Clinical trial NCT02476916
Conditions: Pyruvate Kinase Deficiency
Keywords: Pyruvate Kinase Deficiency Hemolytic anemia Pyruvate Kinase Isoform R (PKR) Drive PK
Sponsor: Agios Pharmaceuticals, Inc.
Start date: 2015-06-26; Completion date: 2025-04-02
Enrollment: 52 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Study AG348-C-003 is a multicenter study designed to evaluate the safety and efficacy of different dose levels of AG-348 (mitapivat) in participants with PK deficiency.

Detailed description:
This is a Phase 2, open label, two arm, multicenter, randomized, dose-ranging study during which adult participants with PK deficiency will receive multiple doses of AG-348 for up to 24 weeks (Core Period); eligible participants may enter an Extension Period to receive AG-348 for up to 8 additional years. Data will be reviewed on a regular basis and study design, dose and schedule will be adapted based on these reviews. The study will evaluate the safety and tolerability of multiple doses of AG-348, pharmacokinetic and pharmacodynamic (PD) profile of AG-348 and early indicators of clinical efficacy.

Arms: arm 1: Participants with PK deficiency received AG-348, 50 milligrams (mg), as initial dose, twice daily (BID) for the Core Period (Week 24). arm 2: Participants with PK deficiency received AG-348, 300 mg, as initial dose, BID for the Core Period (Week 24).
Interventions: intervention 1: AG-348
Intervention descriptions: intervention 1: Participants with PK deficiency were randomized to either receive AG-348, 50 or 300 mg, as initial doses, BID for the Core Period (Week 24). At the Week 24 visit, Core Period participants who had safely tolerated AG-348 and demonstrated clinical activity in response to AG-348 were potentially eligible to immediately roll over to the Extension Period for continued treatment. Participants were assigned to initial doses, however, over the course of the core period were treated across a range of doses due to treatment emergent adverse events (AEs) and hemoglobin (Hb) levels exceeding mid-point of sex-adjusted ranges.

Protocol documents:
Prot_000.pdf: 
 
Official Title: 
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, 
Pharmacokinetic and Pharmacodynamic Study of AG-348 in Adult Patients 
with Pyruvate Kinase Deficiency 
NCT Number: 
NCT02476916 
 
Document Dates: 
 
Protocol Version 6: 30 June 2017 
Protocol Version 5: 30 March 2016 
Protocol Version 4: 10 November 2015 
Protocol Version 3: 05 August 2015 
Protocol Version 2: 02 February 2015 
Protocol Version 1: 05 January 2015 
 
 
 
 
 
 
 
 


1. 
TITLE PAGE 
CLINICAL STUDY PROTOCOL AG348-C-003 
EudraCT No. 2015-000484-13 
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, 
Pharmacokinetic and Pharmacodynamic Study of AG-348 in Adult Patients 
with Pyruvate Kinase Deficiency 
Study Sponsor: 
Agios Pharmaceuticals, Inc. 
88 Sidney Street 
Cambridge, MA 02139-4169  
USA 
Phone: + 1-617-649-8600 
Fax: + 1-617-649-8618 
Sponsor Responsible Medical 
Officer: 
, MD, PhD 
 
 
Agios Pharmaceuticals, Inc. 
Phone: 
 
Email: 
 
Study Medical Monitor: 
, MD 
 
 
Agios Pharmaceuticals, Inc. 
Phone: 
 
Email: 
  
Current Protocol Version (Date): 
Amendment 5, Version 6.0 (30 June 2017), Final 
Previous Protocol Versions 
(Dates): 
Version 1.0 (05 January 2015), Final 
Amendment 1, Version 2.0 (02 February 2015), Final 
Amendment 2, Version 3.0 (05 August 2015), Final 
Amendment 3, Version 4.0 (10 November 2015), Final 
Amendment 4, Version 5.0 (30 March 2016), Final 
This study will be conducted according to the protocol and in compliance with Good Clinical Practice,  
the ethical principles stated in the Declaration of Helsinki, and other applicable regulatory requirements. 
CONFIDENTIALITY NOTE: 
The information contained in this document is confidential and proprietary to Agios Pharmaceuticals, Inc.  
Any distribution, copying, or disclosure is strictly prohibited unless such disclosure is required by federal 
regulations or state law. Persons to whom the information is disclosed must know that it is confidential and that it 
may not be further disclosed by them. 




Clinical Study Protocol AG348-C-003 
Agios Pharmaceuticals, Inc. 
Amendment #5, Version 6.0 
30 June 2017 
Page 3  
INVESTIGATOR’S AGREEMENT 
 
I understand that all documentation provided to me by Agios Pharmaceuticals, Inc. (Agios) 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, the AG-348 
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). No changes will be made to the study protocol without the 
prior written approval of Agios and the IRB, except where necessary to eliminate an immediate 
hazard to the subject. 
I have read, understood, and agree to conduct this study as outlined in the protocol and in 
accordance with the guidelines and all applicable government regulations. 
 
___________________________________  
 
 
 
 
 
 
Investigator Name (Printed)  
 
 
 
 
 
 
 
 
 
 
___________________________________  
 
 
 
 
 
________________________ 
Investigator Signature  
 
 
 
 
 
 
 
 
 
 
 
Date (DD MMM YYYY) 
 
___________________________________________________________________ 
___________________________________________________________________ 
___________________________________________________________________ 
___________________________________________________________________ 
Investigational site or name of institution and location (printed) 


Clinical Study Protocol AG348-C-003 
Agios Pharmaceuticals, Inc. 
Amendment #5, Version 6.0 
30 June 2017 
Page 4  
2. 
SYNOPSIS 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Title of Study:  
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, Pharmacokinetic and 
Pharmacodynamic Study of AG-348 in Adult Patients with Pyruvate Kinase Deficiency 
Study Center(s): 
This study will be conducted at multiple study centers. 
Phase of Development: 2 
Objectives: 
Core Period 
Primary Objective: 
• 
Evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in subjects 
with pyruvate kinase deficiency (PK deficiency). 
Secondary Objectives: 
• 
Evaluate the pharmacokinetics (PK) of AG-348 and the metabolite AGI-8702. 
• 
Evaluate the pharmacodynamic (PD) response of adenosine triphosphate (ATP) and 
2,3-diphosphoglycerate (2,3-DPG) after administration of AG-348. 
• 
Evaluate indicators of clinical activity of AG-348 in subjects with PK deficiency, including 
changes in hemoglobin (Hb), hematocrit (HCT), reticulocyte count, haptoglobin (Hp), 
carboxyhemoglobin (COHb), lactate dehydrogenase (LDH), total and indirect bilirubin, 
erythropoietin (EPO), hepcidin, ferritin, and transferrin saturation (serum iron/ 
iron-binding capacity). 
Extension Period 
Primary Objective: 
• 
Evaluate the safety and tolerability of up to 30 months of AG-348 administration in subjects 
with PK deficiency. 
Secondary Objectives: 
• 
Evaluate the PK of AG-348 and the metabolite AGI-8702. 
• 
Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348. 
• 
Evaluate indicators of clinical activity of AG-348 in subjects with PK deficiency, including 
changes in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, 
hepcidin, ferritin, and transferrin saturation (serum iron/iron-binding capacity). 
• 
Evaluate the optimal maintenance dose of AG-348 for each individual subject during the 
Extension Period. 


Clinical Study Protocol AG348-C-003 
Agios Pharmaceuticals, Inc. 
Amendment #5, Version 6.0 
30 June 2017 
Page 5  
Methodology: 
Study AG348-C-003 is a Phase 2, open label, 2-arm, multicenter, randomized, dose-ranging study in 
adult subjects with PK deficiency; the study will be divided in to a Core Period and an Extension 
Period. During the Core Period, subjects will receive multiple doses of AG-348 for up to 24 weeks; 
subjects who are eligible can enter the Extension Period to receive AG-348 for up to 2 years following 
the end of the Core Period.  
Subjects with PK deficiency confirmed by red blood cell (RBC) PK enzymatic assay performed at 
Screening will be eligible to participate in this study. At the Week 24 visit, subjects who safely 
tolerate AG-348 and demonstrate clinical activity of AG-348 may be eligible to immediately roll over 
to the Extension Period for continued treatment. Subjects who complete treatment at the end of the 
Core Period (24 weeks) will undergo follow-up assessment 4 weeks after the last dose of study drug. 
If a subject discontinues at any other time (including discontinuation during the Core or Extension 
Period), the follow-up assessments will be conducted 4 weeks after discontinuation. Subjects with 
toxicity suspected to be related to study drug will continue follow-up until the adverse event (AE) 
resolves, is declared chronic by the Investigator, or the subject is lost to follow-up.  
For the Core Period, up to 25 subjects will be initially randomized on an open-label 1:1 basis to each 
of two twice-daily (BID) doses of AG-348 (up to 50 subjects total; refer to the Study Schema  
diagram below).  
Study Schema:  Core Period 
 
Abbreviations:  BID (q12h) = twice daily (every 12 hours); DRT = Data Review Team; PKR = pyruvate kinase red blood 
cell isoform; TBD = to be determined; w = week(s). 
The dose of Arm 1 is 300 mg of AG-348 administered orally (PO) every 12 hours (q12h), ie, BID. 
The dose of Arm 2 is 50 mg of AG-348 administered PO BID.  
Randomization will be stratified by PKR mutation in order to maintain balance as much as possible 
across the dose arms for the specific mutations expected to be most frequently enrolled. The PKR 


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mutation stratification factor will consist of 4 levels (R510Q, R486W, R479H, and all other mutations 
as “other”). Mutation status is defined by the presence of at least 1 of the indicated mutations; subjects 
with more than 1 stratified mutation will be assigned based on the Sponsor’s discretion.  
The doses for each arm of the Core Period have been selected from the AG348-C-001 single 
ascending dose (SAD) and AG348-C-002 multiple ascending dose (MAD) studies in healthy adult 
subjects to represent the range of doses/exposures that were safely tolerated and resulted in maximal 
or near-maximal PD effects on 2,3-DPG and ATP.  
Because PK deficiency is a rare disease with a limited eligible subject population and because the 
underlying pathophysiology and clinical phenotype of affected subjects is heterogeneous due to the 
wide variety of mutations in PKR that cause the disease, it is deemed important to focus closely on 
dose findings in this first-in-patient study. Therefore, in addition to initiating this study with 2 
different doses of AG-348 administered BID, a Data Review Team (DRT) will be established to 
review study data on a frequent basis and adapt the study design, dose, and schedule of AG-348  
as indicated.  
The DRT will monitor safety on an ongoing basis and meet at regular intervals of approximately 
every 6 weeks, or ad hoc, as necessary, for as long as any subjects are still in the Core Period to 
review AEs, vital signs (VS), clinical laboratory assessments (hematology, clinical chemistry, 
coagulation, and urinalysis), and electrocardiograms (ECGs). The DRT will also review available 
PK/PD data and indicators of clinical activity (eg, changes from baseline in Hb).  
These DRT meetings will also include data review for all subjects that may be under treatment in the 
Extension Period. If there are no subjects still being treated in the Core Period, and the only subjects 
on treatment are those in the Extension Period, then the frequency of the DRT meetings will reduce to 
approximately every 4 to 6 months in order to match the frequency of subject visits (and new data 
collection) in the Extension Period.  
When all the subjects are in the Extension Period, PK/PD data will no longer be reviewed by the 
DRT. The DRT will be comprised of the study Coordinating Investigator, treating Investigators, 
Medical Monitor, Clinical Pharmacologist, Statistician, and Sponsor’s Responsible Medical Officer.  
Beginning 6 weeks after the first subject is dosed in the Core Period or ad hoc as necessary, and 
proceeding according to the schedule indicated above (approximately every 6 weeks during the Core 
Period, and approximately every 4 to 6 months during the Extension Period once all subjects have 
completed the Core Period), the DRT will review cumulative safety data, available PK/PD data, and 
clinical activity data.  
Based on the DRT’s recurring reviews, the DRT may exercise 1 or more of the following options 
during the Core Period: 
• 
Continue treatment and enrollment in existing arms without change. 
• 
Add 1 new dose arm (Arm 3) to enroll up to 25 subjects at a dose to be determined; the dose 
for Arm 3 may be lower or higher than Arm 1 and Arm 2 doses, but will not exceed 360 mg 
BID; and the dose regimen may be less frequent than BID. 
• 
Terminate or suspend enrollment to allow further review of clinical data in Arm 1 and/or Arm 
2 (and/or potential Arm 3). Enrollment in an arm could be terminated or suspended to allow 
further review, for example, for unacceptable safety/tolerability, poor PD response, or lack of 
signs of clinical activity. 
• 
Re-assign subjects’ doses and schedule in a terminated arm to match the dose and schedule of 
another arm of the study. In this case, the subjects in the terminated arm will remain in their 
original arm, ie, they will not count towards the enrollment quota of the arm whose dose and 
schedule is being adopted. 


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Amendment #5, Version 6.0 
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• 
Implement specific genotype restrictions to enrollment in 1 or more arms to ensure 
representation of subjects with genotypes of greatest clinical relevance. 
 
The DRT may exercise 1 or more of the following options during the Extension Period: 
• 
Continue treatment without change. 
• 
Re-assign subjects’ doses and schedule to an existing dose and schedule that has been 
determined to be safer, and/or have a better PD response, or produce signs of clinical activity. 
• 
Terminate or suspend treatment to allow further review of clinical data (eg, for unacceptable 
safety/tolerability, poor PD response, or lack of signs of clinical activity). 
 
The data that the DRT will review to make these decisions are expected to include, but are not 
necessarily limited to, the following:  
• 
Safety Observations: All AEs, VS, clinical laboratory assessments (hematology, clinical 
chemistry, coagulation, and urinalysis), and ECGs. 
• 
PK and PD Observations: Includes changes in 2,3-DPG and ATP, except when all subjects 
are in the Extension Period 
• 
Indicators of Clinical Activity: Includes changes in Hb, HCT, reticulocyte count, Hp, COHb, 
LDH, EPO, hepcidin, total and indirect bilirubin, ferritin, and transferrin saturation. 
If a third dose arm is implemented, the dose of AG-348 selected will not exceed 360 mg BID, as this 
was the highest dose that demonstrated acceptable safety and tolerance in the 14-day multiple BID 
dosing study in healthy adult subjects.  
The PK/PD sampling schedule in a potential third dosing arm will be determined at the Sponsor’s 
discretion and may follow either the extensive or limited PK/PD sampling schedules as specified. 
The DRT will monitor the data in an ongoing manner as described and may make a decision to 
terminate enrollment in an arm based on evaluation of safety and efficacy.  
All subjects in the Extension Period will undergo a gradual dose-taper regimen to identify the optimal 
maintenance dose for each (defined as the dose that results in ≤ 1.0 g/dL decrease in Hb compared to 
the pretaper Hb value on at least 2 measurements following each dose taper step) on a per-subject 
basis (see Section 9.7.3).  
Due to the potential for AG-348-mediated aromatase inhibition, combined with the known risk of 
osteoporosis in subjects with congenital hemolytic anemias, dual-energy x-ray absorptiometry (DXA) 
scan (hip and spine) will be performed at Screening (if subject has not had prior DXA scan within  
3 months of Day 1) to obtain bone mineral density values and T and Z scores. These data are intended 
to serve as a baseline measure of bone mineral density for all enrolling subjects, and are deemed of 
particular importance for those who enter the longer term Extension Period after completing 24 weeks 
of treatment (Core Period).  
All subjects will have a second DXA scan in the interval between Weeks 24 and 28 for the Core 
Period. Subjects in the Extension Period will have additional DXA scans at Months 12, 18, 24,  
and 30. 
As the number of enrolling arms changes in the study (for example, from 2 to 3), the randomization 
scheme will adjust to enable balanced randomization into each actively accruing arm. Randomization 
and stratification will cease in the event that only a single arm is left enrolling. 
Depending on possible early termination of 1 or both of the initial 2 arms, or the addition of a third 
arm, the study could enroll up to a maximum of 75 subjects. 
An extension study (to be determined) may be offered to subjects who successfully complete the 
Extension Period. 


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Amendment #5, Version 6.0 
30 June 2017 
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Visit Schedule 
Screening assessments will occur within 42 days prior to the first dose of study treatment. During the 
Core Period, subjects will attend visits at baseline (Day 1), weekly through Week 3 (Days 8, 15, and 
22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12), and monthly through Week 
24 (Weeks 16, 20, and 24).  
Subjects who safely tolerate AG-348 through Week 24 (Core Period) and for whom the Investigator 
agrees with continuation of AG-348 treatment may be eligible to immediately enter the Extension 
Period for continued treatment for up to 2 years after completion of the Core Period, upon agreement 
of the treating Investigator and the Medical Monitor or Responsible Medical Officer. 
Study visits for safety and clinical activity assessments will occur approximately every 3 months 
during the Extension Period, except during the dose taper part of the Extension Period when weekly 
visits will be performed. All subjects will undergo a follow-up assessment 4 weeks after the last dose 
of AG-348, regardless of whether this was due to discontinuation, the last dose in the Core Period for 
a subject who chooses not to continue in the Extension Period, or the last dose of the Extension Period 
(except for those who may continue treatment in an extension study [to be designed] after having 
completed the Extension Period).  
Dose Modifications for Safety and/or Increase in Hb Level 
The Investigator will monitor all subjects for safety and tolerability. Modification of an individual 
subject’s dose of AG-348 will be based on AEs and/or observed changes in Hb level as described in 
Section 9.7.1 and Section 9.7.2.  
Additional criteria for dose reduction will apply in the Extension Period, as detailed in Section 9.7.3. 
Number of subjects (planned): Up to approximately 75 subjects. 
Diagnosis and main criteria for inclusion: 
Inclusion Criteria 
Core Period: 
Subjects must meet all of the following criteria to be eligible for inclusion in the Core Period of the 
study: 
1. Have provided signed written informed consent prior to undergoing any study procedure, 
including Screening procedures. 
2. Be male or female, aged 18 years and older. 
3. Have a known medical history of PK deficiency. 
4. Have documented clinical laboratory confirmation of PK deficiency by RBC pyruvate kinase 
enzymatic assay performed at Screening, either by a designated central laboratory or by any 
participating investigative site’s local hematology laboratory. Subjects with prior 
documentation of PK deficiency by RBC enzymatic assay must have reconfirmation of this 
result during Screening as a condition of enrollment. 
NOTES: 
i. 
In the event that a subject’s Screening pyruvate kinase enzymatic assay is negative 
(ie, shows normal pyruvate kinase activity), the subject will be eligible for enrollment 
if the genotyping shows a mutant genotype that has been previously documented in 
the literature to be associated with PK deficiency. 
ii. 
If the genotyping shows a previously undescribed mutation in the PKR gene, the 
subject’s eligibility for enrollment will be determined on an individual case basis by 
the Coordinating Investigator and Medical Monitor in discussion with the 
Investigator. 
iii. 
If no mutation is defined, the subject will not be eligible for enrollment. 
5. Have a blood sample for genotypic characterization of the mutant PKR gene performed by the 
designated central laboratory at Screening.  


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Amendment #5, Version 6.0 
30 June 2017 
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NOTES:  
i. 
The designated central laboratory-determined genotype will generally serve as the 
basis for genotyping for enrollment. However, subjects whose genotype has already 
been determined by another laboratory may be enrolled on the basis of that report, 
with the approval of the Medical Monitor, in the case of an unexpected delay in 
return of the designated central laboratory result during the Screening Period.  
ii. 
Enrollment on the basis of a result from a laboratory other than the designated central 
genotyping laboratory does not relieve the inclusion requirement that ALL subjects 
must have a sample sent to the designated central genotyping laboratory. 
6. Have Hb ≤ 12.0 g/dL (if male) or ≤ 11.0 g/dL (if female). 
7. Be considered transfusion-independent, defined as having had ≤ 3 units of RBCs transfused in 
the 12-month period up to the first day of study drug dosing and no transfusions within 
4 months of the first day of study dosing.  
NOTE: Subjects who have received more transfusion support than described above will be 
evaluated for eligibility on a case-by-case basis by the Medical Monitor. 
8. Have their spleen in place or have undergone splenectomy. Splenectomized subjects must 
meet all of the following conditions: 
i. 
Have undergone the procedure ≥ 6 months prior to Screening. 
ii. 
Be current in their vaccinations for pneumococcal conjugate (PCV13), pneumococcal 
polysaccharide (PPSV23), quadrivalent meningococcal vaccine, and Haemophilus 
influenzae Type B, as recommended by the United States Centers for Disease Control 
and Prevention's Advisory Committee on Immunization Practices (refer to 
http://www.cdc.gov/vaccines/schedules/downloads/adult/adult-schedule.pdf) or, for 
subjects in Canada or the European Union, by immunization advisory groups in those 
locations. 
NOTE: Any missing vaccinations may be administered, starting with the Screening 
Period and continuing throughout the trial, following the initiation of AG-348 dosing 
and as necessary according to recommended vaccination guidance. 
9. Have Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2. 
10. Have been taking ≥ 1 mg of folic acid daily for ≥ 21 days prior to the first dose of study drug 
and agree to continue this regimen during the study. 
11. Have adequate organ function, defined as meeting all of the following conditions: 
i. 
Serum aspartate aminotransferase (AST) ≤ 2.5 × upper limit of normal (ULN), unless 
the increased AST is assessed by the Investigator as due to hemolysis and/or hepatic 
iron deposition, and alanine aminotransferase (ALT) ≤ 2.5 × ULN, unless the 
increased ALT is assessed by the Investigator as due to hepatic iron deposition. 
ii. 
Either normal or elevated levels of serum bilirubin. In subjects with serum bilirubin 
> ULN, the elevation must be attributed to hemolysis with or without Gilbert's 
syndrome and must not be attributed to choledocholithiasis, cholecystitis, biliary 
obstruction, or hepatocellular disease. 
iii. 
Serum creatinine ≤ 1.25 × ULN or, if > 1.25 × ULN, then 24-hour measured or 
calculated (by Cockcroft-Gault) glomerular filtration rate ≥ 60 mL/min. 
iv. 
Absolute neutrophil count (ANC) ≥ 1.0 × 109/L. 
v. 
Platelet count ≥ 100 × 109/L. 
vi. 
Activated partial thromboplastin time (aPTT) and international normalized ratio 
(INR) ≤ 1.25 × ULN, unless the subject is receiving therapeutic anticoagulants. 
12. For women of childbearing potential—defined as females who either have experienced 
menarche and have not undergone successful surgical sterilization (hysterectomy, bilateral 
tubal ligation, and/or bilateral oophorectomy) or who are not postmenopausal (defined as 
either having amenorrhea for ≥ 12 consecutive months without another cause and documented 
serum follicle-stimulating hormone [FSH] level > 35 mIU/mL or being ≥ 62 years of age and 


