A Phase 3, Double-Blind, Randomized, Placebo-Controlled, Multicenter Study Evaluating the Efficacy and Safety of Mitapivat in Subjects With Transfusion-Dependent Alpha- or Beta-Thalassemia (ENERGIZE-T)
Phase: PHASE3
Conditions: Transfusion-dependent Alpha-Thalassemia, Transfusion-dependent Beta-Thalassemia
Sponsor: Agios Pharmaceuticals, Inc.

Official Title:    A Phase 3, Double-blind, Randomized, Placebo-Controlled, Multicenter 
Study Evaluating the Efficacy and Safety of Mitapivat in 
Subjects With Transfusion-Dependent Alpha- or Beta-
Thalassemia (ENERGIZE-T)
NCT Number: 
 NCT04770779 
Document Date: 
Protocol Amendment 2.0: 28-Sep-2022

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CLINICAL STUDY PROTOCOL 
 
A Phase 3, Double-blind, Randomized, Placebo-Controlled, Multicenter Study Evaluating 
the Efficacy and Safety of Mitapivat in Subjects With Transfusion-Dependent 
Alpha- or Beta-Thalassemia (ENERGIZE-T) 
Study AG348-C-018 
 
Study Sponsor: 
Agios Pharmaceuticals, Inc. 
88 Sidney Street 
Cambridge, MA 02139-4169 USA 
Phone: 617-649-8600 
Facsimile: 617-649-8618 
Medical Director: 
 
Medical Director 
Clinical Development, 
Agios Pharmaceuticals, Inc. 
IND Number: 
 
NCT Number: 
NCT04770779 
EudraCT Number: 
2021-000212-34 
Approval Date: 
Original Protocol (27 January 2021) 
Amendment 1 (21 July 2021) 
Amendment 2 (28 September 2022) 
 
This study will be conducted according to the protocol and in compliance with Good Clinical 
Practices, the International Council for Harmonisation of Technical Requirements for 
Registration of Pharmaceuticals for Human Use Guidelines, applicable local regulatory 
requirements, and the spirit of the ethical principles stated in the Declaration of Helsinki. 
 

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SIGNATURE PAGE  
I hereby approve this clinical study protocol on behalf of Agios Pharmaceuticals, Inc. and attest 
that it complies with all applicable regulations and guidelines. 
 
Protocol Title: 
A Phase 3, Double-blind, Randomized, Placebo-Controlled, Multicenter 
Study Evaluating the Efficacy and Safety of Mitapivat in Subjects With 
Transfusion-Dependent Alpha- or Beta-Thalassemia (ENERGIZE-T) 
Approval Date: 
28 September 2022 
 
 Sponsor Signatory: 
, MD 
Medical Director 
 
Name and Title (Printed) 
 
Sign/Date (dd/mmm/yyyy) 
 Ph.D. 
Sr. Director, Biostatistics 
 
Name and Title (Printed) 
 
Sign/Date (dd/mmm/yyyy) 
 

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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 that has not been published 
previously will be kept in strict confidence. This documentation includes the study protocol, 
Investigator’s Brochure, case report forms, 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, in 
compliance with the recommendations in the International Conference on Harmonisation 
Guideline on Good Clinical Practice, and in accordance all applicable regulatory requirements, 
laws, and regulations. 
 
Protocol Title: 
A Phase 3, Double-blind, Randomized, Placebo-Controlled, Multicenter 
Study Evaluating the Efficacy and Safety of Mitapivat in Subjects With 
Transfusion-Dependent Alpha- or Beta-Thalassemia (ENERGIZE-T) 
Approval Date: 
28 September 2022 
 
 Investigator Signatory: 
 
  
 
 
Name and Title 
(Printed) 
 Signature 
 
Date 
(dd Month yyyy) 
 

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PROTOCOL AMENDMENT SUMMARY OF CHANGES 
 
 

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TABLE OF CONTENTS 
CLINICAL STUDY PROTOCOL ..................................................................................................1 
SIGNATURE PAGE .......................................................................................................................2 
INVESTIGATOR’S AGREEMENT ...............................................................................................3 
..........................................................4 
1. 
PROTOCOL SUMMARY ..........................................................................................10 
1.1. 
Synopsis ......................................................................................................................10 
1.2. 
Schedules of Assessments ..........................................................................................18 
2. 
INTRODUCTION ......................................................................................................27 
2.1. 
Background .................................................................................................................27 
2.1.1. 
Thalassemia ................................................................................................................27 
2.1.2. 
Mitapivat .....................................................................................................................29 
2.2. 
Study Rationale ...........................................................................................................32 
2.3. 
Benefit-Risk Assessment ............................................................................................33 
3. 
OBJECTIVES AND ENDPOINTS ............................................................................35 
4. 
STUDY DESIGN .......................................................................................................37 
4.1. 
Overall Design ............................................................................................................37 
4.1.1. 
Screening Period .........................................................................................................37 
4.1.2. 
Double-blind Period ....................................................................................................37 
4.1.3. 
Open-label Extension Period ......................................................................................38 
4.1.4. 
Duration of Treatment and Safety Follow-up .............................................................38 
4.2. 
Study Design Rationale ..............................................................................................38 
4.3. 
Dose Justification ........................................................................................................39 
4.4. 
End of Study Definition ..............................................................................................40 
5. 
STUDY POPULATION .............................................................................................41 
5.1. 
Inclusion Criteria ........................................................................................................41 
5.2. 
Exclusion Criteria .......................................................................................................41 
5.3. 
Lifestyle Considerations .............................................................................................43 
5.4. 
Screen Failures ............................................................................................................43 
6. 
STUDY DRUG ...........................................................................................................44 
6.1. 
Study Drug Administered ...........................................................................................44 
6.2. 
Preparation, Handling, Storage, and Accountability ..................................................45 

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6.3. 
Randomization and Blinding ......................................................................................45 
6.3.1. 
Randomization ............................................................................................................45 
6.3.2. 
Blinding ......................................................................................................................46 
6.3.3. 
Unblinding ..................................................................................................................46 
6.4. 
Study Drug Compliance .............................................................................................46 
6.5. 
Overdose .....................................................................................................................46 
6.6. 
Prior and Concomitant Therapy ..................................................................................46 
6.6.1. 
Prior Therapy ..............................................................................................................46 
6.6.2. 
Permitted Concomitant Therapy .................................................................................47 
6.6.3. 
Prohibited Concomitant Therapy ................................................................................47 
6.7. 
Dose Modifications .....................................................................................................48 
6.7.1. 
Management of Adverse Events and Clinical Laboratory Result Test 
Changes .......................................................................................................................48 
7. 
STUDY DRUG DISCONTINUATION AND SUBJECT WITHDRAWAL 
FROM STUDY ...........................................................................................................50 
7.1. 
Study Drug Discontinuation .......................................................................................50 
7.2. 
Subject Withdrawal From the Study ...........................................................................50 
7.3. 
Lost to Follow-up .......................................................................................................51 
8. 
STUDY ASSESSMENTS AND PROCEDURES ......................................................52 
8.1. 
Informed Consent .......................................................................................................52 
8.2. 
Screening and Eligibility ............................................................................................52 
8.3. 
Demographic Data ......................................................................................................53 
8.4. 
Medical, Procedural, Surgical, and Disease History ..................................................53 
8.5. 
Efficacy Assessments .................................................................................................53 
8.5.1. 
Clinical Laboratory Efficacy Assessments .................................................................53 
 
 
8.6. 
Safety Assessments .....................................................................................................54 
8.6.1. 
Vital Signs and Physical Examinations ......................................................................54 
8.6.2. 
Electrocardiograms .....................................................................................................54 
8.6.3. 
Clinical Laboratory Safety Assessments ....................................................................54 
8.6.4. 
Dual-Energy X-ray Absorptiometry Scans .................................................................55 
8.6.5. 
Adverse Events and Serious Adverse Events .............................................................56 
8.7. 
Red Blood Cell Transfusions and Transfusion Modifications ...................................56 

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8.8. 
Pharmacokinetics and Pharmacodynamics .................................................................57 
8.9. 
Genetics ......................................................................................................................57 
..58 
..58 
..58 
..58 
..59 
..59 
9. 
ADVERSE EVENTS: DEFINITIONS AND PROCEDURES FOR 
RECORDING AND REPORTING ............................................................................60 
9.1. 
Definitions of Adverse Events ....................................................................................60 
9.1.1. 
Definition of an Adverse Event ..................................................................................60 
9.1.2. 
Definition of a Serious Adverse Event .......................................................................60 
9.1.3. 
Abnormal Clinical Laboratory Events ........................................................................61 
9.2. 
Procedures for Recording Adverse Events and Serious Adverse Events ...................61 
9.2.1. 
Recording Adverse Events .........................................................................................61 
9.2.2. 
Severity of Adverse Events ........................................................................................62 
9.2.3. 
Relationship to Study Drug ........................................................................................63 
9.3. 
Procedures for Reporting Adverse Events ..................................................................63 
9.3.1. 
Reporting of Serious Adverse Events .........................................................................63 
9.4. 
Other Safety-Related Issues ........................................................................................64 
9.4.1. 
Overdose or Dose Administration Error .....................................................................64 
9.4.2. 
Pregnancy ...................................................................................................................64 
10. 
STATISTICAL CONSIDERATIONS .......................................................................66 
10.1. 
Statistical Hypotheses .................................................................................................66 
10.2. 
Sample Size Determination ........................................................................................67 
10.3. 
Populations for Analyses ............................................................................................68 
10.4. 
Statistical Analyses .....................................................................................................68 
10.4.1. 
General Considerations ...............................................................................................68 
10.4.2. 
Primary Endpoint ........................................................................................................70 
10.4.3. 
Secondary Efficacy Endpoints ....................................................................................70 
10.4.4. 
Safety Endpoints .........................................................................................................71 

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10.5. 
Interim Analyses .........................................................................................................72 
10.6. 
Independent Data Monitoring Committee ..................................................................72 
11. 
REGULATORY, ETHICAL, AND STUDY OVERSIGHT 
CONSIDERATIONS ..................................................................................................74 
11.1. 
Regulatory and Ethical Considerations ......................................................................74 
11.1.1. 
Temporary Modifications Allowed During Public Health Emergencies ...................74 
11.2. 
Informed Consent Process ..........................................................................................76 
11.3. 
Data Protection ...........................................................................................................77 
11.4. 
Dissemination of Clinical Study Data ........................................................................77 
11.5. 
Quality Compliance ....................................................................................................78 
11.6. 
Source Documents ......................................................................................................78 
11.7. 
Study Termination and Clinical Study Center Closure ..............................................79 
11.8. 
Publication Policy .......................................................................................................79 
12. 
REFERENCES ...........................................................................................................80 
13. 
APPENDICES ............................................................................................................84 
APPENDIX 1. LIST OF ABBREVIATIONS ............................................................................85 
APPENDIX 2. DEFINITION OF WOMEN OF CHILDBEARING POTENTIAL AND 
CONTRACEPTION GUIDANCE .............................................................................88 
 
 
90 
 

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LIST OF TABLES 
Table 1: 
Schedule of Assessments ............................................................................................18 
Table 2: 
Schedule of Assessments (Continued) ........................................................................24 
Table 3: 
Objectives and Endpoints ...........................................................................................35 
Table 4: 
Study Drug Dose and Recommended Dose Taper Regimen ......................................44 
Table 5: 
Study Drug ..................................................................................................................44 
Table 6: 
Dose Modifications for Excessive Hemoglobin Response .........................................49 
Table 7: 
Dose Modifications for Study Drug–Related Adverse Events ...................................49 
Table 8: 
Clinical Laboratory Efficacy Assessments .................................................................54 
Table 9: 
Clinical Laboratory Safety Assessments ....................................................................55 
Table 10: Red Blood Cell Transfusion Modification Guidance .................................................57 
Table 11: Adverse Event Severity ..............................................................................................62 
Table 12: Causal Attribution Guidance ......................................................................................63 
 
LIST OF FIGURES 
Figure 1: 
Study Design ...............................................................................................................37 
Figure 2: 
Statistical Testing Strategy .........................................................................................67 
 

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1. 
PROTOCOL SUMMARY 
1.1. 
Synopsis 
Name of Sponsor/Company: Agios Pharmaceuticals, Inc. 
Name of Investigational Product: mitapivat (also known as mitapivat sulfate, AG-348, and 
PYRUKYND) 
Study Title: A Phase 3, Double-blind, Randomized, Placebo-Controlled, Multicenter Study 
Evaluating the Efficacy and Safety of Mitapivat in Subjects With Transfusion-Dependent 
Alpha- or Beta-Thalassemia (ENERGIZE-T) 
Study Center(s): This multicenter study will be conducted internationally. 
Phase of Development: 3 
Objectives and Endpoints: 
Primary Objective 
• To compare the effect of 
mitapivat versus placebo on 
transfusion burden in subjects 
with α- or β-transfusion-
dependent thalassemia (TDT) 
Primary Endpoint 
• Transfusion reduction response (TRR), defined as a 
≥50% reduction in transfused red blood cell (RBC) 
units with a reduction of ≥2 units of transfused 
RBCs in any consecutive 12-week period through 
Week 48 compared with baseline 
Key Secondary Objective 
• To compare the durability of 
the effect of mitapivat versus 
placebo on transfusion burden 
Key Secondary Endpoints 
• ≥33% reduction in transfused RBC units from 
Week 13 through Week 48 compared with baseline 
(TRR3) 
• ≥50% reduction in transfused RBC units in any 
consecutive 24-week period through Week 48 
compared with baseline (TRR2) 
• ≥50% reduction in transfused RBC units from 
Week 13 through Week 48 compared with baseline 
(TRR4) 
Secondary Objectives 
• To evaluate the effect of 
mitapivat versus placebo on 
additional measures of 
transfusion burden 
Secondary Endpoints 
• Change from baseline in transfused RBC units from 
Week 13 through Week 48 
• Transfusion independence, defined as 
transfusion-free for ≥8 consecutive weeks through 
Week 48 
• To evaluate the effect of 
mitapivat versus placebo on 
iron metabolism 
• Change from baseline in iron, serum ferritin, total 
iron binding capacity, and transferrin saturation 
through Week 48 
• To evaluate the safety of 
mitapivat 
• Type, severity, and relationship of adverse events 
(AEs) and serious adverse events 

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• To evaluate the 
pharmacokinetic and 
pharmacodynamic effects of 
mitapivat 
• Plasma or blood concentrations and pharmacokinetic 
parameters of mitapivat and pharmacodynamic 
parameters, including adenosine triphosphate (ATP) 
and 2,3-diphosphoglycerate (2,3-DPG) 
 
Study Design: Study AG348-C-018 is a Phase 3, double-blind, randomized, placebo-controlled, 
multicenter study evaluating the efficacy and safety of mitapivat versus placebo in adult subjects 
with α- or β-TDT followed by an Open-label Extension Period. Subject eligibility will be 
determined during the (up to 8 weeks) Screening Period. 
The primary objective of the study is to compare the effect of mitapivat versus placebo on 
transfusion burden. Other secondary objectives include the evaluation of markers of iron 
overload, pharmacokinetic and pharmacodynamic parameters, and safety. Safety will be 
evaluated by the incidence, severity, and type of AEs, and by evaluation of vital signs, physical 
examination findings, clinical laboratory results, and bone mineral density scans. 
Subjects will continue to receive appropriate supportive care to manage symptoms and prevent 
secondary complications as clinically indicated and according to applicable guidelines. Dose 
modifications are permitted for excessive hemoglobin response and study drug–related AEs.  
During the 48-week Double-blind Period, study visits will occur approximately every 4 weeks 
for efficacy and safety assessments. Subjects will then be provided the opportunity to receive 
mitapivat for an additional 5 years in the Open-label Extension Period. During the Open-label 
Extension Period, subjects will continue to undergo safety and clinical activity assessments; 
however, the frequency of study visits will be reduced. Subjects will have a Safety Follow-up 
visit approximately 4 weeks after their last dose of study drug. 
Number of Subjects Planned: Approximately 240 subjects are planned to be randomized in this 
study. 
Eligibility Criteria: 
Inclusion Criteria: 
1. ≥18 years of age at the time of providing informed consent. 
2. Documented diagnosis of thalassemia (β-thalassemia with or without α-globin gene 
mutations, HbE/β-thalassemia, or α-thalassemia/HbH disease) based on DNA analysis 
from the subject’s medical record. If this information is not available from the subject’s 
medical record, DNA analysis can be performed by a local laboratory during the 
Screening Period. If a local laboratory is unable to perform the test, results from the 
comprehensive α- and β-globin genotyping performed by the study central laboratory can 
be used. 
3. Transfusion dependent, defined as 6 to 20 RBC units transfused and a ≤6-week 
transfusion-free period during the 24-week period before randomization. 
4. If taking hydroxyurea, the hydroxyurea dose must be stable for ≥16 weeks before 
randomization. 
5. Women of childbearing potential (WOCBP) must be abstinent of sexual activities that 
may induce pregnancy 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 providing 

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informed consent, throughout the study, and for 28 days after the last dose of study drug. 
The second form of contraception can be an acceptable barrier method. 
6. Written informed consent before any study-related procedures are conducted and willing 
to comply with all study procedures for the duration of the study. 
Exclusion Criteria 
1. Pregnant, breastfeeding, or parturient. 
2. Documented history of homozygous or heterozygous HbS or HbC. 
3. Prior exposure to gene therapy or prior bone marrow or stem cell transplantation. 
4. Currently receiving treatment with luspatercept; the last dose must have been 
administered ≥36 weeks before randomization. 
5. Currently receiving treatment with hematopoietic stimulating agents; the last dose must 
have been administered ≥36 weeks before randomization. 
6. History of malignancy (active or treated) ≤5 years before providing informed consent, 
except for nonmelanomatous skin cancer in situ, cervical carcinoma in situ, or breast 
carcinoma in situ.  
7. History of active and/or uncontrolled cardiac or pulmonary disease ≤6 months before 
providing informed consent, including but not limited to: 
a. New York Heart Association Class III or IV heart failure or clinically significant 
dysrhythmia  
b. Myocardial infarction or unstable angina pectoris; hemorrhagic, embolic, or 
thrombotic stroke; deep venous thrombosis; or pulmonary or arterial embolism 
c. Heart rate–corrected QT interval using Fridericia’s method ≥450 milliseconds (males) 
or ≥470 milliseconds (females), except for right or left bundle branch block 
d. Severe pulmonary fibrosis as defined by severe hypoxia, evidence of right-sided heart 
failure, and radiographic pulmonary fibrosis >50% 
e. Severe pulmonary hypertension as defined by severe symptoms associated with 
hypoxia, right-sided heart failure, and oxygen indicated 
8. Hepatobiliary disorders, including but not limited to: 
a. Liver disease with histopathological evidence of cirrhosis or severe fibrosis 
b. Clinically symptomatic cholelithiasis or cholecystitis (prior cholecystectomy is not 
exclusionary) 
c. History of drug-induced cholestatic hepatitis 
d. Aspartate aminotransferase >2.5 × upper limit of normal (ULN); unless due to 
hemolysis and hepatic iron deposition) and alanine aminotransferase >2.5 × ULN 
(unless due to hepatic iron deposition) 
9. Estimated glomerular filtration rate <45 mL/min/1.73 m2 by Chronic Kidney Disease 
Epidemiology Collaboration creatinine equation. 
10. Nonfasting triglycerides >440 mg/dL (5 mmol/L). 
11. Active infection requiring systemic antimicrobial therapy at the time of providing 
informed consent. If antimicrobial therapy is required during the Screening Period, 
screening procedures should not be performed while antimicrobial therapy is being 

