications ................................................................................ 23 
7.5. 
Concomitant Therapies .......................................................................... 23 
7.6. 
Efficacy Analyses .................................................................................. 24 
7.6.1. 
Primary Endpoint ................................................................................... 24 
7.6.1.1. 
Primary Analyses ................................................................................... 24 
7.6.1.2. 
Sensitivity Analyses ............................................................................... 25 
7.6.2. 
Secondary Endpoints ............................................................................. 25 
7.6.2.1. 
Annualized total number of RBC units transfused during the study 
compared with the historical transfusion burden ................................... 25 
7.6.2.2. 
Number of transfusion episodes ............................................................ 25 
7.6.2.3. 
Transfusion-free ..................................................................................... 26 
7.6.2.4. 
Normal Hb concentrations ..................................................................... 26 
7.6.2.5. 
Transfusion trigger ................................................................................. 26 
7.6.3. 
Subgroup Analyses ................................................................................ 26 
7.7. 
Safety Analyses ..................................................................................... 27 
7.7.1. 
Adverse Events ...................................................................................... 27 
7.7.1.1. 
Adverse Events of Special Interest ........................................................ 28 
7.7.1.2. 
Adverse Events Associated with COVID-19 ........................................ 29 
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7.7.2. 
Death ...................................................................................................... 29 
7.7.3. 
Clinical Laboratory Data ....................................................................... 30 
7.7.3.1. 
Hematology ............................................................................................ 31 
7.7.3.2. 
Chemistry ............................................................................................... 31 
7.7.3.3. 
Sex Steroid Tests ................................................................................... 32 
7.7.3.4. 
Pregnancy Test ....................................................................................... 33 
7.7.4. 
Vital Signs and Physical Measurements ................................................ 33 
7.7.5. 
Electrocardiograms ................................................................................ 33 
7.7.6. 
DXA Scans ............................................................................................ 34 
7.7.7. 
Menstrual Cycle Diary ........................................................................... 34 
7.8. 
Exploratory Analyses ............................................................................. 34 
7.8.1. 
Markers of Hemolysis ............................................................................ 34 
7.8.2. 
Erythropoietic Activity Markers ............................................................ 34 
7.8.3. 
Iron Markers .......................................................................................... 35 
7.8.4. 
Liver Iron Concentration (LIC) by Magnetic Resonance Imaging 
(MRI) ..................................................................................................... 35 
7.8.5. 
HRQoL .................................................................................................. 35 
7.8.5.1. 
PKDD .................................................................................................... 35 
7.8.5.2. 
PKDIA ................................................................................................... 35 
7.8.5.3. 
EQ-5D-5L .............................................................................................. 36 
7.8.5.4. 
PGIS ....................................................................................................... 36 
7.9. 
Interim Analyses .................................................................................... 36 
8. 
REFERENCES ...................................................................................... 37 
 
LIST OF TABLES 
Table 1:  
Summary of Major Changes in Statistical Analysis Plan Amendments.. 8 
Table 2:  
Analysis Sets for Each Endpoint ........................................................... 12 
Table 3: 
Power Considerations ............................................................................ 13 
Table 4: 
Subgroup Analyses for TRR .................................................................. 27 
 
 
LIST OF FIGURES 
Figure 1: 
 Study Schema ....................................................................................... 11 
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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation 
Definition 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALP 
Alkaline phosphatase 
ALT 
Alanine aminotransferase 
AST 
Aspartate aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BID 
Twice daily 
BMI 
Body mass index 
CI 
Confidence interval 
CRF 
Case report form 
CSR 
Clinical study report 
CTCAE 
Common Terminology Criteria for Adverse Events 
DXA 
Dual-energy x-ray absorptiometry 
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EOS 
End of study 
EOT 
End of treatment 
EPO 
Erythropoietin 
EQ-5D-5L 
European quality of life five-dimensional descriptive system 
FAS 
Full Analysis Set 
Hb 
Hemoglobin 
HLT 
MedDRA High Level Term 
HRQoL 
Health-related quality of life 
IC 
Informed consent 
LDH 
Lactate dehydrogenase 
LFT 
Liver function test 
LIC 
Liver Iron Concentration 
LLN 
Lower limit of normal 
MedDRA 
Medical Dictionary for Regulatory Activities 
MRI 
Magnetic resonance imaging 
MTF 
Mean transfusion frequency 
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PD 
Pharmacodynamic 
PGIS 
Patient Global Impression of Severity 
PKD 
Pyruvate kinase deficiency 
PKDD 
Pyruvate kinase deficiency diary 
PKDIA 
Pyruvate kinase deficiency impact assessment 
PKR 
Pyruvate kinase isoform R 
PPS 
Per-Protocol Set 
PRO 
Patient-reported outcome 
PT 
Preferred Term 
QD 
Once daily 
QOD 
Every Other Day 
QTc 
Heart rate-corrected QT interval 
QTcB 
Heart rate-corrected QT interval using the Bazett’s formula 
QTcF 
Heart rate-corrected QT interval using the Fridericia’s formula 
RBC 
Red blood cell  
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SD 
Standard deviation 
SOC 
System Organ Class 
TEAE 
Treatment-emergent adverse event 
TIBC 
Total iron binding capacity 
TRR 
Transfusion reduction response 
TT 
Transfusion Trigger 
ULN 
Upper limit of normal 
WBC 
White blood cell 
WHO 
World Health Organization 
 