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having amenorrhea for ≥ 12 consecutive months [no FSH testing required]): 
i. 
Agree to abstain from sexual intercourse or to use an acceptable/effective method of 
contraception (ie, condom plus spermicide, condom plus oral contraceptive, condom 
plus intrauterine device, condom plus diaphragm with spermicide) from as soon as is 
feasible during the Screening period until 30 days following the last dose of AG-348.  
NOTE: Abstinence is an acceptable method only when this is in line with the normal 
lifestyle of the subject, meaning that the subject plans to remain abstinent 
continuously throughout the duration of the study and for ≥ 30 days after the last dose 
of study drug. Periodic abstinence (eg, calendar, symptothermal, and postovulation 
methods) and withdrawal are not acceptable methods of contraception. 
ii. 
Have negative serum or urine pregnancy test within 72 hours before start of AG-348 
dosing. 
iii. 
Not be breastfeeding. 
13. If male (with the exception of subjects who have undergone vasectomy ≥ 6 months prior to 
Screening), agree to abstain from sexual intercourse or, if sexually active, to use a condom 
with spermicide as contraception (regardless of their female partner's childbearing potential  
or their partner's use of their own contraception) from Day 1 of dosing until 30 days following 
the last dose of AG-348.  
NOTE: Abstinence is an acceptable method only when this is in line with the normal life style 
of the subject, meaning that the subject plans to remain abstinent continuously throughout the 
duration of the study and for at least 30 days after the last dose of study drug. Periodic 
abstinence (eg, selective timing of intercourse based on partner’s calendar, symptothermal, 
and postovulation methods) and withdrawal are not acceptable methods of contraception. 
Extension Period 
Subjects must meet all of the following criteria to be eligible for inclusion in the Extension Period of 
the study: 
1. Have provided signed written informed consent prior to undergoing any study procedure 
during the Extension Period. 
2. Have completed 24 weeks of treatment during the Core Period and tolerated AG-348 (defined 
as having completed 24 weeks with or without protocol-permitted dose modifications). 
3. The treating Investigator agrees that there is a potential for clinical benefit to the subject from 
continued treatment and recommends participation in the Extension Period. 
4. The Sponsor's designated Medical Monitor or Responsible Medical Officer approves the 
subject’s participation in the Extension Period. 
5. If applicable, agree to continue to follow the same sexual abstinence/contraception rules as 
stated above for the Core Period (Inclusion Criterion 12 [for females] or 13 [for males]). 
Exclusion criteria: 
Core Period 
Subjects who meet any of the following criteria will be excluded from the Core Period of the study: 
1. Have Hb level > 12.0 g/dL (if male) or > 11.0 g/dL (if female). 
2. Have an additional diagnosis of any other congenital or acquired blood disorder, including 
glucose-6-phosphate-dehydrogenase (G6PD) deficiency, or any other hemolytic anemia 
process, with the exception of mild allo-immunization as a consequence of transfusion 
therapy. 
3. Have iron overload (hemosiderosis or concurrent hemochromatosis) sufficiently severe to 
result in a clinical diagnosis by the Investigator of cardiac, hepatic, or pancreatic 
insufficiency. 
4. Have had prior bone marrow or stem cell transplant. 
5. Have clinically symptomatic cholelithiasis or cholecystitis.  
 


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NOTES:  
i. 
Prior cholecystectomy is not exclusionary.  
ii. 
Subjects with symptomatic cholelithiasis or cholecystitis may be re-screened once the 
disorder has been treated and clinical symptoms have resolved. 
6. Be currently enrolled in another therapeutic clinical trial involving ongoing therapy with any 
investigational or marketed product or placebo. 
NOTE: Concurrent participation in the Pyruvate Kinase Deficiency Natural History Study 
(NCT02053480) is permitted. 
7. Have been exposed to any investigational drug, device, or procedure within 28 days prior to 
Screening or during trial participation     
8. Have any concurrent medical condition that could compromise participation in the study, such 
as: 
i. 
Poorly controlled hypertension (defined as systolic blood pressure [BP] > 150 mm Hg 
or diastolic BP > 90 mm Hg) refractory to medical management. 
ii. 
History of recent (within < 6 months from Screening date) congestive heart failure; 
myocardial infarction or unstable angina pectoris; hemorrhagic, embolic, or 
thrombotic stroke; deep venous thrombosis; or pulmonary or arterial embolism. 
iii. 
Currently active infection requiring the use of parenteral antimicrobial agents or of  
≥ Grade 3 severity (per National Cancer Institute Common Terminology Criteria for 
Adverse Events [CTCAE] v4.03) within 6 months of first dose of study drug. 
iv. 
Pattern or frequency of postsplenectomy sepsis that, in the assessment of the 
Investigator, could reasonably be expected to interfere with the ability of the subject 
to complete participation in the 24-week Core Period of the study. 
v. 
Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) 
antibody with accompanying signs of active hepatitis B or C infection. 
vi. 
Positive test for human immunodeficiency virus (HIV) 1 or 2 antibody. 
vii. 
Diabetes mellitus that is judged to be in poor control by the Investigator or which 
requires > 3 anti-diabetic agents, counting insulin (all insulins are considered 
1 agent). 
NOTE: Use of insulin per se is not exclusionary. 
viii. 
History of any primary malignancy, with the exception of: curatively treated  
nonmelanomatous skin cancer, curatively treated cervical or breast carcinoma in situ, 
or any other primary tumor treated with curative intent, and with no known active 
disease present and no anticancer treatment administered during the last 3 years. 
9. Have undergone major surgery within 6 months of first dose of study drug. 
10. Have currently or have a recent history of a psychiatric disorder that, in the opinion of the 
Investigator or Medical Monitor, could compromise the ability of the subject to cooperate 
with study visits and procedures. 
11. Have used any of the restricted list of products known to strongly inhibit cytochrome P450 
(CYP) 3A4 drug metabolism (Appendix 4.1) within 5 days prior to Day 1 dosing; products 
known to strongly induce CYP3A4 metabolism (Appendix 4.2) within 28 days prior to Day 1 
dosing; products known to strongly inhibit P-glycoprotein (P-gp) transporter (Appendix 4.3) 
within 5 days prior to Day 1 dosing; or digoxin within 5 days prior to Day 1 dosing.  
12. Have serum bilirubin > ULN attributable to factors other than hemolysis and/or Gilbert's 
syndrome. 
13. Have heart-rate corrected QT interval by Fridericia's method (QTcF) > 450 msec (for males) 
or > 470 msec (for females), with the exception of subjects with a left bundle branch block, 
for whom Medical Monitor approval is needed to enroll. 
14. Have cardiac dysrhythmia that is judged clinically significant by the Investigator or which 
requires therapy with drugs that are primarily substrates of CYP3A4. 
 


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15. Have any history of allergy to sulfonamides characterized by acute hemolytic anemia, 
anaphylaxis, or rash of erythema multiforme type or Stevens-Johnson syndrome. 
16. Have any other medical or psychological condition regarded by the Investigator as likely to 
interfere with subject's ability to understand and provide signed written informed consent; 
cooperate with study visits, tests, and procedures; and/or otherwise safely and reliably 
participate in the study. 
Extension Period: 
Subjects who meet this criterion will be excluded from the Extension Period of the study:  
1. Have experienced any AE during the Core Period considered by the treating Investigator or 
the Sponsor’s designated Medical Monitor or Responsible Medical Officer to pose a 
significant safety risk should study treatment be extended. 
Investigational product, dosage, and mode of administration: 
AG-348 sulfate hydrate capsules will be provided as 5 mg, 25 mg, or 100 mg (free-base equivalent) of 
AG-348 sulfate hydrate without excipients in hard gelatin capsules.  
AG-348 will be administered PO BID. The number of capsules per dose will vary by assigned  
dose group.  
AG-348 will be administered with water and may be administered with or without food. 
Reference therapy, dosage and mode of administration:  
Not applicable. 
Duration of treatment:  
The duration of treatment for all subjects in the Core Period will be up to 24 weeks. Subjects who 
safely tolerate AG-348 and for whom the Investigator agrees with continuation of AG-348 treatment 
may be eligible to immediately roll over to the Extension Period for continued treatment (up to 
2 years following completion of the Core Period). 
Criteria for evaluation: 
Safety 
Monitoring of AEs in randomized subjects, including serious AEs (SAEs), AEs of special interest 
(AESIs), and AEs leading to discontinuation (all AEs will be graded using CTCAE, Version 4.03); 
safety laboratory parameters (hematology, chemistry, urinalysis, coagulation); physical examination 
findings (including neurological examination); VS; 12-lead ECGs; and DXA scans.  
Serum sex hormone levels (testosterone [total and free], estrone, and estradiol), bone turnover 
markers (serum osteocalcin-N-mid and serum C-terminal telopeptide [CTX]), 25-hydroxy vitamin D2 
and D3, total cholesterol, high-density lipoprotein-cholesterol (HDL-C), and triglycerides will be 
monitored for evidence of potential inhibition of aromatase by AG-348.  
Menstruating female subjects will also keep a paper-based menstrual cycle diary throughout the study. 
Indicators of Clinical Activity 
Monitoring of potential indicators of clinical activity will include evaluating changes in Hb, HCT, 
reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, hepcidin, ferritin, and 
transferrin saturation.  
Pharmacokinetics 
Approximately the first 10 subjects treated in the Core Period, contingent on clinical site feasibility, 
will undergo extensive PK sampling as detailed in Appendix 1.3. The remainder of treated subjects 
will undergo limited PK sampling as detailed in Appendix 1.4. 
During the Core Period, serial blood sampling for determination of concentration-time profiles of 
AG-348 and its metabolite AGI-8702 will be conducted following the first dose and the morning 
Day 15 dose, and additional trough levels of AG-348 and AGI-8702 will be obtained.  


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During the Extension Period, predose PK samples will be drawn for the measurement of trough levels 
of AG-348 and AGI-8702 at each study visit t (every 3 months; Appendix 1.2). AG-348 and  
AGI-8702 will be analyzed using qualified assays to determine concentrations in plasma.  
Pharmacodynamics 
Pharmacodynamic assessments during the Core Period will include 2,3-DPG, ATP (secondary 
objectives), 
 
  
 
Approximately the first 10 subjects treated during the Core 
Period will undergo extensive PD sampling as detailed in Appendix 1.3. The remainder of treated 
subjects will undergo limited PD sampling as detailed in Appendix 1.4. During the Core Period, serial 
blood sampling for determination of levels of ATP and, 2,3-DPG will be conducted following the first 
dose and the morning Day 15 dose, and additional trough levels of ATP and 2,3-DPG will be 
obtained.  
During the Extension Period, predose PD samples will be drawn for the measurement of trough levels 
of 2,3-DPG, ATP,
 at each study visit (every 3 months; Appendix 1.2). Adenosine 
triphosphate and 2,3 DPG will be analyzed using qualified assays to determine concentrations in 
whole blood. Pharmacodynamic parameters on Day 1 and Day 15 will be computed based on 
observed whole blood ATP and 2,3-DPG concentrations.  
Statistical methods: 
Sample Size 
Due to the rare disease setting, the minimal sample size may be determined by feasibility. Depending 
on possible early termination of 1 or both of the initial 2 arms or the addition of a 3rd dose arm, the 
study could enroll up to a maximum of 75 subjects. 
Analysis Periods 
Analyses of safety and of indicators of clinical activity will be separated for the Core Period, and for 
the Cumulative Period (Core Period and Extension Period), if applicable. Unless specified otherwise, 
safety analysis will be based on the treatment-emergent period defined as from the first dose to  
30 days after the last dose of the corresponding period. Efficacy analysis will be based on the efficacy 
window defined as from the first dose to 1 day after the last dose of the corresponding period. 
Analysis Sets 
Populations for analysis (ie, analysis sets) will include a Safety Analysis Set and an Efficacy Analysis 
Set. The Safety Analysis Set will include all subjects who receive at least 1 dose of study treatment; 
this population will be the primary set for the analysis of safety data and the default analysis set for all 
data analyses unless otherwise stated. Subjects will be classified according to initial treatment group, 
defined as the assigned treatment if it is received at least once, or as the first treatment received if 
assigned treatment is never received.  


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The Efficacy Analysis Set will include all subjects who are enrolled and received any study treatment 
for at least 3 weeks. The Efficacy Analysis Set will be the primary set for the analysis of preliminary 
clinical activity data. Subjects will be classified according to assigned treatment, unless  
specified otherwise.  
Statistical Analysis 
This section presents a summary of the planned statistical analyses of efficacy and safety for  
this study.  
Statistical analysis and presentation details will be provided in the Statistical Analysis Plan (SAP) for 
the study, which will be finalized before the database lock after all subjects have completed the Core 
Period. The results of this analysis will be presented in a clinical study report (CSR).  
Additional data collected during the Extension Period after the Core Period database lock will be 
analyzed for inclusion in a subsequent CSR addendum. 
The primary objective during the Core Period of this Phase 2 study is to evaluate the safety and 
tolerability of up to 24 weeks of AG-348 in subjects with PK deficiency. Therefore, analyses will be 
primarily descriptive in nature; no formal hypothesis testing will be conducted.  
Summaries will be produced for disposition, baseline disease characteristics and demographic data.  
Categorical variables will be summarized by frequency distributions (number and percentages) and 
continuous variables will be summarized by descriptive statistics (mean, standard deviation [SD], 
median, minimum, and maximum).  
All AEs will be coded using the Medical Dictionary for Regulatory Activities (MedDRA), and the 
incidence of AEs in the treatment-emergent period (TEAEs) (new or worsening from baseline) will be 
summarized by primary MedDRA System Organ Class (SOC) and Preferred Term, severity, outcome, 
action taken with study drug, and relationship to study drug.  
Separate summaries will be produced for all TEAEs, treatment-related AEs (ie, those considered by 
the Investigator as at least possibly study drug-related), SAEs, AESIs, AEs leading to treatment 
discontinuation, and AEs ≥ Grade 3 severity. Individual subject listings will be provided for any 
deaths, SAEs, and TEAEs leading to treatment modification, interruption, or discontinuation.  
Descriptive statistics will be provided for clinical laboratory values (eg, hematology, serum chemistry, 
coagulation studies, urinalysis) and VS, presented as both actual values and changes from baseline 
relative to each on-study evaluation. Shift analyses will be conducted for selected laboratory 
parameters based on the baseline CTCAE grade to maximum CTCAE grade. Where applicable 
CTCAE terms do not exist, a grading system based on the upper and/or lower limits of normal will be 
used to classify the results. 
Electrocardiogram analyses will include individual subject listings and summaries of abnormal and 
clinically significant ECG results. Actual values and changes from baseline in the portion of the ECG 
wave from the beginning of the P wave to the beginning of the QRS complex (PR), QRS, heart 
rate-corrected QT intervals (QTc) will be summarized by visit.  
Data collected from the menstrual diaries such as the start and stop dates of the menses and the 
subject reported characteristics of the menses will be presented in a by-subject listing. Additional 
descriptions of the data may also be performed. 
Concomitant medications will be coded using the World Health Organization Drug Dictionary. 
Concomitant medications prior to and after the start of the study drug will be listed by subject, and 
summarized by summarized by Anatomical Therapeutic Chemical Classification System term and 
dose arm. 


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Hormone data, including the actual values and their changes from baseline at each visit will be 
summarized by sex using descriptive statistics (mean, SD, median, min and max). Spaghetti plots will 
be provided by sex.  
Details of analyses to evaluate indicators of potential clinical activity of AG-348 in subjects with PK 
deficiency will be described in the SAP. These will include changes in Hb, HCT, reticulocyte count, 
Hp, COHb, LDH, total and indirect bilirubin, EPO, hepcidin, ferritin, and transferrin saturation 
(serum iron/iron-binding capacity). 
Analysis Related to Dose-Tapering Regimen in the Extension Period 
The number and percentage of subjects at the individual optimal maintenance dose will be 
summarized. If necessary, additional analyses related to the dose-tapering regimen in the Extension 
Period, focusing on the long-term treatment effect in the Cumulative Period, may be conducted. These 
analyses will be described in a separate analysis plan for the Extension Period following the Core 
Period database lock, but before the database lock for the Extension Period.  
PK-PD Analysis 
Descriptive statistics will be used to summarize PK parameters for the parent compound AG-348 and 
the metabolite AGI-8702 for each dose group, and where appropriate, for the entire population. 
Pharmacokinetic parameters will be summarized using the following descriptive statistics: n, mean, 
SD, coefficient of variation %, median, minimum, and maximum, geometric mean, and geometric 
coefficient of variation.  
Descriptive statistics will be used to summarize PD parameters for 2,3-DPG and ATP for each dose 
group, and where appropriate for the entire population. Pharmacodynamic parameters will be 
summarized using the following descriptive statistics: n, mean, SD, coefficient of variation %, 
median, minimum, and maximum, geometric mean, and geometric coefficient of variation %. 
Additional analyses, if conducted, may be described in a separate analysis plan. 
Interim Review 
No formal statistical interim analysis will be conducted. 
Safety data will be reviewed on an ongoing basis by the DRT, who will meet to review safety, PK, 
PD, and preliminary clinical activity data at regular intervals (approximately every 6 weeks during the 
Core Period and approximately every 4 to 6 months during the Extension Period once all subjects 
have completed the Core Period) throughout the duration of the study. When all the subjects are in the 
Extension Period, PK/PD data will no longer be reviewed by the DRT. The composition of the DRT, 
its meeting schedule, the data to be reviewed, and the decisions it is empowered to make have been 
described previously. 
Additional interim reviews of data may be conducted to support decision-making concerning the 
current clinical study, the Sponsor’s development programs in general, or in case of any safety 
concerns.  


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3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF 
FIGURES 
TABLE OF CONTENTS 
1. 
TITLE PAGE ................................................................................................................1 
2. 
SYNOPSIS ...................................................................................................................4 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES ...............16 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS.............................23 
5. 
INTRODUCTION ......................................................................................................26 
5.1. 
Pyruvate Kinase Deficiency .......................................................................................26 
5.2. 
AG-348 .......................................................................................................................27 
 
Summary of Nonclinical Data ....................................................................................27 
5.2.1.
5.2.1.1. 
Pharmacology .............................................................................................................28 
5.2.1.2. 
Safety Pharmacology ..................................................................................................28 
5.2.1.3. 
Pharmacokinetics ........................................................................................................28 
5.2.1.4. 
Toxicology ..................................................................................................................29 
 
Summary of Clinical Data ..........................................................................................30 
5.2.2.
5.2.2.1. 
Pharmacokinetics ........................................................................................................31 
5.2.2.2. 
Pharmacodynamics .....................................................................................................31 
5.2.2.3. 
Safety ..........................................................................................................................32 
Phase 1 Studies AG348-C-001 and AG348-C-002 in Healthy Adult Subjects ..............................32 
Phase 2 Study in Adult Subjects with PK Deficiency .....................................................................33 
 
Summary of Overall Safety Management Plan ..........................................................34 
5.2.3.
 
Risks Associated with AG-348 ...................................................................................35 
5.2.4.
5.2.4.1. 
Risk of Withdrawal Hemolysis ...................................................................................35 
5.2.4.2. 
Risk of Insomnia .........................................................................................................35 
5.2.4.3. 
Risk of Bone Mineral Density Decrease ....................................................................35 
5.2.4.4. 
Potential Risk of Transaminase Increases ..................................................................35 
5.2.4.5. 
Potential Risk of Triglyceride Increases .....................................................................35 
5.2.4.6. 
Potential Risk of Photosensitivity ...............................................................................36 
 
Other Restrictions and Precautions .............................................................................36 
5.2.5.
6. 
STUDY OBJECTIVES AND ENDPOINTS ..............................................................37 
6.1. 
Core Period .................................................................................................................37 


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Primary Objective .......................................................................................................37 
6.1.1.
 
Secondary Objectives .................................................................................................37 
6.1.2.
 
37 
6.2. 
Extension Period .........................................................................................................37 
 
Primary Objective .......................................................................................................37 
6.2.1.
 
Secondary Objectives .................................................................................................37 
6.2.2.
 
38 
6.3. 
Study Measures and Endpoints ...................................................................................38 
 
Safety Measures and Endpoints ..................................................................................38 
6.3.1.
 
Clinical Activity Measures and Endpoints .................................................................38 
6.3.2.
 
Pharmacokinetic and Pharmacodynamic Measures and Endpoints ............................38 
6.3.3.
 
39 
7. 
INVESTIGATIONAL PLAN .....................................................................................40 
7.1. 
Overall Study Design ..................................................................................................40 
7.2. 
Rationale for the Study Design ...................................................................................44 
7.3. 
Rationales Related to Dosing......................................................................................45 
 
Rationale for the Starting Dose...................................................................................45 
7.3.1.
 
Rationale for the Dose Range .....................................................................................45 
7.3.2.
 
Rationale for the Duration of Dosing .........................................................................45 
7.3.3.
 
Rationale for Identification of Individual Optimal Maintenance Dose and 
7.3.4.
Gradual Dose-Taper Regimen ....................................................................................46 
7.4. 
Criteria for Study Termination ...................................................................................47 
8. 
STUDY POPULATION .............................................................................................48 
8.1. 
Number of Subjects ....................................................................................................48 
8.2. 
Inclusion Criteria ........................................................................................................48 
 
Core Period .................................................................................................................48 
8.2.1.
 
Extension Period .........................................................................................................51 
8.2.2.
8.3. 
Exclusion Criteria .......................................................................................................51 
 
Core Period .................................................................................................................51 
8.3.1.
 
Extension Period .........................................................................................................53 
8.3.2.
8.4. 
Subject Identification and Registration ......................................................................53 
8.5. 
Subject Randomization ...............................................................................................53 
8.6. 
Subject Withdrawal Criteria .......................................................................................53 


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8.7. 
Replacement of Subjects .............................................................................................54 
9. 
STUDY TREATMENT ..............................................................................................55 
9.1. 
Description of Study Drug ..........................................................................................55 
9.2. 
Study Drug Packaging and Labeling ..........................................................................55 
9.3. 
Study Drug Storage .....................................................................................................55 
9.4. 
Method of Assigning Subjects to Treatment ..............................................................55 
9.5. 
Blinding ......................................................................................................................55 
9.6. 
Study Drug Preparation and Administration ..............................................................55 
9.7. 
Criteria for Dose Escalation, Dose Modification, or Discontinuation of Study 
Drug ............................................................................................................................56 
 
Dose Modification for Safety .....................................................................................57 
9.7.1.
 