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administered, and the last dose of antimicrobial therapy must be administered ≥7 days 
before randomization. 
12. Positive test for hepatitis C virus (HCV) antibody (Ab) with evidence of active HCV 
infection, or positive test for hepatitis B surface antigen. 
13. Positive test for HIV-1 Ab or HIV-2 Ab. 
14. History of major surgery (including splenectomy) ≤6 months before providing informed 
consent and/or a major surgical procedure planned during the study. 
15. Current enrollment or past participation (≤12 weeks before administration of the first 
dose of study drug or a time frame equivalent to 5 half-lives of the investigational 
treatment, whichever is longer) in any other clinical study involving an investigational 
treatment or device. 
16. Receiving strong cytochrome P450 (CYP)3A4/5 inhibitors that have not been stopped for 
≥5 days or a time frame equivalent to 5 half-lives (whichever is longer), or strong 
CYP3A4 inducers that have not been stopped for ≥4 weeks or a time frame equivalent to 
5 half-lives (whichever is longer), before randomization. 
17. Receiving anabolic steroids that have not been stopped for at least 4 weeks before 
randomization. Testosterone replacement therapy to treat hypogonadism is allowed; the 
testosterone dose and preparation must be stable for ≥12 weeks before randomization. 
18. Known allergy, or other contraindication, to mitapivat or its excipients (microcrystalline 
cellulose, croscarmellose sodium, sodium stearyl fumarate, mannitol, and magnesium 
stearate, Opadry® II Blue [hypromellose, titanium dioxide, lactose monohydrate, 
triacetin, and FD&C Blue #2]). 
19. Any medical, hematological, psychological, or behavioral condition(s) or prior or current 
therapy that, in the opinion of the Investigator, may confer an unacceptable risk to 
participating in the study and/or could confound the interpretation of the study data. Also 
excluded are: 
• Subjects who are institutionalized by regulatory or court order 
• Subjects with any condition(s) that could create undue influence (including but not 
limited to incarceration, involuntary psychiatric confinement, and financial or familial 
affiliation with the Investigator or Sponsor) 
Investigational Product, Dosage, and Mode of Administration: Subjects will receive 100 mg 
twice-daily mitapivat for oral administration. 
Duration of Treatment and End of Study: The duration of participation of a subject in the 
study is up to 6.3 years. The duration of treatment is up to 6.0 years. The end of the study is 
defined as the time at which all subjects complete all study visits, are lost to follow-up, have 
withdrawn consent for further participation in the study, or when the Sponsor terminates the 
study. 
Reference Therapy, Dosage, and Mode of Administration: Subjects will receive matched 
placebo for oral administration. 
Statistical Methods: 
Sample size determination and statistical methodology for the primary analysis of key efficacy 
endpoints, and analyses of safety and pharmacokinetic/pharmacodynamic endpoints, are 

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described below. Further details will be provided in the statistical analysis plan (SAP), which 
will be finalized before database lock and unblinding. 
Randomization and Blinding: Eligible subjects will be randomly assigned in a 2:1 ratio to 
receive study drug (mitapivat or placebo). Randomization will be stratified by geographical 
region and by thalassemia genotype. Study subjects, Investigators, clinical study center 
personnel, pharmacists, and the Sponsor will be blinded to the subject’s treatment assignment. 
During the Double-blind Period, an unblinded Independent Data Monitoring Committee will be 
responsible for ongoing monitoring of the safety of subjects. 
Statistical Testing Strategy: The following statistical hypothesis will be tested to address the 
primary objective: 
H01: 𝑝𝑡1 −𝑝𝑐1 = 0 vs H11: 𝑝𝑡1 −𝑝𝑐1 ≠0 
where 𝑝𝑡1 and 𝑝𝑐1 are the proportion of subjects achieving a TRR in the mitapivat arm and 
placebo arm, respectively. 
Additionally, the following statistical hypotheses will be tested to address the key secondary 
objectives: 
H02: 𝑝𝑡2 −𝑝𝑐2 = 0 vs H12: 𝑝𝑡2 −𝑝𝑐2 ≠0 
H03: 𝑝𝑡3 −𝑝𝑐3 = 0 vs H13: 𝑝𝑡3 −𝑝𝑐3 ≠0 
H04: 𝑝𝑡4 −𝑝𝑐4 = 0 vs H14: 𝑝𝑡4 −𝑝𝑐4 ≠0 
• where 𝑝𝑡2 and 𝑝𝑐2 are the proportion of subjects achieving a TRR2 in the mitapivat 
arm and placebo arm, respectively. 
• 𝑝𝑡3 and 𝑝𝑐3 are the proportion of subjects achieving a TRR3 in the mitapivat arm and 
placebo arm, respectively. 
• 𝑝𝑡4 and 𝑝𝑐4 are the proportion of subjects achieving a TRR4 in the mitapivat arm and 
placebo arm, respectively. 
Overall type I error will be maintained at or below a 2-sided significance level of 0.05 based on a 
graphical gatekeeping approach as illustrated in the figure below: 

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Sample Size Determination: Approximately 240 subjects will be randomized in a 2:1 ratio to 
receive mitapivat or placebo. With the planned sample size, the study will have 95% power to 
detect an increase in TRR rate from 12.5% in the placebo arm to 33.7% in the mitapivat arm at a 
2-sided α-level of 0.05. 

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Populations for Analyses: 
Population 
Description 
All Screened 
Subjects 
All subjects who sign the informed consent form. 
Full Analysis Set 
(FAS) 
All subjects who are randomized. Subjects will be classified according to 
the randomized treatment arm according to the intent-to-treat principle. 
The FAS is the primary analysis set for the evaluation of efficacy 
endpoints, unless otherwise specified. 
Per-Protocol Set 
(PPS) 
The PPS is a subset of the FAS. Subjects who do not receive at least 1 
dose of the randomized treatment will be excluded from the PPS. Other 
criteria leading to exclusion of subjects from the PPS will be prespecified 
in the SAP. 
Safety Analysis 
Set 
All subjects who receive at least 1 dose of the study treatment. Subjects 
will be classified according to the treatment received. If a subject 
randomized to placebo receives at least 1 dose of mitapivat in the Double-
blind Period, then the subject will be classified to the mitapivat arm. 
Pharmacodynamic 
Analysis Set 
A subset of the Safety Analysis Set including all subjects with at least 
1 blood 2,3-DPG or ATP concentration measurement above the lower 
limit of quantitation (LLQ). 
Pharmacokinetic 
Analysis Set 
A subset of the Safety Analysis Set including all subjects with at least 
1 plasma mitapivat concentration measurement above LLQ. 
 
Primary Endpoint: The primary endpoint of TRR is defined as ≥50% reduction in transfused 
RBC units with a reduction of ≥2 units of transfused RBCs in any consecutive 12-week period 
through Week 48 compared with baseline. The proportion of subjects who achieve a TRR will be 
summarized for each treatment arm. The difference in TRR rate between the mitapivat arm and 
the placebo arm will be estimated by a Mantel-Haenszel stratum weighted method adjusting for 
randomization stratification factors. The adjusted difference in TRR rate, along with the 95% CI 
and the 2-sided p-value will be provided. Subjects who are withdrawn from the study before 
Week 12 (Day 85) will be considered nonresponders. 
Key Secondary Endpoints: 
The key secondary endpoints are TRR2, TRR3, and TRR4.  The same analysis method presented 
for the primary endpoint will be used for the analysis of TRR2, TRR3 and TRR4. Subjects who 
are withdrawn from the study before Week 24 will be considered nonresponders in TRR2. 
Subjects who are withdrawn from the study before Week 48 will be considered nonresponders in 
TRR3 and TRR4. 
Pharmacokinetic and Pharmacodynamic Endpoints 
Descriptive statistics of pharmacokinetic (arithmetic and geometric means, standard deviation, 
percent coefficient of variation [CV%], CV% geometric mean, minimum, median, and 
maximum) and pharmacodynamic parameters, including ATP and 2,3-DPG, will be summarized 
as appropriate.  

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Safety Endpoints 
Summaries of safety data from the Double-blind Period will be presented by treatment arm based 
on the Safety Analysis Set. Summaries of safety data from the Open-label Extension Period will 
be presented by treatment arm and overall based on the Safety Analysis Set.  
The on-treatment period begins on the date of the start of study drug and ends 28 days after the 
end of study drug administration (including the recommended dose taper). 
Treatment-emergent adverse events are defined as AEs with a first onset date during the 
on-treatment period or worsening from baseline. Treatment-emergent adverse events will be 
summarized according to the latest version of the Medical Dictionary for Regulatory Activities 
terminology by System Organ Class and/or Preferred Term, severity (based on the National 
Cancer Institute [NCI] Common Terminology Criteria for Adverse Events [CTCAE] 
Version 4.03), seriousness, and relationship to study drug, unless otherwise specified. 
Clinical laboratory test results will be graded according to the NCI CTCAE Version 4.03, as 
applicable, and will be summarized based on the worst CTCAE grade. 
 

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1.2. 
Schedules of Assessments 
 
Table 1: 
Schedule of Assessments 
Study Visit Week: 
Screening 
Period 
On-Treatment Period1 
Double-blind Period 
Open-label Extension Period 
-- 
T02 
-- 
-- 
1 
43 
83 
12 
163 
20 
24 
283 
32 
36 
403 
443 
48 
52 
563 
603 
72 
843 
96 
1083 
Study Visit Day: -56 to  
-8 
-7 to 
-1 
1 
29 
57 
85 113 141 169 197 225 253 281 309 337 365 
393 
421 
505 
589 673 757 
Study Visit Window 
(Days): 
-- 
-- 
-- 
±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 
±12 
±12 
±14 
±14 ±14 ±14 
Procedures: 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Documentation of 
thalassemia 
diagnosis4 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Comprehensive 
α- and β-globin 
genotyping5 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical, procedural, 
surgical, and disease 
history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Transfusion history6 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Prior medications7 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table 1: 
Schedule of Assessments 
Study Visit Week: 
Screening 
Period 
On-Treatment Period1 
Double-blind Period 
Open-label Extension Period 
-- 
T02 
-- 
-- 
1 
43 
83 
12 
163 
20 
24 
283 
32 
36 
403 
443 
48 
52 
563 
603 
72 
843 
96 
1083 
Study Visit Day: -56 to  
-8 
-7 to 
-1 
1 
29 
57 
85 113 141 169 197 225 253 281 309 337 365 
393 
421 
505 
589 673 757 
Study Visit Window 
(Days): 
-- 
-- 
-- 
±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 
±12 
±12 
±14 
±14 ±14 ±14 
Hepatitis and HIV 
screening8 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy testing 
(WOCBP only)9 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomization10 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
BMD by DXA scan11 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
Physical examination 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
Vital signs, weight, 
and height13 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG14 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hematology15 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Clinical chemistry16 
X 
 
X 
X 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
 
X 
X 
X 
X 
X 
Liver tests 
 
17  
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Lipid panel18 
X 
 
  
 
 
  
 
 
  
 
 
  
 
 
X 
 
 
  
  
  
X 
  

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Table 1: 
Schedule of Assessments 
Study Visit Week: 
Screening 
Period 
On-Treatment Period1 
Double-blind Period 
Open-label Extension Period 
-- 
T02 
-- 
-- 
1 
43 
83 
12 
163 
20 
24 
283 
32 
36 
403 
443 
48 
52 
563 
603 
72 
843 
96 
1083 
Study Visit Day: -56 to  
-8 
-7 to 
-1 
1 
29 
57 
85 113 141 169 197 225 253 281 309 337 365 
393 
421 
505 
589 673 757 
Study Visit Window 
(Days): 
-- 
-- 
-- 
±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 
±12 
±12 
±14 
±14 ±14 ±14 
Coagulation tests19 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sex hormones20 
 
 
X 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
 
X 
X 
 
X 
 
Iron panel21 
 
X 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
X 
 
X 
 
Blood sample for 
pharmacodynamic 
analysis 
 
X23 
X23 
 
 
X24 
 
 
X24 
 
 
X25 
 
 
 
 
 
 
 
 
 
 
Blood sample for 
pharmacokinetic 
analysis 
 
 
 
 
 
X24 
 
 
X24 
 
 
X25 
 
 
 
 
 
 
 
 
 
 

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Table 1: 
Schedule of Assessments 
Study Visit Week: 
Screening 
Period 
On-Treatment Period1 
Double-blind Period 
Open-label Extension Period 
-- 
T02 
-- 
-- 
1 
43 
83 
12 
163 
20 
24 
283 
32 
36 
403 
443 
48 
52 
563 
603 
72 
843 
96 
1083 
Study Visit Day: -56 to  
-8 
-7 to 
-1 
1 
29 
57 
85 113 141 169 197 225 253 281 309 337 365 
393 
421 
505 
589 673 757 
Study Visit Window 
(Days): 
-- 
-- 
-- 
±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 ±12 
±12 
±12 
±14 
±14 ±14 ±14 
Study drug 
administration28 
 
 
X (continuous) 
AEs and SAEs29 
X (continuous) 
Concomitant 
medications, 
procedures, and 
surgeries30 
 
 
X (continuous) 
On-treatment 
transfusion record31 
 
 
X (continuous) 
Abbreviations: Ab = antibody; AE = adverse event; ALP = alkaline phosphatase; ALT = alanine aminotransferase; aPTT = activated partial thromboplastin time; AST = aspartate 
aminotransferase; BMD = bone mineral density; BUN = blood urea nitrogen; CO2 = carbon dioxide; DXA = dual-energy x-ray absorptiometry; ECG = electrocardiogram; 
; EOS = End of Study; 
; FSH = follicle stimulating hormone; Hb = hemoglobin; 
; HBsAg = hepatitis B surface antigen; hCG = human chorionic gonadotropin; HCT = hematocrit; HCV = hepatitis C virus; HDL-C = high-density 
lipoprotein cholesterol; 
 INR = international normalized ratio; LCMS = liquid chromatography-mass spectrometry;
; LDL-C = low-density lipoprotein cholesterol; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; MCV = mean 
corpuscular volume; 
 
 RBC = red blood cell; RDW-CV = red cell distribution width coefficient of variation; SAE = serious adverse event; 

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T0 = transfusion zero; Thal = thalassemia; 
 WBC = white blood cell; WOCBP = women of childbearing 
potential. 
Notes: 
 All blood samples must be 
collected before study drug administration and before an RBC transfusion, including T0, if an RBC transfusion is scheduled on the same day (see Section 8.7). 
1 At any time during the study, subjects who discontinue study drug should undergo the recommended dose taper and complete the assessments at the Safety Follow-up visit after 
administration of their last dose of study drug (ie, including the recommended dose taper). Subjects who withdraw from the study should be encouraged to first undergo the 
recommended dose taper (Table 4) and complete the Safety Follow-up visit (Table 2). Subjects who are discontinuing study drug should be monitored for signs of hemolysis 
and worsening of anemia, as clinically indicated. 
2 The first dose of study drug must be administered within 7 days after T0. Transfusion 0 is defined as the last RBC transfusion that was administered before randomization. 
3 If allowed by local institutional guidelines, and at the discretion of the Investigator and the subject, the subject can complete some visits and study-related assessments at home. 
These visits (Investigator Assessment and Home Health) will include a visit conducted by the Investigator (or designee) and a visit at home with a nurse or home health-care 
provider (see Section 8). 
4 Documentation of thalassemia diagnosis by DNA analysis will be recorded from the subject’s medical record. If this documentation is not available from the subject’s medical 
record, the test should be performed during the Screening Period (see Section 8.2). 
5 A blood sample will be collected for comprehensive α- and β-globin genotyping. Results of this analysis are not required for eligibility. 
6 Transfusion history will be recorded for the 24-week period before randomization. Transfusion history includes T0. A pretransfusion Hb concentration threshold will be 
determined for each subject based on transfusion history (see Section 8.7). Definition of an RBC unit and information to be recorded for each RBC transfusion is also in this 
section. 
7 Prior medications are defined as those administered any time ≤4 weeks before providing informed consent until administration of the first dose of study drug, unless otherwise 
specified. Prior use of iron chelation therapy for up to 1 year before administration of the first dose of study drug will be recorded. 
8 A test for HBsAg and HCV Ab will be performed during the Screening Period. Positive results for HCV Ab testing require confirmation by a test for HCV-RNA. A test for 
HIV-1 Ab and HIV-2 Ab will also be performed during the Screening Period. 
9 During the Screening Period, an FSH assessment will be performed for women in the absence of 12 months of amenorrhea to confirm non–reproductive potential (see 
Appendix 2), and a serum (hCG) pregnancy test will be performed for WOCBP. On Day 1, a serum or urine pregnancy test will be performed locally for WOCBP and confirmed 
negative before administration of the first dose of study drug. Thereafter, pregnancy testing should occur more frequently if clinically indicated (ie, if there is suspicion of 
pregnancy) or required by local regulations. 
10 Randomization can occur up to 48 hours before administration of the first dose of study drug on Day 1. Randomization must not occur until T0 has been completed. 
11 DXA scan of the hip and spine. 
 
13 Vital signs include systolic and diastolic blood pressure, pulse rate, and body temperature. Weight will also be recorded. Height will be recorded at screening only. Vital signs 
should be measured after 5 minutes of recumbency, and before blood sample collection. 
14 A single, 12-lead ECG will be recorded and read at the clinical study center after 5 min of recumbency and before blood sample collection. 
15 Hematology includes a complete blood count (Hb, HCT, MCHC, MCH, MCV, RBC count, nucleated RBCs, RDW-CV, % reticulocyte count, absolute reticulocyte count, and 
platelet count) with WBC count and differential (neutrophils, basophils, eosinophils, lymphocytes, and monocytes). 
16 Clinical chemistry includes albumin, BUN or urea, total calcium, CO2 or bicarbonate, chloride, creatinine, 
 magnesium, phosphorus, potassium, 
sodium, total protein, and uric acid. 
17 Liver tests include ALP, ALT/SGPT, AST/SGOT, and bilirubin (direct/conjugated, indirect/unconjugated, and total). 
 
18 Lipid panel includes nonfasting HDL-C, LDL-C, total cholesterol, and triglycerides. 
19 Coagulation tests include fibrinogen, aPTT, and INR. 
20 Sex hormones include nonfasting estradiol, estrone, and testosterone (free and total). If possible, blood samples should be collected in the morning. A highly sensitive LCMS 
assay should be used to analyze samples. 
21 Iron panel includes iron, serum ferritin, transferrin, transferrin saturation, and total iron binding capacity. 
 
23 A blood sample for pharmacodynamic analysis will be collected at T0 (up to 3 days before transfusion) and on Day 1 (≤60 min predose). 

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24 Blood samples for pharmacokinetic and pharmacodynamic analysis will be collected at Week 12 (≤60 min predose) and Week 24 (≤60 min predose). If an RBC transfusion takes 
place during this visit, the blood samples for pharmacokinetic and pharmacodynamic analysis should be collected before the RBC transfusion. 
25 Blood samples for pharmacokinetic and pharmacodynamic analysis will be collected at Week 36 (≤60 min predose, and 0.5 hr [±5 min], 1 hr [±5 min], 3 hr [±15 min], 5 hr 
[±15 min], and 7 hr [±30 min] postdose). If an RBC transfusion takes place during this visit, the blood samples for pharmacokinetic and pharmacodynamic analysis should be 
collected before the RBC transfusion. 
 
 
28 Study drug will be dispensed at every visit. Subjects must return study drug bottles (including empty bottles) and unused tablets at each visit to the clinical study center. The 
morning dose of study drug will be administered at the clinical study center during in-person visits to the clinical study center. During the Double-blind Period, subjects will be 
asked to record study drug dosing in a diary. The last dose of blinded study drug will be administered on the morning of the Week 48 visit. The first dose of open-label mitapivat 
will be administered on the evening of the Week 48 visit. 
29 Adverse events and SAEs will be collected throughout the study. 
30 Concomitant medications, procedures, and surgeries are defined as all medications administered and procedures conducted from the first dose of study drug through 4 weeks 
after administration of the last dose of study drug (ie, EOS visit). 
31 During the study, the subject’s RBC transfusion schedule will be modified according to the guidance in Section 8.7. Information to be recorded for each transfusion is also in this 
section. 
 