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1. 
VERSION HISTORY 
This statistical analysis plan (SAP) describes the analysis associated with protocol AG348-
C-007, Version 3.0 (dated 19-Mar-2019). 
Table 1:  
Summary of Major Changes in Statistical Analysis Plan Amendments 
Version 
Version Date 
Rationale and Summary of Changes 
1.0 
30-Apr-2018 
Original version. 
2.0 
21-Aug-2020 
Amendment rationale: to incorporate changes to the trial reflected in 
protocol amendments, to implement changes due to changes in data 
collection for the study. 
Major changes: 
1. Changes due to protocol amendment 1, protocol v2.0 (15 October 
2018) and protocol amendment 2, protocol v3.0 (19 March 2019) 
a. 
Updated sample size and power calculation. 
b. Added decision rule for hypothesis testing 
c. 
Updated the primary endpoint to be evaluated in the Fixed 
Dose Period only 
d. Updated the primary endpoint to consider subject as non-
responder if discontinued before completing 12 weeks of 
treatment in Fixed Dose Period. 
e. 
IDMC was removed 
f. 
Updated exploratory objectives and endpoints. 
2. Changes due to changes in data collection 
a. 
Analysis of study drug compliance based on days on 
treatment was removed as the diary data was incomplete 
b. Section 7.4.1, treatment compliance derivation was 
corrected to be based on tablets dispensed/returned to align 
with the data collected in the eCRF. 
Other important changes include: 
1. Section 5, added safety analysis set and updated per-protocol set to 
include subjects who completing 12 weeks of treatment in Fixed 
Dose Period. 
2. Section 6.2, updated power calculations and added hypothesis was 
added 
3. Section 6.3.4, updated the baseline definition to align statistical 
methodology, as applicable, with that outlined in the SAP for 
AG348-C-006. 
4. Section 6.3.5, added definition of dosing periods and optimized 
dose 
5. Sections 6.4 and 6.5, updated general methodology and methods to 
handle missing data to align statistical methodology, as applicable, 
with that outlined in the SAP for AG348-C-006 
6. Section 7.6.1, detailed derivations for primary endpoint were added 
7. Section 7.6.2, Regression and GLMM analyses on RBC counts and 
transfusion episodes were removed  
8. Section 7.6.3, subgroup analyses were added. 
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9. Section 7.7, safety analyses were updated to align statistical 
methodology, as applicable, with that outlined in the SAP for 
AG348-C-006 and analyses for AE by dose at onset were added. 
3.0 
25-Sep-2020 
Changes include: 
1. Section 6.3.5, updated definitions of dosing periods to align with 
the definitions outlined in the SAP for AG348-C-006 
2. Section 6.4.5 updated the visit window for DXA and LIC by MRI 
based on the data collection schedule in this study 
3. Sections 7.3.2 and 7.5, added definition of mean transfusion 
frequency as per protocol 
4. Sections 7.6.1, 7.6.1.1 and 7.6.2.1, updated the  “change”  and 
“Percentage change” to “reduction” and “Percentage reduction”, 
respectively, for transfusion burden  
5. Section 7.6.2.3, clarified the definition of transfusion-free in the 
Fixed Dose Period and added the analysis based on FAS 
6. Section 7.8.5.1, corrected the analysis of PKDD to be by visit 
instead of weekly score and Section 6.3.4 corrected the definition of 
baseline to remove the exception for PKDD, based on the data 
collection schedule in this study.  
7. Removed summary on PKR protein levels as it will be covered in a 
separate SAP. 
2. 
INTRODUCTION 
This SAP provides the detailed methodology for summary and statistical analyses of the data 
collected in Study AG348-C-007, except for pharmacokinetic and pharmacodynamic (PD) 
data, which will be described in a separate SAP. This document may modify the plans 
outlined in the protocol. 
The clinical study report (CSR) will include all data up to the End of Study (EOS) for all 
subjects, which is defined as the time at which all subjects have completed the study or are 
lost to follow-up. 
3. 
TRIAL OBJECTIVES AND ENDPOINTS  
3.1. 
Objectives 
3.1.1. 
Primary Objective  
The primary objective of the study is to evaluate the efficacy of treatment with AG-348, as 
assessed by the reduction in transfusion burden. 
3.1.2. 
Secondary Objectives 
The secondary objective of this study is to evaluate the safety of treatment with AG-348. 
3.1.3. 
Exploratory Objectives 
The exploratory objectives of the study are the following: 
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 To determine the effect of AG-348 on markers of hemolysis, erythropoietic 
activity, and other indicators of clinical activity 
 To determine the effect of AG-348 on markers of iron metabolism and indicators 
of iron overload 
 To determine the effect of AG-348 on health-related quality of life (HRQoL), as 
determined using patient reported outcomes (PROs) 
 To evaluate the pharmacokinetics of AG-348 after oral administration 
 To evaluate the relationship of AG-348 pharmacokinetics to indicators of clinical 
activity 
 To evaluate the change in the levels of PKR protein in whole blood 
3.2. 
Endpoints 
3.2.1. 
Primary Endpoint 
The primary endpoint of this study is the proportion of subjects who achieve a reduction in 
transfusion burden, defined as a ≥33% reduction in the number of RBC units transfused 
during the 24 weeks of Part 2 compared with the historical transfusion burden standardized 
to 24 weeks (Standardized Control Period). 
3.2.2. 
Secondary Endpoints 
The secondary endpoints of the study are the following:  
 Annualized total number of RBC units transfused during the study (both Part 1 
and Part 2) compared with the historical transfusion burden 
 Number of transfusion episodes during Part 2 compared with the Standardized 
Control Period  
 Proportion of subjects who become transfusion-free, defined as 0 transfusions 
administered during Part 2 
 Proportion of subjects who achieve Hb concentrations in the normal range at 
least once, 8 weeks or more after a transfusion in Part 2 
 Safety endpoints, including the type, incidence, severity, and relationship to 
treatment of adverse events (AEs) and serious adverse events (SAEs), and AEs 
leading to treatment dose reduction, treatment interruption, and treatment 
discontinuation 
 Laboratory tests over time (eg, serum chemistry, liver function tests [LFTs], 
hematology, coagulation, lipids, sex steroids, urinalysis), physical examination 
(PE) findings, dual-energy X-ray absorptiometry (DXA) scans (hip and lumbar 
spine), vital signs, and 12-lead electrocardiogram (ECG) data 
3.2.3. 
Exploratory Endpoints  
The exploratory endpoints of the study are the following: 
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5. 
ANALYSIS DATA SETS 
Only subjects who sign informed consent (IC) and are screened will be included in the 
analysis sets below. 
 The Full Analysis Set (FAS) will include all subjects who receive at least 1 dose 
of study treatment.  
 The Safety Analysis Set will include all subjects who receive at least 1 dose of 
study treatment. In this non-randomized study, the FAS and the safety analysis 
set are identical. 
 The Per-Protocol Set (PPS) is a subset of the FAS and will include all subjects 
who complete 12 weeks of treatment in the Fixed Dose Period (ie, end date of the 
Fixed Dose Period – start date of the Fixed Dose Period +1 ≥84). See Section 
6.3.5 for definition of start and end date of the Fixed Dose Period.  
Table 2 summarizes the use of the analysis sets. 
Table 2:  
Analysis Sets for Each Endpoint 
Endpoints 
Full Analysis Set (FAS) 
Per-Protocol Set (PPS) 
Safety Analysis Set 
Demographic and other 
baseline characteristics  
 
 
 
Disposition 
 
 
 
Major protocol deviations 
 
 
 
Exposure and concomitant 
therapies 
 
 
 
Efficacy   
 
 (primary and 
transfusion-free 
endpoints only) 
 
Safety 
 
 
 