Dose Modification for Increase in Hemoglobin Level ...............................................59 
9.7.2.
 
Individual Optimal Maintenance Dose-Finding in Extension Period .........................60 
9.7.3.
9.7.3.1. 
Gradual Dose-taper regimen in Extension Period ......................................................60 
 
Stopping Criteria .........................................................................................................62 
9.7.4.
9.8. 
Duration of Subject Participation ...............................................................................62 
9.9. 
Treatment Compliance ................................................................................................62 
9.10. 
Study Drug Accountability .........................................................................................62 
9.11. 
Prior and Concomitant Medications and Treatments .................................................63 
 
Prior Medications and Procedures ..............................................................................63 
9.11.1.
 
Concomitant Therapy .................................................................................................63 
9.11.2.
9.11.2.1. 
Concomitant Therapy to be Avoided (Prohibited) .....................................................63 
9.11.2.2. 
Concomitant Therapy Requiring Careful Monitoring (Use with Caution) ................64 
9.11.2.3. 
Allowed Concomitant Therapy ...................................................................................64 
9.12. 
Management of Nausea, Vomiting, and Diarrhea ......................................................65 
10. 
STUDY ASSESSMENTS ..........................................................................................66 
10.1. 
Schedules of Assessments ..........................................................................................66 
10.2. 
Informed Consent and Confirmation of Eligibility ....................................................67 
10.3. 
Demographic Data, Medical and Medication History ................................................67 
10.4. 
PKR Enzymatic Assay and PKR Genotyping ............................................................68 
10.5. 
Safety Assessments .....................................................................................................68 
 
Physical Examination, Height, and Weight ................................................................68 
10.5.1.
 
Vital Signs ..................................................................................................................68 
10.5.2.


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Electrocardiogram .......................................................................................................68 
10.5.3.
 
DXA Scans .................................................................................................................69 
10.5.4.
 
Safety Laboratory Assessments ..................................................................................69 
10.5.5.
10.5.5.1. 
Hematology, Serum Chemistry, Coagulation Studies, and Urinalysis .......................69 
10.5.5.2. 
Screening Serology .....................................................................................................71 
 
Menstrual Cycle Diary ................................................................................................71 
10.5.6.
 
Adverse Events ...........................................................................................................71 
10.5.7.
10.6. 
Pharmacokinetic Assessments ....................................................................................71 
10.7. 
Pharmacodynamic Assessments .................................................................................72 
10.8. 
Ordering of Blood Sample Collection ........................................................................73 
10.9. 
Sample Processing, Storage, and Shipment ................................................................73 
11. 
ADVERSE EVENTS..................................................................................................74 
11.1. 
Definition of Adverse Events .....................................................................................74 
 
Adverse Event .............................................................................................................74 
11.1.1.
 
Suspected Adverse Reaction .......................................................................................74 
11.1.2.
 
Unexpected Adverse Event .........................................................................................74 
11.1.3.
 
Serious Adverse Event ................................................................................................74 
11.1.4.
 
Adverse Events of Special Interest .............................................................................75 
11.1.5.
11.1.5.1. 
Transaminase Increase ................................................................................................75 
11.2. 
Procedures for Reporting Adverse Events, Adverse Events of Special 
Interest, and Serious Adverse Events .........................................................................75 
11.3. 
Pregnancy Reporting ..................................................................................................77 
12. 
STATISTICAL METHODS .......................................................................................79 
12.1. 
Sample Size and Power ..............................................................................................79 
12.2. 
Analysis Sets ...............................................................................................................80 
12.3. 
Analysis Periods .........................................................................................................80 
12.4. 
Statistical Analysis ......................................................................................................80 
 
General Methods .........................................................................................................80 
12.4.1.
 
Disposition ..................................................................................................................81 
12.4.2.
 
Exposure and Safety Analyses ....................................................................................81 
12.4.3.
 
Sex Hormone Analysis ...............................................................................................82 
12.4.4.
 
Clinical Activity Analyses ..........................................................................................82 
12.4.5.
 
Analysis Specific to the Extension Period ..................................................................82 
12.4.6.


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12.5. 
PK/PD Analysis ..........................................................................................................82 
 
Pharmacokinetic Analysis ..........................................................................................82 
12.5.1.
 
Pharmacodynamic Analysis........................................................................................82 
12.5.2.
 
82 
12.7. 
Interim Analysis ..........................................................................................................82 
13. 
ADMINSTRATIVE REQUIREMENTS ....................................................................84 
13.1. 
Good Clinical Practice ................................................................................................84 
13.2. 
Ethical Considerations ................................................................................................84 
13.3. 
Subject Information and Informed Consent ...............................................................84 
13.4. 
Subject Confidentiality ...............................................................................................84 
13.5. 
Protocol Compliance ..................................................................................................85 
13.6. 
Data Management .......................................................................................................85 
13.7. 
Source Document/Case Report Form Completion .....................................................86 
13.8. 
Direct Access to Source Data .....................................................................................86 
13.9. 
Record Retention ........................................................................................................86 
13.10. 
Liability and Insurance ...............................................................................................86 
13.11. 
Publication of Study Findings and Use of Information ..............................................87 
14. 
LIST OF REFERENCES ............................................................................................88 
15. 
APPENDICES ............................................................................................................90 
APPENDIX 1:  SCHEDULES OF ASSESSMENTS .................................................................91 
Appendix 1.1.  Schedule of Assessments: Core Period ..............................................................91 
Appendix 1.2.  Schedule of Assessments: Extension Period ......................................................96 
Appendix 1.3. 
 Schedule of Assessments: Extensive PK/PD Sampling during the Core 
Period ..................................................................................................................99 
Appendix 1.4.  Schedule of Assessments: Limited PK/PD Sampling during the Core 
Period ................................................................................................................100 
APPENDIX 2:  EASTERN COOPERATIVE ONCOLOGY GROUP PERFORMANCE 
STATUS SCORING .........................................................................................101 
APPENDIX 3: NATIONAL CANCER INSTITUTE COMMON TERMINOLOGY 
CRITERIA FOR ADVERSE EVENTS............................................................102 
APPENDIX 4: CONCOMITANT THERAPY TO BE AVOIDED (PROHIBITED) OR 
REQUIRING CAREFUL MONITORING (USE WITH CAUTION) .............103 
Appendix 4.1.  Strong and Moderate CYP3A4 Inhibitors ........................................................103 
Appendix 4.2.  Strong CYP3A4 Inducers (Prohibited) .............................................................104 


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Appendix 4.3.  Strong P-glycoprotein Inhibitors (Prohibited) ..................................................105 
Appendix 4.4.  Sensitive CYP3A4 Substrates (Substitute or Use with Caution) .....................106 
Appendix 4.5.  Sensitive CYP2B6 Substrates* (Substitute or Use with Caution)....................107 
Appendix 4.6.  Proton-Pump Inhibitors and H2-Receptor Antagonists         (Substitute 
or Use with Caution) .................................................................................................108 
APPENDIX 5:  DECLARATION OF HELSINKI ...................................................................109 
 
 
 


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LIST OF TABLES 
 
Table 1: 
Dose Modification for Adverse Events Deemed at Least Possibly Related  to 
AG-348 .......................................................................................................................58 
Table 2: 
Dose Reduction Table (by Dosing Arm) ....................................................................59 
Table 3: 
Gradual Dose-Taper Regimen (by Actual Dose) ........................................................60 
Table 4:  
Schedule of Assessments for Gradual Dose-Taper Regimen in the Extension 
Period1 .........................................................................................................................61 
Table 5: 
Summary of Assessments when Day 8 and/or Day 22 In-Clinic Visits are 
Performed by Primary Care Physician (Core Period) .................................................67 
Table 6: 
Probability of Observing at Least 1 Specific AE Given Different Underlying 
AE Incidence Rates .....................................................................................................79 
 
LIST OF FIGURES 
 
Figure 1: 
Red Cell Glycolysis ....................................................................................................26 
Figure 2: 
Study Schema:  Core Period .......................................................................................40 
Figure 3: 
PKR Enzymatic Reaction ...........................................................................................72 
 


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4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation or Specialist Term 
Definition 
2,3-DPG 
2,3-diphosphoglycerate 
ADP 
Adenosine diphosphate 
AE 
Adverse event 
AESI 
Adverse event of special interest 
ALC 
Absolute lymphocyte count 
ALT 
Alanine aminotransferase 
ANC 
Absolute neutrophil count 
aPTT 
Activated partial thromboplastin time 
AST 
Aspartate aminotransferase 
ATP 
Adenosine triphosphate 
AUC 
Area under the curve 
AUC0-12hr 
Area under the plasma concentration versus time curve from 0 to  
12 hours 
AUC0 24hr 
Area under the plasma concentration versus time curve from 0 to 
24 hours 
AUC0-∞ 
Area under the plasma concentration versus time curve from 0  
to infinity 
BCRP 
Breast cancer resistance protein 
BID 
Twice daily 
BP 
Blood pressure 
BUN 
Blood urea nitrogen 
CBC 
Complete blood count 
CO2 
Carbon dioxide 
COHb 
Carboxyhemoglobin 
CSR 
Clinical study report 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTX 
Serum C-terminal telopeptide 
CV 
Cardiovascular 
DDI 
Drug-drug interaction 
CYP 
Cytochrome P450 
DLT 
Dose-limiting toxicity 
DRT 
Data review team 


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Abbreviation or Specialist Term 
Definition 
DXA 
Dual-energy x-ray absorptiometry 
ECG 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic case report form 
EDC 
Electronic data capture 
EPO 
Erythropoietin 
FSH 
Follicle-stimulating hormone 
G6PD 
Glucose-6-phosphate-dehydrogenase 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practice 
Hb 
Hemoglobin 
HBsAg 
Hepatitis B surface antigen 
HCT 
Hematocrit 
HCV 
Hepatitis C virus 
HDL-C 
High-density lipoprotein-C 
HIV 
Human immunodeficiency virus 
Hp 
Haptoglobin 
IC50 
Concentration of drug that achieved half-maximal inhibition 
ICH 
International Conference on Harmonisation 
IEC 
Independent Ethics Committee 
INR 
International normalized ratio 
IRB 
Institutional Review Board 
LDH 
Lactate dehydrogenase 
MAD 
Multiple ascending dose 
MCH 
Mean corpuscular hemoglobin 
MCHC 
Mean corpuscular hemoglobin concentration 
MCV 
Mean corpuscular volume 
mPKR 
Pyruvate kinase isoform R mutants 
NCI 
National Cancer Institute 
NOAEL 
No-observed-adverse-effect-level 
P-gp 
P-glycoprotein 
PCV13 
Pneumococcal conjugate 


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Abbreviation or Specialist Term 
Definition 
PD 
Pharmacodynamic 
PEP 
Phosphoenolpyruvate 
PK 
Pharmacokinetic 
PK deficiency 
Pyruvate kinase deficiency 
PKM2 
Pyruvate kinase isoform M2 
PKR 
Pyruvate kinase isoform R 
PO 
Oral(ly) 
PPSV23 
Pneumococcal polysaccharide 
PR 
The portion of the ECG wave from the beginning of the P wave to 
the beginning of the QRS complex 
q12h 
Every 12 hours 
QD 
Once daily 
QTc 
Heart-rate corrected QT interval 
QTcB 
Corrected QT interval – Bazett’s correction formula 
QTcF 
Corrected QT interval – Fridericia’s method 
RBC 
Red blood cell (count) 
RDW 
Red cell distribution width 
SAD 
Single ascending dose 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SD 
Standard deviation 
t½ 
Apparent terminal half-life 
TIBC 
Total iron-binding capacity 
tmax 
Time to maximum plasma concentration 
TEAE 
Treatment-emergent adverse event 
ULN 
Upper limit of normal 
VS 
Vital signs 
WBC 
White blood cell  
WMA 
World Medical Association 
WT 
Wild-type 


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5. 
INTRODUCTION 
5.1. 
Pyruvate Kinase Deficiency 
Pyruvate kinase deficiency (PK deficiency) is a glycolytic enzymopathy that results in  
nonspherocytic hemolytic anemia with a variable clinical presentation, ranging from mild or 
fully compensated forms to life-threatening neonatal anemia and lifelong chronic hemolytic 
anemia associated with severe, debilitating comorbidities. Pyruvate kinase deficiency is caused 
by mutations in the PKLR gene, which results in defective pyruvate kinase isoform R (PKR) in 
the red blood cell (RBC).  
As shown in Figure 1 below, PKR catalyzes the final and irreversible step in glycolysis, 
converting phosphoenolpyruvate (PEP) to pyruvate, and formation of adenosine triphosphate 
(ATP) from adenosine diphosphate (ADP). Mature RBCs rely almost exclusively on the process 
of glycolysis to generate the energy carrier molecule ATP. PKR is thus a key enzyme for 
maintaining energy homeostasis in erythrocytes, and it has been proposed that ATP levels are 
critical for optimally maintaining RBC membrane integrity (van Wijk and van Solinge 2005). 
Figure 1: 
Red Cell Glycolysis 
 
Abbreviations:  1,3 DPG = 1,3 diphosphoglycerate; 2,3 DPG = 2,3 diphosphoglycerate; 3-PG = 3-phosphoglycerate;  
ADP = adenosine diphosphate; ATP = adenosine triphosphate; FBP = fructose 1,6-bisphosphate; PEP = phosphoenolpyruvate;  
PKR = red blood cell-specific form of pyruvate kinase. 
Note: Not all steps in glycolysis are shown. 
The majority of mutations in PKR that have been described have a deleterious effect on PKR 
catalytic activity, protein stability, and/or protein expression. Pyruvate kinase deficiency is 
associated with reduced RBC survival as well as impaired red cell maturation. Mature 
erythrocytes lack mitochondria relying predominantly on glycolysis to generate ATP. It is 
hypothesized that insufficient energy production promotes erythrocyte hemolysis due to impaired 
maintenance of cellular membrane homeostasis. 
Pyruvate kinase deficiency is an autosomal recessive disease, in which both homozygotes and 
compound heterozygotes develop hemolytic anemia. More than 200 different mutations in the 
PKLR gene have been identified in subjects with PK deficiency to date, and the majority of these 
mutations are single nucleotide missense mutations (Zanella, et al. 2005). The prevalence of PK 
deficiency, based on the most common pyruvate kinase mutation in Southern Europe, is 
estimated at 51 cases per million in the Caucasian population (Beutler and Gelbart 2000). 
Some subjects with PK deficiency have a clinically severe disease course that can present as a 
severe hemolytic anemia in early infancy, requiring immediate care which typically includes 
blood transfusion support (Christensen, et al. 2011; Ghidini, et al. 1991; Hennekam, et al. 1990). 
Infants with PK deficiency may have erythroblastosis fetalis with hepatosplenomegaly, 
extramedullary hematopoiesis, and severe indirect hyperbilirubinemia, which is associated with a 
high risk of kernicterus.  
Glucose
Pyruvate
PKR
`
ATP
FBP
1,3-DPG
2,3-DPG
3-PG
PEP
ADP


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Hyperbilirubinemia associated with the hemolytic state often requires supportive treatment with 
intensive phototherapy. For other infants, the hyperbilirubinemia caused by hemolysis can 
require exchange transfusion, as well as supportive transfusion to keep their hematocrit (HCT) 
levels > 60%. 
Adults with PK deficiency are reported to have life-long hemolysis and the subsequent 
associated comorbidities including anemia and transfusion dependence. Other comorbidities 
include frequent miscarriages, aplastic crises, as well as symptoms associated with an acute or 
chronic hemolytic anemia (Rider, et al. 2011). Since unconjugated bilirubin is often chronically 
elevated, pigmented gallstones are common in children and adults. Severe and sometimes life-
threatening iron deposition occurs and is typically progressive. 
The current therapeutic options for subjects with PK deficiency are supportive. Most require life-
long treatment, including blood transfusions at a frequency depending on the disease state. Long-
term surveillance for systemic iron overload, even in transfusion-independent subjects, is 
standard as is the use of chelation therapy. Case reports of cure by allogeneic bone marrow 
transplant have been published (Tanphaichitr, et al. 2000) but have been infrequently performed. 
Splenectomy, while not curative, can reduce the need for supportive blood transfusions and 
decrease bilirubin levels in some subjects. However, this intervention is associated with 
significant morbidity including infection risk, and possible increased risk of thrombosis. 
5.2. 
AG-348 
AG-348 is an orally available, allosteric activator of PKR. It is hypothesized that drug 
intervention with AG-348 restores glycolytic pathway activity and normalizes red cell 
metabolism in vivo. Biochemical experiments demonstrate that AG-348 is a potent, broad-
spectrum activator of many PKR alleles associated with PK deficiency. Pyruvate kinase- 
deficient red cells and their progenitors are characterized by changes in metabolism associated 
with defective glycolysis, including a build-up of PEP and 2,3-diphosphoglycerate (2,3-DPG), 
and lowered ATP levels. Ex vivo treatment of PK deficiency subject red cells with AG-348 
results in increased ATP levels, and reductions in PEP and 2,3-DPG, consistent with 
pharmacological activation of PKR enzyme activity.  
In vitro studies against wild-type (WT) PKR enzymes and ex vivo studies in blood from mice, 
monkeys and humans indicate that AG-348 activates WT PKR with a similar potency and 
efficacy as it does with the mutant versions. In vivo studies in mice confirm the potency of  
AG-348 in increasing WT PKR enzyme activity and modulating the levels of downstream 
markers such as ATP and 2,3-DPG. This therapeutic approach may be an effective way to 
correct the underlying pathology of PK deficiency and, importantly, provide clinical benefit to 
subjects. 
 Summary of Nonclinical Data 
5.2.1.
A series of exploratory pharmacology studies were conducted to characterize the ability of 
AG-348 to activate WT PKR and anemia-associated PKR mutants (mPKR) in vitro, ex vivo, and 
in vivo. Additional details regarding nonclinical data can be found in the AG-348 Investigator’s 
Brochure. 
 


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5.2.1.1. 
Pharmacology 
AG-348 is a potent, broad-spectrum activator of PKR with low nM potency against both WT and 
mutant enzymes. AG-348 is also able to effectively stabilize mPKR enzymes that are 
hypersensitive to thermal denaturation, suggesting that AG-348 may be able to increase PKR 
pathway activity by preventing destabilization and degradation of mPKR enzymes. Its major 
metabolite, AGI-8702, has low potency mixed activity against PKR and mutant isoforms and can 
increase the thermostability of some mPKR, albeit with significantly weaker potency than that of 
AG-348. 
The effect of AG-348 and AGI-8702 on PKR activity and a number of downstream pathway 
markers was evaluated in human RBCs. AG-348 dose-response curves in human whole blood 
showed potent activation of PKR, while AGI-8702 is a weak activator of the PKR enzyme. In 
whole blood from mice treated with AG-348, 13C-glucose labeling through the PKR reaction was 
increased by 80% demonstrating AG-348-mediated activation of the WT PKR enzyme. AG-348 
treatment of RBCs from healthy human donors potently increased levels of the PKR reaction 
product ATP consistent with activation of PKR.  
The effects of AG-348 on PKR activity and RBC metabolism also were assessed in blood 
samples from subjects with PK deficiency. AG-348 activated PKR and induced metabolic 
changes (increased ATP levels and decreased 2,3-DPG levels) consistent with increased 
glycolytic pathway activity in RBCs from PK deficiency subjects with different mutations in the 
PKR enzyme.  
Assessments of binding inhibition potential and enzymatic activity were evaluated in a panel of 
89 receptors, ion channels, and enzymes. Although AG-348 is a histamine H3 antagonist/inverse 
agonist and an aromatase inhibitor, no effects of histamine H3 modulation have been observed in 
safety pharmacology or toxicology studies. Effects consistent with aromatase inhibition were 
seen in toxicology studies (Section 5.2.1.4). 
5.2.1.2. 
Safety Pharmacology 
Safety pharmacology studies demonstrated that neither AG-348 nor AGI-8702 exhibited 
significant inhibition of the human ether-à-go-go related gene (hERG) current (concentration of 
drug that achieved half-maximal inhibition [IC50] 29.4 μM and > 10 μM, respectively). 
Additionally, in the monkey cardiovascular (CV) study, no CV effects were seen at any dose 
level. No effects were seen in AG-348 Good Laboratory Practice (GLP)-compliant Irwin and 
respiratory safety pharmacology studies in Sprague Dawley rats. AG-348 administered via oral 
gavage had a dose-dependent emetic activity in the ferret at ≥ 30 mg/kg. 
5.2.1.3. 
Pharmacokinetics 
Absorption, distribution, metabolism, and excretion studies of AG-348 were performed in 
Sprague Dawley rats, beagle dogs, and cynomolgus monkeys. Both oral (PO) and intravenous 
(IV) routes were evaluated at doses ranging from 1 to 50 mg/kg. A dose of 200 mg (50 mg/kg) 
was administered PO in the food-effect evaluation in monkeys. The PK of AG-348 in animal 
species is characterized by rapid oral absorption, medium to high total body plasma clearance, 
and high volume of distribution at steady-state in rats, dogs, and monkeys. 