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Table 2: 
Schedule of Assessments (Continued) 
Study Visit Week: 
Open-label Extension Period1 
Safety 
Follow- 
up2 
120 132 
144 
1563 
168 
1803 
192 
2043 
216 
2283 
240 
2523 
264 
2763 
288 
3003 
3124 28 Days 
After 
Last 
Dose/ 
EOS 
Study Visit Day: 841 925 1,009 1,093 1,177 1,261 1,345 1,429 1,513 1,597 1,681 1,765 1,849 1,933 2,017 2,101 2,185 
Study Visit Window (Days): ±14 ±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
+4 
Procedures: 
Pregnancy testing 
(WOCBP only)5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
BMD by DXA scan6 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
Physical examination 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
Vital signs and weight8 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Hematology9 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Clinical chemistry10 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Liver tests 
11 
X 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Lipid panel12 
  
  
X 
 
  
 
X 
 
  
 
X 
 
  
 
X 
 
X 
X 
Sex hormones13 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Iron panel14 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 

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Table 2: 
Schedule of Assessments (Continued) 
Study Visit Week: 
Open-label Extension Period1 
Safety 
Follow- 
up2 
120 132 
144 
1563 
168 
1803 
192 
2043 
216 
2283 
240 
2523 
264 
2763 
288 
3003 
3124 28 Days 
After 
Last 
Dose/ 
EOS 
Study Visit Day: 841 925 1,009 1,093 1,177 1,261 1,345 1,429 1,513 1,597 1,681 1,765 1,849 1,933 2,017 2,101 2,185 
Study Visit Window (Days): ±14 ±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
±14 
+4 
Procedures: 
Study drug administration17 
X (continuous) 
X 
 
AEs and SAEs18 
X (continuous) 
X 
Concomitant medications, 
procedures, and surgeries19 
X (continuous) 
X 
On-treatment transfusion 
record20 
X (continuous) 
 
Abbreviations: AE = adverse event; ALP = alkaline phosphatase; ALT = alanine aminotransferase; AST = aspartate aminotransferase; BMD = bone mineral density; BUN = blood 
urea nitrogen; CO2 = carbon dioxide; DXA = dual-energy x-ray absorptiometry; 
 EOS = End of Study; Hb = hemoglobin; 
 
 HCT = hematocrit; HDL-C = high-density lipoprotein cholesterol; 
 
; LDL-C = low-density 
lipoprotein cholesterol; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume; 
 
 RBC = red blood cell; RDW-CV = red cell distribution width coefficient of 
variation; SAE = serious adverse event; 
 WBC = white blood cell; WOCBP = women of childbearing potential. 
Notes: 
 All blood samples must be 
collected before study drug administration and before the RBC transfusion, if an RBC transfusion is scheduled on the same day (see Section 8.7). 
1 At any time during the study, subjects who discontinue study drug should undergo the recommended dose taper and complete the assessments at the Safety Follow-up visit after 
administration of their last dose of study drug (ie, including the recommended dose taper). Subjects who withdraw from the study should be encouraged to first undergo the 
recommended dose taper (Table 4) and complete the Safety Follow-up visit. Subjects who are discontinuing study drug should be monitored for signs of hemolysis and 
worsening of anemia, as clinically indicated. 
2 The Safety Follow-up visit is to be completed after administration of the last of any dose of study drug (ie, including the recommended dose taper). 

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3 If allowed by local institutional guidelines, and at the discretion of the Investigator and the subject, the subject can complete some visits and study-related assessments at home. 
These visits (Investigator Assessment only) will be conducted by the Investigator (or designee) (see Section 8). 
4 After completing the study visit at Week 312, subjects should begin the dose taper. 
5 Pregnancy testing should occur more frequently as clinically indicated (ie, if there is suspicion of pregnancy) or if required by local regulations. A serum or urine pregnancy test 
will be performed locally for WOCBP at the Safety Follow-up visit. 
6 DXA scan of the hip and spine. 
 
 
8 Vital signs include systolic and diastolic blood pressure, pulse rate, and body temperature. Weight will also be recorded. Vital signs should be measured after 5 minutes of 
recumbency, and before blood sample collection. 
9 Hematology includes a complete blood count (Hb, HCT, MCHC, MCH, MCV, RBC count, nucleated RBCs, RDW-CV, % reticulocyte count, absolute reticulocyte count, and 
platelet count) with WBC count and differential (neutrophils, basophils, eosinophils, lymphocytes, and monocytes). 
10 Clinical chemistry includes albumin, BUN or urea, total calcium, CO2 or bicarbonate, chloride, creatinine, 
 magnesium, phosphorus, potassium, 
sodium, total protein, and uric acid. 
11 Liver tests include ALP, ALT/SGPT, AST/SGOT, and bilirubin (direct/conjugated, indirect/unconjugated, and total).
 
12 Lipid panel includes nonfasting HDL-C, LDL-C, total cholesterol, and triglycerides. 
13 Sex hormones include nonfasting estradiol, estrone, and testosterone (free and total). If possible, blood samples should be collected in the morning. A highly sensitive LCMS 
assay should be used to analyze samples. 
14 Iron panel includes iron, serum ferritin, transferrin, transferrin saturation, and total iron binding capacity. 
 
 
17 Study drug will be dispensed at every visit. Subjects must return study drug bottles (including empty bottles) and unused tablets at each visit to the clinical study center. The 
morning dose of study drug will be administered at the clinical study center during in-person visits to the clinical study center. 
18 Adverse events and SAEs will be collected throughout the study. 
19 Concomitant medications, procedures, and surgeries are defined as all medications administered and procedures conducted from the first dose of study drug through 4 weeks 
after administration of the last dose of study drug (ie, EOS visit). 
20 During the study, the subject’s RBC transfusion schedule will be modified according to the guidance in Section 8.7. Definition of an RBC unit and information to be recorded for 
each transfusion is also in this section. 
 
 

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2. 
INTRODUCTION 
2.1. 
Background 
2.1.1. 
Thalassemia 
The thalassemias are a group of disorders in which the normal ratio of α- to β-globin production 
is disrupted due to a disease-causing variant in one or more of the globin genes. Inherited 
hemoglobin (Hb) disorders can be divided into 2 main groups. The first group includes the 
α- and β-thalassemias, which result from the defective synthesis of the α- or β-globin chains of 
adult Hb (HbA). The second group includes structural Hb variants, such as sickle cell 
hemoglobin (HbS), HbC, and HbE (Musallam et al, 2013; Taher et al, 2017). Alpha-globin 
aggregates (found in β-thalassemia) readily precipitate, which disrupts the red blood cell (RBC) 
membrane and results in oxidative stress. Beta-globin tetramers (found in α-thalassemia/HbH 
disease) are generally more soluble but are still unstable and can form precipitates as well 
(Cappellini et al, 2014). 
In thalassemia, the imbalance in globin chain synthesis imposes metabolic stress on the RBCs, 
specifically in the form of excess generation of reactive oxygen species and an increased demand 
on adenosine triphosphate (ATP)-dependent proteolytic mechanisms to clear excess globin 
chains. In thalassemic RBCs, ATP supply appears to be insufficient to maintain RBC membrane 
fitness and clearance of globin precipitates, leading to early and increased death of RBC 
precursors in the bone marrow and in extramedullary sites (Chakraborty et al, 2012; Shaeffer, 
1988; Ting et al, 1994). 
These pathophysiological changes lead to the hallmarks of the disease: ineffective erythropoiesis, 
peripheral hemolysis, and subsequent anemia. Clinical implications of the α- and β-globin 
imbalance include lack of sufficient RBCs and Hb for effective oxygen transport, and ineffective 
erythropoiesis and hemolysis, which can lead to splenomegaly, bone marrow expansion 
(extramedullary hematopoiesis), concomitant bone deformities, and iron overload (Cappellini et 
al, 2014). 
Alpha- and β-thalassemias are genetically heterogeneous and vary by phenotype and severity 
(Galanello, 2012; Galanello and Cao, 2011; Thein, 2013; Vichinsky, 2012; Vichinsky, 2016). 
Clinical management with RBC transfusions is an essential factor in classifying thalassemias as 
either transfusion-dependent thalassemia (TDT) or non–transfusion-dependent thalassemia 
(NTDT). Patients with TDT need life-long regular transfusions for survival. Patients with NTDT 
do not need life-long regular transfusions for survival; however, they may require transfusions 
during times of erythroid stress such as infection, pregnancy, surgery, or aplastic crisis, and may 
transition to requiring regular transfusions and becoming transfusion dependent later in life 
(Musallam et al, 2020; Musallam et al, 2013; Taher, Radwan, et al, 2015). While TDT and 
NTDT are widely used clinical classifications of thalassemia that capture the different 
transfusional needs, the underlying molecular and pathophysiological basis is similar across the 
spectrum of the disease (Galanello, 2012; Galanello and Cao, 2011; Thein, 2013; Vichinsky, 
2012; Vichinsky, 2016). As this clinical study (AG348-C-018) is focused on a population of 
subjects with TDT, the following sections focus more specifically on disease characteristics and 
available treatments for patients with TDT. 

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2.1.1.1. 
Transfusion-Dependent Thalassemia 
Patients with TDT include those with β-thalassemia major or severe forms of β-thalassemia 
intermedia, HbE/β-thalassemia, or α-thalassemia/HbH disease. Patients with β-thalassemia major 
present in early childhood and require life-long regular RBC transfusions for survival. Patients 
with severe forms of β-thalassemia intermedia, HbE/β-thalassemia, or α-thalassemia/HbH 
disease can range from requiring chronic transfusions in early childhood to developing 
transfusion dependence later in adulthood (Cappellini et al, 2014). 
Over time, anemia with or without transfusions can lead to widespread organ damage and 
dysregulated compensatory mechanisms. Regular transfusions are associated with iron overload; 
however, in the absence of RBC transfusions, iron overload can also be caused by chronic 
hemolysis, ineffective erythropoiesis, and hypoxia, ultimately leading to increased intestinal iron 
absorption. Clinical consequences of thalassemia include splenomegaly, extramedullary 
expansion of hematopoiesis, bone deformities, osteoporosis, and potentially, fractures 
(Angastiniotis et al, 2013; Taher et al, 2011; Taher, Musallam, et al, 2015). The death rate of 
adults with TDT remains higher than that of the general United States (US) population (Fung et 
al, 2007), and high morbidity and mortality persist (Borgna-Pignatti et al, 2004; Modell et al, 
2000). Access to transfusions and specialized supportive care varies by region and is challenging 
particularly in resource-constrained countries, creating a gap in access for some patients (Betts et 
al, 2020; Cappellini et al, 2018; Cappellini et al, 2014; Vichinsky, 2012; Weatherall, 2011). 
2.1.1.2. 
Transfusion-Dependent Thalassemia Treatment 
Besides RBC transfusions, most patients with TDT receive supportive care, which may include 
iron chelation therapy and splenectomy (Cappellini et al, 2014). Chronic RBC transfusions are 
associated with iron overload, transfusion reactions, infections, and alloimmunization. Patients 
with TDT often require treatment with iron chelators to avoid long-term complications from 
tissue iron deposition, which may specifically cause hepatic, cardiac, and endocrine toxicity. Iron 
overload–related cardiac events are the leading cause of mortality in patients with TDT. These 
therapies have limitations in efficacy and safety, and access and adherence to treatment are not 
uniform (Cappellini et al, 2018; Taher et al, 2018). Splenectomy increases Hb concentration and 
can lead to improved growth and development (Karimi et al, 2014); however, patients are at 
increased risk of thrombotic and vascular events and infections after splenectomy (Cappellini et 
al, 2000). Despite recent improvements in the clinical management of patients with TDT, 
including increased access to blood supply, reduction of transfusion-associated infections, iron 
chelation therapy, and advanced imaging to evaluate hepatic and cardiac iron concentrations, the 
complication rate remains high (Borgna-Pignatti et al, 2004; Modell et al, 2000). 
Recently, targeted therapies have become available for patients with TDT, but they either have 
substantial restrictions associated with their administration or address only a subpopulation of 
patients with β-thalassemia. Hematopoietic stem cell transplantation (HSCT) and gene therapy 
are potentially curative treatments (Angelucci et al, 2014; Schuessler-Lenz et al, 2020). 
Zynteglo® (autologous CD34+ cells encoding βA-T87Q-globin gene) was approved in the 
European Union in 2019 for patients 12 years and older with β-thalassemia (non-(β0/β0) 
genotype) who require regular RBC transfusions and for whom HSCT is appropriate, but a donor 
is not available (Zynteglo Summary of Product Characteristics, 2019). Reblozyl® (luspatercept), 
an activin receptor fusion protein, was approved in the US in 2019 and the European Union in 

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2020 to treat anemia in adults with β-thalassemia who require regular RBC transfusions 
(Reblozyl (luspatercept-aamt) USPI, 2019). 
Mitapivat offers the option of oral administration with a favorable safety profile and has the 
potential to treat anemia, improve iron homeostasis, and serve a broad portion of the population 
of patients with thalassemia. 
2.1.2. 
Mitapivat 
Mitapivat (also known as mitapivat sulfate, AG-348, and PYRUKYND) is a first-in-class, orally 
bioavailable, potent, allosteric activator of wild-type RBC-specific form of pyruvate kinase 
(PKR) and a range of PKR mutant enzymes (Kung et al, 2017). The RBC-specific form of 
pyruvate kinase is 1 of 4 pyruvate kinase isoenzymes expressed in human tissues from 2 separate 
genes, liver-specific form of pyruvate kinase (PKL) and pyruvate kinase muscle isozyme (PKM). 
Both PKR and PKL are splice isoforms of the PKLR gene, while PKM1 and PKM2 are both 
expressed from the PKM gene. Mitapivat is an allosteric activator of the PKR, PKL, and PKM2 
isoenzymes, with similar potency against each. 
Mitapivat acts by allosterically binding to the PKR tetramer and enhancing its affinity for 
phosphoenolpyruvate (PEP), thereby increasing the conversion of PEP + adenosine diphosphate 
to pyruvate + ATP. 
Mitapivat (PYRUKYND) was approved by the US Food and Drug Administration (FDA) on 
17 February 2022 for the treatment of hemolytic anemia in adults with pyruvate kinase 
deficiency (Pyrukynd (mitapivat) USPI, 2022). 
Additional information is in the current edition of the Investigator’s Brochure (IB). 
2.1.2.1. 
Pharmacokinetics and Pharmacodynamics Experience 
The pharmacokinetics and pharmacodynamic effects of mitapivat have been characterized in 
healthy subjects. Results from single- and multiple-ascending dose studies and a drug-drug 
interaction study are summarized. 
In a single-ascending dose study, mitapivat doses ranging from 30 to 2,500 mg were evaluated. 
Mitapivat was rapidly absorbed as indicated by the short time to maximum (peak) concentration 
(tmax) (ranged from 0.77 to 2.5 hours with doses from 30 to 1,400 mg). Dose-normalized area 
under the plasma concentration × time curve (AUC) remained constant over the dose range 
studied, suggesting that mitapivat total exposure increased in a dose-proportional manner. The 
mean half-life (t1/2) ranged from 17.8 to 20.4 hours when samples were collected through 
72 hours and from 50.3 to 79.3 hours when samples were collected through 120 hours. This 
terminal elimination phase contributed little to the overall exposure of mitapivat, as indicated by 
the small difference between AUC from time 0 to 12 hours and AUC from time 0 to infinity 
(AUC0-∞), suggesting a shorter effective t1/2 of approximately 3 to 6 hours.  
After single-ascending doses of mitapivat, a dose-dependent decrease in 2,3-diphosphoglycerate 
(2,3-DPG) concentrations was observed starting at 3 hours postdose, reaching a minimum at 
24 hours postdose, and then returning to values similar to baseline by 72 to 120 hours postdose. 
This decrease was accompanied by a minimal increase in ATP concentrations observed at 24 to 
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The effect of food on the pharmacokinetics of mitapivat (700 mg) showed that food did not alter 
the pharmacokinetics of mitapivat. As a result, mitapivat can be taken with or without food. 
In a multiple-ascending dose study, mitapivat doses ranging from 15 to 700 mg twice daily 
(BID) and 120 mg once daily (QD) were evaluated. At doses of 120 mg BID or higher, mitapivat 
exposure, as measured by both maximum (peak) concentration (Cmax) and AUC, was lower on 
Day 14 compared with Day 1. This observation is attributed to the cytochrome P450 (CYP) 3A 
autoinduction effect of mitapivat.  
Changes in 2,3-DPG concentrations after multiple-ascending doses were similar to those 
observed after single-ascending doses, with concentrations approaching baseline approximately 
72 hours after the final dose of mitapivat. The effect of mitapivat on decreasing 2,3-DPG blood 
concentrations reached 90% of maximal response between 120 mg and 360 mg BID. There were 
no increases in ATP concentrations for 12 hours after the first dose; however, mean ATP 
concentrations increased from baseline, reaching steady state by Day 10. Adenosine triphosphate 
concentrations just before the last dose on Day 14 were similar to predose concentrations on 
Day 10. Mean ATP concentrations remained above baseline values for 120 hours after the last 
dose on Day 14. The effect of mitapivat on increasing ATP concentrations also reached 90% of 
maximal response between 120 and 360 mg BID. 
In a drug-drug interaction study, the pharmacokinetics of mitapivat was evaluated in the presence 
and absence of itraconazole, a strong CYP3A and P-glycoprotein (P-gp) inhibitor, and in the 
presence and absence of rifampin, a strong CYP3A inducer. Systemic mitapivat exposure in the 
presence of itraconazole was higher compared with mitapivat alone; geometric mean AUC from 
time 0 to time t (AUC0-t), AUC0-∞, and Cmax ratios in the presence and absence of itraconazole 
were 4.7, 4.9, and 1.7, respectively. Systemic mitapivat exposure in the presence of rifampin was 
lower compared with mitapivat alone; geometric mean AUC0-t, AUC0-∞, and Cmax ratios in the 
presence and absence of rifampin were 0.09, 0.09, and 0.23, respectively. Similar decreases were 
observed in physiologically-based pharmacokinetic modelling simulations with multiple doses of 
mitapivat and concurrent itraconazole administration. 
Additional information is in the current edition of the IB. 
2.1.2.2. 
Nonclinical Experience 
Information on the use of mitapivat in nonclinical studies is in the current edition of the IB. 
2.1.2.3. 
Clinical Experience 
2.1.2.3.1. Pyruvate Kinase Deficiency 
Mitapivat has been evaluated in 4 studies in adult subjects with pyruvate kinase deficiency, 
another hemolytic anemia. A Phase 2, open-label, safety and efficacy study (AG348-C-003 
[DrivePK]) is ongoing. A Phase 3, double-blind, randomized, placebo-controlled study 
evaluating efficacy and safety in subjects who are not regularly transfused (AG348-C-006 
[ACTIVATE]) has completed. Additionally, 2 open-label studies are ongoing: a Phase 3 study 
evaluating efficacy and safety in subjects who are regularly transfused (AG348-C-007 
[ACTIVATE-T]) and a Phase 3 extension study for subjects who were previously enrolled in 
mitapivat studies (AG348-C-011). 

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In Study AG348-C-003, data from the initial 24 weeks of treatment (Core Period) indicated that 
approximately 50% of subjects (N=26) who received mitapivat (doses ranged from 50 to 300 mg 
BID) achieved maximum increases in Hb concentration that were >1 g/dL. Most increases were 
rapid. The median time to the first observation of an Hb concentration increase >1 g/dL above 
baseline was 10 days (range: 7 to 187 days). The Hb response was sustained in all 19 subjects 
who continued to receive mitapivat in the Extension Period. The median duration of exposure 
was 29 months (range: 22 to 35 months). Common adverse events (AEs), including headache 
and insomnia, occurred at the time of drug initiation and were transient; approximately 92% of 
the episodes of headache and 47% of the episodes of insomnia resolved within 7 days. The most 
common serious adverse events (SAEs), hemolytic anemia and pharyngitis, each occurred in 
2 patients (4%) (Grace, Rose, et al, 2019). Additionally, AEs during the Extension Period were 
similar to those reported during the initial 24-week Core Period, indicating that safety was 
consistent with extended mitapivat administration (Grace, Layton, et al, 2019). 
In Study AG348-C-006, 80 subjects randomized 1:1 to mitapivat or placebo received escalating 
doses of mitapivat to a maximum of 50 mg BID. Recently, data from this study demonstrated 
that approximately 40% of the 40 subjects who received mitapivat achieved a sustained Hb 
concentration increase of ≥1.5 g/dL compared with placebo (2-sided p-value <0.0001). 
Additionally, treatment with mitapivat demonstrated statistically significant improvements 
compared with placebo in key secondary endpoints including markers of hemolysis (indirect 
bilirubin, haptoglobin, and lactate dehydrogenase [LDH]) and markers of hematopoietic activity 
(reticulocyte percentages). The safety data from Study AG348-C-006 were consistent with data 
observed in Study AG348 C-003, and there were no AEs leading to study drug discontinuation in 
either the mitapivat or placebo arm. Safety data from Studies AG348-C-007 and AG348-C-011 
have been consistent with those in Study AG348-C-006, and no new safety signals have been 
observed.    
2.1.2.3.2. Non–Transfusion-Dependent Thalassemia (Study AG348-C-010) 
Study AG348-C-010 is an ongoing Phase 2, open-label study to determine the efficacy, safety, 
pharmacokinetics, and pharmacodynamics of mitapivat in adult subjects with α- or β-NTDT 
(EudraCT 2018-002217-35; ClinicalTrials.gov NCT03692052). Subjects with a baseline Hb 
concentration ≤10.0 g/dL received an initial dose of 50 mg BID mitapivat, which was increased 
to 100 mg BID at Week 6 in the absence of drug-related AEs or excessive Hb response. 
The study completed enrollment with 20 subjects (5 with α-thalassemia and 15 with 
β-thalassemia) who received mitapivat, and the Core Period of the study (initial 24 weeks of 
treatment) has completed; data from the Core Period are presented here, unless otherwise noted. 
Nineteen subjects completed the Core Period and 1 subject was withdrawn from the study due to 
an unrelated SAE. Seventeen subjects were continuing to receive mitapivat in the Extension 
Phase of the study (1 subject withdrew consent from the study and 1 subject did not meet criteria 
for participation in the Extension Phase). 
Of the 20 subjects who received mitapivat, the median age of subjects was 44 years and 
15 subjects were female. The mean baseline Hb concentration was 7.94 g/dL (range: 5.13 to 
9.80 g/dL). The mean baseline Hb concentrations in the α- and β-thalassemia subgroups were 
7.89 g/dL (range: 5.13 to 9.80 g/dL) and 8.12 g/dL (range: 6.67 to 9.20 g/dL), respectively. 