6. 
GENERAL STATISTICAL CONSIDERATIONS 
6.1. 
Randomization, Blinding, Unblinding, and Crossover 
Not applicable. This is a non-randomized, open-label study. 
6.2. 
Sample Size Determination and Decision Rules 
6.2.1. 
Sample Size Determination 
The following statistical hypothesis will be tested to address the primary objective: 
𝐻1: 𝜆𝑡≤0.1 vs  𝐻1: 𝜆𝑡> 0.1 
where 𝜆𝑡 is the transfusion reduction response (TRR) rate. 
Due to the rarity of the disease and the small patient population, the sample size is largely 
driven by feasibility and the study will enroll a minimum of 20, with up to 40, subjects.  The 
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power to reject H0 at a 1-sided α=0.025 for different sample sizes and under different 
assumptions for the true TRR rate is shown in Table 3.  
Table 3: 
Power Considerations 
Sample Size 
True TRR Rate 
25% 
30% 
35% 
20 
0.38 
0.58 
0.75 
30 
0.49 
0.72 
0.88 
40 
0.70 
0.89 
0.97 
6.2.2. 
Decision Rules 
The study will have demonstrated the efficacy of mitapivat if the 1-sided p-value of the exact 
test for TRR rate is <0.025 and the TRR rate is >0.1. 
6.3. 
Definitions 
6.3.1. 
Study Drug and Study Treatment 
Study drug and study treatment are both defined as mitapivat. 
6.3.2. 
Start and End Dates of Study Drug and Study Treatment 
The start of study treatment is the earliest date/time of administration of a non-zero dose of 
the study treatment.  
The end of study treatment is the latest date/time of administration of a non-zero dose of 
the study treatment on or before the EOS date. 
6.3.3. 
Study Day 
The study day for assessments or events occurring on or after the start of study treatment 
(eg, AE onset, laboratory assessment) will be calculated as: 
Study day=Date of the assessment or event−start of study treatment+1.  
The study day for assessments or events occurring before the start of study treatment 
(eg, laboratory assessment during the Screening Period, medical history) will be negative 
and calculated as: 
Study day=Date of the assessment or event−start of study treatment. 
There is no study day 0. The study day will be displayed in data listings.  
6.3.4. 
Baseline  
Efficacy Evaluations  
Baseline for transfusion burden will be determined based on the historical transfusion data 
during the 52 weeks before IC. 
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For HRQOL assessments, the last measurement before the start of study treatment will be 
used as the baseline. 
For exploratory efficacy hematology laboratory parameters including Hb, markers of 
hemolysis, erythropoietic activity, iron metabolism and indicators of iron overload, the most 
recent non-missing measurements before Transfusion 0 (the most recent transfusion 
occurring in the Screening Period 2-7 days before the start of study treatment on Day 1) will 
be defined as the baseline to minimize the impact of transfusion on these hematology 
laboratory parameters. 
Safety Evaluations 
For alanine aminotransferase (ALT) and aspartate aminotransferase (AST), baseline is 
defined as the average of all assessments collected within 56 days before the start of study 
treatment. Baseline will be derived based on central laboratory data; if no central laboratory 
data are available before the start of study treatment, then local laboratory data will be used 
to derive the baseline.  
For other laboratory assessments: 
 Prior to deriving the baseline, 
o  If there are multiple records with the same assessment day and time from the 
same laboratory, the average value will be used  
o If there are multiple records with the same assessment day and time from 
different laboratories, the value from the central laboratory will be used   
 The baseline will then be the last value on or before the start of study treatment. 
Triplicate ECGs are collected in this study; the baseline for each ECG measurement is the 
average of the last predose replicate measurements on or before the start of study treatment. 
Unscheduled assessments will not be included in the calculation of the average. The average 
of the replicate measurements will be determined after the derivation of the individual 
parameter at each time point. 
For all other safety parameters, the last assessment on or before the start of study treatment 
will be used as the baseline.   
If, per protocol, an assessment (efficacy or safety) is to be performed on study day 1, before 
the first dose of study treatment, and the assessment time, time of first dose of study 
treatment, or both, is missing (or not collected), it will be assumed that the assessment is 
performed before study treatment administration. Unscheduled assessments will be used in 
the determination of baseline; however, an unscheduled assessment on study day 1 will be 
considered to have been obtained after study treatment administration. 
If no assessment meets the definition of baseline for an evaluation (efficacy or safety), the 
baseline will be set to missing. 
6.3.5. 
On-Treatment Period and Optimized Dose 
The on-treatment period starts on the date of the start of study treatment and ends 28 days 
after the end of study treatment. 
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Within the on-treatment period the following dosing periods are defined: 
 Dose Optimization Period (Part 1) starts on the date of start of study treatment 
and ends  
o on the earliest date of EOS, the end of the on-treatment period, and the first 
day of the dose taper prescription if the subject discontinues study treatment 
before reaching the Part 2 Day 1 visit.  
o on the day of the Part 2 Day 1 visit, otherwise  
 Fixed Dose Period (Part 2) starts 1 day after the end of the Dose Optimization 
Period and ends  
o on the first day of the dose taper prescription, if the subject enters the dose 
taper period, or  
o 4 days after the end of study treatment, otherwise. 
 Dose Taper Period starts 1 day after the first day of the dose taper prescription 
and ends on the earlier date of EOS and the end of on-treatment period.  This 
period is only applicable to subjects who go through the dose taper. The first day 
of the dose taper prescription is captured in the prescribed dose eCRF page as 
“planned treatment discontinuation”. 
Data listings will include all assessments and events, with those that occur outside of the 
on-treatment period flagged. 
The optimized dose is defined as the dose prescribed at the Part 2 Day 1 visit (5 mg BID, 
20 mg BID, or 50 mg BID). 
6.4. 
General Methods 
6.4.1. 
Data Handling After Cutoff Date 
Not applicable. 
6.4.2. 
Standard Derivations and Reporting Conventions 
The following conversion factors will be used to convert days into weeks, months or years: 
1 week=7 days, 1 month=30.4375 days, and 1 year=365.25 days. 
The following derivations will be implemented. 
 Age (years)=(year of given informed consent − year of birth), since only year of 
birth is collected in the eCRF.  
The integer part of the calculated age will be used for reporting purposes.  
 Body mass index (BMI; kg/m2)=weight (kg)/height (m)2 
 Duration (in days) from a reference date (eg, start date of study treatment) = 
o date of event − reference date + 1, if the date of the event is on or after the 
reference date  
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o date of event − reference date, if the date of the event is before the reference 
date 
Reporting conventions will be as follows:  
 Mean and median will be displayed to one more decimal place than the raw data. 
 SD will be displayed to two more decimal places than the raw data. 
 Percentages will be displayed to 1 decimal place (however, percentages 
corresponding to 0 counts will be reported as 0 rather than 0.0 and 100 percent 
will be reported as 100 rather than 100.0). 
 p-values will be reported with 4 decimal places; all p-values should be specified 
to be 1-sided or 2-sided. 
 Unless otherwise specified, rounding will be performed to the closest integer/first 
decimal using the common mid-point between the two consecutive values, eg, 
5.11 to 5.14 will be rounded to 5.1, and 5.15 to 5.19 will be rounded to 5.2. 
o Non-zero percentages that are <0.1 before rounding will be displayed as 
“<0.1”, eg, 0.09 will be reported as <0.1 rather than as 0.1. 
o p-values<0.0001 before rounding will be displayed as “<0.0001”, eg, a 
p-value of 0.00009 will be displayed as <0.0001 rather than as 0.0001. 
6.4.3. 
Pooling of Data Across Sites 
In order to provide overall estimates of treatment effects, data will be pooled across sites. 
The “site” factor will not be considered in statistical models or subgroup analyses given the 
high number of participating sites in contrast to the anticipated small number of subjects 
treated at each site. 
6.4.4. 
Continuous and Categorical Variables 
Continuous variables will be summarized using descriptive statistics, ie, number of non-
missing values, mean, SD, median, quartiles, minimum, and maximum.  
Categorical variables will be summarized by frequency distributions (number and 
percentage of subjects within a given category in the analysis data set). Unless otherwise 
specified, the calculation of percentages will include the “missing” category. Therefore, 
counts of missing observations will be included in the denominator and presented as a 
separate category. For summaries by visit, percentages will be based on the number of 
subjects with data available for that visit, unless otherwise specified. 
6.4.5. 
Unscheduled Visits 
Generally, data collected at unscheduled visits will be included and summarized for both 
safety and efficacy analyses in the same manner as the data collected at scheduled visits. 
Data collected at unscheduled visits will be included in by-subject listings together with the 
data collected at scheduled visits. 
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Summaries of outliers [eg, worst value, worst change from baseline, worst Common 
Terminology Criteria for Adverse Events (CTCAE) grade] during the on-treatment period 
for safety endpoints such as AEs, laboratory measurements and ECG parameters will 
include data from both scheduled and unscheduled visits. 
Individual longitudinal plots for laboratory measurements during the on-treatment period 
will include data from both scheduled and unscheduled visits.    
Descriptive statistics (mean, SD, median, quartiles, minimum, maximum) by visit will be 
provided for DXA and MRI results with windows derived based on the rules described 
below.  
Analysis Visit Windows   
For the evaluation of HRQOL endpoints, the analysis visit windows will be derived based 