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AG-348 has a medium to long apparent terminal half-life (t1/2) in the rat, dog, and monkey. 
Medium to high oral bioavailability was observed for AG-348 in animals. A high-fat diet led to 
lower oral absorption of AG-348 in monkeys. 
AG-348 showed low brain penetration in rats (brain/plasma ratio of 5.7% to 9.1%) based on area 
under the plasma concentration versus time curve from 0 to 24 hours (AUC0-24hr) following 
single and repeated-dose administration, respectively. Metabolism appears to be the major 
elimination pathway for AG-348 with minimal biliary or urinary excretion.  
Both in vitro and in vivo, the predominant metabolite was the N-dealkylated product, AGI-8702. 
There were no metabolites unique to human liver microsomes and no direct Phase 2 metabolites. 
The plasma metabolite profile of AG-348 did not change with repeat or ascending dosing, 
suggesting that AG-348 is not susceptible to metabolic switching with increasing or prolonged 
dose administration. 
AG-348 is a weak direct inhibitor of cytochrome P450 (CYP) 2C8, CYP2C9, CYP2C19, 
CYP2D6, and CYP3A4/5 (testosterone 6β-hydroxylation). There was no direct inhibition of 
CYP1A2, CYP2B6, or CYP3A4/5 (midazolam 1′-hydroxylation) by AG-348. There was 
evidence of metabolism-dependent inhibition of CYP2C19 (largely reversible) and CYP3A4 
(largely irreversible) by AG-348. AG-348 is considered a weak time-dependent CYP3A4 
inhibitor. AGI-8702 is a weak direct inhibitor of CYP2C9 and CYP2C19. AG-348 has the 
potential to cause an induction-related drug-drug interaction (DDI) with sensitive CYP2B6 and 
CYP3A4 substrates. 
The routes of metabolism for AG-348 are via multiple CYPs with CYP3A4 contributing > 70% 
of the total. CYP1A2, CYP2C9, and CYP2C8 contribute approximately 6%, 10%, and 7% to the 
remaining metabolism of AG-348; other isoforms contribute < 4% each. 
AG-348 is a substrate for P-glycoprotein (P-gp), but not breast cancer resistance protein (BCRP). 
AG-348 is an inhibitor of P-gp (91% and 99% inhibition at 41 and 411 µM, respectively), but 
does not inhibit BCRP. AGI-8702 is not an inhibitor of P-gp or BCRP under the concentrations 
tested (5 and 100 μM). 
5.2.1.4. 
Toxicology 
The toxicity profile of AG-348 was evaluated in bacterial reverse mutation assays, the in vitro 
human peripheral blood micronucleus assay, the in vivo micronucleus assay, and in vivo in 
Sprague Dawley rats, New Zealand White rabbits, Beagle dogs, and cynomolgus monkeys. The 
toxicity of the major metabolite of AG-348, AGI-8702, was also evaluated through metabolic 
formation both in vitro in S9 fractions and in vivo. 
AG-348 was nonmutagenic and nonclastogenic in GLP-compliant assays including bacterial 
reverse mutation assays, an in vitro micronucleus assay in human peripheral blood lymphocytes, 
and an in vivo micronucleus assay in male and female Sprague Dawley rats.  
In dogs, following single oral dose administration, clinical observations consistent with 
anaphylactoid reactions were seen, and the maximum tolerated dose was 62.5 mg/kg, which was 
associated with an AUC0-12hr value of 8576 ng × h/mL. In monkeys, single oral doses of AG-348 
up to 1000 mg/kg did not result in adverse findings, and were associated with an AUC0-12hr value 
of 105,000 ng × h/mL.  


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In the GLP-compliant 28-day monkey study, the high dose of 150 mg/kg/day (75 mg/kg/dose) 
was the no-observed-adverse-effect-level (NOAEL). Effects were limited to increased liver 
weights without serum chemistry or microscopic correlate. 
In the 13-week repeat-dose study in monkeys, no adverse effects were identified at doses up to 
200 mg/kg/day (100 mg/kg/dose; Day 90 AUC0-12hr values of 10,400 and 14,600 ng × h/mL in 
males and females, respectively), and no new effects were identified when compared to the 
28-day repeat-dose study. In the 9-month repeat-dose monkey toxicology study, a previously 
unidentified adverse finding of minimal subcapsular inflammation/hepatocellular pressure 
necrosis resulting from nonadverse adaptive hepatocellular hypertrophy secondary to CYP 
enzyme induction was observed in the liver of males at 100 and 200 mg/kg/day (50 and 
100 mg/kg/dose) and in females at 200 mg/kg/day (100 mg/kg/dose). No other adverse findings 
were observed.  
In rats (Sprague Dawley), single oral doses of AG-348 up to 2000 mg/kg did not result in 
adverse findings, and were associated with AUC0-12hr values up to 789,327 ng × h/mL. In the 
GLP-compliant 28-day rat study, the NOAEL in males was the highest dose tested, 
600 mg/kg/day (300 mg/kg/dose), and in females was the lowest dose tested, 20 mg/kg/day 
(10 mg/kg/dose). 
In the 13-week repeat-dose rat study, adverse effects in males were identified in the testes and 
consisted of seminiferous tubular degeneration, spermatid retention, and Leydig cell 
hypertrophy. These effects were defined as adverse at dose levels at which they are expected to 
impair fertility. In the 13-week repeat-dose rat study, adverse effects in females were identified 
in the uterus and consisted of uterine atrophy and increased folding of the luminal surface. 
Similar to males, these effects were defined as adverse at the dose level at which they are 
expected to impair fertility. In the 6-month repeat-dose rat study, the highest dose tested was 
300 mg/kg/day (150 mg/kg/dose) in males and 200 mg/kg/day (100 mg/kg/dose) in females, 
which resulted in no test article-related deaths. 
AG-348 inhibits human aromatase activity with an IC50 of 2050 nM (based on human placental 
microsomes) and rat aromatase with an IC50 of 493 nM (based on rat ovarian microsomes). The 
potential adverse effects observed in the male and female reproductive tracts of rats in the  
6-month toxicology study were consistent with aromatase inhibition and mirror findings in the 
previous 13-week toxicology study. AGI-8702 is not an aromatase inhibitor. In GLP-compliant 
embryo-fetal development studies in rats and rabbits, developmental toxicities were observed in 
rats but not rabbits. Fetal adverse effects in rats were considered likely due to aromatase 
inhibition. 
For additional details of toxicology study findings, refer to the Investigator’s Brochure. 
 Summary of Clinical Data 
5.2.2.
To date, 72 healthy adult subjects have been exposed to AG-348 in 2 clinical studies, a single 
ascending dose (SAD) study and a multiple ascending dose (MAD) study, with 31 of these 
subjects exposed to a single AG-348 dose under fasted conditions; 5 exposed to 2 doses of 
AG-348 under fasted and fed conditions; and 36 exposed to repeated doses of AG-348 for up to 
14 days.  


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Except as otherwise specified, the following discussion of clinical data refers only to healthy 
adult subjects, as this is the first clinical trial in which subjects with PK deficiency will be treated 
with AG-348. 
5.2.2.1. 
Pharmacokinetics 
The pharmacokinetics (PK) of AG-348 showed low to moderate variability between subjects. 
After single doses of AG-348 from 30 to 2500 mg, AG-348 was rapidly absorbed as indicated by 
the short time to maximum plasma concentration (tmax). There appeared to be some lengthening 
of the period of absorption at higher doses as indicated by higher tmax and a less-than-
proportional increase in maximum plasma concentration.  
Dose-normalized area under the curve (AUC) generally remained constant over the dose range 
studied, suggesting that AG-348 total exposure increased in a dose-proportional manner. The 
mean t1/2 ranged from 17.8 to 20.4 hours when samples were collected through 72 hours and 
from 50.3 to 79.3 when samples were collected through 120 hours. However, this terminal 
elimination phase contributed little to overall exposure of AG-348, as indicated by the small 
difference between AUC0-12hr and area under the plasma concentration versus time curve from  
0 to infinity (AUC0-∞), suggesting a shorter effective half-life of approximately 3 to 6 hours.  
AG-348 was extensively distributed (mean apparent volume of distribution range of 271 to 
1148 L) and had a moderate rate of clearance (geometric mean clearance range of 10.3 to 
13.9 L/hr). The fraction of AG-348 excreted in urine ranged from 0.0145 to 0.0209 across the 
dose levels, suggesting that renal excretion plays a minor role in the systemic elimination of 
AG-348. 
The preliminary repeat-dose PK of AG-348 at doses ranging from 15 mg every 12 hours (q12h), 
ie, twice daily (BID), to 700 mg BID also showed an increase in AG-348 exposure in a  
dose-proportional manner after the first dose. AG-348 exposure observed after repeated dosing 
for 14 days at doses of 120 mg every 24 hours, ie, once daily (QD) to 700 mg BID were lower 
than observed on Day 1 after the first dose. The magnitude of the effect was greater at higher 
doses; lower doses of 15 mg BID and 60 mg BID did not show this effect. This observation may 
be related to the nonclinical finding that AG-348 is a potential inducer of human CYP3A4, an 
enzyme which biotransforms AG-348. It is possible that multiple doses of AG-348 leads to an 
increased rate of its own metabolism. 
Evaluation of the effect of food on the PK of a single 700 mg dose of AG-348 in 5 subjects who 
were administered the drug while fasting and then, after an appropriate washout period, were 
re-administered the drug following ingestion of a standard United States Food and Drug 
Administration high-fat meal, showed that food likely has a minimal effect on the PK 
of AG-348. 
5.2.2.2. 
Pharmacodynamics 
After a single dose of AG-348, a decrease in the concentration of 2,3-DPG was observed at 
3 hours postdose, decreased in a dose-dependent manner to a minimum at 24-hour postdose, and 
then returned to values similar to baseline by 72 to 120 hours postdose. The mean decrease at 
24 hours was approximately 300 µg/mL at the 700 through 2500 mg dose levels. Similar 
decreases were observed after the first dose of multiple doses and prior to doses in the MAD 


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study. After the final dose of multiple doses, the concentration of 2,3-DPG returned to values 
similar to baseline between 72 and 120 hours post dose. 
After a single dose of AG-348, a minimal increase in the concentration of ATP was observed at 
24 to 120 hours postdose.  
In the MAD study, no increase was observed at 12 hours after the first dose; the concentration of 
ATP was increased on Day 8 to concentrations greater than in the SAD study, continued to trend 
upward on Day 11, and remained at a similar level through Day 14. The concentration of ATP 
remained elevated through 120 hours after the last dose on Day 14. The magnitude of the 
increase in ATP was similar across the dose range from 60 mg BID to 700 mg BID. 
5.2.2.3. 
Safety 
Overall, AG-348 has been well-tolerated among healthy adult subjects at doses that produced 
strong pharmacodynamic (PD) effects on 2,3-DPG and ATP. 
Phase 1 Studies AG348-C-001 and AG348-C-002 in Healthy Adult Subjects 
After a single AG-348 dose, adverse events occurring in the treatment-emergent period (TEAEs) 
reported by > 1 subject at any time on study (either under fasted or fed conditions) included 
headache (22%), nausea (14%), and contact dermatitis and vomiting (each 6%). After repeated 
dosing of AG-348 for 14 days, TEAEs that occurred in > 5% of all AG-348-treated subjects 
across all cohorts included headache (13.9%), nausea (13.9%), vomiting (8.3%), decreased 
appetite (8.3%), feeling hot (8.3%), restlessness (8.3%), and dizziness, fatigue, vessel puncture 
site bruise, hyperhidrosis, dermatitis allergic, and drug eruption (5.6% each). 
A dose-relationship was apparent with regard to the incidence of gastrointestinal events, 
primarily nausea and vomiting, with the incidence of such events increasing with increasing 
dose. Nausea and/or vomiting were observed only at doses ≥ 1400 mg in the SAD study and only 
at doses ≥ 700 BID in the MAD study. Nausea and vomiting were not observed at any dose 
≤ 360 mg in either the SAD or MAD studies. 
All but 1 TEAE reported to date have been mild or moderate (Grade 1 or 2) in intensity. The 
only Grade 3 TEAE was elevated liver transaminases (alanine aminotransferase [ALT]; aspartate 
aminotransferase [AST]) in a single subject in the MAD study treated with 700 mg AG-348 BID. 
The event was considered to be possibly related to AG-348, was declared a dose-limiting toxicity 
(DLT), and led to study drug discontinuation, after which the elevated liver transaminases 
resolved. Altogether, 3 of 6 subjects treated with AG-348 at 700 mg BID discontinued study 
drug in the MAD study: the subject described above with Grade 3 elevated transaminases, and 
2 others who withdrew themselves for nausea and vomiting. A fourth subject treated with 
AG-348 at 60 mg BID was discontinued from the study for a drug eruption. No AG-348-treated 
subject discontinued in the SAD study due to an adverse event (AE). 
Furthermore, no DLTs were observed after a single administration of AG-348 at doses of 30 mg 
to 2500 mg or after repeat administration of AG-348 for 14 days at daily doses of 15 mg to 
700 mg BID except for the event of Grade 3 elevated liver function tests described above. 
Due to preclinical observations pertaining to the potential for inhibition of the aromatase enzyme 
(see Section 5.2.1.4), the AG348-C-002 (MAD) study included assessment of baseline and serial 
measures of free and total serum testosterone and serum estradiol and estrone. In males treated 


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with AG-348, compared with placebo-treated males, the aromatase-dependent hormone 
assessments demonstrated an increase in total and free testosterone mean serum concentrations 
and decreased concentrations of estradiol and estrone at all doses of AG-348, including the 
lowest dose of 15 mg BID.  
Most of the increases in total and free testosterone remained within the reference range, except at 
the 360 mg BID dose where the dose group means on Day 8 and Day 14 exceeded the upper 
limit of normal (ULN). Most of the estradiol concentrations observed in males remained within 
the reference range, but the mean male estrone concentrations dropped to the lower limit of 
quantification in all dose groups except 15 mg BID.  
These changes in aromatase-dependent hormone levels in the male subjects treated with AG-348 
are consistent with inhibition of human aromatase, and were reversible within 14 days upon 
cessation of dosing. The study did not enroll enough female subjects to draw any definitive 
conclusions regarding serial changes in aromatase-dependent hormones over time. 
Phase 2 Study in Adult Subjects with PK Deficiency 
As of a data cutoff of 27 March 2017, among the 52 subjects with PK deficiency who had 
received AG-348 in this study, 50 (96.2%) subjects had experienced at least 1 AE. Adverse 
events that occurred in ≥ 10% of subjects across both cohorts included headache (23 [44.2%] 
subjects); insomnia (20 [38.5%] subjects); nausea (19 [36.5%] subjects); viral upper respiratory 
tract infection (9 [17.3%] subjects); arthralgia and fatigue (8 [15.4%] subjects each); back pain, 
cough, dizziness, hot flush, and vomiting (7 [13.5%] subjects each); and diarrhea and influenza 
(6 [11.5%] subjects each). 
Thirty-seven of 52 (71.2%) subjects who had received AG-348 experienced at least 1 AE that 
was considered by the Investigator to be study drug-related. Related AEs that occurred in ≥ 10% 
of AG-348-treated subjects included insomnia (17 [32.7%] subjects), nausea (15 [28.8%] 
subjects), headache (13 [25.0%] subjects), and hot flush (6 [11.5%] subjects). 
The majority of AEs were assessed by the Investigator as National Cancer Institute Common 
Terminology Criteria for Adverse Events (CTCAE) Grade 1 or 2 in severity. There were 
12 (23.1%) subjects with at least 1 reported AE that was CTCAE Grade ≥ 3. Adverse events that 
were Grade ≥ 3 included hypertriglyceridemia (3 [5.8%] subjects); anemia and insomnia 
(2 [3.8%] subjects each); and hemolysis, colitis, mesenteric vein thrombosis, influenza, 
pharyngitis, postprocedural hemorrhage, dizziness, and hypertension (1 [1.9%] subject each).    
Of these, 6 (11.5%) subjects had at least 1 Grade ≥ 3 AE that was considered study drug-related, 
including anemia and hypertriglyceridemia (2 [3.8%] subjects each) as well as hemolysis, 
pharyngitis, dizziness, and insomnia (1 [1.9%] subject each). 
No deaths have been reported in this study.  
A total of 9 (17.3%) subjects had experienced at least 1 SAE as of the data cutoff date. A total of 
12 SAEs were reported in 9 subjects, including anemia in 2 (3.8%) subjects; and hemolysis, 
colitis, enteritis, mesenteric vein thrombosis, influenza, pharyngitis, cholelithiasis, 
postprocedural hemorrhage, osteoporosis, and musculoskeletal weakness in 1 (1.9%) subject 
each. Six SAEs were reported as being possibly or probably related to AG-348: osteoporosis, 
hemolysis, anemia (2 events), pharyngitis, and musculoskeletal weakness (the latter of which 
was determined to be nonserious after the data cutoff date). In addition, an SAE of 


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hypertriglyceridemia was reported as probably related to AG-348 but was not captured as 
treatment-emergent in the clinical database as of the cutoff date. 
 Summary of Overall Safety Management Plan 
5.2.3.
Measures to minimize the risks to subjects enrolled in the Core and Extension Periods have been 
taken with respect to the following study design elements: 
• The initial doses for Arms 1 and 2 have been selected on the basis of safety and 
tolerability observations already carefully made in 2 predecessor clinical trials in 
adult healthy male and female subjects. 
• The specified inclusion/exclusion criteria have been carefully considered to avoid 
enrollment of subjects for whom exposure to the study drug might pose a hazard. 
• A designated Data Review Team (DRT) consisting of the overall study Coordinating 
Investigator, treating Investigators, Medical Monitor, Clinical Pharmacologist, 
Statistician, and Sponsor’s Responsible Medical Officer has been established to meet 
regularly at approximately 6-week intervals throughout the duration of the Core 
Period, and approximately every 4-6 months during the Extension Period once all 
subjects have completed the Core Period, to review the accumulating study data. The 
DRT will exercise options to suspend enrollment to 1 or both of the initial 2 study 
dose arms, discontinue enrollment to 1 or both of the initial 2 study dose arms, adjust 
the dose of subjects in 1 or both of the initial 2 study arms, and/or implement 1 new 
study dose arm.  
If a new dosing arm is implemented in the Core Period, the dose selected will not 
exceed 360 mg BID, the highest dose that demonstrated acceptable safety and 
tolerance in the 14-day, multiple-dose (BID) study in healthy adult subjects.  
Group cohort stopping rules for terminating enrollment into an arm based on the 
severity (CTCAE v4.03 grade) and frequency of AEs are defined. 
• Dose modification and stopping rules are defined for individual subjects. 
• Guidance for permitted, prohibited, and cautionary concomitant medications is 
specified based on the estimated potential for DDIs from hepatic cytochrome enzyme 
interactions with AG-348. 
• Due to the potential for AG-348-mediated aromatase inhibition, bone mineral density 
will be monitored using dual-energy x-ray absorptiometry (DXA) scans (hip and 
spine) at Baseline (if subject has not had prior DXA scan within 3 months of Day 1) 
and between Week 24 and Week 28. 
In the event that any clear and unequivocal, previously unidentified/unexpected toxicities occur 
in preclinical toxicology studies, the Sponsor will notify the Investigators, Institutional Review 
Boards (IRBs)/Independent Ethics Committees (IECs), and regulatory authorities. The Sponsor 
will also report these to the DRT for review and discussion of appropriate actions, which may 
include, but are not necessarily limited to 1 or more of the following:  
• Amending the protocol to adjust the inclusion/exclusion criteria (eg, to exclude 
subjects with certain at-risk concurrent conditions). 


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• Amending the protocol to adjust safety monitoring procedures (eg, to require 
additional monitoring of specified AEs, physical examinations, clinical laboratory 
testing, electrocardiogram [ECG] monitoring, or other testing as appropriate). 
• Adjusting the dose of an arm of the study as appropriate. 
• Adjusting the dose modification and/or stopping rules (Section 9.7). 
• Adjusting the subject withdrawal criteria (Section 8.6). 
• Terminating the trial. 
 Risks Associated with AG-348 
5.2.4.
5.2.4.1. 
Risk of Withdrawal Hemolysis 
Acute hemolysis upon sudden withdrawal of AG-348 has been observed in subjects with a 
marked and rapid increase in Hb. Investigators are advised to not discontinue dosing of AG-348 
without first speaking with the study medical monitor for guidance on appropriate dose 
adjustment to avoid withdrawal hemolysis in subjects who experience a sustained increase in Hb. 
Any dose modification should be per protocol (Table 1).  
5.2.4.2. 
Risk of Insomnia 
As discussed in Section 5.2.1.1 of this protocol and in the AG-348 Investigator’s Brochure,  
AG-348 has been identified as a histamine H3 receptor antagonist/inverse agonist which has 
been documented to affect wakefulness and cognition in humans. No effects of histamine H3 
modulation have been observed in safety pharmacology or toxicology studies (Schwartz 2011). 
Insomnia has been classified as an identified risk of AG-348 treatment, as described in the 
Investigator’s Brochure.  
5.2.4.3. 
Risk of Bone Mineral Density Decrease 
AG-348 inhibits human aromatase activity, as shown in studies in human placental microsomes 
and rat ovarian microsomes. In this study, 1 subject from 52 treated subjects experienced a 
medically important event of osteoporosis (Grade 2), which was assessed as likely due to 
aromatase inhibition. 
5.2.4.4. 
Potential Risk of Transaminase Increases 
Transient increases in transaminases have been observed with AG-348, as described in the 
Investigator’s Brochure. No SAEs or cases of suspected drug-induced liver injuries have been 
reported. Liver function test results should be monitored per protocol. Transaminase increase is 
an AE of special interest (AESI) for AG-348 (refer to Section 11.1.5 for details). 
5.2.4.5. 
Potential Risk of Triglyceride Increases 
Transient increases in triglycerides have been observed with AG-348, as described in the 
Investigator’s Brochure. One observed case was an SAE that recovered after AG-348 
discontinuation. No cases of pancreatitis have been reported.  


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5.2.4.6. 
Potential Risk of Photosensitivity 
AG-348 may cause sensitivity to direct and indirect sunlight. Subjects should be warned to avoid 
direct sun exposure. When exposure to sunlight is anticipated for longer than 15 minutes, the 
subject should be instructed to apply Sun Protection Factor 30 or higher sunscreen to exposed 
areas and wear protective clothing and sunglasses. 
 Other Restrictions and Precautions 
5.2.5.
Subjects should be advised to refrain from altering their normal exercise routine for the first 
28 days of treatment with AG-348, as symptoms of anemia could theoretically temporarily 
worsen before the eventual potential beneficial effect of AG-348 on the Hb level becomes 
evident.  
The elevated blood levels of 2,3-DPG commonly seen in subjects with PK deficiency may 
produce a right shift in the Hb-O2 dissociation curve. The effect of this is to enhance oxygen 
delivery at the tissue level, thus counteracting to some extent the physiologic consequences of 
the anemia. AG-348 was shown to produce rapid decreases (within the first 12 hours following a 
single dose) in 2,3-DPG in 2 prior clinical trials with healthy adult male and female subjects. In 
subjects with PK deficiency who have elevated 2,3-DPG levels, it is theoretically possible that 
decreases in 2,3-DPG that precede correction of the anemia could result in a temporary decrease 
of oxygen delivery at the tissue level resulting in clinical symptoms (eg, increased fatigue). 
Subjects should not discontinue dosing without first speaking with the treating Investigator; 
abrupt discontinuation of AG-348 dosing in a subject who experience a substantial increase in 
Hb may result in withdrawal hemolysis. 
 