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A total of 16 subjects (80.0%; 5 with α-thalassemia and 11 with β-thalassemia) met the primary 
endpoint of Hb response, defined as an increase in Hb concentration of ≥1.0 g/dL from baseline 
at ≥1 assessment between Week 4 and Week 12. The time to the first Hb response (ie, ≥1.0 g/dL 
from baseline) was observed as early as Week 2, with a median of 3.3 weeks (range: 2.1 to 
12.1 weeks). The mean (±standard deviation [SD]) increase from baseline in Hb concentration 
over a continuous 12-week interval (Week 4 to Week 12) was 1.15±0.07 g/dL (range: -0.24 to 
2.52 g/dL). Of the 16 subjects who met the primary endpoint, 13 subjects (65.0%) had a 
sustained Hb response; these subjects met the primary endpoint and had an increase in Hb 
concentration ≥1.0 g/dL from baseline at 2 of the 4 assessments from Week 12 to Week 24. Of 
the 4 subjects who did not meet the primary endpoint, 2 subjects demonstrated a delayed Hb 
response, defined as an increase in Hb concentration of ≥1.0 g/dL from baseline at ≥1 assessment 
from Week 16 to Week 24. 
In the 19 subjects who completed 24 weeks of mitapivat treatment, the mean (±SD) increase 
from baseline in Hb concentration over a continuous 12-week interval (Week 12 to Week 24) 
was 1.3±6.28 g/dL (range: -0.03 to 2.56 g/dL). The average increase from baseline in Hb 
concentration was similar between subjects with α-thalassemia and β-thalassemia. Fourteen of 
20 subjects (70.0%; 4 with α-thalassemia, 10 with β-thalassemia) had a ≥1.0 g/dL increase in 
average Hb concentration from Week 12 through Week 24 compared with baseline. 
Among all 20 subjects, hemolytic and erythropoietic markers relevant to the pathophysiology of 
the disease showed trends for improvement as early as Week 2. Baseline mean indirect bilirubin, 
LDH, and erythropoietin was 31.49±27.536 μmol/L, 269.35±113.055 U/L, and 
870.53±2,659.950 IU/L, respectively. Over a continuous 12-week interval (Week 12 to 
Week 24), the mean (±SD) change from baseline in indirect bilirubin, LDH, and erythropoietin 
was -17.72±18.861 μmol/L, -40.68±123.699 U/L, and -627.00±2404.460 IU/L, respectively. 
The safety profile of mitapivat in subjects with NTDT is consistent with that in healthy subjects 
and in subjects with pyruvate kinase deficiency, another hemolytic anemia with similar clinical 
manifestations. No new safety signals were identified when mitapivat was administered in 
subjects with thalassemia. There were no differences in the type and frequency of AEs between 
subjects with α- and β-thalassemia. During the Core Period, 17 (85.0%) of the 20 subjects who 
received at least 1 dose of mitapivat had at least 1 AE. Commonly reported AEs (occurring in 
≥20% or ≥4 subjects) included cough, dizziness, dyspepsia, fatigue, headache, initial insomnia, 
nasal congestion, and upper respiratory tract infection. One subject experienced a National 
Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) Grade 1 
adverse event of special interest of aspartate aminotransaminase increased. One subject had a 
related nonserious Grade 3 event of initial insomnia that led to a (permanent) dose reduction to 
50 mg BID, and 1 subject had an unrelated SAE of renal impairment that led to study drug 
discontinuation. Overall, when compared with 50 mg BID, 100 mg BID dosing did not result in 
additional AEs despite the higher dose and longer duration of follow-up. 
2.2. 
Study Rationale 
Data from the ongoing Phase 2 study (AG348-C-010) demonstrate that mitapivat has a 
manageable safety profile, improves anemia, as measured by an increase in Hb concentration and 
a reduction in hemolytic parameters, and results in favorable trends in erythropoietic parameters 
relevant to thalassemia. While subjects with TDT were not evaluated in this study, the study 

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included 4 subjects with a baseline Hb concentration below 7 g/dL, a threshold that renders 
patients eligible for chronic transfusions based on current treatment guidelines (Cappellini et al, 
2014), with 3 of them achieving a ≥1.0 g/dL increase in average Hb concentration from Week 12 
through Week 24 compared with baseline. The clinical benefit observed in Study AG348-C-010 
is considered relevant to subjects with transfusion-dependence as well, because the 
pathophysiology of the disease is the same regardless of transfusion status (see Section 2.1.1). 
The goal for thalassemia treatment in general, independent of transfusion status, is to decrease 
the ineffective erythropoiesis and hemolysis and subsequently improve chronic anemia to 
prevent downstream clinical sequelae (Taher et al, 2018). PKR activation by mitapivat (see 
Section 2.1.2) in AG348-C-010 resulted in improvement in hemolytic and erythropoietic markers 
(see Section 2.1.2.3.2). Therefore, the data from the Phase 2 study are supportive of the 
development of mitapivat in patients with TDT.  
A similar approach was taken in the development of mitapivat for patients with PK deficiency. 
The knowledge gained from Study AG348-C-003 in a non-transfusion dependent PK deficiency 
population allowed for the development of 2 Phase 3 studies, one in patients who do not require 
regular transfusions (AG348-C-006) and the other in patients who require regular transfusions 
(AG348-C-007). Preliminary review of safety data from Study AG348-C-007 has not 
demonstrated a change in the safety profile from that observed in Studies AG348-C-006 or 
AG348-C-003. 
Study AG348-C-018 is a Phase 3, double-blind, randomized, placebo-controlled, multicenter 
study designed to demonstrate the clinical efficacy and safety of mitapivat in subjects with α- or 
β-TDT. The primary objective of the study is to compare the effect of mitapivat versus placebo 
on transfusion burden. Secondary objectives include the evaluation of markers of iron overload 
as well as safety and pharmacokinetic and pharmacodynamic parameters. 
Although recently approved therapies offer new options for patients with β-TDT, many patients 
do not respond, luspatercept is not approved for patients with α-thalassemia, and thus is ample 
reason to test mitapivat based on data from the Phase 2 study. Luspatercept, indicated for β-TDT, 
requires subcutaneous administration every 3 weeks by a health-care provider and includes a 
warning for hypertension and thromboembolic events (Reblozyl (luspatercept-aamt) USPI, 
2019). In addition to luspatercept, betibeglogene autotemcel (EMA, 2019) and HSCT are 
potential treatment options for a limited group of patients with β-TDT, but these options require 
access to specialized treatment centers and are also associated with substantial clinical risks. 
Therefore, a serious unmet medical need still exists for patients with transfusions-dependent 
α- or β-thalassemia. Mitapivat, has the potential to improve the transfusion burden in patients 
with TDT with the added benefit of oral administration. 
The study design rationale and dose justification are provided in Section 4.2 and Section 4.3, 
respectively. 
2.3. 
Benefit-Risk Assessment 
The Sponsor has conducted a risk assessment for concomitant use of a COVID-19 vaccine with 
mitapivat with specific consideration for the trial population, and determined that the COVID-19 
vaccine given to a study subject is considered a simple concomitant medication with no 

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interaction that requires advice on timing of the vaccine or other aspects that need to be 
mitigated. 
The COVID-19 public health emergency is expected to be ongoing when this study is initiated. 
To ensure subject safety, maintain compliance with Good Clinical Practice (GCP), and minimize 
risks to study integrity while conducting this study during a pandemic, this protocol includes the 
allowance for temporary modifications to protocol-specified procedures (see Section 11.1.1). 
These modifications are consistent with ensuring subject safety and continued access to study 
drug while minimizing risk to study subjects, Investigators, and clinical study center personnel. 
The importance of conducting this study despite an ongoing pandemic was assessed, and this 
study is considered a critical evaluation of a study drug for a serious unmet medical need in 
patients with thalassemia (Section 2.1.1.1). COVID-19 is not expected to affect the disease of 
thalassemia. While some patients with thalassemia needing transfusions may have more limited 
access to these in certain regions of the world because of pandemic restrictions or disruptions, 
the Investigator can exclude patients via the last exclusion criterion (#19), if this situation is 
relevant to the patient and if this creates a medical condition that does not allow for safe 
participation. Therefore, the COVID-19 public health emergency is not considered to negatively 
affect the risk of subject participation or the assessment of study objectives. Overall, with the 
potential allowance for temporary modifications in place, the benefits of conducting this study 
despite the challenges due to COVID-19 outweigh the potential risks to patients with 
thalassemia. Throughout the study, this assessment will be reevaluated by the Sponsor and 
Investigator. 
Additionally, the benefit-risk assessment for mitapivat in clinical studies is in the current edition 
of the IB.  

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3. 
OBJECTIVES AND ENDPOINTS 
The study objectives and endpoints are provided in Table 3. 
Table 3: 
Objectives and Endpoints 
Primary Objective 
• 
To compare the effect of mitapivat 
versus placebo on transfusion 
burden in subjects with α- or 
β-transfusion-dependent 
thalassemia 
Primary Endpoint 
• 
Transfusion reduction response, defined as a ≥50% 
reduction in transfused red blood cell (RBC) units with 
a reduction of ≥2 units of transfused RBCs in any 
consecutive 12-week period through Week 48 compared 
with baseline 
Key Secondary Objective 
• 
To compare the durability of the 
effect of mitapivat versus placebo 
on transfusion burden 
Key Secondary Endpoints 
• 
≥33% reduction in transfused RBC units from Week 13 
through Week 48 compared with baseline 
• 
≥50% reduction in transfused RBC units in any 
consecutive 24-week period through Week 48 compared 
with baseline 
• 
≥50% reduction in transfused RBC units from Week 13 
through Week 48 compared with baseline 
Secondary Objectives 
• 
To evaluate the effect of mitapivat 
versus placebo on additional 
measures of transfusion burden 
Secondary Endpoints 
• 
Change from baseline in transfused RBC units from 
Week 13 through Week 48 
• 
Transfusion independence, defined as transfusion-free 
for ≥8 consecutive weeks through Week 48 
• 
To evaluate the effect of mitapivat 
versus placebo on iron metabolism 
• 
Change from baseline in iron, serum ferritin, total iron 
binding capacity, and transferrin saturation through 
Week 48 
• 
To evaluate the safety of mitapivat 
• 
Type, severity, and relationship of adverse events and 
serious adverse events 
• 
To evaluate the pharmacokinetic 
and pharmacodynamic effects of 
mitapivat 
• 
Plasma or blood concentrations and pharmacokinetic 
parameters of mitapivat and pharmacodynamic 
parameters, including adenosine triphosphate and 
2,3-diphosphoglycerate 

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4. 
STUDY DESIGN 
4.1. 
Overall Design 
This is a Phase 3, double-blind, randomized, placebo-controlled, multicenter study evaluating the 
efficacy and safety of mitapivat versus placebo in adult subjects with α- or β-TDT followed by 
an Open-label Extension Period. Approximately 240 subjects are planned to be randomized in 
this study. This multicenter study will be conducted internationally. 
An overview of the study design is presented in Figure 1. Study assessments will be performed at 
the time points in the Schedules of Assessments (see Section 1.2). 
Figure 1: 
Study Design 
 
Abbreviations: BID = twice daily; EOS = End of Study. 
4.1.1. 
Screening Period 
Subject eligibility will be determined during the Screening Period (see Section 5). Baseline 
disease characteristics, including transfusion history from the 24-week period before 
randomization, will be recorded (see Section 8.7). The Investigator will confirm the subject’s 
eligibility before randomization. Screening extensions will not be granted. If a subject requires 
rescreening, screening assessments that do not need to be repeated are in Section 8.2. 
4.1.2. 
Double-blind Period 
Eligible subjects will be randomly assigned in a 2:1 ratio to receive study drug (mitapivat or 
placebo) for BID oral administration. Randomization will be stratified by thalassemia genotype 
and geographical region (see Section 6.3.1). Study subjects, Investigators, clinical study center 
personnel, and the Sponsor will be blinded to the subject’s treatment assignment (see 
Section 6.3.2). Subjects will receive either mitapivat or matched placebo for 48 weeks. During 
the study, subjects will receive RBC transfusions according to the guidance in Section 8.7. Study 
visits will occur approximately every 4 weeks for efficacy and safety assessments and will be 
aligned, if possible, to occur with the subject’s routine transfusion schedule. If allowed by local 
institutional guidelines, subjects will have the option for at-home study visits as designated in the 
Schedules of Assessments. Subjects who discontinue study drug before completing the 
Double-blind Period are not permitted to receive mitapivat in the Open-label Extension Period. 

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4.1.3. 
Open-label Extension Period 
Subjects will be provided the opportunity to receive mitapivat for an additional 5 years in the 
Open-label Extension Period. Treatment assignment from the Double-blind Period will remain 
blinded until the study is unblinded for the analysis of the primary endpoint (see Section 6.3.2). 
During the Open-label Extension Period, subjects will continue to undergo safety and clinical 
activity assessments; however, the frequency of study visits will be reduced. Study visits will 
continue to be aligned, if possible, with the subject’s routine transfusion schedules and modified 
according to the guidance in Section 8.7. If allowed by local institutional guidelines, subjects 
will have the option for at-home study visits as designated in the Schedules of Assessments. 
4.1.4. 
Duration of Treatment and Safety Follow-up 
The duration of participation of a subject in the study is up to 6.3 years. The duration of 
treatment is up to 6.0 years. Subjects will have a Safety Follow-up visit approximately 4 weeks 
after their last dose of study drug (including the recommended dose taper).  
4.2. 
Study Design Rationale 
Study AG348-C-018 is a Phase 3, double-blind, randomized, placebo-controlled, multicenter 
study designed to demonstrate the clinical efficacy and safety of mitapivat in subjects with α- or 
β-TDT followed by an Open-label Extension Period.  
The double-blind study design minimizes assessment bias. The proposed use of placebo as a 
comparator will allow an objective evaluation of the efficacy and safety of mitapivat. The choice 
of placebo control is supported by the lack of widely available and disease-specific, approved 
treatments for patients with α-TDT and patients with β-TDT. 
The primary endpoint, in conjunction with the key secondary endpoints, will allow for a 
consistent and reliable measurement of the intended effect of mitapivat on transfusion burden. 
Eligible subjects will be randomized 2:1 to receive mitapivat or matched placebo, providing an 
increased chance to receive active study drug. Placebo administration will be limited to the 
48-week Double-blind Period, and subjects, including those who were randomized to placebo, 
will have the opportunity to receive mitapivat during the Open-label Extension Period. This 
allows for a longer-term assessment of safety while providing access to active drug. During the 
study, an Independent Data Monitoring Committee (IDMC) will be responsible for ongoing 
monitoring of the safety of subjects. 
Randomization will be stratified by thalassemia genotype to ensure that the comparison reflects a 
balanced allocation of subjects to mitapivat or placebo within each genotype, and geographical 
region to allow for a characterization of the effect across areas with regional care variations.  
Additionally, during the study, subjects will continue to receive appropriate supportive care to 
manage symptoms and prevent secondary complications as clinically indicated and according to 
applicable guidelines. Study visit windows were developed to facilitate alignment with the 
subjects’ routine transfusion schedules for flexibility and convenience. 
Overall, the study design will allow for an evaluation of the efficacy and safety of mitapivat in 
subjects with α- or β-TDT while subjects continue receiving appropriate supportive care to 
manage symptoms and prevent secondary complications. 

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4.3. 
Dose Justification 
Based on pharmacokinetic, pharmacodynamic, efficacy, and safety data from subjects with 
NTDT in Study AG348-C-010, a dose of 100 mg BID mitapivat has been selected for further 
evaluation in subjects with thalassemia. Data from this study indicate that a dose of 100 mg BID 
was associated with more subjects who achieved a Hb responses. Further, safety data 
demonstrated that at 100 mg BID mitapivat, AEs were manageable and were consistent with 
those in other studies across the clinical development program (see Section 2.1.2.3.2). 
Preliminary pharmacokinetic, pharmacodynamic, and exposure-response analyses were 
conducted for 14 subjects with NTDT (data cutoff date: 03 March 2020). Per protocol, subjects 
received an initial dose of 50 mg BID mitapivat and had their dose increased to 100 mg BID at 
Week 6. Exposure to mitapivat (as assessed by AUC from time 0 to the last measurable 
concentration [AUClast]) was approximately 30% higher at 100 mg BID compared with the 
exposure estimated from a single 50 mg dose on Day 1. The pharmacokinetics of mitapivat after 
a single 50 mg dose was similar to that observed at steady state after multiple 50 mg BID doses. 
This observation is consistent with previous studies where autoinduction was observed at 120 mg 
BID or higher in healthy subjects and at 300 mg BID in subjects with pyruvate kinase deficiency; 
autoinduction was not observed at the next lower doses of 60 mg BID in healthy subjects or 
50 mg BID in subjects with pyruvate kinase deficiency. The mean ATP percent change from 
baseline was 82.7% (N=9) at Week 6 when subjects were still receiving 50 mg BID, increasing 
to 86.7% (N=12) and 92.3% (N=5) at Weeks 12 and 24, respectively, at which point subjects 
were receiving 100 mg BID.  
A sequential population pharmacokinetic-pharmacodynamic modelling approach was used to 
characterize the exposure-efficacy relationship of mitapivat using Hb as the efficacy endpoint. 
The time course of Hb response was described by an indirect-response model, and the effect of 
mitapivat on Hb formation rate was described by a maximum effect model. 
 
 
 
 
 
 
 
Although mitapivat was administered sequentially in Study AG348-C-010, and duration of 
exposure varied by dose (ie, several subjects received 100 mg BID for longer than 50 mg BID), 
there were no apparent differences in the spectrum of AEs reported while subjects were receiving 
either 50 mg BID or 100 mg BID (see Section 2.1.2.3.2).  
Based on data from nonclinical and clinical studies, insomnia is an identified risk associated with 
mitapivat administration. An increased incidence of insomnia was observed in subjects with 
pyruvate kinase deficiency who received 300 mg BID compared with 50 mg BID (Study 
AG348-C-003). The 6-fold difference in dose resulted in an approximately 3.2-fold difference in 
total exposure at steady state. A logistic regression analysis was performed to examine the 
relationship between AEs and mitapivat exposure. 
 
 
 

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 Recently, data from the 
randomized, placebo-controlled study (AG348-C-006) in which subjects received a maximum 
dose of 50 mg BID, AEs of insomnia as defined by Preferred Term (initial insomnia, insomnia, 
middle insomnia, and terminal insomnia) were observed with similar frequency between the 
mitapivat and placebo groups (17.5% vs 17.9%). 
 
 
 
 
 
 
In summary, 100 mg BID mitapivat has been shown to have a manageable safety profile for 
chronic dosing. 
 
 
 Based on these findings, a dose of 100 mg BID of mitapivat has been selected for subjects 
with NTDT (Study AG348-C-017). Because the underlying molecular and pathophysiological 
basis for TDT and NTDT are similar, the same dose (100 mg BID of mitapivat) has been 
selected for this current study. 
Dose modifications are presented in Section 6.7. 
4.4. 
End of Study Definition 
The end of the study is defined as the time at which all subjects complete all study visits, are lost 
to follow-up, have withdrawn consent for further participation in the study, or when the Sponsor 
terminates the study. Study completion is the date of the last visit of the last subject in the study. 