on the target study day for the scheduled visits as follows. Note that based on the 
scheduled of assessments, a Week 4 Visit, for example, will have a target study day of 
1+(4×7)=29.  
 Visit windows will be implemented for scheduled visits after Day 1.  
 For analysis visit(n):   
o Start day of visit window =1+end day of window for visit(n−1). If n=1, start 
day of the visit window is study day 2   
o End day of visit window = [(target day for analysis visit(n) + target day for 
analysis visit(n+1))/2]–1 for visits before the Fixed Dose Period Week 24 
visit. The end day of the visit window for the Fixed Dose Period Week 24 
visit is the end of the Fixed Dose Period.  
For DXA scan results and LIC by MRI, the analysis visit window for  
 the Part 2 Day 1 Visit will start 85 days before the start of the Fixed Dose Period 
and end on day 85 of the Fixed Dose Period or EOS, whichever is earlier  
 the Fixed Dose Period Week 24 Visit will start on day 86 ofthe Fixed Dose 
Period and end on the EOS.  
 Results outside visit windows will not be summarized.  
Derivation of Values at Scheduled Postbaseline Visits Based on Analysis Visit 
Windows  
For HRQOL data, assessments (scheduled or unscheduled) within the visit windows and on 
or before the end of the Fixed Dose Period will be used.   
If multiple assessments are identified within a visit window for a parameter, the following 
rules will be applied:  
 The assessment measured closest to the target study day of the scheduled visit 
will be used   
 If there are multiple assessments equidistant to the target study day   
o the average value will be used for efficacy laboratory parameters  
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o the later assessment will be used for HRQOL endpoints  
6.5. 
Methods for Handling Missing Data 
6.5.1. 
Adverse Event and Concomitant Medication Start Dates 
If the end date is non-missing and the imputed start date is after the end date, the end date 
will be used as the start date. 
(1) Missing day only 
 If the month and year are the same as the month and year of the date of the start 
of study treatment, the date of the start of study treatment will be used. 
 If the month and year are before the month and year of the date of the start of 
study treatment, the last day of the month will be used. 
 If the month and year are after the month and year of the date of the start of study 
treatment, the first day of the month will be used. 
(2) Missing day and month 
 If the year is the same as the year of the date of the start of study treatment, the 
date of the start of study treatment will be used. 
 If the year is before the year of the date of the start of study treatment, 
31 December will be used. 
 If the year is after the year of the date of the start of study treatment, 01 January 
will be used. 
(3) Missing day, month, and year 
 The date of the start of study treatment will be used. 
6.5.2. 
Adverse Event and Concomitant Medication End Dates 
If the start date is non-missing and the imputed end date is before the start date, the start date 
will be used as the end date. If an imputation for an AE end date results in an AE end date 
that is after the EOS date, the AE will be considered as ongoing at the EOS date. 
(1) Missing day only 
 The last day of the month will be used. 
(2) Missing day and month 
 31 December will be used. 
(3) Missing day, month, and year 
 The event will be regarded as ongoing. 
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6.5.3. 
Exposure 
No imputation will be done for the date of the first dose of study drug. 
If the date of the last dose of study drug is missing or partially missing, it will be imputed as 
follows (separately for each study drug): 
 If the last date of study drug is completely missing and there is no End of 
Treatment Disposition eCRF page for the study drug AND there is no death date, 
the subject should be considered to be ongoing and the EOS date for the analysis 
will be used as the last dosing date. 
 If the last date of study drug is completely or partially missing and there is 
EITHER an End of Treatment Disposition eCRF page for the study drug OR a 
death date (on or before the EOS date), then the imputed last dose date is: 
=Last day of the year, if only the year is available and Year <Year of min (EOT 
date, death date)  
=Last day of the month, if both the year and month are available and Year=Year 
of min (EOT date, death date) and Month <Month of min (EOT date, death date) 
=min (EOT date, death date), for all other cases 
7. 
STATISTICAL ANALYSES 
7.1. 
Subject Disposition 
For all subjects screened in the study, the following will be summarized: 
 Number of subjects screened in the study  
 Frequency (number and percentage) of subjects who discontinued the study 
before start of study treatment, overall and by reason for discontinuation. 
Percentages will be calculated based on the number of subjects screened in the 
study.  
In addition, the frequency of subjects in each of the analysis sets described in Section 5 will 
be summarized. Percentages will be calculated only for analysis sets that are a subset of the 
FAS or a subset of the safety analysis set. 
The following summaries will be presented based on the FAS:  
 Frequency of subjects treated in each geographic region, country, and site 
 Frequency of subjects with study drug ongoing 
 Frequency of subjects who discontinued study drug, overall and by the reason for 
discontinuation of study drug  
 Frequency of subjects who completed the study 
 Frequency of subjects ongoing in the study 
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 Frequency of subjects who discontinued the study, overall and by the reason for 
study discontinuation 
The frequency of subjects with disposition reason, in each epoch, due to reasons associated 
with COVID-19 will further be summarized under the main reason for discontinuation. 
Disposition for all screened subjects will be provided in by-subject listings. 
7.2. 
Protocol Deviations 
All major protocol deviations that impact the safety of the subjects, the conduct of the study, 
or the evaluation of the study results will be reported based on the FAS. These will include: 
 Subjects treated despite not satisfying the eligibility criteria 
 Subjects who develop withdrawal criteria while on the study but are not 
withdrawn 
 Subjects who receive an excluded concomitant medication 
In addition, for each category of major protocol deviations, those related to COVID-19 will 
be summarized. 
Major protocol deviations will be provided in a by-subject listing. 
7.3. 
Demographic and Other Baseline Characteristics 
The following summaries will be presented based on the FAS, unless otherwise specified. 
7.3.1. 
Demographics and Physical Measurements 
Demographic characteristics and physical measurements at baseline will be summarized as 
follows: 
 Demographic characteristics  
o Sex: male, female (child-bearing potential status will be summarized for 
female subjects) 
o Race: American Indian or Alaska Native, Asian, Black or African American, 
Native Hawaiian or other Pacific Islander, White, other, unknown  
o Ethnic origin: Hispanic or Latino, not Hispanic or Latino, not reported 
o Age (years): summary statistics  
 Age categories:  
o <65, ≥65 years 
o <35, ≥35 years 
 Physical measurements 
o Height (cm) 
o Weight (kg) 
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o BMI (kg/m2) 
Demographic data for all screened subjects will be provided in a by-subject listing. 
7.3.2. 
Disease Characteristics 
The following baseline characteristics of the underlying disease will be summarized based 
on the data entered in the eCRF: 
 Baseline Hb concentration 
 Baseline ferritin level 
 DXA scan results by location (femoral total and adjusted spine): bone mineral 
density (BMD) and their corresponding T-scores and Z-scores.  Frequency of 
subjects with T-scores in 3 categories (≤-2.5, >-2.5-<-1, ≥-1.0) 
 UGT1A1 genotype 
 Prior splenectomy status (Yes, No; if Yes, age of splenectomy) 
 Prior cholecystectomy status (Yes, No; if Yes, age of cholecystectomy) 
 Prior chelation status (Yes, No); the status is “Yes” if a subject has received 
chelation therapy within 52 weeks before the first dose of study treatment. 
 PKR genotype (missense/missense, missense/non-missense)  
 Transfusion History during the 52 weeks before IC:  
o Number of transfusion episodes 
o Number of transfusion episodes standardized to 24 weeks and categories (≤6, 
>6) 
o Number of RBC units transfused 
o Number of RBC units transfused standardized to 24 weeks and categories 
(≤6, >6) 
Value standardized to 24 weeks = value for the 52-week period×24/52 
o Mean Transfusion Frequency (MTF) = 52 weeks / number of transfusion 
episodes 
o Average duration between transfusions = (last transfusion date – first 
transfusion date +1 – number of transfusions) /(number of transfusions –1) 
o Average RBC units transfused (unit/transfusion) 
o Individual Transfusion Trigger (TT) as reported by the investigator 
 Transfusions on or after IC and before start of study treatment 
o Number of transfusion episodes 
o Time from last transfusion to start of study treatment = start of study 
treatment – date of last transfusion 
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o Number of RBC units transfused 
o Average of RBC units transfused (unit/transfusion) 
In the calculations of number of transfusion episodes, transfusions received over up to 3 
consecutive days will be counted as 1 episode.  
Data on disease characteristics will be provided in by-subject listings. 
7.3.3. 
Medical History 
Medical history will be summarized in frequency tabulations according to the latest version 
of the Medical Dictionary for Regulatory Activities (MedDRA) by System Organ Class 
(SOC) and Preferred Term (PT). 
Medical history will be provided in by-subject listings. 
7.3.4. 
Prior Therapies 
The following summaries will be presented based on the safety analysis set. 
Prior medications are defined as medications (from the Prior and Concomitant Medications 
eCRF) that are started before the start of study treatment.  
All non-study medications will be coded according to the Anatomical Therapeutic Chemical 
(ATC) code and PT using the latest version of the World Health Organization (WHO) Drug 
Dictionary. All prior medications will be summarized in frequency tabulations according to 
the WHO ATC third level and PT.  
Prior medications will be provided in a by-subject listing. 
Prior procedures are defined as procedures (from the Surgical History and Concomitant 