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6. 
STUDY OBJECTIVES AND ENDPOINTS 
6.1. 
Core Period 
 Primary Objective 
6.1.1.
The primary objective of the Core Period of the study is to: 
• Evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in 
subjects with PK deficiency. 
 Secondary Objectives 
6.1.2.
The secondary objectives of the Core Period of the study are to: 
• Evaluate the PK of AG-348 and the metabolite AGI-8702. 
• Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348. 
• Evaluate indicators of clinical activity of AG-348 in subjects with PK deficiency, 
including changes in Hb, HCT, reticulocyte count, haptoglobin (Hp), 
carboxyhemoglobin (COHb), lactate dehydrogenase (LDH), total and indirect 
bilirubin, erythropoietin (EPO), hepcidin, ferritin, and transferrin saturation (serum 
iron/iron-binding capacity). 
6.2. 
Extension Period 
 Primary Objective 
6.2.1.
The primary objective of the Extension Period of the study is to: 
• Evaluate the safety and tolerability of up to 30 months of AG-348 administration in 
subjects with PK deficiency. 
 Secondary Objectives 
6.2.2.
The secondary objectives of the Extension Period are to: 
• Evaluate the PK of AG-348 and the metabolite AGI-8702. 
• Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348. 
• Evaluate indicators of clinical activity of AG-348 in subjects with PK deficiency, 
including changes in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and 


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indirect bilirubin, EPO, hepcidin, ferritin, and transferrin saturation (serum iron/iron-
binding capacity). 
• Evaluate the optimal maintenance dose of AG-348 for each individual subject during 
the Extension Period. 
6.3. 
Study Measures and Endpoints 
 Safety Measures and Endpoints 
6.3.1.
Safety will be evaluated by all of the following: 
• Monitoring of AEs, including SAEs, AESIs, and AEs leading to discontinuation.  
All AEs will be graded using CTCAE, Version 4.03. 
• Safety laboratory parameters (hematology, chemistry, urinalysis, coagulation). 
• Physical examination findings (including neurological examination). 
• Vital signs (VS). 
• 12-lead electrocardiograms (ECGs). 
• DXA scans. 
• Serum sex hormone levels (testosterone [total and free], estrone, and estradiol), bone 
turnover markers (serum osteocalcin-N-mid and serum C-terminal telopeptide 
[CTX]), 25-hydroxy vitamin D2 and D3, total cholesterol, high-density lipoprotein-
cholesterol (HDL-C), and triglycerides will be monitored for evidence of potential 
inhibition of aromatase by AG-348. 
• Menstruating female subjects will also keep a paper-based menstrual cycle diary 
throughout the Core and Extension Periods. 
 Clinical Activity Measures and Endpoints 
6.3.2.
• Monitoring of potential indicators of clinical activity will include evaluating changes 
in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, 
hepcidin, ferritin, and transferrin saturation. 
 Pharmacokinetic and Pharmacodynamic Measures and Endpoints 
6.3.3.
The PK and PD profile of AG-348 will be evaluated by all of the following: 


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• Approximately the first 10 subjects treated during the Core Period, contingent on 
clinical site feasibility, will undergo extensive PK sampling as detailed in 
Appendix 1.3. The remainder of treated subjects will undergo limited PK sampling as 
detailed in Appendix 1.4. 
• During the Core Period, serial blood sampling for determination of concentration-
time profiles of AG-348 and its metabolite AGI-8702 will be conducted following the 
first dose and the morning Day 15 dose, and additional trough levels of AG-348 and 
AGI-8702 will be obtained. 
• During the Extension Period, predose PK samples will be drawn for the measurement 
of trough levels of AG-348 and AGI-8702 at each study visit (every 3 months; 
Appendix 1.2). AG-348 and AGI-8702 will be analyzed using qualified assays to 
determine concentrations in plasma.    
• Pharmacodynamic assessments during the Core Period will include 2,3-DPG, ATP 
(secondary objectives), 
 
 
  
 
Approximately the first 10 subjects treated during the Core Period will undergo 
extensive PD sampling as detailed in Appendix 1.3. The remainder of treated subjects 
will undergo limited PD sampling as detailed in Appendix 1.4. 
• During the Core Period, serial blood sampling for determination of levels of ATP 
and, 2,3-DPG will be conducted following the first dose and the morning Day15 dose, 
and additional trough levels of ATP and 2,3-DPG will be obtained. 
• During the Extension Period, predose PD samples will be drawn for the measurement 
of trough levels of 2,3-DPG, ATP, 
 at each study visit (every 
3 months; Appendix 1.2). Adenosine triphosphate and 2,3-DPG will be analyzed 
using qualified assays to determine concentrations in whole blood.   


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7. 
INVESTIGATIONAL PLAN 
7.1. 
Overall Study Design 
Study AG348-C-003 is a Phase 2, open-label, 2-arm, multicenter, randomized, dose-ranging 
study in adult subjects with PK deficiency; the study will be divided in to a Core Period and an 
Extension Period. During the Core Period, subjects will receive multiple doses of AG-348 for up 
to 24 weeks; subjects who are eligible can enter the Extension Period to receive AG-348 for up 
to 2 years following the end of the Core Period.  
Subjects with PK deficiency confirmed by RBC PK enzymatic assay performed at Screening will 
be eligible to participate in this study. At the Week 24 visit, subjects who safely tolerate AG-348 
and demonstrate clinical activity of AG-348 may be eligible to immediately roll over to the 
Extension Period for continued treatment. Subjects who complete treatment at the end of the 
Core Period (24 weeks) will undergo follow-up assessment 4 weeks after the last dose of study 
drug. If a subject discontinues at any other time (including discontinuation during the Core or 
Extension Period), follow-up assessments will be conducted 4 weeks after discontinuation. 
Subjects with toxicity suspected to be related to study drug will continue follow-up until the AE 
resolves, is declared chronic by the Investigator, or the subject is lost to follow-up. 
For the Core Period, up to 25 subjects will be initially randomized on an open-label, 1:1 basis to 
each of 2 BID doses of AG-348 (up to 50 subjects; Figure 2).  
Figure 2: 
Study Schema:  Core Period 
 
Abbreviations:  BID (q12h) = twice-daily (every 12 hours); DRT = data review team; PKR = pyruvate kinase red blood cell 
isoform; TBD = to be determined; w = weeks.  


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The dose of Arm 1 is 300 mg of AG-348 administered PO q12h (ie, BID). The dose of Arm 2 is 
50 mg of AG-348 administered PO BID. Randomization will be stratified by PKR mutation in 
order to maintain balance as much as possible across the dose arms for the specific mutations 
expected to be most frequently enrolled. The PKR mutation stratification factor will consist of 
4 levels (R510Q, R486W, and R479H, and all other mutations as “other”). Mutation status is 
defined by the presence of at least 1 of the indicated mutations; subjects with more than 
1 stratified mutation will be assigned based on Sponsor’s discretion. 
The doses for each arm of the Core Period have been selected from the AG348-C-001 SAD 
study and AG348-C-002 MAD studies in healthy adult subjects to represent the range of 
doses/exposures that were safely tolerated and resulted in maximal or near maximal PD effects 
on 2,3-DPG and ATP. 
Because PK deficiency is a rare disease with a limited eligible subject population and because 
the underlying pathophysiology and clinical phenotype of affected subjects is heterogeneous due 
to the wide variety of mutations in PKR that cause the disease, it is deemed important to focus 
closely on dose findings in this first-inpatient study. Therefore, in addition to initiating this study 
with 2 different doses of AG-348 administered BID, a DRT will be established to review study 
data on a frequent basis and adapt the study design, dose and schedule of AG-348 as indicated.  
The DRT will monitor safety on an ongoing basis and meet at regular intervals of approximately 
every 6 weeks, or ad hoc as necessary, for as long as any subjects are still in the Core Period to 
review AEs, VS, clinical laboratory (hematology, clinical chemistry, coagulation, and 
urinalysis), and ECGs. The DRT will also review available PK/PD data and indicators of clinical 
activity (eg, changes from baseline in Hb). These DRT meetings will also include data review for 
all subjects that may be under treatment in the Extension Period.  
If there are no subjects still being treated in the Core Period, and the only subjects on treatment 
are those in the Extension Period, then the frequency of the DRT meetings will reduce to 
approximately every 4 to 6 months in order to match the frequency of subject visits (and new 
data collection) in the Extension Period. When all the subjects are in the Extension Period, 
PK/PD data will no longer be reviewed by the DRT. The DRT will be comprised of the study 
Coordinating Investigator, treating Investigators, Medical Monitor, Clinical Pharmacologist, 
Statistician, and Sponsor’s Responsible Medical Officer. 
Beginning 6 weeks after the first subject is dosed in the Core Period or ad hoc as necessary, and 
proceeding according the schedule indicated above (approximately every 6 weeks during the 
Core Period, approximately every 4 to 6 months during the Extension Period once all subjects 
have completed the Core Period), the DRT will review cumulative safety data, available PK/PD 
data, and clinical activity data.  
Based on the DRT’s recurring, the DRT may exercise 1 or more of the following options during 
the Core Period: 
• Continue treatment and enrollment in existing arms without change. 
• Add 1 new dose arm (Arm 3) to enroll up to 25 subjects at a dose to be determined; 
the dose for Arm 3 may be lower or higher than Arm 1 and Arm 2 doses, but will not 
exceed 360 mg BID; and the dose regimen may be less frequent than BID. 


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• Terminate or suspend enrollment to allow further review of clinical data in Arm 1 
and/or Arm 2 (and/or potential Arm 3).  
Enrollment in an arm could be terminated or suspended to allow further review, for 
example, for unacceptable safety/tolerability, poor PD response, or lack of signs of 
clinical activity. 
• Re-assign subject’s doses and schedule in a terminated arm to match the dose and 
schedule of another arm of the study. In this case, the subjects in the terminated arm 
will remain in their original arm, ie, they will not count towards the enrollment quota 
of the arm whose dose and schedule is being adopted. 
• Implement specific genotype restrictions to enrollment in 1 or more arms to ensure 
representation of subjects with genotypes of greatest clinical relevance. 
The DRT may exercise 1 or more of the following options during the Extension Period: 
• Continue treatment without change. 
• Re-assign subjects’ doses and schedule to an existing dose and schedule that has been 
determined to be safer, and/or have a better PD response, or produce signs of clinical 
activity. 
• Terminate or suspend treatment to allow further review of clinical data (eg, for 
unacceptable safety/tolerability, poor PD response, or lack of signs of clinical 
activity). 
The data that the DRT will review to make these decisions are expected to include, but are not 
necessarily limited to, the following:  
• Safety Observations: All AEs, VS, clinical laboratory assessments (hematology, 
clinical chemistry, coagulation, and urinalysis), and ECGs. 
• PK and PD Observations: Includes changes in 2,3-DPG and ATP, except when all 
subjects are in the Extension Period. 
• Indicators of Clinical Activity: Includes changes in Hb, HCT, reticulocyte count, Hp, 
COHb, LDH, EPO, hepcidin, total and indirect bilirubin, ferritin, and transferrin 
saturation.  
If a third dose arm is implemented, the dose of AG-348 selected will not exceed 360 mg BID, as 
this was the highest dose that demonstrated acceptable safety and tolerance in the 14-day 
multiple BID dosing study in healthy adult subjects. The PK/PD sampling schedule in a potential 
third dosing arm will be determined by Sponsor’s discretion and may follow either the extensive 
or limited PK/PD sampling schedules as specified. 
Subjects in the Extension Period will undergo a gradual dose-taper regimen to identify the 
optimal maintenance dose for each (defined as the dose that results in ≤ 1.0 g/dL decrease in Hb 
compared to the pre-taper Hb value on at least 2 measurements following each dose taper step) 
on a per-subject basis (Section 9.7.3). 
 


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Due to the potential for AG-348-mediated aromatase inhibition, combined with the known risk 
of osteoporosis in subjects with congenital hemolytic anemias (Steer, et al. 2017; Wong, et al. 
2014), DXA scan (hip and spine) will be performed at Screening (if subject has not had prior 
DXA scan within 3 months of Day 1) to obtain bone mineral density and T and Z scores. These 
data are intended to serve as a baseline measure of bone mineral density for all enrolling 
subjects, and are deemed of particular importance for those who may enter the longer-term 
Extension Period after completing 24 weeks of treatment (Core Period). All subjects will have a 
second DXA scan in the interval between Weeks 24 and 28 for the Core Period. Subjects in the 
Extension Period will have additional DXA scans at Months 12, 18, 24, and 30. 
As the number of enrolling arms changes in the study (eg, from 2 to 3), the randomization 
scheme will adjust to enable balanced randomization into each actively accruing arm. 
Randomization and stratification will cease in the event that only a single arm is left enrolling. 
Depending on possible early termination of 1 or both of the initial 2 arms, or the addition of a 
third arm, the study could enroll up to a maximum of 75 subjects. 
The Investigator will monitor all subjects for safety and tolerability. Modification of an 
individual subject’s dose of AG-348 will be based on AEs and observed changes in Hb levels as 
detailed in Section 9.7.1 and Section 9.7.2. 
Screening assessments will occur within 42 days prior to the first dose of study treatment. During 
the Core Period, subjects will attend visits at baseline (Day 1), weekly through Week 3 (Days 8, 
15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12), and monthly 
through Week 24 (Weeks 16, 20, and 24). Subjects who safely tolerate AG 348 through Week 24 
(Core Period) may be eligible to immediately enter the Extension Period for continued treatment.  
Study visits for safety and clinical activity assessments will occur approximately every 3 months 
during the Extension Period for up to 2 years after the end of the Core Period, except during the 
dose taper part of the Extension Period when weekly visits will be performed (see Section 9.7.3.1 
for details). All subjects will undergo a follow-up assessment 4 weeks after the last dose of 
AG-348, regardless of whether this was due to discontinuation, the last dose in the Core Period 
for a subject who chooses not to continue in the Extension Period, or the last dose of the 
Extension Period except for those who may continue treatment in an extension study (to be 
designed) after having completed the Extension Period. 
Safety assessments will include monitoring of AEs, including SAEs, AESIs, and AEs leading to 
discontinuation; safety laboratory parameters (eg, hematology, serum chemistry, coagulation 
studies, and urinalysis); physical examination findings (including neurological examination); VS; 
12-lead ECGs, and DXA scans. Additional safety assessments will include monitoring of sex 
hormone levels (testosterone [total and free], estrone, and estradiol), bone turnover markers 
(osteocalcin-N-mid and CTX), 25-hydroxy vitamin D2 and D3 levels, total cholesterol, HDL-C, 
and triglycerides.  
Follow-up assessments will be conducted on Day 197 (Week 28) for subjects who do not enter 
the Extension Period and will include physical examination, weight, performance status, VS, 
12-lead ECGs, laboratory evaluations (hematology, Hp, EPO levels, serum chemistry, 
coagulation studies, urinalysis; lipids, hormonal testing), AEs, and transfusion record.  


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These follow-up assessments will be performed approximately 4 weeks after discontinuation of 
AG-348 for subjects who discontinue prior to completion of the Core Period and for those who 
discontinue in the Extension Period. Menstruating female subjects will also be required to keep a 
paper-based menstrual cycle diary throughout the study.  
Pharmacokinetic assessments will include serial blood sampling for PK profiles of AG-348 and 
its metabolite AGI-8702.  
Pharmacodynamic evaluations will include serial blood sampling for determination of levels of 
ATP and 2,3 DPG. Extensive PK/PD sampling will be conducted on the first approximately 
10 subjects total treated in Arms 1 and 2 of the Core Period (Appendix 1.3) while limited PK/PD 
sampling will be conducted on the remainder of treated subjects (Appendix 1.4). Limited trough 
sampling will be conducted every 3 months during the Extension Period (Appendix 1.2). 
 
 
 
 
7.2. 
Rationale for the Study Design 
The primary and secondary objectives of this study are to evaluate the safety, tolerability, PK 
and PD, and indicators of clinical activity of AG-348 in subjects with PK deficiency. The choice 
of dose and schedule of administration of AG -348 for Arms 1 and 2 was based on the highest 
safely tolerated dose (Arm 1: 300 mg BID) and the lowest dose with potentially relevant PD 
activity (Arm 2: 50 mg BID) from the forerunner AG348-C-002 MAD study in healthy adult 
subjects.  
Decisions regarding continuing enrollment and treatment in these initial dose arms and/or 
implementation of an additional dose arm will be based on DRT review of safety, PK and PD 
data, and indicators of clinical activity collected from all subjects treated in Arm 1 and Arm 2. 
This design was chosen to minimize risk to subjects while allowing evaluation of safe and 
pharmacologically active dose levels of AG-348, and to allow the necessary flexibility to adjust 
dose and schedule should the safety, tolerability, PK, and/or PD be different in subjects with PK 
deficiency compared with healthy adult subjects.  
Additional safety measures intended to minimize risk to subjects include monitoring of AEs by 
the DRT and specified provisions for individual subject dose modification as needed for safety 
and (potentially) large increases in Hb level (Section 9.7.1 and Section 9.7.2). Measures intended 
to maximize the opportunity for subjects with demonstrated safety and tolerability to continue to 
derive benefit from any observed clinical activity of AG-348 include the option for continued 
treatment in the Extension Period.  
A comprehensive series of safety evaluations, including laboratory parameters, physical 
examinations (including neurological examination), VS, 12-lead ECGs, and monitoring for SAEs 
and AEs, will be conducted to evaluate the safety profile of AG-348 and to aid in the 
determination of the recommended dose for continued development. 
The study includes serial blood sampling across at least 2 different doses of the study drug to 
assess its PK and PD profiles.  


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Consistent with the design of many Phase 2 studies, preliminary evaluation of the potential PD 
and clinical activity of AG-348 are secondary objectives of this study. The latter will include 
assessments of the PK/PD relationship between AG-348 and the biomarkers ATP and 2,3-DPG, 
 
7.3. 
Rationales Related to Dosing 
 Rationale for the Starting Dose 
7.3.1.
Prior to execution of this study, the Sponsor conducted 2 clinical studies of AG-348 in healthy 
adult subjects, including a SAD study (AG348-C-001) and a MAD (14-day, BID) study 
(AG348-C-002). Available details of these studies are discussed in the current Investigator’s 
Brochure (IB). Between these 2 studies, 72 healthy human subjects have been dosed with 
AG-348. In vitro investigations, also reported in the Investigator’s Brochure, had previously 
demonstrated that AG-348 increased the activity of WT PKR approximately to the same extent 
as it did a series a recombinant mPKRs. Therefore it was deemed reasonable to study the safety, 
tolerability, PK, and PD of AG-348 in healthy subjects in a controlled Phase 1 setting as a more 
efficient means of obtaining information than in the actual rare disease population of subjects 
with PK deficiency.  
The MAD study demonstrated that the exposures produced by AG-348 doses from 60 mg BID to 
360 mg BID (including 120 mg QD) resulted in maximal changes from baseline for the PD 
markers 2,3-DPG (reduction from baseline) and ATP (increase from baseline). The exposures 
resulting from doses less than 60 mg BID were of lesser magnitude and the exposures resulting 
from doses greater than 360 mg BID were of no greater magnitude than the aforementioned 
range. Therefore the starting doses for this first dose ranging study in subjects with PK 
deficiency were selected to be 300 mg BID (Arm 1) and 50 mg BID (Arm 2). These doses were 
demonstrated to be safe and tolerable in the healthy adult subject studies.  
 Rationale for the Dose Range 
7.3.2.
The availability of ATP is proposed as being critical for optimally maintaining RBC membrane 
integrity (Section 5.1). The dose ranges from 50 mg BID to 300 mg BID may result in clinically 
effective modulation of PKR in PK deficiency subjects if the mutated enzyme is responsive to 
AG-348 in a similar manner to the WT enzyme in healthy subjects. However, there are many 
different mutations in PKR that result in PK deficiency, and these mutations produce variable 
effects on the enzyme in terms of catalytic activity and thermal stability. It is not known if 
different mutations will respond clinically in a similar manner to the same exposure to AG-348. 
Therefore, it is prudent to study the range of safe and pharmacodynamically relevant doses as 
specified in this study, and to allow flexibility for the DRT to analyze the evolving study data to 
adapt the dose and schedule of administration of AG-348 to produce the optimal combination of 
safety, tolerability, and PD, and, potentially, clinical response. 
 Rationale for the Duration of Dosing  
7.3.3.
The initial treatment duration of 24 weeks (6 months) for the Core Period was chosen for this 
proof-of-concept trial for 2 principal reasons: 1) to begin establishing a safety database 
addressing the chronic administration of AG-348; and 2) to allow sufficient time for clinical 
response to treatment to appear.  


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It is anticipated that this treatment, if successful, may be taken for life, as PK deficiency is a 
genetically determined inborn error of metabolism. Therefore, it is important to begin to 
investigate the long-term safety of the treatment. As will be discussed below, the safety package 
supports the treatment duration of 6 months.  
Red blood cell turnover is typically 120 days (4 months), although it may be shorter in some 
populations of pyruvate kinase deficient RBCs (Mentzer, et al. 1971). Different PKR mutations 
produce a variety of physiologic consequences. In mutations where normal or nearly normal 
levels of PKR protein persist, but the protein catalytic function is impaired, an improvement in 
PKR functional activity might be seen relatively quickly upon exposure to AG-348. However, in 
cases where the PKR mutation results in an unstable mutant, PKR protein levels may be low and 
additional time may be required for stabilization of the newly synthesized mutant enzyme in 
developing bone marrow erythroblasts so that eventually more mature RBCs may be produced 
with more nearly normal levels of functional PKR protein.  
This study plans up to 24 weeks of dosing with AG-348 during the Core Period with the 
possibility of continued dosing beyond 24 weeks during the Extension Period, up to 2 years, in 
subjects for whom AG-348 is safely tolerated and demonstrates clinical activity.  
The International Conference on Harmonisation (ICH) Guideline M3(R2) on Nonclinical Safety 
Studies for the Conduct of Human Clinical Trials and Marketing Authorisation for 
Pharmaceuticals recommends that clinical trials of a duration between 2 weeks and 6 months 
should be supported by repeat-dose toxicity studies of at least equivalent duration, and clinical 
trials lasting beyond 6 months should be supported by 6-month rodent and 9-month nonrodent 
studies. This guidance allows for clinical trials of longer duration to be initiated based on 3 
months of nonclinical data, and for clinical dosing in serious or life-threatening indications to be 
extended based on complete chronic rodent data and in-life and necropsy data in the nonrodent 
(and complete histopathology in the nonrodent provided within an additional 3 months).  
For the current investigational product (AG-348), 13-week, repeat-dose toxicology studies have 
been completed and are summarized in Section 5.2.1.4 of this protocol and in the current 
Investigator’s Brochure. Considering that PK deficiency is a serious condition with an unmet 
medical need, it is appropriate to initiate this clinical study with the available nonclinical data.  
 Rationale for Identification of Individual Optimal Maintenance Dose and Gradual 
7.3.4.
Dose-Taper Regimen 
AG-348 has proven efficacious across a range of doses in this study, with several subjects having 
achieved and maintained clinically meaningful increases in Hb at doses < 50 mg BID. 
Previously, dose decreases have been implemented in this study only for safety/tolerability 
reasons and in subjects with Hb values reaching > 13.5 g/dL (for women) or > 15.0 g/dL  
(for men).  
In subjects who have a sustained increase in Hb while continuing to tolerate their randomized 
dose of either 50 mg BID or 300 mg BID, there is no provision in the current protocol for dose 
de-escalation. However, it is possible and even likely that some subjects may maintain improved 
Hb values at a lower dose of AG-348.  