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5. 
STUDY POPULATION 
Protocol deviations to recruitment and enrollment criteria, also known as protocol waivers or 
exemptions, are not permitted. 
5.1. 
Inclusion Criteria 
Subjects are eligible to be included in the study only if all the following criteria apply: 
1. ≥18 years of age at the time of providing informed consent. 
2. Documented diagnosis of thalassemia (β-thalassemia with or without α-globin gene 
mutations, HbE/β-thalassemia, or α-thalassemia/HbH disease) based on DNA analysis 
from the subject’s medical record. If this information is not available from the subject’s 
medical record, DNA analysis can be performed by a local laboratory during the 
Screening Period. If a local laboratory is unable to perform the test, results from the 
comprehensive α- and β-globin genotyping performed by the study central laboratory can 
be used (see Section 8.2 and Section 8.9). 
3. Transfusion dependent, defined as 6 to 20 RBC units transfused and a ≤6-week 
transfusion-free period during the 24-week period before randomization. 
4. If taking hydroxyurea, the hydroxyurea dose must be stable for ≥16 weeks before 
randomization. 
5. Women of childbearing potential (WOCBP) must be abstinent of sexual activities that 
may induce pregnancy 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 providing 
informed consent, throughout the study, and for 28 days after the last dose of study drug. 
The second form of contraception can be an acceptable barrier method (see Appendix 2 
for the definition of WOCBP and acceptable contraception methods). 
6. Written informed consent before any study-related procedures are conducted and willing 
to comply with all study procedures for the duration of the study. 
5.2. 
Exclusion Criteria 
Subjects are excluded from the study if any of the following criteria apply: 
1. Pregnant, breastfeeding, or parturient. 
2. Documented history of homozygous or heterozygous HbS or HbC. 
3. Prior exposure to gene therapy or prior bone marrow or stem cell transplantation. 
4. Currently receiving treatment with luspatercept; the last dose must have been 
administered ≥36 weeks before randomization. 
5. Currently receiving treatment with hematopoietic stimulating agents; the last dose must 
have been administered ≥36 weeks before randomization. 
6. History of malignancy (active or treated) ≤5 years before providing informed consent, 
except for nonmelanomatous skin cancer in situ, cervical carcinoma in situ, or breast 
carcinoma in situ.  

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7. History of active and/or uncontrolled cardiac or pulmonary disease ≤6 months before 
providing informed consent, including but not limited to: 
a. New York Heart Association Class III or IV heart failure or clinically significant 
dysrhythmia  
b. Myocardial infarction or unstable angina pectoris; hemorrhagic, embolic, or 
thrombotic stroke; deep venous thrombosis; or pulmonary or arterial embolism 
c. Heart rate–corrected QT interval using Fridericia’s method ≥450 milliseconds (males) 
or ≥470 milliseconds (females), except for right or left bundle branch block 
d. Severe pulmonary fibrosis as defined by severe hypoxia, evidence of right-sided heart 
failure, and radiographic pulmonary fibrosis >50% 
e. Severe pulmonary hypertension as defined by severe symptoms associated with 
hypoxia, right-sided heart failure, and oxygen indicated 
8. Hepatobiliary disorders, including but not limited to: 
a. Liver disease with histopathological evidence of cirrhosis or severe fibrosis 
b. Clinically symptomatic cholelithiasis or cholecystitis (prior cholecystectomy is not 
exclusionary) 
c. History of drug-induced cholestatic hepatitis 
d. Aspartate aminotransferase (AST) >2.5 × upper limit of normal (ULN); unless due to 
hemolysis and hepatic iron deposition) and alanine aminotransferase (ALT) >2.5 × 
ULN (unless due to hepatic iron deposition) 
9. Estimated glomerular filtration rate <45 mL/min/1.73 m2 by Chronic Kidney Disease 
Epidemiology Collaboration creatinine equation (Levey et al, 2009). 
10. Nonfasting triglycerides >440 mg/dL (5 mmol/L). 
11. Active infection requiring systemic antimicrobial therapy at the time of providing 
informed consent. If antimicrobial therapy is required during the Screening Period, 
screening procedures should not be performed while antimicrobial therapy is being 
administered, and the last dose of antimicrobial therapy must be administered ≥7 days 
before randomization. 
12. Positive test for hepatitis C virus (HCV) antibody (Ab) with evidence of active HCV 
infection, or positive test for hepatitis B surface antigen (HBsAg). 
13. Positive test for HIV-1 Ab or HIV-2 Ab. 
14. History of major surgery (including splenectomy) ≤6 months before providing informed 
consent and/or a major surgical procedure planned during the study. 
15. Current enrollment or past participation (≤12 weeks before administration of the first 
dose of study drug or a time frame equivalent to 5 half-lives of the investigational 
treatment, whichever is longer) in any other clinical study involving an investigational 
treatment or device. 
16. Receiving strong CYP3A4/5 inhibitors that have not been stopped for ≥5 days or a time 
frame equivalent to 5 half-lives (whichever is longer), or strong CYP3A4 inducers that 
have not been stopped for ≥4 weeks or a time frame equivalent to 5 half-lives (whichever 
is longer), before randomization. 

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17. Receiving anabolic steroids that have not been stopped for at least 4 weeks before 
randomization. Testosterone replacement therapy to treat hypogonadism is allowed; the 
testosterone dose and preparation must be stable for ≥12 weeks before randomization. 
18. Known allergy, or other contraindication, to mitapivat or its excipients (microcrystalline 
cellulose, croscarmellose sodium, sodium stearyl fumarate, mannitol, and magnesium 
stearate, Opadry® II Blue [hypromellose, titanium dioxide, lactose monohydrate, 
triacetin, and FD&C Blue #2]). 
19. Any medical, hematological, psychological, or behavioral condition(s) or prior or current 
therapy that, in the opinion of the Investigator, may confer an unacceptable risk to 
participating in the study and/or could confound the interpretation of the study data. Also 
excluded are: 
• Subjects who are institutionalized by regulatory or court order 
• Subjects with any condition(s) that could create undue influence (including but not 
limited to incarceration, involuntary psychiatric confinement, and financial or familial 
affiliation with the Investigator or Sponsor) 
5.3. 
Lifestyle Considerations 
Not applicable 
5.4. 
Screen Failures 
Screen failures are defined as subjects who provide informed consent to participate in the clinical 
study but who do not meet the eligibility criteria for participation in this study and who are not 
subsequently randomized. Information to be collected for screen failures includes the reason for 
screen failure, demography, eligibility criteria, SAEs, and deaths.  
Individuals who do not meet the criteria for participation in this study (screen failure) may be 
rescreened. See Section 8.2 for criteria for which rescreening is permitted. 

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6. 
STUDY DRUG 
6.1. 
Study Drug Administered 
Study drug is defined as mitapivat or matched placebo. Study drug is for investigational use only 
and to be used only within the context of this study protocol.  
Subjects will receive 100 mg BID mitapivat or matched placebo for oral administration. Tablets 
are to be swallowed whole with water and may be taken with or without food. Tablets are not to 
be crushed, chewed, or dissolved in water. If a dose of mitapivat is missed by ≤4 hours, the dose 
should be taken as soon as possible. If a dose of mitapivat is missed by >4 hours, the dose should 
be skipped and taken at the time of the next scheduled dose. The regular dosing schedule should 
resume the following day. 
Subjects who discontinue study drug should undergo the recommended dose taper and be 
monitored as clinically indicated for signs and symptoms of acute hemolysis and worsening 
anemia. If immediate or abrupt study drug discontinuation is required for an AE or medical 
emergency, subjects should be monitored as clinically indicated for signs of acute hemolysis or 
worsening anemia. Study drug dose modifications are provided in Section 6.7. 
Study drug dose and recommended dose taper are provided in Table 4. 
Table 4: 
Study Drug Dose and Recommended Dose Taper Regimen 
Level 
Dose 
Schedule 
Full dose 
100 mg 
BID 
Taper dose 
100 mg 
QD for 7 days, then discontinue study drug 
Abbreviations: BID = twice daily; QD = once daily. 
Study drug will be supplied according to the specifications in Table 5. 
Table 5: 
Study Drug 
Treatment Arm: 
Active 
Matched Placebo 
Study drug name (INN) 
Mitapivat (AG-348)  
NA 
Dose formulation 
Tablet 
Tablet 
Tablet unit strength(s) 
100 mg 
NA 
Appearance 
Oblong blue tablet 
Oblong blue tablet 
Route of administration 
Oral 
Oral 
Sourcing 
Provided by the Sponsor 
Provided by the Sponsor 
Packaging and labeling 
Provided in containers with 
child-resistant closures and labeled 
as required per local regulations 
Provided in containers with 
child-resistant closures and labeled 
as required per local regulations 
Abbreviations: INN = international nonproprietary name; NA = not applicable. 
Additional information will be provided in the Pharmacy Manual. 

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6.2. 
Preparation, Handling, Storage, and Accountability 
Only subjects enrolled in the study may receive study drug, and only authorized clinical study 
center personnel may supply or administer study drug. 
All study drug must be stored in a secure, environmentally controlled, and monitored (manual or 
automated) area in accordance with the labeled storage conditions with access limited to the 
Investigator and authorized clinical study center personnel. 
Accountability for the study drug at the clinical study center 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. 
The Investigator (or designee) will maintain accurate drug accountability records indicating the 
study drug delivery date to the clinical study center, inventory at the center, use by each subject, 
and return to the Sponsor (or designee), or disposal of study 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 Sponsor (or designee) will review study drug accountability records at the 
study center on an ongoing basis during monitoring visits. 
Study drug must not be used for any purpose other than the present clinical study. Study drug 
that has been dispensed to a subject and returned unused must not be redispensed to a different 
subject. 
All used, unused, or expired study drug should be retained at the study center until inventoried 
and verified by study center personnel and/or the study monitor. All used, unused, or expired 
study drug will be returned to the Sponsor (or designee) or if authorized, disposed of at the study 
center per the study center standard operating procedures (SOPs) and documented. All material 
containing mitapivat or matched placebo will be treated and disposed of as hazardous waste in 
accordance with governing regulations. 
Additional information will be provided in the Pharmacy Manual. 
6.3. 
Randomization and Blinding 
6.3.1. 
Randomization 
In this study, eligible subjects will be randomly assigned in a 2:1 ratio to receive study drug 
(mitapivat or placebo). Randomization will be stratified by geographical region (North America 
and Europe; Asia-Pacific; and Rest of World) and by thalassemia genotype (see Section 8.2). 
Randomization assignment will be generated and implemented by an interactive voice/web 
response system (IXRS) provider. Mitapivat and matched placebo will be packaged and labeled 
identically to maintain the study blind. Randomization can occur up to 48 hours before 
administration of the first dose of study drug on Day 1. 

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6.3.2. 
Blinding 
Study subjects, Investigators, clinical study center personnel, pharmacists, and the Sponsor will 
be blinded to the subject’s treatment assignment. After completing the Double-blind Period, 
subjects will be provided the opportunity to receive mitapivat in the Open-label Extension 
Period. At the last study visit of the Double-blind Period, subjects who continue in the 
Open-label Extension Period will be provided with active mitapivat; however, study subjects, 
Investigators, clinical study center personnel, and the Sponsor will continue to remain blinded to 
the randomized treatment assignment during the previous Double-blind Period until the study is 
unblinded for the analysis of the primary endpoint. 
6.3.3. 
Unblinding 
The IXRS will be programmed with blind-breaking instructions. In the event of a medical 
emergency or confirmed pregnancy in a female subject, if knowledge of the investigational 
product assignment is critical to the subject’s management, the Investigator has the sole 
responsibility for determining if unblinding of a subject’s treatment assignment is warranted. 
Subject safety must always be the first consideration in making such a determination. If the 
Investigator decides that unblinding is warranted, the Investigator should make every effort to 
contact the Medical Monitor before unblinding a subject’s treatment assignment unless this could 
delay emergency treatment of the subject. If a subject’s treatment assignment is unblinded, the 
Medical Monitor must be notified within 24 hours after breaking the blind. The Medical Monitor 
must remain blinded to the subject’s treatment assignment. The date and reason that the blind 
was broken must be recorded in the IXRS. 
6.4. 
Study Drug Compliance 
Subjects will receive instructions for home administration of study drug. Subjects must return 
study drug bottles (including empty bottles) and unused tablets at each visit to the clinical study 
center. Compliance with the study drug dosing regimen will be assessed. Deviation(s) from the 
assigned dose or dose regimen should be recorded in the electronic case report form (eCRF). 
6.5. 
Overdose 
Overdose is the accidental or intentional use of the study drug in an amount higher than the dose 
being studied. No information is available on the treatment of an overdose of mitapivat. In the 
event of an overdose, observation and general supportive measures should be instituted, and the 
Medical Monitor should be contacted. Any study drug overdose or incorrect administration of 
study drug should be recorded on the eCRF. 
See the current edition of the IB for details. See Section 9.4.1 for reporting of AEs associated 
with an overdose. 
6.6. 
Prior and Concomitant Therapy 
6.6.1. 
Prior Therapy 
All medications administered and procedures and surgeries conducted ≤4 weeks before 
providing informed consent until administration of the first dose of study drug are to be recorded 

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on the eCRF. This includes over-the-counter or prescription medicines, vitamins, and herbal 
supplements.  
Use of iron chelation therapy for up to 1 year before administration of the first dose of study drug 
will be recorded on the eCRF. 
Transfusion history will also be recorded (see Section 8.7). 
6.6.2. 
Permitted Concomitant Therapy 
Transfusions and other supportive care therapies are permitted as clinically indicated, unless 
otherwise specified in Section 6.6.3. Concomitant administration of iron chelation therapy will 
be recorded on the eCRF. Red blood cell transfusions are discussed in Section 8.7. 
6.6.3. 
Prohibited Concomitant Therapy 
Concomitant use of investigational treatments or devices is not permitted during study 
participation.  
Concomitant administration of strong CYP3A4/5 inhibitors is prohibited during the study. These 
therapies must be discontinued ≥5 days or a time frame equivalent to 5 half-lives (whichever is 
longer) before randomization. 
Products known to inhibit CYP3A4 (eg, grapefruit or grapefruit juice) are prohibited during the 
study. 
Concomitant administration of strong CYP3A4 inducers is prohibited during the study. These 
therapies must be discontinued ≥4 weeks or a time frame equivalent to 5 half-lives (whichever is 
longer) before randomization. 
Concomitant administration of luspatercept is prohibited during the study; the last dose must 
have been administered ≥36 weeks before randomization. 
Concomitant administration of erythropoietin is prohibited during the study; the last dose must 
have been administered ≥36 weeks before randomization. 
Concomitant administration of anabolic steroids, including testosterone preparations, is 
prohibited during the study; the last dose must have been administered at least 4 weeks before 
randomization. However, continuation of testosterone replacement therapy to treat 
hypogonadism is allowed if the dose and preparation has been stable for at least 12 weeks before 
randomization. It is desirable to maintain a stable dose during the Double-blind Period.  
Subjects requiring a prohibited concomitant therapy will be discontinued from study drug and 
withdrawn from the study. 
See the current edition of the IB for examples of prohibited concomitant therapies. 
6.6.3.1. 
Concomitant Therapy to be Used With Caution or Replaced 
Medications under the following categories should be replaced with alternative treatments before 
administration of the first dose of study drug: 
• Corticosteroids 
• Sensitive CYP3A4 substrates 

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• Moderate CYP3A4 inhibitors 
• Moderate inducers of CYP3A4 
• Gastric acid–reducing agents, namely proton-pump inhibitors and histamine 
2-receptor antagonists. (Concomitant use of antacids such as magnesium hydroxide 
and aluminum hydroxide is permitted.) 
If alternative treatments are not possible, subjects receiving these medications concomitantly 
with study drug should be carefully monitored. Investigators must monitor subjects for lack of 
efficacy of the prescribed medication or for side effects arising from the medication and make 
appropriate modifications to the dose of the prescribed medication or find alternatives to the 
prescribed medication. 
In vitro data suggest that mitapivat has the potential to induce uridine 
5ʹ-diphospho-glucuronosyltransferase (UGT) enzymes and therefore has the potential to decrease 
systemic exposure of UGT enzyme substrates. Iron chelators that are substrates of UGT enzymes 
may be administered concomitantly with mitapivat. Subjects receiving these medications 
concomitantly should, however, be closely monitored. 
See the current edition of the IB for examples of concomitant therapies to be used with caution. 
6.6.3.2. 
Rescue Medicine 
Not applicable 
6.7. 
Dose Modifications 
Dose modifications are permitted for excessive Hb response and study drug–related AEs. Dose 
modification decisions should be made by the Investigator based on a clinical evaluation of the 
subject. The Investigator should notify the Medical Monitor of any dose modifications that are 
planned or instituted. 
Subjects who discontinue study drug should undergo the recommended dose taper (Table 4) and 
be monitored as clinically indicated for signs and symptoms of acute hemolysis and worsening 
anemia. If immediate or abrupt study drug discontinuation is required for an AE or medical 
emergency, subjects should be monitored as clinically indicated for signs of acute hemolysis or 
worsening anemia. 
6.7.1. 
Management of Adverse Events and Clinical Laboratory Result Test Changes 
6.7.1.1. 
Dose Modifications for Excessive Hb Response 
Study drug dose modification guidance is provided for excessive Hb response, in the absence of 
RBC transfusions, to avoid abrupt discontinuation of mitapivat. Excessive Hb response is 
defined as an increase in Hb concentration that is greater than the ULN (by sex), in the absence 
of RBC transfusions. 
During the study, Investigators will monitor changes in Hb concentration levels with study drug 
administration and modify the subject’s RBC transfusion schedule according to guidelines in 
Section 8.7. If a subject has not required an RBC transfusion for ≥4 weeks and their Hb 

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concentration is greater than the ULN (by sex), dose modification guidelines for excessive Hb 
response in Table 6 should be followed. 
Table 6: 
Dose Modifications for Excessive Hemoglobin Response 
Clinical Laboratory Test Result 
Study Drug Modification 
Hb concentration >ULN (by sex), 
in the absence of RBC 
transfusions for ≥4 weeks 
Reduce to 100 mg QD.  
• 
If Hb concentration decreases to ≤ULN (by sex) within 
28 days of the dose reduction to 100 mg QD, increase to 
100 mg BID. 
• 
If Hb concentration does not decrease to <ULN (by sex) 
within 28 days of the dose reduction to 100 mg QD, contact 
the Medical Monitor. 
Abbreviations: BID = twice daily; Hb = hemoglobin; QD = once daily; RBC = red blood cell; ULN = upper limit of 
normal. 
6.7.1.2. 
Dose Modifications for Study Drug–Related Adverse Events 
Dose modifications for study drug–related AEs are provided in Table 7. 
Table 7: 
Dose Modifications for Study Drug–Related Adverse Events 
AE Severity1 
Study Drug Modification 
Grade 1 (mild) 
None required 
Grade 2 (moderate) 
None required 
Grade 3 (severe) 
First 
occurrence  
Reduce to 100 mg QD. 
• 
If the event resolves to Grade ≤2 within 28 days of the 
dose reduction, increase to 100 mg BID. 
• 
If the event does not resolve to Grade ≤2 within 28 days 
of the dose reduction, discontinue study drug. 
Second 
occurrence 
Reduce to 100 mg QD for 7 days, then discontinue study 
drug. 
Grade 4 (life-threatening) 
Reduce to 100 mg QD for 7 days, then discontinue study drug. 
Abbreviations: BID = twice daily; QD = once daily. 
1 Adverse event severity assessed as mild, moderate, or severe, or according to National Cancer Institute Common 
Terminology Criteria for Adverse Events. 

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7. 
STUDY DRUG DISCONTINUATION AND SUBJECT 
WITHDRAWAL FROM STUDY 
7.1. 
Study Drug Discontinuation 
In some instances, it may be necessary for a subject to discontinue study drug. Subjects may be 
discontinued from study drug for the following reasons: 
• Completed 
• Adverse event 
• Death 
• Lost to follow-up 
• Physician decision 
• Pregnancy 
• Protocol deviation 
• Study terminated by sponsor 
• Withdrawal by subject 
• Other 
The primary reason for study drug discontinuation will be recorded in the eCRF. All efforts will 
be made to complete and report protocol-defined study observations as thoroughly as possible. 
Subjects who are discontinuing study drug should undergo the recommended dose taper and be 
monitored as clinically indicated for signs and symptoms of acute hemolysis and worsening 
anemia. Assessments to be completed at the time of study drug discontinuation are specified in 
the Schedules of Assessments (Table 2). 
7.2. 
Subject Withdrawal From the Study 
A subject may withdraw consent from participation in the study at any time at the subject’s 
request or may be withdrawn from the study at any time at the discretion of the Investigator for 
safety, behavioral, compliance, or administrative reasons. Subjects may be withdrawn from the 
study for the following reasons: 
• Death 
• Lost to follow-up 
• Withdrawal by subject 
• Study terminated by sponsor 
• Other 
The primary reason for withdrawal from the study will be recorded in the eCRF. If a subject 
withdraws consent from study participation, no further evaluations should be performed, and no 
attempts should be made to collect additional data. The subject will be discontinued from study 
drug and withdrawn from the study at that time.  