Procedures eCRF) that are started before the start of study treatment.   
The prior procedures will be coded according to the latest version of the MedDRA by SOC 
and PT and will be summarized in frequency tabulations by SOC and PT.  
Prior procedures and surgical history of splenectomy will be provided in by-subject listings. 
Prior transfusions are collected in the Transfusion History eCRF and will be summarized as 
described in Section 7.3.2. 
7.4. 
Exposure to Study Drug and Compliance 
The following summaries will be presented based on the safety analysis set. 
7.4.1. 
Treatment Duration and Exposure 
The frequency of subjects with an optimized dose of 5 mg BID, 20 mg BID or 50 mg BID 
will be summarized by treatment arm. 
Duration of exposure will be summarized as a continuous variable as well as in categories 
(>0-8, >8-16, >16-24, >24-32, >32-40, and >40 weeks), where  
Duration of exposure = end date of study drug – start date of study drug +1.  
Study drug compliance will be summarized based on percentage of tablets taken, where   
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 Percentage of tablets taken = 100×(total number of tablets administered)/(total 
number of tablets intended)  
 Total number of tablets administered = total number of tablets dispensed – tablets 
returned 
 Duration of prescription = end date of prescription – start date of prescription +1 
 Number of tablets intended during each prescription: for each new prescription, 
prescribed dosing frequency × duration of the prescription. Prescribed dosing 
frequency takes value of 0.5, 1, and 2 for every other day (QOD), once daily 
(QD) and twice daily (BID), respectively.  
 Total number of tablets intended=sum of number of tablets intended over all 
prescriptions 
Percentage of tablets taken will be summarized. The frequency of subjects whose 
compliance is <80% , 80-100%, >100-120%, and >120% will be summarized.  
7.4.2. 
Dose Modifications 
The summary of dose modifications will include:  
 The frequency of subjects with at least 1 dose reduction 
 Summary of reasons for dose reduction 
Dose reduction is defined as the prescribed dose being decreased from the previous dose 
collected in the Prescribed Dose eCRF. Dose prescriptions and modifications will be 
provided in a by-subject listing.  
7.5. 
Concomitant Therapies 
The following summaries will be presented based on the safety analysis set. 
Concomitant medications are defined as non-study medications (from the Prior and 
Concomitant Medications eCRF) that are started during the on-treatment period or are 
started before the start of the study treatment and end or remain ongoing during the on-
treatment period.  
All non-study medications will be coded according to ATC code and PT using the latest 
version of the WHO Drug Dictionary. All concomitant medications will be summarized in 
frequency tabulations according to WHO ATC third level and PT. 
Concomitant procedures are defined as procedures (from the Prior and Concomitant 
Procedures eCRF) that are started during the on-treatment period or are started before the 
start of the study treatment and end or remain ongoing during the on-treatment period. 
The concomitant procedures will be coded by the latest version of MedDRA by SOC and PT 
and will be summarized in frequency tabulations by SOC and PT.  
Concomitant transfusions are collected in the “On Study Transfusions” eCRF page.  
On-study transfusions in each dosing period (Dose Optimization Period, Fixed Dose Period, 
Dose Taper Period) will be summarized using descriptive statistics:   
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 Number of transfusion episodes 
 Number of RBC units transfused 
 MTF(weeks) = (end of dosing period – the start of dosing period +1)/[7×(number 
of transfusions in the dosing period)] 
 Average duration between transfusions = [last transfusion date in the dosing 
period– first transfusion date in the dosing period +1 – number of transfusions in 
the dosing period) /(number of transfusions in the dosing period –1) 
 Average RBC units transfused unit/transfusion) 
In the calculations of number of transfusion episodes, transfusions received over up 
to 3 consecutive days will be counted as 1 episode. 
7.6. 
Efficacy Analyses 
The following analyses will be based on the FAS, unless otherwise specified.  
7.6.1. 
Primary Endpoint 
The primary endpoint is the reduction in transfusion burden (transfusion reduction response, 
TRR), defined as a ≥33% reduction in total number of RBC units transfused during the  
Fixed Dose Period (on-study transfusion burden) standardized to 24 weeks compared with 
the historical transfusion burden standardized to 24 weeks.   
 Historical transfusion burden standardized to 24 weeks (units/24-weeks) = total 
number of transfused RBC units during the 52 weeks before IC ×24/52.  
 On-study (Fixed Dose Period) transfusion burden standardized to 24 weeks = 
24×total number of transfused RBC units in the Fixed Dose Period / (total 
number of days in the Fixed Dose Period/7).  
Transfusion reduction response will be evaluated based on percentage reduction in 
transfusion burden standardized to 24 weeks: 
 Percentage reduction in transfusion burden standardized to 24 weeks =                           
[Historical transfusion burden standardized to 24 weeks – On-study (Fixed Dose 
Period) transfusion burden standardized to 24 weeks] / historical transfusion 
burden standardized to 24 weeks.   
Subjects who complete at least 12 weeks of treatment in the Fixed Dose Period (ie, end date 
of the Fixed Dose Period - start date of the Fixed Dose Period +1 ≥84) will be consider 
responders if the percentage reduction in transfusion burden is ≥33%. Subjects who 
discontinue the study before completing 12 weeks of treatment in the Fixed Dose Period will 
be considered non-responders. 
7.6.1.1. 
Primary Analyses 
The frequency of subjects with TRR will be summarized based on the FAS along with the 
1-sided p-value of binomial exact test and the 2-sided 95% exact CI using the Clopper-
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Pearson method (exact CI for a binomial proportion as computed by default by the FREQ 
procedure using the EXACT option). 
The frequency of subjects with TRR and the 2-sided 95% exact CI will also be summarized 
by optimized dose. If the number of subjects with an optimized dose is small (<10% of 
subjects), the results by optimized dose may not be interpretable. 
Historical transfusion burden standardized to 24 weeks, on-study (Fixed Dose Period) 
transfusion burden standardized to 24 weeks and its reduction and percentage reduction 
from historical transfusion burden standardized to 24 weeks will be summarized. 
The frequency of subjects with different categories of percentage reduction will be provided 
(<0, 0 to <20, 20 to <33, 33 to <50, ≥50%). 
7.6.1.2. 
Sensitivity Analyses  
To evaluate the impact of early discontinuation in the primary analysis a sensitivity analysis 
will be conducted based on the PPS. The frequency of subjects with TRR will be 
summarized along with the 2-sided 95% exact CI using the Clopper-Pearson method.  
7.6.2. 
Secondary Endpoints  
7.6.2.1. 
Annualized total number of RBC units transfused during the study 
compared with the historical transfusion burden 
Annualized historical transfusion burden = total number of RBC units transfused during the 
52 weeks before IC. 
Annualized total number of RBC units transfused (units/52-weeks) during the study will be 
evaluated based on two calculations: 
 Including data up to EOS: 
=52× total number of RBC units transfused during the entire study/ [(date of EOS – 
date of start of study treatment + 1)/7]. 
 Including data up to the end of Fixed Dose Period: 
=52× total number of RBC units transfused during the Dose Optimization and Fixed 
Dose Periods combined/ [(end date of the Fixed Dose Period – date of start of study 
treatment + 1)/7]. 
The annualized historical transfusion burden, the annualized total number of RBC units 
transfused and its reduction and percentage reduction from the annualized historical 
transfusion burden will be summarized.  
The frequency of subjects with different categories in percentage reduction will be provided 
(<0, 0 to <20, 20 to <33, 33 to <50, ≥50%). 
7.6.2.2. 
Number of transfusion episodes 
Number of historical transfusion episodes standardized to 24 weeks = total number of 
transfusion episodes in 52 weeks before IC ×24/52.  
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Number of transfusion episodes during the Fixed Dose Period standardized to 24 weeks = 
24×total number of transfusion episodes in the Fixed Dose Period / (total number of days in 
the Fixed Dose Period/7).  
In these calculations, transfusions received over up to 3 consecutive days will be counted as 
1 episode. 
The number of historical transfusion episodes standardized to 24 weeks, the number of 
transfusion episodes during the Fixed Dose Period standardized to 24 weeks and its change 
and percent change from the number of historical transfusion episodes standardized to 24 
weeks will be summarized. 
7.6.2.3. 
Transfusion-free 
A subject is considered to become transfusion-free in a dosing period if he/she does not 
receive transfusions during that period.   
The frequency of subjects who become transfusion-free in the Fixed Dose Period will be 
summarized along with the 95% exact CI using the Clopper-Pearson method. Subjects who 
did not complete 12 weeks of treatment in the Fixed Dose Period will not be considered as 
transfusion-free. This analysis will be conducted based on the FAS and PPS.  
7.6.2.4. 
Normal Hb concentrations 
The frequency of subjects who achieve Hb concentrations in the normal range at least once 
in the Fixed Dose Period if the normal Hb sample is taken ≥8 weeks after the latest 
transfusion before the date of sample collection, will be summarized along with the 95% 
exact CI using the Clopper-Pearson method.  
7.6.2.5. 
Transfusion trigger 
Subjects should be transfused when the subject has reached his/her individual Transfusion 
Trigger (TT), as calculated by the investigator at screening and recorded in the eCRF.   
When a subject’s Hb concentration is below the upper margin of his/her individual TT but 
did not receive a transfusion, the reason (clinically asymptomatic, scheduling challenges, 
patient decision, waiting for additional assessments, other) will be collected in the 
transfusion trigger eCRF. 
To assess transfusion compliance, the following summaries will be provided: 
 