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A general principle in medicine is to administer the lowest efficacious dose of a drug. 
Furthermore, it is expected that administration of lower doses of AG-348 will be associated with 
reduced frequency and severity of AEs.  
Based upon these considerations, the protocol will now include the practice of attempting to 
decrease each subject’s dose while maintaining a desirable Hb level. 
Subjects remaining on study are receiving AG-348 across a wide range of doses (from 300 mg 
BID to 5 mg QD). These observations indicate that the optimal maintenance dose of AG-348 is 
likely to vary by subject and cannot be determined in advance. Consequently, individual optimal 
maintenance doses will be determined empirically by an individualized gradual dose taper while 
monitoring Hb and Hp levels.  
An additional justification for implementing a gradual dose taper is the finding that abrupt 
cessation of treatment with AG-348, which was implemented for 2 responsive subjects in the 
Core Period with Hb values over the protocol-defined threshold, resulted in withdrawal 
hemolysis and anemia. Therefore, it is not known whether a sharp decrease in AG-348 dosing 
(eg, from 300 mg BID to 50 mg BID) would have the same effect as a treatment interruption; 
however, the scenario is theoretically possible in subjects with an individual optimal 
maintenance dose > 50 mg BID. 
7.4. 
Criteria for Study Termination 
This study may be prematurely terminated if, in the opinion of the Sponsor, there is sufficient 
reasonable cause. In the event of such action, written notification documenting the reason for 
study termination will be provided to each Investigator. 
Circumstances that may warrant termination include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to subjects. 
• Insufficient adherence to protocol requirements. 
• Plans to modify, suspend, or discontinue the development of the study drug. 
• Other administrative reasons. 
Should the study be closed prematurely, all study materials must be returned to the Sponsor or 
the Sponsor’s designee. Subjects who have participated in the Extension Period may be able to 
screen for eligibility in a study for a second generation allosteric PKR activator, if such a 
compound is available at that time, and after a suitable washout period has been observed. 


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8. 
STUDY POPULATION 
8.1. 
Number of Subjects 
Up to approximately 75 subjects may be enrolled in this study. 
8.2. 
Inclusion Criteria 
 Core Period 
8.2.1.
Subjects must meet all of the following criteria to be eligible for inclusion in the Core Period of 
the study: 
1. Have provided signed written informed consent prior to undergoing any study procedure, 
including Screening procedures. 
2. Be male or female, aged 18 years and older. 
3. Have a known medical history of PK deficiency. 
4. Have documented clinical laboratory confirmation of PK deficiency by RBC pyruvate 
kinase enzymatic assay performed at Screening, either by a designated central laboratory 
or by any participating investigative site’s local hematology laboratory. Subjects with 
prior documentation of PK deficiency by RBC enzymatic assay must have reconfirmation 
of this result during Screening as a condition of enrollment. 
 
NOTES: 
i. 
In the event that a subject’s Screening pyruvate kinase enzymatic assay is 
negative (ie, shows normal pyruvate kinase activity), the subject will be eligible 
for enrollment if the genotyping shows a mutant genotype that has been 
previously documented in the literature to be associated with PK deficiency. 
ii. 
If the genotyping shows a previously undescribed mutation in the PKR gene, the 
subject’s eligibility for enrollment will be determined on an individual case basis 
by the Coordinating Investigator and Medical Monitor in discussion with the 
Investigator. 
iii. 
If no mutation is defined, the subject will not be eligible for enrollment. 
5. Have a blood sample for genotypic characterization of the mutant PKR gene performed 
by the designated central laboratory at Screening.  
 
NOTES:  
i. 
The designated central laboratory-determined genotype will generally serve as the 
basis for genotyping for enrollment. However, subjects whose genotype has 
already been determined by another laboratory may be enrolled on the basis of 
that report, with the approval of the Medical Monitor, in the case of an 
unexpected delay in return of the designated central laboratory result during the 
Screening Period.  


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ii. 
Enrollment on the basis of a result from a laboratory other than the designated 
central genotyping laboratory does not relieve the inclusion requirement that ALL 
subjects must have a sample sent to the designated central genotyping laboratory. 
6. Have Hb ≤ 12.0 g/dL (if male) or ≤ 11.0 g/dL (if female). 
7. Be considered transfusion-independent, defined as having had ≤ 3 units of RBCs 
transfused in the 12-month period up to the first day of study drug dosing and no 
transfusions within 4 months of the first day of study dosing.  
 
NOTE: Subjects who have received more transfusion support than described above will 
be evaluated for eligibility on a case-by-case basis by the Medical Monitor. 
8. Have their spleen in place or have undergone splenectomy. Splenectomized subjects must 
meet all of the following conditions: 
i. 
Have undergone the procedure ≥ 6 months prior to Screening. 
ii. 
Be current in their vaccinations for pneumococcal conjugate (PCV13), 
pneumococcal polysaccharide (PPSV23), quadrivalent meningococcal vaccine, 
and Haemophilus influenzae Type B, as recommended by the United States 
Centers for Disease Control and Prevention's Advisory Committee on 
Immunization Practices (refer to 
http://www.cdc.gov/vaccines/schedules/downloads/adult/adult-schedule.pdf) or, 
for subjects in Canada or the European Union, by immunization advisory groups 
in those locations. 
NOTE: Any missing vaccinations may be administered, starting with the 
Screening Period and continuing throughout the trial, following the initiation of 
AG-348 dosing and as necessary according to recommended vaccination 
guidance. 
9. Have Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2. 
10. Have been taking ≥ 1 mg of folic acid daily for ≥ 21 days prior to the first dose of study 
drug and agree to continue this regimen during the study. 
11. Have adequate organ function, defined as meeting all of the following conditions: 
i. 
Serum AST ≤ 2.5 × ULN, unless the increased AST is assessed by the 
Investigator as due to hemolysis and/or hepatic iron deposition, and ALT  
≤ 2.5 × ULN, unless the increased ALT is assessed by the Investigator as due to 
hepatic iron deposition. 
ii. 
Either normal or elevated levels of serum bilirubin. In subjects with serum 
bilirubin > ULN, the elevation must be attributed to hemolysis with or without 
Gilbert's syndrome and must not be attributed to choledocholithiasis, 
cholecystitis, biliary obstruction, or hepatocellular disease. 
iii. 
Serum creatinine ≤ 1.25 × ULN or, if > 1.25 × ULN, then 24-hour measured or 
calculated (by Cockcroft-Gault) glomerular filtration rate ≥ 60 mL/min. 
iv. 
Absolute neutrophil count (ANC) ≥ 1.0 × 109/L. 


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v. 
Platelet count ≥ 100 × 109/L. 
vi. 
Activated partial thromboplastin time (aPTT) and international normalized ratio 
(INR) ≤ 1.25 × ULN, unless the subject is receiving therapeutic anticoagulants. 
12. For women of childbearing potential—defined as females who either have experienced 
menarche and have not undergone successful surgical sterilization (hysterectomy, 
bilateral tubal ligation, and/or bilateral oophorectomy) or who are not postmenopausal 
(defined as either having amenorrhea for ≥ 12 consecutive months without another cause 
and documented serum follicle-stimulating hormone [FSH] level > 35 mIU/mL or being 
≥ 62 years of age and having amenorrhea for ≥ 12 consecutive months [no FSH testing 
required]): 
i. 
Agree to abstain from sexual intercourse or to use an acceptable/effective method 
of contraception (ie, condom plus spermicide, condom plus oral contraceptive, 
condom plus intrauterine device, condom plus diaphragm with spermicide) from 
as soon as is feasible during the Screening period until 30 days following the last 
dose of AG-348.  
 
NOTE: Abstinence is an acceptable method only when this is in line with the 
normal lifestyle of the subject, meaning that the subject plans to remain abstinent 
continuously throughout the duration of the study and for ≥ 30 days after the last 
dose of study drug. Periodic abstinence (eg, calendar, symptothermal, and 
postovulation methods) and withdrawal are not acceptable methods of 
contraception. 
ii. 
Have negative serum or urine pregnancy test within 72 hours before start of 
AG-348 dosing. 
iii. 
Not be breastfeeding. 
13. If male (with the exception of subjects who have undergone vasectomy ≥ 6 months prior 
to Screening), agree to abstain from sexual intercourse or, if sexually active, to use a 
condom with spermicide as contraception (regardless of their female partner's 
childbearing potential or their partner's use of their own contraception) from Day 1 of 
dosing until 30 days following the last dose of AG-348.  
 
NOTE: Abstinence is an acceptable method only when this is in line with the normal life 
style of the subject, meaning that the subject plans to remain abstinent continuously 
throughout the duration of the study and for at least 30 days after the last dose of study 
drug. Periodic abstinence (eg, selective timing of intercourse based on partner’s calendar, 
symptothermal, and postovulation methods) and withdrawal are not acceptable methods 
of contraception. 
 
 


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 Extension Period 
8.2.2.
Subjects must meet all of the following criteria to be eligible for inclusion in the Extension 
Period of the study: 
1. Have provided signed written informed consent prior to undergoing any study procedure 
during the Extension Period. 
2. Have completed 24 weeks of treatment during the Core Period and tolerated AG-348 
(defined as having completed 24 weeks with or without protocol-permitted dose 
modifications). 
3. The treating Investigator agrees that there is a potential for clinical benefit to the subject 
from continued treatment and recommends participation in the Extension Period. 
4. The Sponsor's designated Medical Monitor or Responsible Medical Officer approves the 
subject’s participation in the Extension Period. 
5. If applicable, agree to continue to follow the same sexual abstinence/contraception rules 
as stated in Section 8.2.1, Inclusion Criterion 12 (for females) or 13 (for males). 
8.3. 
Exclusion Criteria 
 Core Period 
8.3.1.
Subjects who meet any of the following criteria will be excluded from the Core Period of the 
study: 
1. Have Hb level > 12.0 g/dL (if male) or > 11.0 g/dL (if female). 
2. Have an additional diagnosis of any other congenital or acquired blood disorder, 
including glucose-6-phosphate-dehydrogenase (G6PD) deficiency, or any other 
hemolytic anemia process, with the exception of mild allo-immunization as a 
consequence of transfusion therapy. 
3. Have iron overload (hemosiderosis or concurrent hemochromatosis) sufficiently severe to 
result in a clinical diagnosis by the Investigator of cardiac, hepatic, or pancreatic 
insufficiency. 
4. Have had prior bone marrow or stem cell transplant. 
5. Have clinically symptomatic cholelithiasis or cholecystitis.  
 
NOTES:  
i. 
Prior cholecystectomy is not exclusionary.  
ii. 
Subjects with symptomatic cholelithiasis or cholecystitis may be re-screened once 
the disorder has been treated and clinical symptoms have resolved. 
6. Be currently enrolled in another therapeutic clinical trial involving ongoing therapy with 
any investigational or marketed product or placebo. 
NOTE: Concurrent participation in the Pyruvate Kinase Deficiency Natural History 
Study (NCT02053480) is permitted. 


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7. Have been exposed to any investigational drug, device, or procedure within 28 days prior 
to Screening or during trial participation. 
8. Have any concurrent medical condition that could compromise participation in the study, 
such as: 
i. 
Poorly controlled hypertension (defined as systolic blood pressure [BP] 
> 150 mm Hg or diastolic BP > 90 mm Hg) refractory to medical management. 
ii. 
History of recent (within < 6 months from Screening date) congestive heart 
failure; myocardial infarction or unstable angina pectoris; hemorrhagic, embolic, 
or thrombotic stroke; deep venous thrombosis; or pulmonary or arterial embolism. 
iii. 
Currently active infection requiring the use of parenteral antimicrobial agents or 
of ≥ Grade 3 severity (per CTCAE v4.03) within 6 months of first dose of study 
drug. 
iv. 
Pattern or frequency of postsplenectomy sepsis that, in the assessment of the 
Investigator, could reasonably be expected to interfere with the ability of the 
subject to complete participation in the 24-week Core Period of the study. 
v. 
Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) 
antibody with accompanying signs of active hepatitis B or C infection. 
vi. 
Positive test for human immunodeficiency virus (HIV) 1 or 2 antibody. 
vii. 
Diabetes mellitus that is judged to be in poor control by the Investigator or which 
requires > 3 anti-diabetic agents, counting insulin (all insulins are considered 
1 agent). 
 
NOTE: Use of insulin per se is not exclusionary. 
viii. 
History of any primary malignancy, with the exception of: curatively treated 
nonmelanomatous skin cancer, curatively treated cervical or breast carcinoma in 
situ, or any other primary tumor treated with curative intent, and with no known 
active disease present and no anticancer treatment administered during the last 
3 years. 
9. Have undergone major surgery within 6 months of first dose of study drug. 
10. Have currently or have a recent history of a psychiatric disorder that, in the opinion of the 
Investigator or Medical Monitor, could compromise the ability of the subject to cooperate 
with study visits and procedures. 
11. Have used any of the restricted list of products known to strongly inhibit cytochrome 
P450 (CYP) 3A4 drug metabolism (Appendix 4.1) within 5 days prior to Day 1 dosing; 
products known to strongly induce CYP3A4 metabolism (Appendix 4.2) within 28 days 
prior to Day 1 dosing; products known to strongly inhibit P-glycoprotein (P-gp) 
transporter (Appendix 4.3) within 5 days prior to Day 1 dosing; or digoxin within 5 days 
prior to Day 1 dosing.  


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12. Have serum bilirubin > ULN attributable to factors other than hemolysis and/or Gilbert's 
syndrome. 
13. Have heart-rate corrected QT interval by Fridericia's method (QTcF) > 450 msec (for 
males) or > 470 msec (for females), with the exception of subjects with a left bundle 
branch block (LBBB), for whom Medical Monitor approval is needed to enroll. 
14. Have cardiac dysrhythmia that is judged clinically significant by the Investigator or 
which requires therapy with drugs that are primarily substrates of CYP3A4. 
15. Have any history of allergy to sulfonamides characterized by acute hemolytic anemia, 
anaphylaxis, or rash of erythema multiforme type or Stevens-Johnson syndrome. 
16. Have any other medical or psychological condition regarded by the Investigator as likely 
to interfere with subject's ability to understand and provide signed written informed 
consent; cooperate with study visits, tests, and procedures; and/or otherwise safely and 
reliably participate in the study. 
 Extension Period 
8.3.2.
Subjects who meet this criterion will be excluded from the Extension Period of the study:  
1. Have experienced any AE during the Core Period considered by the treating Investigator 
or the Sponsor’s designated Medical Monitor or Responsible Medical Officer to pose a 
significant safety risk should study treatment be extended. 
8.4. 
Subject Identification and Registration 
Subjects who are candidates for enrollment into the study will be evaluated for eligibility by the 
Investigator to ensure that the inclusion and exclusion criteria (see Section 8.2 and Section 8.3, 
respectively) have been satisfied and that the subject is eligible for participation in this clinical 
study. The site will submit to the Sponsor an Eligibility form for each eligible subject and the 
Medical Monitor will confirm eligibility for all subjects prior to receipt of the first dose of 
AG-348. 
8.5. 
Subject Randomization 
Subjects who have been confirmed as eligible will be randomized in an equal ratio to a treatment 
arm (eg, 1:1 or 1:1:1 depending on which arms are open). The site will provide a request for 
randomization form (including the subject’s confirmed genotype) to the study Medical Monitor. 
The randomization will be stratified by PKR mutation in order to maintain balance across the 
dose arms for the specific mutations expected to be most frequently enrolled. The PKR mutation 
stratification factor will consist of 4 levels (R510Q, R486W, and R479H, and all other mutations 
as “other”). Since this is an open label study, randomization will not be blinded. 
Please refer to the study manual for the randomization procedure. 
8.6. 
Subject Withdrawal Criteria 
Subjects have the right to withdraw from the study at any time for any reason. Subjects will be 
withdrawn from study-related procedures and treatments under any of the following conditions: 
 


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• Withdrawal of consent. 
• Experiences unacceptable toxicity. 
• Development of an intercurrent medical condition that precludes further participation 
in the trial. 
• Subject requires use of a prohibited concomitant medication (Section 9.11.2.1). 
• Investigator decision 
• Protocol violation (nonadherence to protocol requirements). 
• Pregnancy. 
• Lost to follow-up. 
Should a subject decide to withdraw, all efforts will be made to complete and report the protocol-
defined study observations up to the time of the subject’s withdrawal as completely as possible 
and to determine the reason for withdrawal. 
In the event a subject is withdrawn from the study, the Medical Monitor must be informed. If 
there is a medical reason for withdrawal, the subject will remain under the supervision of the 
Investigator until the AE resolves, is declared chronic by the Investigator, or the subject is lost to 
follow-up. 
When a subject withdraws from the study, the primary reason for discontinuation, if known, 
must be recorded in the appropriate section of the electronic case report form (eCRF) and all 
efforts will be made to complete and report final study observations as thoroughly as possible. 
All AEs should be followed until resolution or for a period of 30 days from the last dose of study 
drug, whichever is shorter. Subjects with toxicity suspected to be related to study drug will 
continue follow-up until the AE resolves, is declared chronic by the Investigator, or the subject is 
lost to follow-up. 
8.7. 
Replacement of Subjects 
Subjects who drop out of the Core Period prior to completing the first 12 weeks of assigned 
dosing for reasons other than AEs may be replaced at the Sponsor’s discretion. 


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9. 
STUDY TREATMENT 
9.1. 
Description of Study Drug 
AG-348 sulfate hydrate capsules will be provided as 5 mg, 25 mg, or 100 mg (free-base 
equivalent) of AG-348 sulfate hydrate drug substance without excipients in hard gelatin 
capsules. 
All study drugs are for investigational use only and are to be used only within the context of this 
study. All study drug products will be supplied by the Sponsor. The Sponsor reserves the right to 
discontinue the supply of any specific capsule strength should the evolving trial experience 
demonstrate that the specific capsule strength fills no additional need beyond the other capsule 
strengths available. Please see the Investigator’s Brochure for further details regarding study 
drug. 
9.2. 
Study Drug Packaging and Labeling 
AG-348 sulfate hydrate capsules are packaged in white, high-density polyethylene (HDPE) 
induction sealed bottles with a child-resistant screw cap. 
Packaging and labeling will be prepared to meet all regulatory requirements. 
9.3. 
Study Drug Storage 
The recommended storage condition and expiry (where required) are stated on the product label. 
All study drug products must be stored in a secure, limited-access location and may be dispensed 
only by the Investigator or by a member of the staff specifically authorized by the Investigator. 
9.4. 
Method of Assigning Subjects to Treatment 
Up to a maximum of 25 subjects will be randomized to any 1 of the dosing arms in this study. 
Randomization will be balanced 1:1 or 1:1:1, depending on whether 2 or potentially 3 arms are 
open, and will be stratified by PKR mutation (see Section 8.5). The dose and schedule of 
AG-348 each subject receives will be dependent upon which dose arm is open for enrollment 
when the subject qualifies for and is randomized into the study. Subjects in the Extension Period 
will continue on the dose they were randomized to in the Core Period, unless the DRT had 
reason to establish a different dose/schedule during the course of the Core Period (the DRT will 
not propose a dose higher than 360 mg BID).  
9.5. 
Blinding 
This is an open-label study; no blinding methods will be used. 
9.6. 
Study Drug Preparation and Administration 
For the initial 2 treatment arms, (Arm 1 and Arm 2) in the Core Period, AG-348 will be 
administered PO BID (approximately q12h, with a minimum of 10 hours between doses) over a 
24-week treatment period. Starting with Day 1, dosing will be continuous; there will be no rest 
periods. Subjects who do not meet any of the treatment withdrawal criteria (see Section 8.6) may 
continue treatment for the entire 24-week treatment period. 


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Subjects will be dispensed the appropriate number of Sponsor-packaged, labeled bottles to allow 
for dosing until the next scheduled visit. The amount of study drug dispensed should be 
sufficient to provide an adequate reserve supply of AG-348 to ensure uninterrupted dosing in the 
event of an unexpected delay for the next scheduled study visit (seven extra days of dosing 
supply are recommended during the Core Period; 14 extra days is recommended during the 
Extension Period). 
Subjects will be given a dosing diary to be used for each 28-day dosing period. They should 
record relevant information regarding their study drug in the diary (eg, confirmation that each 
daily dose was taken, reasons for missed doses). 
Treatment compliance will be assessed based on return of unused drug and the dosing diary (see 
Section 9.9). 
Subjects should be instructed to take their daily dose at approximately the same times each day 
except for dosing on in-clinic visiting days.  
Subjects who undergo extensive PK/PD sampling during the Core Period (see Appendix 1.3) 
should be instructed from Week 3 on to bring the AM dose with them for in-clinic visits and to 
ingest the dose following PK/PD blood draws.  
Subjects receiving limited PK/PD sampling during the Core Period (see Appendix 1.4) should be 
instructed to bring the AM dose with them for all in-clinic visits and to take the AM dose 
following PK/PD blood draws.  
Subjects receiving extensive PK/PD sampling on Day 1 and 15 will also have limited PK/PD on 
other visit days. As a general rule, regardless of extensive or limited schedule, subjects will bring 
in the AM dose for all visits and take this dose following PK/PD blood draws. Subjects not 
continuing into the Extension Period are not required to take the Week 24/Day 169 morning dose 
of AG-348 after the required PK/PD blood samples are collected, as these subjects will be 
discontinuing the study. 
A minimum of 10 hours between the AM and PM dose will be required on those dosing days. 
Each dose should be taken with a glass of water and consumed over as short a time as possible. 
AG-348 may be taken with or without food. Subjects should be instructed to swallow capsules 
whole and to not chew the capsules. For subjects who have difficulty swallowing tablet(s), the 
Medical Monitor should be contacted to discuss administration. 
Subjects will receive their first dose of AG-348 in the clinic on Day 1 and then may take the 
remaining doses on an outpatient basis (except for in-clinic visit days, as described above). 
9.7. 
Criteria for Dose Escalation, Dose Modification, or Discontinuation 
of Study Drug 
Intra-subject dose escalations will be permitted in this study under 2 circumstances. First, the 
DRT may decide to re-assign subjects’ doses and schedule in a terminated arm to match the dose 
and schedule of another arm of the study. In this case, the subjects in the terminated arm will 
remain in their originally assigned arm; ie, they will not count towards the enrollment quota of 
the arm whose dose and schedule is being adopted.  