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Subjects who withdraw consent for further participation in the study will not be replaced.  
If the subject withdraws consent for disclosure of future information, the Sponsor may retain and 
continue to use any data collected before withdrawal of consent.  
If a subject withdraws consent for further participation in the study, the subject may request 
destruction of any samples taken and not tested, and the Investigator must document this in the 
clinical study center records. 
7.3. 
Lost to Follow-up 
A subject will be considered lost to follow-up if the subject repeatedly fails to attend study visits 
(at the clinical study center or at home) and is unable to be contacted by the clinical study center. 
If a subject fails to attend a study visit, the clinical study center must attempt to contact the 
subject to reschedule the missed visit as soon as possible, counsel the subject on the importance 
of maintaining the assigned visit schedule, and ascertain whether the subject wishes to and/or 
should continue in the study. Before a subject is considered lost to follow-up, the Investigator (or 
designee) must make every effort to regain contact with the subject. These contact attempts 
should be documented in the subject’s medical record. Should the subject continue to be 
unreachable, the subject will be deemed lost to follow-up and withdrawn from the study.  

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8. 
STUDY ASSESSMENTS AND PROCEDURES 
Study procedures and their timing are outlined in the Schedules of Assessments in Section 1.2. 
Protocol waivers or exemptions are not allowed. 
Details on the processes for collection and handling of blood samples will be provided in the 
Laboratory Manual. 
If allowed by local institutional guidelines, and at the discretion of the Investigator and the 
subject, the subject can complete some visits and study-related assessments at home. The 
designated time points and assessments for these at-home study visits, instead of in-person visits 
to the clinical study center, are in the Schedules of Assessments. 
An at-home visit consists of 1 or 2 of the following components:  
1. Investigator Assessment: A visit conducted by the Investigator (or designee) either via 
video conference or telephone call (telemedicine) or in person at home for assessments 
that can be completed via this mode (eg, AEs and concomitant medications). 
2. Home Health: A visit at home with a nurse or home health-care provider for assessments 
that can be completed via this mode (eg, vital signs and blood collection for clinical 
laboratory assessments). Patient-reported outcome (PRO) measures may also be 
administered during at-home study visits, as applicable. 
During the Double-blind and Open-Label Extension Periods through Week 108, the at-home 
visits will include both Investigator Assessment and Home Health components. Starting at 
Week 156 during the Open-Label Extension Period, the visits will consist of the Investigator 
Assessment component only. 
8.1. 
Informed Consent 
Informed consent will be obtained before any study procedures are performed. 
8.2. 
Screening and Eligibility 
All screening evaluations must be completed and documented by the Investigator in the eCRF 
within the specified screening window (see Schedules of Assessments; Table 1). The 
Investigator will confirm the subject’s eligibility before randomization. Screening extensions 
will not be granted. 
A blood sample will be collected for comprehensive α- and β-globin genotyping during the 
Screening Period for analysis by the study central laboratory. Results from the comprehensive 
α-and β-globin genotyping are not required for eligibility if conclusive DNA analysis results are 
available in the subject’s medical record or can be obtained by a local laboratory during the 
Screening Period. 
Randomization will be stratified by thalassemia genotype as either subjects who do not have a 
β0 mutation at both alleles of the β-globin gene (non-[β0/β0]), including subjects with 
HbE/β-thalassemia and α-thalassemia/HbH disease; or subjects who have a β0 mutation at both 
alleles of the β-globin gene (β0/β0). If it is unclear how the subject should be classified for 
stratification, the Investigator should contact the Sponsor to facilitate a review of the testing 
results before randomization. If a subject’s screening window expires before the availability of 

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genetic testing results from local or central laboratory analyses, or before the determination of 
their stratification class, the subject may be rescreened. 
If a subject is ineligible due to a transient condition (eg, prohibited concomitant medication, 
curable medical condition), the subject can be rescreened after the criterion that the subject failed 
to meet has resolved. If a subject was 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. 
Rescreened subjects will be assigned a different subject number than for the previous screening. 
Rescreened subjects need to re-sign the informed consent form (ICF). Each subject may be 
rescreened a maximum of 2 times (ie, a subject may be screened a total of 3 times). 
If a subject requires rescreening, the following screening assessments do not need to be repeated: 
demographics, documentation of thalassemia diagnosis, and comprehensive α- and β-globin 
genotyping. If the magnetic resonance imaging (MRI) and dual-energy x-ray absorptiometry 
(DXA) scan are within 6 months of randomization, these screening assessments do not need to 
be repeated. 
8.3. 
Demographic Data 
Demographic data will be collected during the Screening Period and include sex, year of birth, 
race, and ethnicity, according to local regulatory requirements. 
8.4. 
Medical, Procedural, Surgical, and Disease History 
A complete medical, procedural, surgical, and disease history will be obtained during the 
Screening Period. The medical history is to include all relevant medical history and all current 
medical conditions. 
8.5. 
Efficacy Assessments 
8.5.1. 
Clinical Laboratory Efficacy Assessments 
Study-required clinical laboratory efficacy assessment samples will be analyzed by a central 
laboratory. If samples for local laboratory analysis are collected, the study-required samples for 
central laboratory analysis must also be collected at the same time. 
Clinical laboratory efficacy assessment parameters are provided in Table 8. 

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Table 8: 
Clinical Laboratory Efficacy Assessments 
Assessment 
Parameters 
Anemia 
Hb 
 
 
 
 
soluble transferrin receptor 
Iron metabolism 
Iron, serum ferritin, transferrin, hepcidin, TSAT, and TIBC1 
Abbreviations: Hb = hemoglobin; 
; 
; TIBC = total iron binding 
capacity; TSAT = transferrin saturation. 
Note: Study-required clinical laboratory efficacy assessment samples will be analyzed by a central laboratory. 
1 TSAT and TIBC will be calculated. 
Clinical laboratory safety assessment parameters are provided in Section 8.6.3. 
8.6. 
Safety Assessments 
8.6.1. 
Vital Signs and Physical Examinations 
Vital signs should be measured after 5 minutes of recumbency and before blood sample 
collection. Vital signs include systolic and diastolic blood pressure, pulse rate, and body 
temperature. Weight will also be recorded.  
Height will be measured and recorded during the Screening Period. 
8.6.2. 
Electrocardiograms 
A single, 12-lead electrocardiogram (ECG) will be recorded and read at the clinical study center 
after 5 minutes of recumbency and before blood sample collection. 
8.6.3. 
Clinical Laboratory Safety Assessments 
Study-required clinical laboratory assessment samples will be analyzed by a central laboratory. 
Local laboratory analysis is allowed, at the discretion of the Investigator, if faster turnaround 
time is required to support clinical decision-making. If samples for local laboratory analysis are 
collected, the study-required samples for central laboratory analysis must also be collected at the 
same time. Subjects will be monitored for potential aromatase inhibition by assessments of sex 
hormone levels. 

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Clinical laboratory safety assessment parameters are provided in Table 9. 
Table 9: 
Clinical Laboratory Safety Assessments 
Assessment 
Parameters 
Hematology 
• 
Complete blood count (Hb, HCT, MCHC, MCH, MCV, RBC count, 
nucleated RBCs, RDW-CV, % reticulocyte count, absolute reticulocyte 
count, and platelet count) with WBC count and differential (neutrophils, 
basophils, eosinophils, lymphocytes, and monocytes) 
Clinical chemistry 
• 
Albumin, BUN or urea, total calcium, CO2 or bicarbonate, chloride, 
creatinine, 
 magnesium, phosphorus, 
potassium, sodium, total protein, and uric acid 
Liver tests 
• 
Liver tests include ALP, ALT/SGPT, AST/SGOT, and bilirubin 
(direct/conjugated, indirect/unconjugated, and total).  
 
 
Coagulation tests 
• 
Fibrinogen, aPTT, and INR 
Lipid panel 
• 
Nonfasting HDL-C, LDL-C, total cholesterol, and triglycerides 
Sex hormones 
• 
Nonfasting estradiol, estrone, and testosterone (total and free) 
Spot urine sample 
• 
Albumin to creatinine ratio 
Screening virology 
• 
HBsAg, HCV Ab (positive results for HCV Ab testing require 
confirmation by a test for HCV-RNA), HIV-1 Ab, and HIV-2 Ab 
Screening FSH and 
serum hCG 
• 
FSH (for women in the absence of 12 months of amenorrhea to confirm 
non–reproductive potential) and a serum (hCG) pregnancy (for WOCBP) 
Abbreviations: Ab = antibody; ALP = alkaline phosphatase; ALT = alanine aminotransferase; aPTT = activated 
partial thromboplastin time; AST = aspartate aminotransferase; BUN = blood urea nitrogen; CO2 = carbon dioxide; 
 FSH = follicle stimulating hormone; Hb = hemoglobin; 
HBsAg = hepatitis B surface antigen; hCG = human chorionic gonadotropin; HCT = hematocrit; HCV = hepatitis C 
virus; HDL-C = high-density lipoprotein cholesterol; INR = international normalized ratio; LCMS = liquid 
chromatography–mass spectrometry; 
; LDL-C = low density lipoprotein cholesterol; 
MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; MCV = mean 
corpuscular volume; RBC = red blood cell; RDW-CV = red cell distribution width coefficient of variation; 
WBC = white blood cell; WOCBP = women of childbearing potential. 
Note: Study-required clinical laboratory assessment samples will be analyzed by a central laboratory. 
 
 
 
 
Clinical laboratory efficacy assessment parameters are provided in Section 8.5.1. 
8.6.4. 
Dual-Energy X-ray Absorptiometry Scans  
Subjects will be monitored for potential aromatase inhibition by assessments of bone mineral 
density. Bone mineral density will be assessed by DXA scans of the hip and spine performed 
according to the instructions provided by the central-read vendor. The DXA scans will be read 
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8.6.5. 
Adverse Events and Serious Adverse Events 
The definitions of and reporting requirements for AEs and SAEs are provided in Section 9. 
8.7. 
Red Blood Cell Transfusions and Transfusion Modifications 
The first dose of study drug must be administered within 7 days after Transfusion 0. 
Transfusion 0 is defined as the last RBC transfusion that was administered before randomization. 
Randomization must not occur until T0 has been completed. 
 
 All blood samples must be 
collected before administration of the study drug and before an RBC transfusion, including 
Transfusion 0, if an RBC transfusion is scheduled on the same day. 
Transfusion history will be recorded for the 24-week period before randomization. Transfusion 
history includes T0. On-treatment transfusions will also be recorded. The transfusion history and 
on-treatment transfusion record includes transfusion dates, number of RBC units transfused, and 
pretransfusion Hb concentration. An RBC unit refers to packed red blood cell (pRBC) 
preparations. Due to hematocrit concentration differences in blood products used per local 
standards of care, 2 units of whole blood are equivalent to 1 unit of pRBCs and constitute an 
RBC unit. If available, the volume and the hematocrit of each transfused RBC unit should also 
be recorded. All RBC transfusions, including those received outside of the clinical study center, 
must be recorded. 
A pretransfusion Hb threshold will be determined for each subject based on transfusion history. 
The pretransfusion Hb threshold is defined as the mean of all documented pretransfusion Hb 
concentration values recorded for the RBC transfusions administered during the 24-week 
transfusion history.  
During the study, Investigators will monitor changes in Hb concentration and modify the 
subject’s RBC transfusion schedule according to the guidelines in Table 10. Study drug dose 
modification guidance for excessive Hb response is provided in Section 6.7.1.1. 
Subjects may receive RBC transfusions at the discretion of the Investigator for acute symptoms 
related to anemia or other comorbidities requiring immediate treatment (eg, infection or 
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 Table 10: 
Red Blood Cell Transfusion Modification Guidance 
Pretransfusion Hb Concentration1 
RBC Transfusion Modification 
Hb concentration ≥1 g/dL above the 
pretransfusion Hb threshold and 
≤ULN (by sex) 
Delay RBC transfusion or decrease the number of RBC units to 
be transfused. 
>ULN (by sex) 
Delay RBC transfusion. 
• 
Recheck Hb concentration within 28 days. 
• 
If Hb concentration remains >ULN (by sex) for ≥4 weeks, 
in the absence of RBC transfusions, modify the study drug 
dose as described in Section 6.7.1.1. 
Abbreviations: Hb = hemoglobin; RBC = red blood cell; ULN = upper limit of normal. 
1 The pretransfusion Hb concentration is the Hb concentration obtained before a planned transfusion event to guide 
its transfusion prescription, irrespective of whether a transfusion is subsequently delivered or deferred. 
8.8. 
Pharmacokinetics and Pharmacodynamics 
Blood samples will be collected for measurement of concentrations of mitapivat and other 
pharmacokinetic parameters as appropriate, and for pharmacodynamic measurement of 2,3-DPG 
and ATP. 
On days when there are pharmacokinetic or pharmacodynamic assessments, the morning dose of 
study drug will be administered at the clinical study center. 
The actual date and time (24-hour clock time) of sample collection will be recorded in the source 
documents or eCRF. An explanation should be provided in the source documents for any missed 
or mishandled pharmacokinetic samples, and for any samples collected outside the time 
windows. The actual date and time (24-hour clock time) of study drug administration for the 
morning dose and the last dose taken the previous evening will also be recorded in the source 
documents or eCRF. 
Pharmacokinetic samples will be analyzed using a validated liquid chromatography-tandem mass 
spectrometry (LC-MS/MS) method. Plasma pharmacokinetic parameters will be computed from 
measured plasma concentrations and using actual sample collection times.  
Pharmacodynamic samples will be analyzed for 2,3-DPG and ATP using an LC-MS/MS method. 
Samples may be retained for a maximum of 5 years (or according to local regulations) after the 
end of the study at a facility selected by the Sponsor for further analysis of pharmacokinetic or 
pharmacodynamic responses to mitapivat for subjects who consent to this optional exploratory 
research in the ICF (see Section 11.2). 
8.9. 
Genetics 
Blood samples collected for comprehensive α- and β-globin genotyping 
 
 will be analyzed by the study central laboratory. Samples will be maintained 
in a secure storage facility with adequate measures to protect subject confidentiality. Samples 
will be retained for a maximum of 10 years (or according to local regulations). 

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9. 
ADVERSE EVENTS: DEFINITIONS AND PROCEDURES FOR 
RECORDING AND REPORTING 
9.1. 
Definitions of Adverse Events 
9.1.1. 
Definition of an Adverse Event 
An AE is any untoward medical occurrence in a patient or clinical investigation subject 
administered a pharmaceutical product that does not necessarily have a causal relationship with 
this treatment. An AE can therefore be any of the following: 
• Any unfavorable and unintended sign (eg, an abnormal laboratory finding), symptom, 
or disease temporally associated with the use of a study drug, whether or not 
considered related to the study drug. 
• Any new disease or exacerbation of an existing disease (ie, a worsening in the 
character, frequency, or severity of a known condition) 
• Recurrence of an intermittent medical condition (eg, headache) not present at baseline 
• Any deterioration in a laboratory value or other clinical test (eg, ECG, x-ray) that is 
associated with clinical signs and symptoms, leads to a modification or 
discontinuation of study drug or concomitant therapy, requires medical or surgical 
intervention, or is considered by the Investigator to be clinically significant 
• AEs that are related to a protocol-mandated intervention, including those that occur 
before assignment of study drug (eg, invasive screening procedures such as biopsies) 
9.1.2. 
Definition of a Serious Adverse Event 
An SAE is any AE or suspected adverse reaction that:  
• Results in death 
• Is immediately life-threatening 
− Life-threatening means that the subject was at immediate risk of death from the 
reaction as it occurred (ie, it does not include a reaction which hypothetically 
might have caused death had it occurred in a more severe form). 
• Requires inpatient hospitalization or prolongation of existing hospitalization 
− Planned hospital admissions or surgical procedures for an illness or disease which 
existed before the subject was enrolled in the study or before study drug was 
administered are not to be considered AEs unless the condition deteriorated in an 
unexpected manner during the study (eg, surgery was performed earlier or later 
than planned). 
• Results in persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions 
• Results in congenital anomaly/birth defect 

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• Is considered an 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 or may 
require medical or surgical intervention to prevent one 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 the development of drug dependency or drug abuse. 
The terms “severe” and “serious” are not synonymous. Severity refers to the intensity of an AE 
(mild, moderate, or severe, or according to NCI CTCAE); the event itself may be of relatively 
minor medical significance (eg, severe headache without any further findings). Severity and 
seriousness need to be independently assessed for each AE recorded on the eCRF. 
9.1.3. 
Abnormal Clinical Laboratory Events  
A clinical laboratory test result should be reported as an AE if it meets any of the following 
criteria: 
• Associated with clinical signs and symptoms 
• Results in a dose modification or study drug discontinuation 
• Requires medical or surgical intervention or a change in concomitant therapy 
• Clinically significant in the Investigator’s judgment 
The Investigator is responsible for reviewing all clinical 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 or symptom of a disease or syndrome, 
only the diagnosis should be recorded on the AE eCRF. 
If a clinically significant laboratory abnormality is not a sign or symptom of a disease or 
syndrome, the abnormality itself should be recorded on the AE eCRF, along with a descriptor 
indicating if the test result is above or below the normal range. 
9.2. 
Procedures for Recording Adverse Events and Serious Adverse 
Events 
9.2.1. 
Recording Adverse Events  
Adverse events and SAEs that occur from the time of providing informed consent until 28 days 
after the administration of the last dose of study drug will be recorded on the appropriate pages 
of the eCRF. 
Adverse events spontaneously reported by the subject and/or in response to open nonleading 
questions (eg, “How are you feeling?”) from study personnel or revealed by observation, 
physical examination, or other diagnostic procedures will be collected at every visit and recorded 
in the subject’s medical record and in the appropriate pages of the eCRF. 

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Adverse events will be followed until 28 days after administration of the last dose of study drug 
or until study termination. Subjects will be assessed at the Safety Follow-up visit to determine if 
any new AEs have occurred. Adverse events that are ongoing at the time of study drug 
discontinuation should be followed through the Safety Follow-up visit. After this period, 
Investigators should report only SAEs that are considered related to study drug. Subjects who are 
withdrawn from the study due to a study drug related SAE will continue to be followed until the 
SAE resolves or is declared chronic by the Investigator, or until the subject is lost to follow-up. 
Any clinically significant deterioration in clinical laboratory assessments (see Section 9.1.3) or 
other clinical finding that is considered an AE must be recorded in the eCRF. When possible, 
signs and symptoms indicating a common underlying pathology should be noted as 
one comprehensive event. 
Information to be reported in the description of each AE includes:  
• A medical diagnosis of the event (if a medical diagnosis cannot be determined, a 
description of each sign or symptom characterizing the event should be recorded)  
• Event onset date  
• Event resolution date  
• Whether the event is serious (Section 9.1.2) 
• Severity of the event (Section 9.2.2)  
• Relationship of the event to study drug (Section 9.2.3) 
• Action taken with study drug 
• Event outcome 
9.2.2. 
Severity of Adverse Events 
The severity of all AEs, including clinically significant treatment-emergent clinical laboratory 
abnormalities, will be graded by the Investigator according to Version 4.03 of the NCI CTCAE 
on a 5-point severity scale (Grade 1 through Grade 5). Adverse events not specified by the NCI 
CTCAE will be graded according to Table 11.  
Table 11: 
Adverse Event Severity 
Severity 
Definition 
NCI CTCAE 
Mild 
The event is noticeable to the subject but does not interfere with 
routine activity. 
Grade 1 
Moderate  
The event interferes with routine activity but responds to 
symptomatic therapy or rest. 
Grade 2 
Severe  
The event significantly limits the subject’s ability to perform 
routine activities despite symptomatic therapy. 
Grade 3 
Life-threatening  The subject was at risk of death at the time of the event. 
Grade 4 
Fatal  
An event that results in the death 
Grade 5 
Abbreviations: NCI CTCAE = National Cancer Institute Common Terminology Criteria for Adverse Events. 

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An AE of severe intensity may not be considered serious. Severity is a measure of intensity, 
whereas seriousness is defined as described in Section 9.1.2. 
9.2.3. 
Relationship to Study Drug 
Investigators must determine whether an AE is considered to be related to the study drug, 
indicating “yes” or “no” according to the causal attribution guidance in Table 12. 
Table 12: 
Causal Attribution Guidance 
Is the AE suspected to be caused by the study drug based on facts, evidence, science-based 
rationales, and clinical judgment? 
Yes 
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 the AE follows a known pattern of 
response to the study drug; and/or the AE abates or resolves upon discontinuation of the 
study drug or dose reduction and, if applicable, reappears upon rechallenge. 
No 
Adverse events will be considered related unless evidence exists that the AE has an etiology 
other than the study drug (eg, preexisting medical condition, underlying disease, intercurrent 
illness, or concomitant therapy); and/or the AE has no plausible temporal relationship to 
study drug administration (eg, cancer diagnosed 2 days after first dose of study drug). 
Abbreviation: AE = adverse event. 
When determining a causal attribution to study drug, the following guidance should be 
considered: 
• Temporal relationship of event onset to the initiation of study drug 
• Course of the event, considering especially the effects of dose reduction, study drug 
discontinuation, or study drug rechallenge, as applicable 
• Known association of the event with the study drug or with similar treatments 
• Known association of the event with the disease under study 
• Presence of risk factors for the subject or use of concomitant therapies known to 
increase the occurrence of the event 
• Presence of non–treatment-related factors that are known to be associated with the 
occurrence of the event 
9.3. 
Procedures for Reporting Adverse Events 
9.3.1. 
Reporting of Serious Adverse Events 
All SAEs, regardless of relationship to study drug, that occur from the time of providing 
informed consent until 28 days after administration of the last dose of study drug must be 
reported to the Sponsor within 24 hours from the time when the Investigator becomes aware of 
the SAE. This 24-hour notification applies to the initial SAE information and all follow-up SAE 
information. 