Frequency of subjects who had missed transfusions after reaching TT, overall 
and by reason 
 Frequency of subjects who received a transfusion without reaching TT, overall 
and by reason (adverse event, other)  
7.6.3. 
Subgroup Analyses  
Subgroup analyses to be performed for the primary endpoint based on the FAS are presented 
in Table 4. 
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Table 4: 
Subgroup Analyses for TRR 
Subgroup  
Categories 
PKR genotype 
missense/missense, missense/non-missense 
Baseline individual TT 
<8.5 g/dL, ≥8.5 g/dL 
Historical transfusion episodes during the 52 weeks before IC 
standardized to 24 weeks 
≤ 6, > 6 
Number of RBC units transfused during the 52 weeks before 
IC standardized to 24 weeks 
≤6 units, >6 units 
Splenectomy at baseline 
Yes, No 
TRR rate will be summarized for each category within each subgroup along with the 95% 
exact CI using the Clopper-Pearson method. 
Efficacy analyses in subgroups will be purely exploratory and are intended to evaluate the 
consistency of treatment effect. If there is a low number of subjects within a category (≤10% 
of the subjects in the FAS), the subgroup may not be summarized. 
7.7. 
Safety Analyses 
Summaries of safety data will be presented based on the safety analysis set.  
7.7.1. 
Adverse Events 
Treatment-emergent adverse events (TEAEs) are AEs with a first onset date during the on-
treatment period or worsening from baseline. All summaries described below will be based 
on TEAEs, if not otherwise specified.  
All AEs will be listed by subject and AEs with onset outside of the on-treatment period will 
be flagged in the listings. Unless otherwise specified, TEAEs will be summarized according 
to the latest version of MedDRA by SOC and/or PT, severity (based on CTCAE v4.03 
grading), seriousness, and relation to study treatment in decreasing frequency based on the 
frequencies observed for all subjects. 
Each subject will be counted only once within each SOC or PT. If a subject experiences 
multiple TEAEs under the same PT within a SOC for the same summary period, only the 
TEAE assessed as related or with the worst severity, as applicable, will be included in the 
summaries of relationship and severity. If a subject has TEAEs with missing and non-
missing grades, the maximum of the non-missing grades will be displayed. No imputation of 
missing grades will be performed. 
The following will be summarized: 
 TEAEs by SOC and PT 
 TEAEs by SOC, PT, and worst grade 
 Most common TEAEs and Grade ≥3 TEAEs by PT; these will include TEAEs 
(any grade) reported in ≥10% of all subjects or Grade ≥3 TEAEs reported in ≥5% 
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of all subjects. These thresholds may be changed based on the observed data 
without an amendment to this SAP. 
 Treatment-related TEAEs, by SOC and PT  
 Treatment-related TEAEs, by SOC, PT, and worst grade 
 Grade ≥3 TEAEs, by SOC and PT 
 Treatment-related Grade ≥3 TEAEs, by SOC and PT  
 Serious TEAEs, by SOC and PT 
 Treatment-related Serious TEAEs, by SOC and PT  
 TEAEs leading to discontinuation of study drug, by SOC and PT  
 TEAEs leading to interruption of study drug, by SOC and PT  
 TEAEs leading to dose reduction, by SOC and PT 
 TEAEs leading to death, by SOC and PT 
 Treatment-related TEAEs leading to death, by SOC and PT  
In addition, the following will be summarized by prescribed dose at TEAE onset: 
 First occurrence of TEAEs by PT 
 First occurrence of serious TEAEs by PT 
 TEAEs by PT 
 Serious TEAEs by PT 
7.7.1.1. 
Adverse Events of Special Interest 
Transaminase increase is an AESI for mitapivat and will be reported by the investigator in 
the AESI eCRF page if there is a transaminase increase of >2.5×baseline or an increase in 
AST or ALT to Grade ≥2 in severity, whichever is lower.    
Additional TEAEs of interest for mitapivat are as follows:   
 AEs of endocrinological interest (identified based on the criteria outlined in the 
mitapivat program specified Safety Search Criteria)  
 Insomnia (PTs under HLT of “Disturbances in Initiating and Maintaining Sleep” 
or identified based on the criteria outlined in the mitapivat program specified 
Safety Search Criteria)  
The following will be summarized for AESIs and the additional TEAEs of interest: 
 AESIs/TEAEs of interest by PT 
 AESIs/TEAEs of interest by PT and worst grade 
 Grade ≥3 AESIs/TEAEs of interest by PT 
 AESIs/TEAEs of interest leading to discontinuation of study drug by PT  
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 Serious AESIs/TEAEs of interest by PT  
 AESIs/TEAEs of interest leading to death by PT 
In addition, the following will be summarized by prescribed dose at TEAE onset for the 
additional AE of interest “Insomnia”: 
 First occurrence of TEAEs by PT 
 First occurrence of serious TEAEs by PT 
 TEAEs by PT 
 Serious TEAEs by PT 
7.7.1.2. 
Adverse Events Associated with COVID-19 
The selection of AEs associated with COVID-19 will be based on the MedDRA MSSO list 
of PTs. The following will be summarized: 
 TEAEs associated with COVID-19, by SOC and PT 
 Grade ≥3 TEAEs associated with COVID-19, by SOC and PT 
 Serious TEAEs associated with COVID-19, by SOC and PT 
 TEAEs associated with COVID-19 leading to discontinuation of study drug, by 
SOC and PT  
 TEAEs associated with COVID-19 leading to interruption of study drug, by SOC 
and PT  
 TEAEs associated with COVID-19 leading to dose reduction, by SOC and PT 
 TEAEs associated with COVID-19 leading to death, by SOC and PT 
7.7.2. 
Death 
The frequency of subjects in the safety analysis set who died will be tabulated based on 
information from the EOS eCRF. Deaths will be summarized for the following categories: 
 On-treatment death: Deaths within 28 days after the last dose of study treatment 
(ie, deaths during the on-treatment period) 
 Post-treatment death: Deaths more than 28 days after the last dose of study 
treatment (ie, deaths after the end of the on-treatment period) 
 Overall: All deaths 
In addition, deaths related to COVID-19 will be summarized. 
Deaths for all screened subjects will be provided in a by-subject listing. 
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7.7.3. 
Clinical Laboratory Data 
Clinical laboratory test results will be expressed in SI units. Preferred unit (g/dL) will also 
be used for Hb in efficacy analysis. 
For each laboratory test (chemistry, hematology, coagulation) performed in the study, a by-
subject listing of laboratory test results will be presented with the corresponding CTCAE 
grades (if applicable), laboratory normal ranges, and flags for values below lower limit of 
normal (LLN) or above upper limit of normal (ULN).   
Parameters with CTCAE grades available:  
Clinical laboratory test results will be graded according to CTCAE v4.03 as applicable. 
Grading will be derived based on the numerical thresholds defined by the CTCAE criteria. 
Non-numerical qualifiers will not be taken into consideration in the derivation of CTCAE 
grading.  
Laboratory test results classified according to CTCAE will be described using the worst 
grade. For parameters graded with 2 separate toxicity criteria, such as potassium 
(hypokalemia/hyperkalemia), the toxicities will be summarized separately. Low direction 
toxicity (eg, hypokalemia) grades at baseline and postbaseline will be set to 0 when the 
variables are derived for summarizing high direction toxicity (eg, hyperkalemia), and vice 
versa. 
The frequency of subjects with laboratory toxicities during the on-treatment period will be 
tabulated as follows. The denominator used to calculate percentages for each laboratory test 
is the number of subjects evaluable for CTCAE grading for that parameter (ie, those subjects 
for whom a Grade of 0, 1, 2, 3 or 4 can be derived). 
 The summary of laboratory parameters by CTCAE grade will include the number 
and percentage of subjects with Grade 1, 2, 3, 4; Grade 3-4; and Any Grade 
(Grades 1-4) during the on-treatment period. The highest CTCAE grade during 
the on-treatment period is considered the worst grade. 
 The shift table will summarize baseline CTCAE grade versus worst CTCAE grade 
during the on-treatment period. The highest CTCAE grade during the on-
treatment period is considered the worst grade.  
 Newly occurring or worsening laboratory abnormalities (Any Grade, Grade 3-4) 
during the on-treatment period will also be summarized. 
Parameters with CTCAE grades not available: 
Results of laboratory tests that are not part of CTCAE will be presented according to the 
following categories: below the LLN, within normal limits, and above the ULN according to 
the laboratory normal ranges.   
Shift tables will display the frequency of subjects with shifts from baseline missing, <LLN, 
normal, or >ULN to each of <LLN, normal or >ULN during the on-treatment period. 
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7.7.3.1. 
Hematology 
For WBC differential counts [total neutrophil, lymphocyte, monocyte, eosinophil, and 
basophil counts], the absolute value will be used when reported. When only percentages are 
available (relevant primarily for neutrophils and lymphocytes, because the CTCAE grading 
is based on the absolute counts), the absolute value is derived as follows:  
Derived differential absolute count=(WBC count)×(Differential % value/100) 
If the range for the differential absolute count is not available (ie, the range is only available 
for the percentage) then Grade 1 will be attributed as follows:  
 Lymphocyte count decreased:   
o Derived absolute count does not meet Grade 2-4 criteria, and 
o % value <% LLN value, and 
o Derived absolute count ≥800/mm3 
 Neutrophil count decreased:  
o Derived absolute count does not meet Grade 2-4 criteria, and 
o % value<% LLN value, and 
o Derived absolute count ≥1,500/mm3 
7.7.3.2. 
Chemistry 
Liver function tests: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), 
alkaline phosphatase (ALP), and total bilirubin are used to assess possible drug-induced 
liver toxicity. The ratios of test result to ULN will be calculated and categorized for these 
parameters during the on-treatment period.    
The summary of liver function tests will include the following categories. The frequency of 
subjects with each of the following during the on-treatment period will be summarized: 
 ALT >3×ULN, ALT >5×ULN, ALT >10×ULN, ALT >20×ULN 
 AST >3×ULN, AST >5×ULN, AST >10×ULN, AST >20×ULN 
 (ALT or AST) >3×ULN, (ALT or AST) >5×ULN, (ALT or AST) >10×ULN, 
(ALT or AST) >20×ULN 
 total bilirubin >2×ULN 
 Concurrent ALT >3×ULN and total bilirubin >2×ULN 
 Concurrent AST >3×ULN and total bilirubin >2×ULN 
 Concurrent (ALT or AST) >3×ULN and total bilirubin >2×ULN  
 Concurrent (ALT or AST) >3×ULN and total bilirubin >2×ULN and 
ALP ≥2×ULN 
 Concurrent (ALT or AST) >3×ULN and total bilirubin >2×ULN and 
(ALP <2×ULN or missing) 
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Concurrent measurements are those occurring on the same date. 
Categories will be cumulative, ie, a subject with an AST >10×ULN will also appear in the 
categories >5×ULN and >3×ULN. Liver function test elevation and possible Hy’s Law 
cases will be summarized using frequency counts and percentages.  
An evaluation of Drug-Induced Serious Hepatotoxicity (eDISH) plot will be created, by 
graphically displaying: 
 Peak serum ALT (/ULN) vs peak total bilirubin (/ULN) including reference lines 
at ALT=3×ULN and total bilirubin=2×ULN  
 Peak serum AST (/ULN) vs peak total bilirubin (/ULN) including reference lines 
at AST=3×ULN and total bilirubin=2×ULN  
In addition, the following individual longitudinal plots and by-subject listings will be 
provided: 
 Individual longitudinal plot of ALT including subjects with at least one ALT 
during the on-treatment period >2.5×baseline or worsening to CTCAE Grade ≥2 
during the on-treatment period  
 Individual longitudinal plot of AST including subjects with at least one AST 
during the on-treatment period >2.5×baseline or worsening to CTCAE Grade ≥2 
during the on-treatment period 
 Listing of all total bilirubin, ALT, AST, and ALP values for subjects with a 
postbaseline total bilirubin >2×ULN, ALT >3×ULN, or AST >3×ULN 
 Listing of all total bilirubin, indirect bilirubin, ALT, AST and ALP values for 
subjects with a postbaseline ALT >ULN or AST >ULN 
In addition, a shift table from baseline to the worst CTCAE grade of ALT and AST during 
the on-treatment period will be provided. For each subject:  
 If the worst CTCAE grade of ALT is worse than that of AST during the on-
treatment period, the baseline CTCAE grade of ALT will be used  
 