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Second, a treating Investigator, with Medical Monitor approval, may elect to increase the dose 
for a subject in Arm 2 (50 mg BID) or a potential third arm of the study (if < 300 mg BID) to 
300 mg BID if the subject is adequately tolerating his/her current dose and if their Hb has not 
reached at least the lower limit of the normal gender-adjusted reference range as specified by the 
designated central laboratory (male: 13.0 g/dL; female: 11.6 g/dL) after at least 12 weeks of 
treatment in the Core Period. The subject must have completed the 12-week visit during the Core 
Period and had all assigned tests/procedures for that visit before an intra-subject dose escalation 
will be allowed. An intra-subject dose escalation may also be made later than the 12-week visit 
in the Core Period.  
Subjects in the Extension Period who have not experienced a robust and sustained increase in 
hemoglobin by the next scheduled visit, in particular after a dose increase, should be encouraged 
to discontinue AG-348 if the only benefit of continued treatment is reduction in 
jaundice/bilirubin levels and/or improved feeling of wellbeing. 
Dosing modifications, as outlined below, will be implemented following discussions with the 
Medical Monitor.  
Subjects should be advised not to discontinue dosing without first speaking with the treating 
Investigator—abrupt discontinuation of AG-348 dosing in a subject who experience a substantial 
increase in Hb may result in withdrawal hemolysis. 
 Dose Modification for Safety 
9.7.1.
The Investigator will monitor all subjects for safety and tolerability. Modification of an 
individual subject’s dose of AG-348 will be based on AEs and observed changes in Hb levels 
(see Section 9.7). 


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Table 1: 
Dose Modification for Adverse Events Deemed at Least Possibly Related  
to AG-348 
Adverse Event(s) 
Severity1 
AG-348 Dose Adjustment 
Grade 1 
None required. 
Grade 2 
None required; use Investigator and Medical Monitor judgment to manage, as for  
Grade 3 events (see below). 
Grade 3 
Suspend dosing2. 
If event resolves to Grade 1 or baseline within approximately 14 days of suspension, 
resume dosing with at least a 1 dose-level reduction and discuss the advisability of 
additional dose modification with the Medical Monitor (see Table 2). 
If event does not resolve to Grade 1 or baseline within approximately 14 days of 
suspension, permanently discontinue dosing, unless the benefits outweigh the risks of 
resuming treatment in the opinion of the Investigator as agreed upon by the Medical 
Monitor. 
Grade 4 
Permanently discontinue dosing, unless the benefits outweigh the risks of resuming 
treatment in the opinion of the Investigator as agreed upon by the Medical Monitor. 
1. 
Per National Cancer Institute Common Terminology Criteria for Adverse Events. 
2. 
A related adverse event of Grade 3 severity would generally be expected to require suspension of dosing with AG-348.   
  
However, in some circumstances in which the hemoglobin concentration has normalized prior to the onset of the related  
  
adverse event, the Investigator may elect to discuss with the Medical Monitor tapering the dose of AG-348 in order to   
  
mitigate potential withdrawal hemolysis (see Section 5.2.4.1 and Section 9.7.2). 
Dose modifications for Grade 2 and 3 AEs should be maintained long enough for the 
Investigator to be confident that the reduced dose is being well-tolerated before considering 
contacting the Medical Monitor to approve re-escalation to the former dose level. Dosing for an 
individual subject will be discontinued permanently for Grade 3 and Grade 4 AEs that do not 
resolve to Grade 1 or baseline within approximately 14 days of suspension of dosing, unless the 
benefits outweigh the risks of resuming treatment and are approved by the Medical Monitor.  
No subjects may be re-escalated to their former dose level after a dose modification without 
discussion with the Medical Monitor. If, following the first dose reduction for a Grade 3 AE, the 
subject experiences a second occurrence of the same Grade 3 AE, then treatment with AG-348 
must be immediately and permanently discontinued. However, in any subject having experienced 
a robust and sustained increase in Hb, AG-348 discontinuation should be done after progressive 
dose decrease (assuming safety considerations allow it) to avoid withdrawal hemolysis. 
It should be noted that if the initial dose of 300 mg BID selected for Arm 1 demonstrates an 
unacceptable safety profile resulting in multiple subjects undergoing dose modifications, the 
DRT may exercise its option to re-assign these subjects’ dose and schedule to match the dose and 
schedule of another study arm (eg, Arm 2 of the study, or to match the dose and schedule of a 
[potential] Arm 3, if implemented). 


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 Dose Modification for Increase in Hemoglobin Level 
9.7.2.
It is presently unknown to what magnitude, how rapidly, or even whether, AG-348 will result in 
increased Hb levels in subjects with PK deficiency across the variety of potential PKR mutations 
that may be encountered during this study. Moreover, it is unknown whether any potential 
increases in Hb experienced by subjects in this study may be a safety risk.  
As a conservative measure, this study incorporates guidance for dose modification based on 
potential large increases in Hb levels that exceed the midpoint of the typical normal range by 
gender. The intent of this guidance is to strike a balance between abruptly stopping AG-348 
treatment, thereby potentially risking withdrawal hemolysis, versus reducing the dose 
sufficiently to allow an opportunity for Hb to settle more gradually to a level less than the 
midpoint of the typical normal range by gender.  
The Investigator will monitor all subjects for changes in Hb levels and should adjust the dose of 
AG-348 as outlined in Table 2, per the following guidelines and with Medical Monitor approval. 
• Males: If Hb > 15.0 g/dL and confirmed with a second test, reduce dose by at least 
1 dose level (Table 2) and discuss the advisability of additional dose modification or 
suspension with the Medical Monitor. 
• Females: If Hb > 13.5 g/dL and confirmed with a second test, reduce dose by at least 
1 dose level (Table 2) and discuss the advisability of additional dose modification or 
suspension with the Medical Monitor. 
• The treating Investigator will discuss with the Medical Monitor questions relating to 
additional dose modifications and the need for additional unscheduled Hb monitoring 
on an as needed basis.  
Table 2: 
Dose Reduction Table (by Dosing Arm) 
Dosing Arm 
Starting Dose 
1st Dose Reduction 
2nd Dose Reduction 
Arm 1 
300 mg BID 
200 mg BID 
100 mg BID 
Arm 2 
50 mg BID 
25 mg BID 
TBD1 
Potential Arm 3 
TBD 
To approximately 50-66% 
of initial dose. 
To approximately 25-33% 
of initial dose. 
1.  Dose to be determined by Medical Monitor. 
Abbreviations: BID = twice daily; TBD = to be determined. 
Hemoglobin levels above the ULN (by gender) should be reported as AEs and graded per the 
CTCAE v4.03, according to the guidance provided in Section 11.2. 
 
 


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 Individual Optimal Maintenance Dose-Finding in Extension Period 
9.7.3.
9.7.3.1. 
Gradual Dose-taper regimen in Extension Period 
All subjects in the Extension Period who are receiving doses > 25 mg BID will undergo an 
individual and gradual dose-taper regimen to identify their individual optimal maintenance 
doses, defined as the dose that results in ≤ 1.0 g/dL decrease in hemoglobin compared to the 
pretaper Hb value on at least 2 weekly measurements following each dose taper step. The 
minimum dose target is 25 mg BID.  
Table 3 shows the various dose levels to be used. Other dose levels should not be used during the 
dose taper. 
Table 3: 
Gradual Dose-Taper Regimen (by Actual Dose) 
Starting Dose 
1st Dose Reduction 
2nd Dose Reduction 
3rd Dose Reduction 
4th Dose Reduction 
300 mg BID 
200 mg BID 
100 mg BID 
50 mg BID 
25 mg BID 
200 mg BID 
100 mg BID 
50 mg BID 
25 mg BID 
N/A 
100 mg BID 
50 mg BID 
25 mg BID 
N/A 
N/A 
50 mg BID 
25 mg BID 
N/A 
N/A 
N/A 
Abbreviations: BID = twice daily; N/A = not applicable. 
After each dose reduction, the subject will remain on the reduced dose for a period of 3 weeks. 
Hb will be measured 1 week and 2 weeks after the start of the 1st reduced dose. If Hb (mean of 
these 2 Hb levels) has decreased by ≤ 1.0 g/dL from the pretaper value, the dose should be 
decreased to the next level at the 3 week visit, and the process repeated. If, at any step during this 
process, Hb (mean of levels after 1 week and 2 weeks at a reduced dose) has decreased by 
> 1.0 g/dL below the pretaper Hb value, the subject’s dose should be increased to the next higher 
dose level.  
Table 4 shows the Schedule of Assessments during the dose-taper regimen. The Hb values used 
to guide dose taper decisions (pre-dose taper, after 1 and 2 weeks of dose taper at each step) 
should be those provided by the central laboratory. In exceptional cases when these central 
laboratory Hb values are not available at the visit 3 weeks after the start of a tapering step, Hb 
values from the local laboratory may be used, provided that the pre-dose taper value from the 
same local laboratory is available for comparison. 
Blood sampling for weekly Hb monitoring during the gradual dose-taper regimen may be 
performed at the subject’s home by a visiting nurse, at the local physician’s office, or at a local 
laboratory, with the samples being shipped promptly to the central laboratory.  
 
 


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Table 4:  
Schedule of Assessments for Gradual Dose-Taper Regimen in the Extension Period1 
 
1st dose reduction 
2nd dose reduction 
(if applicable) 
3rd dose reduction 
(if applicable) 
4th dose reduction 
(if applicable) 
Study day 
Next 
scheduled 
visit in 
Extension 
Period 
7 days 
after 1st 
dose 
reduction2 
14 days 
after 1st 
dose 
reduction2 
21 days 
after 1st 
dose 
reduction 
7 days 
after 2nd 
dose 
reduction2 
14 days 
after 2nd 
dose 
reduction2 
21 days 
2nd dose 
reduction 
7 days 
after 3rd 
dose 
reduction2 
14 days 
after 3rd 
dose 
reduction2 
21 days 
after 3rd 
dose 
reduction 
7 days 
after 4th 
dose 
reduction2 
14 days 
after 4th 
dose 
reduction2 
21 days after 
4th dose 
reduction 
Visit window 
± 2 
weeks 
± 2 days 
± 2 days 
± 2 days 
± 2 days 
± 2 days 
± 2 days 
± 2 days 
± 2 days 
± 2 days 
± 2 days 
± 2 days 
± 2 days 
Procedure 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dose 
reduction 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
 
Hematology/ 
haptoglobin3 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
1. 
All subjects in the Extension Period will undergo a gradual dose-taper regimen starting at the next scheduled clinic visit, as per Table 3 in Section 9.7.3.1. Follow the Schedule of Assessments 
 
for the appropriate assessments to be conducted along with the 1st dose reduction visit. 
2. 
For greater subject convenience, blood samples at the time points displayed in shaded columns may be performed at home by qualified study personnel (eg, home health care  nurse), at the subject’s 
 
local  physician office, or at a local lab, with the samples being shipped promptly to the central lab. 
3. 
Hematology parameters include haptoglobin and complete blood count (CBC), with the latter to include hematocrit, hemoglobin (Hb), red blood cell count, absolute reticulocyte count, percent 
 
reticulocyte count, mean corpuscular volume, mean corpuscular Hb, mean corpuscular Hb concentration, red cell distribution width, nucleated red blood cell count, white blood cell count with 
 
differential, absolute neutrophil count, absolute lymphocyte count, and platelet count.


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 Stopping Criteria 
9.7.4.
Dosing for an individual subject will be discontinued permanently for Grade 3 AEs that do not 
resolve to Grade 1 or baseline within approximately 14 days of suspension of dosing, and for 
Grade 4 AEs, unless the benefits outweigh the risks of resuming treatment and are approved by 
the Medical Monitor (Section 9.7.1). Other reasons for treatment termination are provided in 
Section 8.6. 
9.8. 
Duration of Subject Participation 
The duration of treatment for all subjects will be up to 24 weeks in the Core Period. Subjects 
who safely tolerate AG-348 and for whom the Investigator agrees with continuation of AG-348 
treatment may be eligible to immediately roll over to the Extension Period for continued 
treatment (up to 2 years following completion of the Core Period). 
9.9. 
Treatment Compliance 
During in-clinic visits, doses of AG-348 will be ingested by the subject under the supervision of 
clinical facility personnel. For at-home dosing, subjects will be given a dosing diary to be used 
for the duration of the 24-week Core Period; the diary will also be used by subjects who roll over 
to the Extension Period. Subjects should record relevant information regarding their study drug 
in the diary (eg, confirmation that each daily dose was taken, reasons for missed doses) and 
return the diary at each study visit. 
9.10. 
Study Drug Accountability 
Accountability for the study drug at the clinical facility is the responsibility of the Investigator. 
The Investigator will ensure that the study drug is used only in accordance with this protocol. 
Where allowed, the Investigator may choose to assign drug accountability responsibilities to a 
pharmacist or other appropriate individual. 
The Investigator or delegate will maintain accurate drug accountability records indicating the 
drug’s delivery date to the site, inventory at the site, use by each subject, and return to the 
Sponsor or its designee (or disposal of the drug, if approved by the Sponsor). These records will 
adequately document that the subjects were provided the doses as specified in the protocol and 
should reconcile all study drug received from the Sponsor. Accountability records will include 
dates, quantities, batch/serial numbers, expiration dates (if applicable), and subject numbers. A 
monitor will review drug accountability at the site on a schedule agreed to by the Sponsor. 
Study drug must not be used for any purpose other than the present study.  
All unused and used study drug will be retained at the site until it is inventoried by the Study 
Monitor after database lock has occurred. All used, unused or expired study drug will be returned 
to the Sponsor or its designee or, if authorized, disposed of at the study site per the site’s 
Standard Operating Procedures and documented. All material containing AG-348 will be treated 
and disposed of as hazardous waste in accordance with governing regulations. 


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9.11. 
Prior and Concomitant Medications and Treatments 
 Prior Medications and Procedures 
9.11.1.
All medications administered and procedures conducted within 28 days prior to, or from 
Screening Visit #1 until the first day of study drug administration, whichever interval is longer, 
are to be recorded on the source documentation and included in the eCRF. 
 Concomitant Therapy 
9.11.2.
All concomitant medications and procedures administered from 28 days prior to, or from 
Screening Visit #1 until, the first day of study drug administration (whichever interval is longer) 
through the last Follow-up Visit must be recorded in the appropriate section of the source 
documentation and eCRF, along with dosage information, dates of administration, and reason for 
use.  
Investigational drugs must be discontinued no less than 28 days prior to the first dose of study 
drug. 
In vitro studies using human liver microsomes and recombinant CYP enzymes have shown that 
AG-348 is primarily metabolized by CYP3A4 (>70%), with minor contributions from CYP2C9, 
CYP2C8, and CYP1A2. In addition, AG-348 has been shown to be a weak time-dependent 
CYP3A4 inhibitor and a potential inducer of CYP3A4 and CYP2B6 in vitro. In vitro transporter 
studies have shown that AG-348 is a substrate and inhibitor of P-gp. Based on these results, 
below is a list of concomitant therapy to be avoided and concomitant therapy requiring careful 
monitoring. 
Since AG-348 exhibits pH-dependent solubility, proton-pump inhibitors and H2-receptor 
antagonists may decrease the absorption of AG-348. 
9.11.2.1. Concomitant Therapy to be Avoided (Prohibited) 
The following therapies are contraindicated during this study: 
• Strong inhibitors of CYP3A4 (listed in Appendix 4.1). 
• Products known to inhibit CYP3A4, such as grapefruit or grapefruit juice. 
• Strong inducers of CYP3A4 (listed in Appendix 4.2). 
• Strong inhibitors of P-gp (listed in Appendix 4.3). 
• Digoxin, a P-gp transporter-sensitive substrate. 
If a subject is taking any medication listed in Appendices 4.1, 4.2, or 4.3 and/or digoxin prior to 
enrolling in the study, the medication must be discontinued within 5 days prior to Day 1 dosing. 
• Hematopoietic stimulating agents (eg, EPOs, granulocyte colony stimulating factors, 
thrombopoietins) must be discontinued no fewer than 28 days prior to the first dose of 
study drug. (Folic acid 1 mg PO per day is required for all subjects. B12 injections are 
permitted for subjects with a prior diagnosis of B12 deficiency syndromes, but must 
be repleted to stability of the Hb and mean corpuscular volume [MCV] prior to 
enrollment in the study). 


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• Anabolic steroids, including testosterone preparations, administered for anemia must 
be discontinued no less than 28 days prior to the first dose of study drug. 
• As the target population for this study consists of transfusion independent subjects 
and transfusion of blood products could confound key endpoints of the study, blood 
transfusions of any type must be strictly avoided except in cases of compelling 
medical need. If medical circumstances permit, the Medical Monitor should be 
contacted for discussion before any transfusions are administered. 
9.11.2.2. Concomitant Therapy Requiring Careful Monitoring (Use with Caution) 
The following therapies should be avoided and replaced with alternative treatments. If this is not 
possible, subjects receiving these drugs should be adequately monitored. 
• Corticosteroids (sensitive substrates of CYP3A4 and weak CYP3A4 inducers). 
• Sensitive substrates of CYP3A4 (listed in Appendix 4.4). 
• Moderate inhibitors of CYP3A4 (listed in Appendix 4.1). 
• Sensitive substrates of CYP2B6 (listed in Appendix 4.5). 
• Proton-pump inhibitors and H2-receptor antagonists (listed in Appendix 4.6) should be 
used with caution. Antacids such as magnesium hydroxide and aluminum hydroxide can 
be used with AG-348. 
• AG-348, being a potential CYP3A4 inducer, has the potential to reduce the effectiveness 
of oral contraceptives. Therefore, women using oral contraceptives must also utilize a 
barrier method while enrolled in the study and till at least 30 days after their last dose of 
study drug, as specified in Inclusion Criterion 12 (see Section 8.2). 
• Drugs that displace unconjugated bilirubin from albumin (including some common sulfa 
antibiotics [including sulfamethoxazole/trimethoprim], cephalosporins, salicylates, and 
aminophylline) should be used with caution with the understanding that subjects with 
elevated levels of unconjugated bilirubin may potentially be at risk for kernicterus 
syndrome (Strauss, et al. 2006). 
9.11.2.3. Allowed Concomitant Therapy 
Medications and treatments other than those specified above are permitted during the study. All 
intercurrent medical conditions will be treated at the discretion of the Investigator according to 
acceptable local standards of medical care. The expected subject comedications deferoxamine, 
deferasirox, deferiprone, and oral penicillin are not expected to interact with AG-348. Subjects 
may receive analgesics, anti-emetics, anti-infectives (including penicillins), and antipyretics as 
medically indicated and consistent with the guidance in the previous 2 sections. Subjects must 
continue taking at least 1 mg of folic acid for the duration of the study. 
All concomitant medications, including transfusions of blood products, procedures performed 
during the study, including those used to treat AEs, will be recorded on the eCRF.  


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9.12. 
Management of Nausea, Vomiting, and Diarrhea 
As the primary objective of this study is to assess the safety and tolerability of up to 24 weeks of 
AG-348, routine use of prophylactic anti-emetic and antidiarrheal or other classes of medications 
is prohibited. However, if subjects experience AEs relating to nausea, vomiting, or diarrhea, 
these may be treated at the Investigator's clinical discretion with recommended medications as 
follows: 
• Nausea/vomiting: allowed agents include standard clinical dosing with palonosetron 
(Aloxi), dolasetron (Anzemet), or promethazine (Phenergen). Not recommended are 
aprepitant (Emend; CYP3A4 inhibitor), ondansetron (Zofran; CYP3A4 inducer), 
chlorpromazine (Thorazine; CYP3A4 inducer), prochlorperazine (Compazine; 
CYP3A4 substrate, QT prolongation has been reported), and granisetron (Kytril; 
CYP3A4 substrate, QT prolongation has been reported). 
• Diarrhea: recommended management includes standard clinical dosing with 
Kaopectate or other nonabsorbable anti-diarrheals, diphenoxylate/atropine (Lomotil), 
or loperamide (Imodium). Loperamide is the least preferred choice because it is both 
a substrate and inhibitor for CYP3A4, a substrate for CYP2B6, and a substrate for 
P-gp. 
• For the use of any medications not specifically mentioned above the Investigator may 
confer with the Sponsor’s Medical Monitor. 


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10. 
STUDY ASSESSMENTS 
10.1. 
Schedules of Assessments 
The Schedules of Assessments for this study are provided in Appendix 1.   
After obtaining written informed consent, subjects will undergo screening evaluations. The 
Screening visit is to be conducted within approximately 42 days prior to first dose of study 
treatment.  
During the Core Period, subjects will attend visits at baseline (Day 1), weekly through Week 3 
(Days 8, 15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12) and 
monthly through Week 24 (Weeks 16, 20, and 24). Subjects who safely tolerate AG-348 through 
Week 24 (Core Period) and for whom the Investigator agrees with continuation of AG-348 
treatment may be eligible to immediately enter the Extension Period for continued treatment 
upon agreement of the treating Investigator and the Medical Monitor or Responsible Medical 
Officer. Study visits for safety and clinical activity assessments will occur approximately every 
3 months during the Extension Period. All subjects will undergo a follow-up assessment 4 weeks 
after the last dose of AG-348, regardless of whether this was due to discontinuation, the last dose 
in the Core Period for a subject who chooses not to continue in the Extension Period, or the last 
dose of the Extension Period, except for those who may continue treatment in an extension study 
(to be designed) after having completed the Extension Period. 
Although not encouraged, as a convenience for subjects who travel long distances to the study 
site, in-clinic visits on Day 8 and Day 22 may be performed by the subject’s primary care 
physician if necessary and must be approved by the Sponsor on a case-by-case basis. For details, 
please refer to Appendix 1.1. For subjects having their Day 8 and/or Day 22 visits performed by 
the primary care physician, the principal investigator will him or herself, or have a qualified 
research nurse or other designated site staff member, make telephone contact with the subject to 
inquire about any AEs. These must be recorded as if the subject appeared in the main study 
center. The Principal Investigator will exercise prudent clinical judgment in determining any 
clinical course of action to take based on any AEs discovered. These telephone contacts on Day 
8 and Day 22 must be explained to the subject in advance and scheduled in advance to maximize 
the likelihood of successfully making contact.  
Having in-clinic visits on Days 8 and/or Day 22 performed by the primary care physician will 
necessitate re-scheduling certain assessments that the primary care physician’s office may not be 
reasonably expected to perform. Table 5 summarizes the details of the re-scheduling of these 
assessments as described in Appendix 1.1. 
During the Extension Period, all scheduled visits, with the exception of blood draws for those 
subjects undergoing gradual dose-taper regimen, must be conducted by the Investigator and at 
the participating authorized investigative site; local primary care visits will not be allowed. 