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Serious adverse events that occur more than 28 days after administration of the last dose of study 
drug that are considered by the Investigator to be related to study drug must be reported to the 
Sponsor any time the Investigator becomes aware of such an event. 
The SAE eCRF must be completed and will include the following information: subject number, a 
narrative description of the event, and an assessment by the Investigator of the severity of the 
event and its relationship to study drug. Additional follow-up information on the SAE may be 
requested by the Sponsor or Medical Monitor.  
If the electronic data capture (EDC) system is unavailable for more than 24 hours, SAEs must be 
reported using the EDC Downtime SAE Report Form. The SAE Report Form and completion 
instructions, a Safety Cover Letter, as well as a list of country-specific fax numbers are provided 
by the Sponsor. Completed SAE reports may be submitted to Agios via email at 
Safety_Operations@agios.com. 
If there are suspected unexpected serious adverse reactions associated with the use of mitapivat, 
the Sponsor will notify the appropriate regulatory agency(ies) and all participating Investigators 
on an expedited basis. The local Institutional Review Board (IRB)/Independent Ethics 
Committee (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. 
Any on-treatment deaths, regardless of relationship to study drug, must be recorded on the 
appropriate eCRF and reported to the Sponsor within 24 hours. Deaths that occur during the 
protocol-specified SAE reporting period that are attributed by the Investigator solely to disease 
under study should not be reported as SAEs and should only be recorded on the End of Study 
eCRF page. 
9.4. 
Other Safety-Related Issues 
9.4.1. 
Overdose or Dose Administration Error 
Overdose is the accidental or intentional use of the study drug in an amount higher than the dose 
being studied. An overdose or incorrect administration of study drug is not an AE unless it 
results in untoward medical effects. All AEs associated with an overdose or incorrect 
administration of study drug should be recorded on the AE eCRF. If the associated AE fulfills 
the criteria for an SAE, the event must be reported as specified in Section 9.3.1. 
9.4.2. 
Pregnancy 
Pregnancy is not reported as an AE unless there is a suspicion that an investigational product 
may have interfered with the effectiveness of a contraceptive medication or a complication 
relating to the pregnancy occurs that may qualify as an SAE (eg, spontaneous abortion). The 
Investigator must report any pregnancy that occurs during the treatment period and within 
28 days after administration of the last dose of study drug, even in the absence of an AE. 
Pregnancy must be reported to the Sponsor within 24 hours of learning of its occurrence using a 
Pregnancy Report Form. Abnormal pregnancy outcomes (eg, maternal serious complications, 
spontaneous or therapeutic abortion, ectopic pregnancy, stillbirth, neonatal death, congenital 
anomaly, or birth defect) are considered SAEs and must be reported as specified in Section 9.3.1.  

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9.4.2.1. 
Pregnancies in Female Subjects 
Female subjects who become pregnant while participating in the study will be immediately 
discontinued from study drug. Subjects should be encouraged to undergo the assessments at the 
Safety Follow-up visit. All AEs that occur during this time, including those related to the 
pregnancy, will be reported. 
Investigators should counsel the subject regarding the risks of continuing the pregnancy and the 
possible effects on the fetus. The Investigator must follow-up and document the course and 
outcome of all pregnancies. Monitoring of the subject should continue until conclusion of the 
pregnancy. After completion of the pregnancy, attempts should be made to collect and report 
details of the outcome of the pregnancy and development of the child. 
9.4.2.2. 
Pregnancy Follow-up 
All outcomes of pregnancy from a female subject must be reported by the Investigator to the 
Sponsor or Medical Monitor in the pregnancy outcome section of the paper Pregnancy Reporting 
Form immediately after becoming aware of the outcome of the pregnancy. Any termination of 
the pregnancy will be reported regardless of fetal status (presence or absence of anomalies) or 
indication for the procedure. 
The Sponsor will attempt to collect follow-up information where possible and allowable by local 
practice and regulations (ie, with the consent of the subject) regarding the course and outcome of 
the pregnancy, including any postnatal sequelae in the infant and the development of the child in 
order to perform an independent medical assessment of the reported pregnancy. 
After study termination, the Investigator may report any outcomes or AEs directly to the sponsor 
on a paper SAE form. 

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10. 
STATISTICAL CONSIDERATIONS 
10.1. 
Statistical Hypotheses 
The following statistical hypothesis will be tested to address the primary objective: 
H01: 𝑝𝑡1 −𝑝𝑐1 = 0 vs H11: 𝑝𝑡1 −𝑝𝑐1 ≠0 
where 𝑝𝑡1 and 𝑝𝑐1 are the proportion of subjects achieving a transfusion reduction response 
(TRR), defined as ≥50% reduction in transfused RBC units with a reduction of ≥2 units of 
transfused RBCs in any consecutive 12-week period through Week 48 compared with baseline, 
in the mitapivat arm and placebo arm, respectively. 
Additionally, the following statistical hypotheses will be tested to address the key secondary 
objectives: 
H02: 𝑝𝑡2 −𝑝𝑐2 = 0 vs H12: 𝑝𝑡2 −𝑝𝑐2 ≠0 
H03: 𝑝𝑡3 −𝑝𝑐3 = 0 vs H13: 𝑝𝑡3 −𝑝𝑐3 ≠0 
H04: 𝑝𝑡4 −𝑝𝑐4 = 0 vs H14: 𝑝𝑡4 −𝑝𝑐4 ≠0 
• where 𝑝𝑡2 and 𝑝𝑐2 are the proportion of subjects achieving a ≥50% reduction in 
transfused RBC units in any consecutive 24-week period through Week 48 compared 
with baseline (referred to as TRR2) in the mitapivat arm and placebo arm, 
respectively. 
• 𝑝𝑡3 and 𝑝𝑐3 are the proportion of subjects achieving a ≥33% reduction in transfused 
RBC units from Week 13 through Week 48 compared with baseline (referred to as 
TRR3) in the mitapivat arm and placebo arm, respectively. 
• 𝑝𝑡4 and 𝑝𝑐4 are the proportion of subjects achieving a ≥50% reduction in transfused 
RBC units from Week 13 through Week 48 compared with baseline (referred to as 
TRR4) in the mitapivat arm and placebo arm, respectively. 
Overall type I error will be maintained at or below a 2-sided significance level of 0.05 based on a 
graphical gatekeeping approach as illustrated in Figure 2. 

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Figure 2: 
Statistical Testing Strategy 
 
The primary endpoint will be tested at a 2-sided α-level of 0.05. Then, the first 2 key secondary 
endpoints will be tested at an α-level of 0.032 and 0.018, respectively, if the null hypothesis for 
the primary endpoint is rejected. The last key secondary endpoint will be tested at an α-level of 
0.018 (if only H02 is rejected), 0.032 (if only H03 is rejected), or 0.05 (if both H02 and H03 are 
rejected). 
The study will have met its primary objective if the primary endpoint is statistically significant at 
the time of the final analysis at the 2-sided α-level of 0.05. 
10.2. 
Sample Size Determination 
Approximately 240 subjects will be randomly assigned in a 2:1 ratio to receive study drug 
(mitapivat or placebo). With the planned sample size, the study will have 95% power to detect an 
increase in TRR rate from 12.5% in the placebo arm to 33.7% in the mitapivat arm at a 2-sided 
α-level of 0.05. 
The conservative assumptions for the TRR rate were informed by the results of a Phase 3 clinical 
trial in patients with β-TDT where 12.5% was the upper bound of the 95% CI for TRR rate in the 
placebo arm, and 33.7% was the lower bound of the 95% CI for the TRR rate for the active arm 
(Cappellini et al, 2020). 

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10.3. 
Populations for Analyses 
The following analysis data sets will be evaluated and used for presentation of the data: 
Population 
Description 
All Screened 
Subjects 
All subjects who sign the ICF. 
Full Analysis Set 
(FAS) 
All subjects who are randomized. Subjects will be classified according to the 
randomized treatment arm according to the ITT principle. The FAS is the 
primary analysis set for the evaluation of efficacy endpoints, unless otherwise 
specified. 
Per-Protocol Set 
(PPS) 
The PPS is a subset of the FAS. Subjects who do not receive at least 1 dose of 
the randomized treatment will be excluded from the PPS. Other criteria leading 
to exclusion of subjects from the PPS will be prespecified in the statistical 
analysis plan. 
Safety Analysis Set 
All subjects who receive at least 1 dose of the study treatment. Subjects will be 
classified according to the treatment received. If a subject randomized to placebo 
receives at least 1 dose of mitapivat in the Double-blind Period, then the subject 
will be classified to the mitapivat arm. 
Pharmacodynamic 
Analysis Set 
A subset of the Safety Analysis Set including all subjects with at least 1 blood 
2,3-DPG or ATP concentration measurement above LLQ. 
Pharmacokinetic 
Analysis Set 
A subset of the Safety Analysis Set including all subjects with at least 1 plasma 
mitapivat concentration measurement above LLQ. 
Abbreviations: 2,3-DPG = 2,3-diphosphoglycerate; ATP = adenosine triphosphate; ITT = intent-to-treat; 
LLQ = lower limit of quantitation. 
10.4. 
Statistical Analyses 
The statistical analysis plan (SAP) will be finalized before database lock and unblinding and will 
include a more detailed description of the statistical analyses described in this section. 
10.4.1. 
General Considerations 
10.4.1.1. Baseline Definitions 
Efficacy Evaluations 
Baseline transfusion burden is defined as the total number of RBC units transfused during the 
24-week period before randomization for subjects randomized and not dosed or before the start 
of study treatment for subjects randomized and dosed. Baseline transfusion burden will be 
standardized to match the postbaseline assessment duration specified for each endpoint 
associated with reduction of transfusion burden from baseline. 
For other efficacy endpoints (markers of iron overload and iron metabolism), baseline is defined 
as the last assessment before Transfusion 0. Baseline will be derived based on central laboratory 
data; if no central laboratory data are available, then local laboratory data will be used to derive 
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Baseline Characteristics 
For summaries of baseline characteristics based on the FAS, baseline will be defined as follows: 
• For subjects randomized and not dosed: the last assessment before randomization 
• For subjects randomized and dosed: the last assessment before the start of study 
treatment 
Safety Evaluations 
For clinical laboratory assessments the following applies: 
• Before deriving the baseline: 
− If there are multiple records with the same assessment day and time from the 
same laboratory, the average value will be used. 
− 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 before the start of study treatment. 
10.4.1.2. On-Treatment Period 
The on-treatment period begins on the date of the start of study drug and ends 28 days after the 
end of study drug administration (including the recommended dose taper). 
10.4.1.3. Pooling of Data Across Clinical Study Centers 
To provide overall estimates of treatment effects, data will be pooled across clinical study 
centers (“sites”). The “site” factor will not be considered in statistical models or subgroup 
analyses given the high number of participating centers in contrast to the anticipated small 
number of subjects randomized at each study center. 
10.4.1.4. Continuous and Categorical Variables 
Continuous variables will be summarized using descriptive statistics (ie, number of nonmissing 
values, mean, SD, median, quartiles, minimum, and maximum). 
Categorical variables will be summarized by frequency distributions (ie, 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. 
10.4.1.5. 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. Descriptive 
statistics (mean, SD, median, quartiles, minimum, and maximum) by nominal visit or time point 
for safety endpoints (eg, clinical laboratory measurements and vital signs) will include only data 
from scheduled visits. Summaries of outliers (eg, worst value, worst change from baseline, worst 

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severity grade) during the on-treatment period for safety endpoints (eg, AEs and clinical 
laboratory measurements) will include data from both scheduled and unscheduled visits. 
10.4.2. 
Primary Endpoint 
10.4.2.1. Transfusion Reduction Response 
The primary endpoint of TRR (as defined in Section 10.1) will be tested using the 
Mantel-Haenszel stratum weighted method adjusting for randomization stratification factors. 
Subjects who are withdrawn from the study before Week 12 (Day 85) will be considered 
nonresponders. The proportion of subjects who achieved a TRR will be summarized for each 
treatment arm. The adjusted difference in TRR rate between the mitapivat arm and placebo arm, 
along with the 95% CI and the 2-sided p-value, will be provided. 
10.4.2.2. Sensitivity Analyses 
TRR will be summarized using the methodology described for the primary analysis but based on 
PPS. 
Additional sensitivity analyses may be performed and will be prespecified in the SAP. 
10.4.2.3. Subgroup Analyses 
Transfusion reduction response rate will be summarized for subgroups defined by each 
randomization stratification factor. Within each subgroup, TRR rate will be summarized by 
treatment arm, and the difference in TRR rate between the mitapivat arm and the placebo arm 
will be estimated (difference in Hb response rate and 95% CI) using an unstratified method.  
Transfusion reduction response may be summarized for other subgroups prespecified in the SAP. 
10.4.3. 
Secondary Efficacy Endpoints 
10.4.3.1. Key Secondary Efficacy Endpoints 
The key secondary endpoints (TRR2, TRR3, and TRR4, as defined in Section 10.1) will be 
tested using the Mantel-Haenszel stratum weighted method adjusting for randomization 
stratification factors. The response rate based on different definitions of response will be 
summarized for each treatment arm. The adjusted differences in response rate between the 
mitapivat arm and placebo arm, along with the 95% CI and the 2-sided p-value, will be provided. 
Subjects who are withdrawn from the study before Week 24 will be considered nonresponders in 
TRR2. Subjects who are withdrawn from the study before Week 48 will be considered 
nonresponders in TRR3 and TRR4. 
10.4.3.2. Other Secondary Efficacy Endpoints 
The change from baseline in transfused RBC units from Week 13 through Week 48 will be 
summarized. The frequency of subjects within the following percentage reduction categories will 
also be provided: <0, 0 to <20, 20 to <33, 33 to <50, and ≥50%. 
The frequency of subjects achieving transfusion independence will be summarized for each 
treatment arm. Transfusion independence is defined as transfusion-free for ≥8 consecutive weeks 

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through Week 48. The adjusted difference in the proportion of subjects who achieve transfusion 
independence between the mitapivat arm and the placebo arm using a Mantel-Haenszel stratum 
weighted method adjusting for randomization stratification factors, along with the 95% CI, will 
be provided. 
The change from baseline in iron, serum ferritin, transferrin saturation, and total iron binding 
capacity will be summarized by treatment arm and visit. 
10.4.3.3. Pharmacokinetic and Pharmacodynamic Endpoints 
Descriptive statistics of pharmacokinetic (arithmetic and geometric means, SD, percent 
coefficient of variation [CV%], CV% geometric mean, minimum, median, and maximum) and 
pharmacodynamic parameters, including ATP and 2,3-DPG, will be summarized as appropriate.  
10.4.4. 
Safety Endpoints 
Summaries of safety data from the Double-blind Period will be presented by treatment arm based 
on the Safety Analysis Set. Summaries of safety data from the Open-label Extension Period will 
be presented by treatment arm and overall based on the Safety Analysis Set. 
10.4.4.1. Adverse Events  
Treatment-emergent adverse events are defined as AEs with a first onset date during the 
on-treatment period or worsening from baseline. All summaries will be based on 
treatment-emergent adverse events, unless otherwise specified. Treatment-emergent adverse 
events will be summarized according to the latest version of Medical Dictionary for Regulatory 
Activities terminology by System Organ Class and/or Preferred Term, severity (based on NCI 
CTCAE Version 4.03 grading), seriousness, and relationship to study drug, unless otherwise 
specified. 
10.4.4.2. Clinical Laboratory Assessments 
The actual values and the changes from baseline will be summarized by study visit for clinical 
laboratory tests. 
10.4.4.2.1. Parameters With CTCAE Grades Available 
Clinical laboratory test results will be graded according to the CTCAE as applicable. Grading 
will be derived based on the numerical thresholds defined by the CTCAE. Nonnumerical 
qualifiers will not be taken into consideration in the derivation of CTCAE grading. Clinical 
laboratory test results classified according to the CTCAE will be described using the worst 
grade.  
The frequency of subjects with clinical laboratory toxicities during the on-treatment period will 
be tabulated as follows.  
• Summaries of clinical laboratory parameters by CTCAE grade will include the 
number and percentage of subjects with Grade 1, 2, 3, 4; Grades 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. 

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• Shift tables 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, Grades 3-4) 
during the on-treatment period will also be summarized. 
The denominator used to calculate percentages for each clinical laboratory test is the number of 
subjects evaluable for CTCAE grading for that parameter (ie, subjects for whom a Grade of 0, 1, 
2, 3, or 4 can be derived). 
10.4.4.2.2. Parameters With CTCAE Grades Not Available 
Clinical laboratory test results that are not specified by the CTCAE will be presented according 
to the following categories: < the lower limit of normal (LLN), within normal limits, and >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. 
10.4.4.3. Dual-Energy X-ray Absorptiometry Scans 
Results from bone mineral density on DXA scan will be summarized. 
10.4.4.4. Vital Signs and Physical Examinations 
The actual values and the changes from baseline will be summarized by study visit for vital signs 
assessments. Clinically significant changes in physical examinations will also be presented. 
10.5. 
Interim Analyses  
An interim analysis is not planned. 
10.6. 
Independent Data Monitoring Committee 
An unblinded IDMC will be responsible for ongoing monitoring of the safety of subjects 
according to the IDMC Charter. The recommendations for study conduct (eg, continue as 
planned, continue with modifications, terminate) made by the IDMC will be provided to the 
Sponsor (or designee) for the final decision. The Sponsor (or designee) will forward such 
decisions, which may include summaries of aggregate analyses of endpoint events and of safety 
data that are not endpoints, to regulatory authorities, as appropriate. 
The IDMC will perform the first review of safety data after approximately 30 subjects have been 
randomized in the study. Subsequently, the IDMC will convene to monitor safety in the study 
approximately every 3 months until the study is unblinded for the analysis of the primary 

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endpoint. Recommendations for study conduct (continue as planned, continue with 
modifications, terminate) will be conveyed to Agios by the IDMC chair. 

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11. 
REGULATORY, ETHICAL, AND STUDY OVERSIGHT 
CONSIDERATIONS 
11.1. 
Regulatory and Ethical Considerations 
This study will be conducted in accordance with the protocol and with the following: 
• Consensus ethical principles derived from international guidelines including the 
Declaration of Helsinki and Council for International Organizations of Medical 
Sciences International Ethical Guidelines 
• Applicable International Council for Harmonisation (ICH) GCP Guidelines 
• Applicable laws and regulations 
The protocol, ICF, IB, and other relevant documents (eg, advertisements) must be reviewed and 
approved by an IRB/IEC before the study is initiated.  
Any amendments to the protocol will require IRB/IEC approval before implementation of 
changes, except for changes necessary to eliminate an immediate hazard to study subjects in 
which case approval will be obtained as soon as possible following implementation.  
The Investigator will be responsible for the following: 
• Providing written summaries of the study status to the IRB/IEC in accordance with 
the requirements, policies, and procedures established by the IRB/IEC (at least 
annually and at study closeout) 
• Notifying the IRB/IEC of SAEs or other significant safety findings as required  
• Overseeing the conduct of the clinical study at the study center and adhering to all 
applicable regulations 
11.1.1. 
Temporary Modifications Allowed During Public Health Emergencies 
In the event of a declared public health emergency or natural disaster that affects a geographic 
area (eg, state, province, country, region, continent) and impedes adherence to protocol-specified 
procedures, certain modifications (see Section 11.1.1.1) are temporarily allowable to ensure 
subject safety, maintain compliance with GCP, and minimize risks to study integrity; the 
protocol must be followed to the fullest extent possible. 
These modifications are only allowable 1) when consistent with applicable regulations and 
guidance and 2) for the duration of the declared public health emergency or natural disaster. 
During this period, the need for all implemented modifications will be reassessed and the 
Sponsor will no longer allow these modifications once the situation resolves. 
Examples of declared public health emergencies and natural disasters are: 
• The public health emergency related to COVID-19 declared by the US Secretary of 
Health and Human Services in 2020 
• The Australian Bushfires Disaster declared by the Australian Attorney General in 
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Documented approval from the Sponsor is required before these modifications can be 
implemented. 
11.1.1.1. Allowable Temporary Modifications 
The following temporary modifications are allowed in the event of a declared public health 
emergency or natural disaster and must be reported as protocol deviations; refer to the Schedules 
of Assessment (Section 1.2) for the timing of assessments. 
• Alternative distribution of study drug 
− Study drug may be shipped to a local health-care provider or pharmacy or, if 
necessary, directly to a subject. The quantity to be shipped must be reviewed and 
approved in advance by the Sponsor (or designee), in agreement with the 
Investigator. 
− Secure, trackable delivery methods (delivery service companies [eg, DHL], 
couriers, and hand delivery) must be used. 
− Sponsor (or designee) approval is required before each shipment. Shipment will 
be permitted only if, at minimum, a telemedicine visit has been conducted that 
incorporates appropriate safety assessments. 
• Returning unused study drug and empty study drug packaging 
− Return of unused study drug and empty study drug packaging may be delayed 
until the subject’s next visit to the study site. In certain circumstances, the nature 
of the return process may vary (eg, personal protective equipment may be 
required). 
• Telemedicine visits for assessments other than PROs 
− Telemedicine visits, preferably via video conference, are permissible for all 
assessments that can be completed via this mode (eg, medical history, 
concomitant medications, review of AEs). 
• Telemedicine for collection of PROs 
− Sponsor agreement is needed before implementing alternative solutions for PROs 
that were intended to be collected at the site. 
• Use of laboratories and health-care providers not specified in the clinical study 
documentation (must be reported as a protocol deviation) 
− For assessments that cannot be completed via telemedicine, the use of health-care 
providers and laboratories that are not specified in the clinical trial documentation 
(eg, an imaging facility, clinic, or local practice that is more readily accessible by 
the subject) is permissible for all assessments that can be completed via this mode 
(eg, blood collection for laboratory assessments, ECG, physical examinations, 
imaging). 