If the worst CTCAE grade of AST is worse than that of ALT during the on-
treatment period, the baseline CTCAE grade of AST will be used  
 
If AST and ALT have the same worst CTCAE grade during the on-treatment 
period, the lower baseline CTCAE grade of ALT and AST will be used. 
For calcium, CTCAE grading is based on corrected calcium and ionized calcium. Corrected 
Calcium is calculated from albumin and calcium as follows: 
 
Corrected calcium (mmol/L)=measured total calcium (mmol/L)+0.02×[40−serum 
albumin (g/L)]     
7.7.3.3. 
Sex Steroid Tests 
For sex steroid test results, shift tables will display the frequency of subjects with shifts from 
baseline missing, < LLN, normal, > ULN to each of < LLN, normal or > ULN during the 
on-treatment period.    
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In addition, individual longitudinal plots will be provided for each sex hormone by sex.  
7.7.3.4. 
Pregnancy Test 
Pregnancy test results will be presented in a by-subject listing. 
7.7.4. 
Vital Signs and Physical Measurements 
All physical measurements and vital sign assessments (height, weight, BMI, systolic blood 
pressure, diastolic blood pressure, pulse rate, temperature) will be presented in a by-subject 
listing.  
7.7.5. 
Electrocardiograms 
All ECG summaries and listings will be based on the central reading results. 
ECG summaries will include all ECG assessments from the on-treatment period.  
Selecting Primary QT Interval Correction for Heart Rate 
The analysis of QT interval data is complicated by the fact that the QT interval is highly 
correlated with heart rate. Because of this correlation, formulas are routinely used to obtain a 
corrected QT interval, denoted QTc, which is independent of heart rate. This QTc is 
intended to represent the QT interval at a standardized heart rate. Several correction 
formulas have been proposed in the literature. For this analysis several of those methods of 
correction will be used, as described below. The QT interval corrected for heart rate by the 
Bazett’s formula, QTcB, is defined as  
RR
QT
QTcB 
  
and the QT interval corrected for heart rate by the Fridericia’s formula, QTcF, is defined as  
3 RR
QT
QTcF 
 , 
where RR represents the RR interval of the ECG, in seconds.    
Although Bazett’s correction is the historical standard, it does not perform well when heart 
rate fluctuates. Fridericia’s formula may perform better under these conditions. 
ECG Summaries 
The following analyses will be performed for each applicable ECG parameter (RR, PR, 
QRS, QT, and QTc) during the on-treatment period. The denominator used to calculate 
percentages for each category is the number of subjects evaluable for the category. 
 Pearson correlation between QT and RR interval, QTc (QTcF, QTcB) and RR 
interval using individual (non-averaged) baseline assessments  
 Frequency of subjects with notable ECG values, defined as those in the following 
categories: 
o QT/QTc interval increase from baseline >30 ms, >60 ms 
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o QT/QTc interval >450 ms, >480 ms, >500 ms 
o PR interval >200 ms 
o QRS duration >120 ms  
All ECG assessments and qualitative ECG abnormalities will be presented in by-subject 
listings. 
7.7.6. 
DXA Scans 
DXA scan results including bone mineral density (BMD), T-scores, Z-scores during the on-
treatment period will be summarized by location (total femur and adjusted spine), and visit. 
For T-scores, shift from baseline to Week 24 in the Fixed Dose Period by category (≤-2.5, 
>-2.5 to <-1.0, ≥-1.0) will be provided.    
All DXA scan results will be presented in a by-subject listing.    
7.7.7. 
Menstrual Cycle Diary 
Menstrual cycle diary data collected from women of childbearing potential during the on-
treatment period will be summarized by regular contraceptive status (oral contraceptives or 
depot injection). The following summaries will be included: 
 Total number of menstrual cycles reported 
 Total number of abnormal menstrual cycles in the following categories: heavier, 
lighter, longer, shorter, sooner and later than usual.  
Menstrual cycle diary data will be presented in a by-subject listing with regular 
contraceptive status flagged. 
7.8. 
Exploratory Analyses 
Unless otherwise specified, summaries for exploratory endpoints will be presented based on 
the FAS. 
7.8.1. 
Markers of Hemolysis 
Markers of hemolysis include bilirubin, LDH, and haptoglobin.   
By-subject longitudinal plots will be presented with markers of hemolysis at baseline and 
postbaseline, transfusions, and prescribed dose over time. The plots will further include 
optimized dose, age, sex, race, PKLR gene mutation category, baseline individual TT, prior 
splenectomy status, baseline chelation status, and postbaseline chelation status. 
Markers of hemolysis will be presented in a by-subject listing 
7.8.2. 
Erythropoietic Activity Markers 
Erythropoietic markers include erythropoietin (EPO), soluble transferrin receptor, 
erythroferrone, and reticulocyte.  
By-subject longitudinal plots will be presented with erythropoietic markers at baseline and 
postbaseline, transfusions, and prescribed dose over time. The plots will further include 
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optimized dose, age, sex, race, PKLR gene mutation category, baseline individual TT, prior 
splenectomy status, baseline chelation status, and postbaseline chelation status. 
Erythropoietic markers will be presented in a by-subject listing. 
7.8.3. 
Iron Markers 
Iron markers include serum iron, serum ferritin, total iron-binding capacity (TIBC), 
transferrin saturation, and hepcidin.  
By-subject longitudinal plots will be presented with iron markers at baseline and 
postbaseline, transfusions, and prescribed dose over time. The plots will further include 
optimized dose, age, sex, race, PKLR gene mutation category, baseline individual TT, prior 
splenectomy status, baseline chelation status, and postbaseline chelation status. 
Iron markers including non-transferrin bound iron (NTBI), and C-reactive protein (CRP) 
will be provided in listings. 
7.8.4. 
Liver Iron Concentration (LIC) by Magnetic Resonance Imaging (MRI) 
Liver MRI results will be listed and summarized by visit. 
7.8.5. 
HRQoL 
PRO data are collected with an electronic device outside of the clinic, hence the reported 
nominal visit will not be used. The analysis visit windows detailed in Section 6.4.5 will be 
used. When there are multiple assessments within the same visit window, the one closest to 
the target study day will be used; when there are more than 2 assessment of the same 
distance to the target day, the last assessment will be used. 
Given the nature of the PROs and their limitations in this open label study, no additional 
modelling will be conducted.  
7.8.5.1. 
PKDD 
PKDD is a self-administered, daily, 7-item PRO measure of the core signs and symptoms of 
PKD in adults. The PKDD daily scores will be calculated based on the subject’s response to 
the PKDD questionnaire.  
The PKDD scores up to the end of the Fixed Dose Period will summarized descriptively at 
each visit. 
7.8.5.2. 
PKDIA 
The PKDIA is a 12-item PRO measure of the common impacts of PK deficiency on 
activities of daily living. Subjects rate how PK deficiency has impacted aspects of daily 
living in the past 7 days, including impacts on relationships; perceived appearance; work 
performance; and leisure, social, mental, and physical activities. 
The PKDIA score at each visit will be calculated and provided by the instrument developer 
(Endpoint Outcomes) based on the subject’s response to the PKDIA questionnaire.   
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The following summaries will be provided for PKDIA scores up to the end of the Fixed 
Dose Period at each visit:  
 Summaries of scores for each item. For questions 9a, 11a and 12, the frequency 
of subjects with answers in each category will be summarized. For questions 9b 
and 11b, scores from patients who answered “Yes” will be summarized. For all 
the other questions, responses will be treated as continuous variables and 
summarized descriptively.   
 Descriptive summaries for PKDIA score and change from baseline  
7.8.5.3. 
EQ-5D-5L 
For EQ-5D-5L results, the following descriptive summaries will be provided at each visit, 
including: 
 Index score and change from baseline. 
 VAS score and change from baseline. 
 Frequency of subjects in each level for each of the 5 dimensions.  
7.8.5.4. 
PGIS 
Frequency of subjects with answers in each category for each question will be summarized. 
7.9. 
Interim Analyses  
There is no interim analysis planned for this study. 
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8. 
REFERENCES 
Clopper, C., & Pearson, E. S. (1934). The use of confidence or fiducial limits illustrated in 
the case of the binomial. Biometrika, 26(4), 404-413. 
 
 
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