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− Use of a laboratory or health-care provider not specified in the clinical study 
documentation requires coordination between the subject, the Investigator, and 
the subject’s local health-care provider. 
− The Investigator must document their review of the results provided by 
laboratories and health-care providers not specified in the clinical study 
documentation. 
• Home health study support 
− For assessments that cannot be completed via telemedicine, home health-care 
provider visits are permissible for all assessments that can be completed via this 
mode (eg, physical examination, collection of laboratory samples). 
− The Investigator must document their review of the results of home health-care 
provider visits. 
• Virtual informed consent/reconsent in lieu of in-person informed consent/reconsent 
− Reconsent (ie, consenting to an amended version of the protocol) may be 
completed virtually and documented in the relevant subject medical records. In 
these instances, reconsent may be completed virtually where allowed by the 
applicable regulations, and documented in the relevant subject medical records. 
− The other allowable modifications described in this section may require consent 
from the subject because their implementation requires a variation from the 
specifications in the protocol to which the subject has consented (eg, consent for a 
home visit, consent to provide name and address to a third-party delivery service, 
consent to a new mode of completing study procedures and receiving study drug). 
In these instances, consent may be completed virtually where allowed by the 
applicable regulations, and documented in the relevant subject medical records. 
11.2. 
Informed Consent Process 
The Investigator (or designee) will explain the nature of the study to subjects (or their legally 
authorized representative) and answer all questions regarding the study. Subjects will be 
informed that their participation is voluntary.  
Subjects (or their legally authorized representative) will be required to sign a statement of 
informed consent that meets the requirements of Title 21 Code of Federal Regulations Part 50, 
local regulations, ICH guidelines, Health Insurance Portability and Accountability Act of 1996 
requirements, where applicable, and the IRB/IEC. The authorized person obtaining the informed 
consent must also sign the ICF. A copy of the ICF will be provided to subjects (or their legally 
authorized representative). 
The medical record must include a statement that written informed consent was obtained before 
the subject was enrolled in the study and the date the written informed consent was obtained. 
During participation in the study, subjects (or their legally authorized representative) must 
provide written informed consent to the most current and IRB/IEC-approved version of the study 
ICF.  

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The ICF will contain a separate section that addresses the use of remaining mandatory samples 
for optional exploratory research. The Investigator (or designee) will explain to each subject the 
objectives of the exploratory research. Subjects will be told that they are free to refuse to 
participate and may withdraw their consent to allow any remaining samples to be used for 
optional exploratory research at any time and for any reason during the storage period. A 
separate signature will be required to document a subject’s agreement to allow any remaining 
specimens to be used for exploratory research. Subjects who decline to participate in this 
optional research will not provide this separate signature.  
11.3. 
Data Protection 
Subjects will be assigned a unique identifier by the Sponsor (or designee). Any subject records or 
datasets transferred to the Sponsor will be labeled with only this unique identifier; neither subject 
names nor other information that readily enables subject identification will be transferred. The 
key needed to connect the unique identifier with the subject’s name will be held by the Principal 
Investigator and approved research staff at the clinical study center.  
The subject will be informed that personal study-related data will be used by the Sponsor in 
accordance with applicable data protection laws. Where required by applicable data protection 
laws, the subject will be required to provide consent for their data to be used as described in the 
informed consent.   
The subject must be informed that his/her medical records and personal data collected in the 
study may be examined by Clinical Quality Assurance auditors or other authorized personnel 
appointed by the Sponsor, by appropriate IRB/IEC members, and by inspectors from regulatory 
authorities. The subject will also be informed that his/her information will be disclosed to certain 
other parties working for or with the Sponsor in conducting the study (eg, contract research 
organizations [CROs], central laboratories, and research collaborators).   
Should the Sponsor become aware of a serious data breach, the Sponsor will evaluate the breach 
to determine whether notification to regulatory authorities or study subjects is appropriate in 
accordance with applicable law. 
11.4. 
Dissemination of Clinical Study Data 
All information regarding study drug 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 part of the Investigator to provide the Sponsor 
with the complete data obtained during the study. Such information will be used in the clinical 
development of the study drug and may be disclosed to regulatory authorities, other 
Investigators, corporate partners, or consultants, as required. 

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11.5. 
Quality Compliance 
All subject data relating to the study will be recorded on a printed CRF or eCRF unless 
transmitted to the Sponsor (or designee) electronically (eg, clinical laboratory data). The 
Investigator is responsible for verifying that data entries are accurate and correct by physically or 
electronically signing the CRF.  
The Investigator must maintain accurate documentation (source data) supporting the information 
entered in the CRF. 
The Investigator must permit study-related monitoring, audits, IRB/IEC review, and regulatory 
agency inspections and provide direct access to source data documents. 
Monitoring details describing strategy (eg, risk management and mitigation strategies and 
analytical risk-based monitoring), methods, responsibilities and requirements, including handling 
of noncompliance issues and monitoring techniques (eg, central, remote, or on-site monitoring), 
are in the Monitoring Plan. 
The Sponsor (or designee) is responsible for the data management of this study including quality 
checking of the data.  
The Sponsor assumes accountability for actions delegated to other individuals (eg, CROs). 
Study monitors will perform ongoing source data verification to confirm that data entered into 
the CRF by authorized clinical study center personnel are accurate, complete, and verifiable from 
source documents; that the safety and rights of subjects are being protected; and that the study is 
being conducted in accordance with the currently approved protocol, applicable SOPs, work 
instructions, policies and any other study agreements, GCP (including but not limited to ICH, the 
European Medicines Agency, and the US FDA), and all applicable regulatory requirements.  
Essential documents pertaining to the conduct of this study, including signed ICFs, must be 
retained by the Investigator until at least 2 years after the last approval of a marketing application 
for the investigational product or until at least 2 years have elapsed since the formal 
discontinuation of the clinical development of the investigational product. These documents 
should be retained for a longer period if required by applicable regulatory requirements, by an 
agreement with the Sponsor, or by local regulations or institutional policies. No records may be 
destroyed during the retention period without the written approval of the Sponsor. No records 
may be transferred to another location or party without written notification to the Sponsor. 
Record destruction is to be documented, comply with applicable laws and regulations, and 
ensure subject confidentiality. 
11.6. 
Source Documents 
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 (or on the paper CRF, if applicable). 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 treatment, assessments, and study 

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procedures. Any outstanding entries must be completed after the final study visit. An explanation 
should be provided for all missing data. 
11.7. 
Study Termination and Clinical Study Center Closure 
Clinical study centers will be closed upon study completion. A study center is considered closed 
when all required documents and study supplies have been collected and a study center closure 
visit has been performed. 
The Sponsor reserves the right to terminate the study or close the clinical study center at any 
time for any reason at the sole discretion of the Sponsor.  
The Investigator may initiate study center closure at any time, provided there is reasonable cause 
and enough notice is provided to the Sponsor in advance of the intended termination. 
Reasons for study termination or the early closure of a clinical study center by the Sponsor or 
Investigator may include but are not limited to the following: 
• Failure of the Investigator to comply with the protocol, the requirements of the 
IRB/IEC or local health authorities, the Sponsor’s procedures, or GCP guidelines 
• Inadequate recruitment of subjects by the Investigator 
• Discontinuation of further study drug development 
11.8. 
Publication Policy 
The results of this study may be published or presented at scientific meetings. If this is foreseen, 
the Investigator agrees to submit all manuscripts or abstracts to the Sponsor before submission. 
This allows the Sponsor to protect proprietary information and to provide comments.  
The Sponsor will comply with the requirements for publication of study results. In accordance 
with standard editorial and ethical practice, the Sponsor will generally support publication of 
multicenter studies only in their entirety and not as individual clinical study center data. In this 
case, a coordinating Investigator will be designated by mutual agreement. 
Authorship will be determined by mutual agreement and in line with International Committee of 
Medical Journal Editors authorship requirements. 

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12. 
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Betts M, Flight PA, Paramore LC, Tian L, Milenkovic D, Sheth S. Systematic literature review 
of the burden of disease and treatment for transfusion-dependent beta-thalassemia. Clin Ther. 
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Borgna-Pignatti C, Rugolotto S, De Stefano P, et al. Survival and complications in patients with 
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Cappellini M, Viprakasit V, Taher A, et al. The Believe Trial: results of a phase 3, randomized, 
double-blind, placebo-controlled study of luspatercept in adult beta-thalassemia patients who 
require regular red blood cell (RBC) transfusions. Blood. 2018;132(suppl 1):163. 
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Cappellini MD, Cohen A, Perter J, Taher A, Viprakasit V. Guidelines for the Management of 
Transfusion Dependent Thalassemia (TDT). 3rd ed: Thalassemia International Federation; 2014. 
Cappellini MD, Robbiolo L, Bottasso BM, Coppola R, Fiorelli G, Mannucci AP. Venous 
thromboembolism and hypercoagulability in splenectomized patients with thalassaemia 
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Cappellini MD, Viprakasit V, Taher AT, et al. A phase 3 trial of luspatercept in patients with 
transfusion-dependent beta-thalassemia. N Engl J Med. 2020;382(13):1219-1231. 
doi:10.1056/NEJMoa1910182 
Chakraborty I, Mishra R, Gachhui R, Kar M. Distortion of beta-globin chain of hemoglobin 
alters the pathway of erythrocytic glucose metabolism through band 3 protein. Arch Med Res. 
2012;43(2):112-116. doi:10.1016/j.arcmed.2012.02.003 
EMA. Zynteglo (betibeglogene autotemcel) - Assessment report. European Medicines Agency; 
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Fung EB, Harmatz P, Milet M, et al. Morbidity and mortality in chronically transfused subjects 
with thalassemia and sickle cell disease: A report from the multi-center study of iron overload. 
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Galanello R. Recent advances in the molecular understanding of non-transfusion-dependent 
thalassemia. Blood Rev. 2012;26(suppl 1):S7-S11. doi:10.1016/S0268-960X(12)70004-8 
Galanello R, Cao A. Gene test review. Alpha-thalassemia. Genet Med. 2011;13(2):83-88. 
doi:10.1097/GIM.0b013e3181fcb468 
Grace R, Layton D, Galactéros F, et al. Long-term safety and efficacy of mitapivat (AG-348), a 
pyruvate kinase activator, in patients with pyruvate kinase deficiency: The DRIVE PK study. 
Blood. 2019;134(suppl 1):3512-3512. doi:10.1182/blood-2019-123406 
Grace RF, Rose C, Layton DM, et al. Safety and efficacy of mitapivat in pyruvate kinase 
deficiency. N Engl J Med. 2019;381(10):933-944. doi:10.1056/NEJMoa1902678 
 
 
 
 
 
 
Karimi M, Cohan N, De Sanctis V, Mallat NS, Taher A. Guidelines for diagnosis and 
management of Beta-thalassemia intermedia. Pediatr Hematol Oncol. 2014;31(7):583-596. 
doi:10.3109/08880018.2014.937884 
 
 
 
Kung C, Hixon J, Kosinski PA, et al. AG-348 enhances pyruvate kinase activity in red blood 
cells from patients with pyruvate kinase deficiency. Blood. 2017;130(11):1347-1356. 
doi:10.1182/blood-2016-11-753525 
Levey AS, Stevens LA, Schmid CH, et al. A new equation to estimate glomerular filtration rate. 
Ann Intern Med. 2009;150(9):604-612. doi:10.7326/0003-4819-150-9-200905050-00006 
Modell B, Khan M, Darlison M. Survival in beta-thalassaemia major in the UK: data from the 
UK Thalassaemia Register. Lancet. 2000;355(9220):2051-2052. doi:10.1016/s0140-
6736(00)02357-6 
Musallam KM, Cappellini MD, Viprakasit V, Kattamis A, Rivella S, Taher AT. Revisiting the 
non-transfusion-dependent (NTDT) vs. transfusion-dependent (TDT) thalassemia classification 
10 years later. Am J Hematol. 2020. doi:10.1002/ajh.26056 
Musallam KM, Rivella S, Vichinsky E, Rachmilewitz EA. Non-transfusion-dependent 
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Pyrukynd (mitapivat). USPI. Agios Pharmaceuticals; 2022. 
Reblozyl (luspatercept-aamt). USPI. Celgene; 2019. 
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Shaeffer JR. ATP-dependent proteolysis of hemoglobin alpha chains in beta-thalassemic 
hemolysates is ubiquitin-dependent. J Biol Chem. 1988;263(27):13663-13669.  
Taher A, Musallam K, Cappellini M. Guidelines for the Management of Non-Transfusion 
Dependent Thalassaemia (NTDT). 2nd ed: Thalassaemia International Federation; 2017. 
Taher AT, Musallam KM, Cappellini MD, Weatherall DJ. Optimal management of beta 
thalassaemia intermedia. Br J Haematol. 2011;152(5):512-523. doi:10.1111/j.1365-
2141.2010.08486.x 
Taher AT, Musallam KM, Saliba AN, Graziadei G, Cappellini MD. Hemoglobin level and 
morbidity in non-transfusion-dependent thalassemia. Blood Cells Mol Dis. 2015;55(2):108-109. 
doi:10.1016/j.bcmd.2015.05.011 
Taher AT, Radwan A, Viprakasit V. When to consider transfusion therapy for patients with non-
transfusion-dependent thalassaemia. Vox Sang. 2015;108(1):1-10. doi:10.1111/vox.12201 
Taher AT, Weatherall DJ, Cappellini MD. Thalassaemia. Lancet. 2018;391(10116):155-167. 
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Thein SL. The molecular basis of beta-thalassemia. Cold Spring Harb Perspect Med. 
2013;3(5):a011700. doi:10.1101/cshperspect.a011700 
Ting YL, Naccarato S, Qualtieri A, Chidichimo G, Brancati C. In vivo metabolic studies of 
glucose, ATP and 2,3-DPG in beta-thalassaemia intermedia, heterozygous beta-thalassaemic and 
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Vichinsky E. Advances in the treatment of alpha-thalassemia. Blood Rev. 2012;26 Suppl 1:S31-
34. doi:10.1016/S0268-960X(12)70010-3 
Vichinsky E. Non-transfusion-dependent thalassemia and thalassemia intermedia: epidemiology, 
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Weatherall DJ. The challenge of haemoglobinopathies in resource-poor countries. Br J 
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Zynteglo. Summary of Product Characteristics. bluebird bio (Netherlands) B.V.; 2019. 
 

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13. 
APPENDICES 

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APPENDIX 1. LIST OF ABBREVIATIONS 
Abbreviation 
Term 
2,3-DPG 
2,3-diphosphoglycerate 
Ab 
antibody 
AE 
adverse event 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
ATP 
adenosine triphosphate 
AUC 
area under the plasma concentration × time curve 
AUC0-∞ 
area under the plasma concentration × time curve from time 0 to infinity 
AUClast 
area under the plasma concentration × time curve from time 0 to the last 
measurable concentration 
AUC0-t 
area under the plasma concentration × time curve from time 0 to time t 
BID 
twice daily 
Cmax  
maximum (peak) concentration 
COVID-19 
coronavirus disease 2019 
CRO 
contract research organization 
CTCAE 
Common Terminology Criteria for Adverse Events 
CV% 
percent coefficient of variation 
CYP 
cytochrome P450 
DXA 
dual-energy x-ray absorptiometry 
ECG 
electrocardiogram 
eCRF 
electronic case report form 
EDC 
electronic data capture 
FDA 
Food and Drug Administration 
FSH 
follicle stimulating hormone 
GCP 
Good Clinical Practice 
Hb 
hemoglobin 
HbA 
adult hemoglobin 
HbS 
sickle cell hemoglobin 
HBsAg 
hepatitis B surface antigen 

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Abbreviation 
Term 
HCV 
hepatitis C virus 
HRT 
hormone replacement therapy 
HSCT 
hematopoietic stem cell transplantation  
IB 
Investigator’s Brochure 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
IDMC 
Independent Data Monitoring Committee 
IEC 
Independent Ethics Committee 
IRB 
Institutional Review Board 
IXRS 
interactive voice/web response system 
LC-MS/MS 
liquid chromatography-tandem mass spectrometry 
LLN 
lower limit of normal 
NCI 
National Cancer Institute 
NTDT 
non–transfusion-dependent thalassemia 
PEP 
phosphoenolpyruvate 
P-gp 
P-glycoprotein 
PKL 
liver-specific form of pyruvate kinase 
PKM 
pyruvate kinase muscle isozyme 
PKR 
red blood cell–specific form of pyruvate kinase 
pRBC 
packed red blood cell 
PRO 
patient-reported outcome 
QD 
once daily 
RBC 
red blood cell 
SAE 
serious adverse event 
SAP 
statistical analysis plan 
SD 
standard deviation 

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Abbreviation 
Term 
SOP 
standard operating procedure 
t1/2 
half-life 
TDT 
transfusion-dependent thalassemia 
tmax  
time to maximum (peak) concentration 
TRR 
transfusion reduction response 
UGT 
uridine 5ʹ-diphospho-glucuronosyltransferase 
ULN 
upper limit of normal 
US 
United States 
WOCBP 
women of childbearing potential 
 

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APPENDIX 2. DEFINITION OF WOMEN OF CHILDBEARING 
POTENTIAL AND CONTRACEPTION GUIDANCE 
Definition of Women of Childbearing Potential 
A woman is considered fertile after menarche and until becoming postmenopausal unless 
permanently sterile. If fertility is unclear (eg, amenorrhea in adolescents or athletes) and a 
menstrual cycle cannot be confirmed before administration of the first dose of study drug, 
additional evaluation should be considered. 
A woman is not considered a WOCBP if any of the following apply: 
1. Premenarchal 
2. Premenopausal female with 1 of the following: 
• Documented hysterectomy 
• Documented bilateral salpingectomy 
• Documented bilateral oophorectomy 
For individuals with permanent infertility due to an alternate medical cause other than the 
above (eg, Mullerian agenesis, androgen insensitivity), Investigator discretion should be 
applied to determining study eligibility. 
Documentation can come from the subject’s medical records, medical examination, or 
medical history interview. 
3. Postmenopausal female 
• A postmenopausal state is defined as no menses for 12 months without an alternative 
medical cause.  
− A high follicle stimulating hormone (FSH) level in the postmenopausal range may 
be used to confirm a postmenopausal state in women not using hormonal 
contraception or hormone replacement therapy (HRT); however, in the absence of 
12 months of amenorrhea, confirmation with >1 FSH measurement is required.  
• Females on HRT and whose menopausal status is in doubt will be required to use 1 of 
the nonestrogen hormonal highly effective contraception methods if they wish to 
continue their HRT during the study. Otherwise, they must discontinue HRT to allow 
confirmation of postmenopausal status before study enrollment. 
Acceptable Contraception Methods 
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. 
Hormonal contraception being used as a highly effective form of contraception must be 
accompanied by an acceptable barrier method. 

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An acceptable barrier method of contraception includes male or female condoms with or without 
spermicide, and cervical cap, diaphragm, or sponge with spermicide.  

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