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Table 5: 
Summary of Assessments when Day 8 and/or Day 22 In-Clinic Visits are 
Performed by Primary Care Physician (Core Period) 
 
Day 8 Visit by Primary Care Physician 
Day 22 Visit by Primary Care Physician 
Primary 
care office 
Main 
study 
site 
Assessment 
to move to 
Day 15 
(main study 
site) 
Assessments not 
required 
Primary 
care office 
Main 
study 
site 
Assessment 
to move to 
Day 43 
(main 
study site) 
Assessments not 
required 
Hematology 
to central 
laboratory 
Phone 
contact 
with 
subject 
12-lead 
ECG 
VS; serum 
chemistry 
 
Hematology 
sample to 
central 
laboratory 
Phone 
contact 
with 
subject 
12-lead 
ECG 
VS; serum chemistry; 
coagulation; 
haptoglobin; EPO 
level; carboxy-
hemoglobin; PK/PD 
Abbreviations: ECG = electrocardiogram; EPO = erythropoietin; PK/PD = pharmacokinetics/pharmacodynamics; VS = vital 
signs. 
Whenever more than 1 assessment is scheduled for the same nominal time, the assessments 
should be performed in the order of least invasive to most invasive assessment (eg, VS first, 
ECG, blood draw). The timing of these assessments should allow the PK blood draw to occur at 
the exact nominal time. The order of procedures may be revised with prior discussion between 
Sponsor and Site. 
Minor adjustments to the timing, number of planned safety monitoring procedures (eg, VS, ECG, 
blood draw), and PK/PD assessments may be made during the course of the study based on 
collected data to ensure appropriate safety monitoring and will not require a protocol 
amendment. These minor changes will require prior approval from the Sponsor’s Medical 
Monitor (or Responsible Medical Officer) as well as appropriate documentation in the study 
records. The addition of new safety monitoring procedures or other assessments will require a 
protocol amendment. 
10.2. 
Informed Consent and Confirmation of Eligibility 
A complete description of the study is to be presented to each potential subject and a signed and 
dated informed consent is to be obtained before any study specific procedures are performed. 
Separate informed consent forms will be used for the Core and Extension Periods. 
Subject’s eligibility will be confirmed at Screening and within 24 hours prior to study treatment 
dose. If a subject is determined to be ineligible, the subject will be excluded from participation. 
10.3. 
Demographic Data, Medical and Medication History 
Subject demographic data, including gender, date of birth, age, race, and ethnicity, will be 
obtained at Screening. Collection of demographic data will be modified by country regulatory 
requirements, as appropriate.  
Medication history, including all relevant prior medical history and current medical conditions, 
will be obtained at the Screening assessment and on Day -1. 


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All concomitant medications administered and procedures conducted within 28 days prior to, or 
from Screening Visit #1 until the first day of study drug administration, whichever interval is 
longer, should be reported in the source documentation and eCRF.  
Investigators will be asked to provide information on the subject’s history of any medical 
diagnoses (eg, iron overload) and surgical procedures (eg, splenectomy, cholecystectomy) 
pertaining to their diagnosis of PK deficiency and prior available CBCs over the preceding 
6 months and transfusion history over the preceding 12 months prior to the date of signing 
informed consent.  
10.4. 
PKR Enzymatic Assay and PKR Genotyping 
Assessments for PKR enzymatic activity and PKR genotyping will be performed at Screening 
only for confirmation of study eligibility. PKR enzymatic assays will be conducted at 
 
 or any participating investigative site’s local hematology 
laboratory. PKR genotyping will be conducted at 
. 
10.5. 
Safety Assessments 
  
Physical Examination, Height, and Weight 
10.5.1.
A complete physical examination (including neurological examination; genital and rectal 
examinations will be performed at the discretion of the Investigator) will be obtained at 
Screening, Baseline, Week 12, and the Follow up Visit (Week 28). The neurological examination 
must include an assessment of general orientation and mental status including level of alertness 
(assess as normal or abnormal and specify any abnormality).  
Limited, focused physical examinations will be performed at all other visits during the Core 
Period. Focused physical examinations, including neurological examination, will continue every 
3 months during the Extension Period; a complete physical examination will be performed at 
Month 30.  
Any findings will be recorded on the eCRF.  
Height will be collected at Screening only. 
  
Vital Signs 
10.5.2.
Vital signs, including systolic and diastolic BP, heart rate, respiratory rate, and temperature, will 
be obtained according the Schedules of Assessments (Appendix 1). 
 
Vital sign measurements will be repeated if clinically significant or machine/equipment errors 
occur. Out of range BP or heart rate measurements will be repeated at the Investigator’s 
discretion. Any confirmed, clinically significant vital sign measurements will be recorded as 
AEs. 
  
Electrocardiogram  
10.5.3.
A 12-lead ECG will be obtained according to the Schedules of Assessments (Appendix 1). The 
ECGs will be measured using an ECG machine that reports the heart rate and the portion of the 
ECG wave from the beginning of the P wave to the beginning of the QRS complex (PR), QRS, 


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QT, QTcB (Bazett correction formula; may also be calculated), and QTcF intervals (may also be 
calculated). Only QTcF (not QTcB) will be used for determination of eligibility. 
The 12-lead ECGs should be obtained following 5 minutes of recumbency. ECGs will be 
repeated if clinically significant abnormalities are observed, if artifacts are present, or if 
machine/equipment errors occur. Any confirmed, clinically significant ECG findings will be 
recorded as AEs. 
  
DXA Scans 
10.5.4.
DXA scans (hip and spine) will be performed at Screening to obtain T and Z scores and bone 
mineral density that will serve as a baseline measure for all enrolling subjects. An additional 
DXA scan for the Core Period will be conducted in the interval between Week 24 and Week 28, 
and scans will be conducted at Months 12, 18, 24, and 30 during the Extension Period as 
indicated in the Schedules of Assessments (Appendix 1.2). All redacted DXA scan reports must 
be held at the study site and will be collected by the Sponsor. 
  
Safety Laboratory Assessments 
10.5.5.
10.5.5.1. Hematology, Serum Chemistry, Coagulation Studies, and Urinalysis 
Laboratory values obtained prior to Screening and RBC antibodies obtained at Screening will be 
performed at a local laboratory. On-study clinical laboratory evaluations are to be performed by 
a central laboratory. If Investigators believe that it is clinically indicated to obtain safety 
laboratory results from their own local laboratories on the day of the subject's visit, and before 
the results are returned from the designated central laboratory, they are free to exercise their 
discretion to do so. Investigators should be aware that since the Hb level is a secondary endpoint 
of the study, it behooves them to minimize blood volumes drawn, and samples for the central lab 
must still be collected, since it will serve as the official lab result for this study. 
Clinical laboratory evaluations are to be collected according to the Schedules of Assessments 
(Appendix 1). In addition, all clinically significant laboratory abnormalities noted on testing will 
be followed by repeat testing and further investigated according to the judgment of the 
Investigator. Please note that serum estradiol, free and total testosterone, and CBC will be 
collected in the AM at any 2 time points during Screening at least 2 days apart in addition to 
Baseline/Day 1 (total of 3 time points prior to Day 1 dosing).  
The safety laboratory parameters listed below are to be determined. 
 


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Hematology: 
HCT, Hb, RBC count, absolute reticulocyte count, percent 
reticulocyte count, MCV, mean corpuscular Hb (MCH), mean 
corpuscular Hb concentration (MCHC), red cell distribution width 
(RDW), white blood cell (WBC) count with automated (or manual, 
as indicated) differential, ANC, and absolute lymphocyte count 
(ALC), and platelet count.  
 
In the event that the designated central laboratory for the study is 
unable to provide a valid result for any specific component of the 
defined CBC for a specific subject, the site may be asked to have the 
test performed at their local laboratory. The result of the local CBC 
will be entered into the study database along with the local normal 
reference range.  
 
G6PD (may be conducted at Mayo Medical Laboratories [Rochester, 
MN] or any participating investigative site’s local hematology 
laboratory) and RBC antibody screen will be performed at Screening 
only. 
Other 
EPO, Hp, COHb, hepcidin, 25-hydroxy vitamin D2 and D3. 
Serum Chemistry: 
Alkaline phosphatase (ALP), sodium, potassium, chloride, calcium, 
magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, 
albumin, total protein, glucose, blood urea nitrogen (BUN), 
creatinine, uric acid, LDH, ALT, AST, total bilirubin, indirect 
bilirubin (and estimated creatinine clearance or glomerular filtration 
rate for Screening only, as appropriate). 
Sex Hormones: 
Testosterone (total and free), estrone, and estradiol. FSH will only 
be performed at Screening for female subjects only for confirmation 
of postmenopausal status. 
Bone Turnover: 
Serum osteocalcin-N-mid and CTX. 
Lipids: 
Total cholesterol, HDL-C, triglycerides. 
Iron Panel: 
Iron, total iron-binding capacity (TIBC), transferrin saturation, 
ferritin. 
Coagulation Studies: 
Fibrinogen, aPTT, INR. 
Urinalysis: 
Color and appearance, pH, specific gravity, ketones, protein, 
glucose, bilirubin, nitrite, urobilinogen, pregnancy screen (dipstick), 
occult blood. Microscopic inspection of sediment will only be 
performed for cause or to investigate an abnormal dipstick finding 
per the Investigator’s discretion. 


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10.5.5.2. Screening Serology 
A blood sample for serology, including HBsAg, HCV Ab screen, and HIV1 and HIV2 Ab, is to 
be collected from all subjects at Screening.  
  
Menstrual Cycle Diary 
10.5.6.
Menstruating female subjects will be required to fill out a paper-based menstrual cycle diary 
each month in order to monitor any changes. Diaries will be dispensed and collected as indicated 
in the Schedules of Assessments (Appendix 1). Subjects will record the start date, stop date, and 
any notable characteristics of each menstrual cycle. 
  
Adverse Events 
10.5.7.
Each subject will be carefully monitored for the development of any AEs, including AESIs 
(Section 11.1.5), throughout the study, from signing of the informed consent through all 
scheduled study follow-up visits, or withdrawal of consent, whichever occurs first. In addition, 
SAEs (Section 11.1.4) that are assessed as possibly or probably related to study treatment that 
occur > 30 days post-treatment also are to be reported. 
Adverse events will be evaluated by the Investigator and recorded as described in the Schedules 
of Assessments. On dosing visits, all AEs (elicited and spontaneously reported) will be 
continuously evaluated by the Investigator and recorded. At any nondosing day visit, AEs will be 
evaluated by the Investigator and recorded.  
Any AEs already documented at a previous assessment and designated as ongoing will be 
reviewed at subsequent visits or assessment time points as necessary. If these AEs have resolved, 
this will be documented. 
All AEs will be graded using the CTCAE v4.03 grading system (Appendix 3). 
Complete details of AE monitoring are provided in Section 11. 
10.6. 
Pharmacokinetic Assessments 
The first approximately 10 subjects treated in the Core Period, contingent on clinical site 
feasibility, will undergo extensive PK sampling as detailed in Appendix 1.3. The remainder of 
treated subjects will undergo limited PK sampling as detailed in Appendix 1.4.  
The in-clinic visit on Day 22 may be performed by the subject’s primary care physician if 
necessary and must be approved by the Sponsor on a case by case basis. In this instance, PK 
sampling will not be required on Day 22. (Additional details regarding Day 8 and Day 22 visits 
performed by the subject’s primary care physician can be found in Table 5.)  
During the Extension Period, predose PK samples will be drawn for the measurement of trough 
levels of AG-348 and AGI-8702 at each study visit (every 3 months; see Appendix 1.2). 
The collection times for postdose PK samples will start from the time that dosing is completed; 
eg, a PK draw at 30 minutes will be collected 30 minutes after the last capsule has been ingested. 
The completion time of each dose will be recorded. 
Procedures for sample collection and processing will be provided in a separate study manual. 
The actual time point of each sample collection will be recorded. 


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Samples for PK and PD assessments may be retained for up to 2 years from collection.  
10.7. 
Pharmacodynamic Assessments 
The first approximately 10 subjects treated in the Core Period, contingent on clinical site 
feasibility, will undergo extensive PD sampling for 2,3-DPG and ATP as detailed in Appendix 
1.3. The remainder of treated subjects will undergo limited PD for 2,3-DPG and ATP sampling 
as detailed in Appendix 1.4. During the Extension Period, predose PD samples will be drawn for 
the measurement of trough levels of 2,3-DPG, ATP, 
 at each study visit (every 
3 months; see Appendix 1.2). 
The collection times for postdose PD samples will start from the time that dosing is completed; 
eg, a PD draw at 30 minutes will be collected 30 minutes after the last capsule has been ingested. 
The completion time of each dose will be recorded. 
Procedures for sample collection and processing will be provided in a separate study manual. 
The actual time point of each sample collection will be recorded. 
The in-clinic visit on Day 22 may be performed by the subject’s primary care physician if 
necessary and must be approved by the Sponsor on a case by case basis. In this instance, PD 
sampling will not be required on Day 22. (Additional details regarding Day 8 and Day 22 visits 
performed by the subject’s primary care physician are provided in Table 5.) 
Figure 3 provides a brief schematic outlining the PKR reaction and how each of these PD 
assessments fits into a complete mechanistic understanding of the action of AG-348. 
Figure 3: 
PKR Enzymatic Reaction 
 
Abbreviations: 2,3-DPG = 2,3 diphosphoglycerate; ATP = adenosine triphosphate; PEP = phosphoenolpyruvate;  
PKR = pyruvate kinase isoform R. 
 
The PKR enzyme catalyzes the PEP to pyruvate reaction, with concomitant formation of ATP. 
• Binding of AG-348 to the PKR tetramer can be assessed through an ex vivo 
biochemical assay of cell lysates from AG-348 treated subjects. Because WBCs 
contain a high level of pyruvate kinase from a nonPKR pyruvate kinase isoform, 
WBCs are first removed by filtration before the purified red cells are frozen.  
 
 
 


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 It has been reported 
in the literature that there may be compensatory expression of PKM2 in the RBCs of 
some subjects with PKR deficiency (Black, et al. 1979; Kahn, et al. 1975; Rijksen, et 
al. 1990). Therefore, levels of PKM2 and appropriate reference proteins (eg actin, 
GAPDH) may be evaluated in these whole blood samples. 
• 
 
 
 
 
  
 
 
 
 
• AG-348 target engagement 
 activity has been 
shown in preclinical models and healthy adult subject clinical studies to result in 
accumulation of ATP and depletion of the upstream metabolite 2,3-DPG. Therefore, 
levels of these metabolites will be measured by mass spectrometry in frozen whole 
blood samples. 
Blood samples will be stored at the site and regularly transported at -80°C (±10 °C) to the 
bioanalytical laboratory for analysis. Procedures for sample collection and processing will be 
provided in a separate study manual. 
10.8. 
Ordering of Blood Sample Collection 
When more than 1 blood sample is collected at the same nominal time, the samples will be 
collected in the following order: 
1. Safety laboratory assessments. 
2. PD (2,3 DPG, ATP). 
 
4. PK. 
 
 
10.9. 
Sample Processing, Storage, and Shipment 
Instructions for the processing, storage and shipment of all study samples for central analysis 
will be provided in a separate study manual. 


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11. 
ADVERSE EVENTS 
Monitoring of AEs, including SAEs, AESIs, and AEs leading to discontinuation, will be 
conducted throughout the study. Adverse events and SAEs will be recorded in the source 
documentation and eCRF from time of the signing informed consent through the Follow-up 
Visits for randomized subjects. All AEs should be monitored until they are resolved or are 
clearly determined to be due to a subject’s stable or chronic condition or intercurrent illness(es). 
All SAEs will be followed through 30 days after the last dose of study treatment or until the SAE 
has resolved. Any SAEs that are assessed as possibly or probably related to study treatment that 
occur > 30 days post-treatment also are to be reported. 
11.1. 
Definition of Adverse Events 
  
Adverse Event 
11.1.1.
An AE is any untoward medical occurrence associated with the use of a drug in humans, whether 
or not considered study drug-related. An AE (also referred to as an adverse experience) can be 
any unfavorable and unintended sign (eg, an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a drug, without any judgment about causality. An AE can 
arise from any use of the drug (eg, off-label use, use in combination with another drug) and from 
any route of administration, formulation, or dose, including an overdose. 
  
Suspected Adverse Reaction 
11.1.2.
A suspected adverse reaction is any AE for which there is a reasonable possibility that the drug 
caused the AE. For the purposes of expedited safety reporting, ‘reasonable possibility’ means 
that there is evidence to suggest a causal relationship between the drug and the AE.  
  
Unexpected Adverse Event 
11.1.3.
An unexpected AE is one for which the nature or severity of the event is not consistent with the 
applicable product information, eg, the Investigator’s Brochure. 
  
Serious Adverse Event 
11.1.4.
An AE or suspected adverse reaction is considered serious (SAE) if, in the view of either the 
Investigator or Sponsor, it results in any of the following outcomes: 
• Death. 
• Life-threatening (meaning 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). 
• Inpatient hospitalization or prolongation of existing hospitalization. Hospitalization 
admissions and/or surgical operations scheduled to occur during the study period, but 
planned prior to study entry are not considered AEs if the illness or disease existed 
before the subject was enrolled in the study, provided that it did not deteriorate in an 
unexpected manner during the study (eg, surgery performed earlier than planned). 
• A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions. 


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• Congenital anomaly/birth defect. 
• Important medical event. An important medical event is an event that may not result 
in death, be life-threatening, or require hospitalization but may be considered an SAE 
when, based upon appropriate medical judgment, it may jeopardize the subject or 
subject and may require medical or surgical intervention to prevent 1 of the outcomes 
listed in the definitions for SAEs. Examples of such medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood 
dyscrasias or convulsions that do not result in inpatient hospitalization, or the 
development of drug dependency or drug abuse. 
  
Adverse Events of Special Interest  
11.1.5.
An AESI can be serious or nonserious. Ongoing monitoring and rapid communication (within 
24 hours) by the Investigator to the Sponsor is required to allow for further characterization and 
reporting to regulatory authorities. 
11.1.5.1. Transaminase Increase 
Transaminase increase is an AESI for AG-348. Transaminase increases of > 2.5 × baseline or 
those that have increased to ≥ Grade 2 in severity should be reported as an AESI within 24 hours. 
The increase should subsequently be investigated as the Investigator deems appropriate.  
 
Transaminase levels should then be monitored weekly until they have decreased to 
< 2.5 × baseline. If a dose adjustment is being considered, the Investigator should consult with 
the Medical Monitor and refer to Section 9.7.1. 
In the event of a transaminase increase of > 2.5 × baseline or 1 that has increased to ≥ Grade 2 in 
severity, the study site should report this occurrence to the Sponsor using the SAE form within 
24 hours of their first knowledge of the event.  
11.2. 
Procedures for Reporting Adverse Events, Adverse Events of Special 
Interest, and Serious Adverse Events  
Each subject must be carefully monitored for the development of any AEs. This information 
should be obtained in the form of nonleading questions (eg, “How are you feeling?”) and from 
signs and symptoms detected during each examination, observations of study personnel, and 
spontaneous reports from subjects. 
All AEs (serious and nonserious) spontaneously reported by the subject and/or in response to an 
open question from study personnel or revealed by observation, physical examination, or other 
diagnostic procedures will be recorded in the source documentation and eCRF. Any clinically 
relevant deterioration in laboratory assessments or other clinical findings is considered an AE 
and must be recorded in the appropriate source documentation and eCRF.  
Treatment-emergent abnormal clinical laboratory results should generally be reported as AEs if 
there are accompanying symptoms; if additional diagnostic evaluations or medical (including 
drug therapy) or surgical interventions are undertaken; if a change in study drug dosing or study 
drug discontinuation is required; or, if the laboratory result is considered clinically significant by 
the Investigator.  


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Although it is an objective of this study to determine if treatment with AG-348 results in 
increased Hb levels in subjects with PK deficiency, overshoot of the Hb level above the ULN by 
gender is not recommended and should be reported as an AE, graded per the CTCAE v4.03. 
When possible, signs and symptoms indicating a common underlying pathology should be noted 
as 1 comprehensive event. 
All AESIs and SAEs that occur during the course of the study must be promptly reported by the 
Investigator to 
 Global Safety and Pharmacovigilance (see contact information 
below). Any deaths and any AEs assessed as life-threatening are to be reported immediately. 
Any AESIs and any SAEs that are not life-threatening/do not result in death are to be reported 
within 24 hours from the point in time when the Investigator first becomes aware of the event. 
All AESI and SAEs must be reported, regardless of whether they are considered causally related 
to AG-348.  
 
For each AESI or SAE, an SAE form is to be completed, to include subject number, a narrative 
description of the event, and an assessment by the Investigator as to the severity of the event and 
its relatedness to study drug. Follow-up information about the event may be requested by the 
Sponsor or Medical Monitor. 
Contact Information: 
 Global Safety and Pharmacovigilance 
SAE Hotline (United States): 
 
SAE Fax (United States): 
 
SAE Hotline (France): 
 
SAE Fax (France): 
  
SAE Hotline (Netherlands): 
 
SAE Fax (Netherlands): 
 
SAE Hotline (Italy): 
 
SAE Fax (Italy): 
 
SAE Hotline (United Kingdom): 
 
SAE Fax (United Kingdom): 
 
Email: 
 
If there are serious, unexpected adverse drug reactions associated with the use of AG-348, the 
Sponsor will notify the appropriate regulatory agency(ies) and all participating Investigators on 
an expedited basis. The local IRB/IEC will be promptly notified based on local regulations 
where required by the IRB/IEC of all serious, unexpected adverse drug reactions involving risk 
to human subjects. 
All AEs, whether serious or not, will be described in the source documents in the database. All 
new events, as well as those that worsen in intensity or frequency relative to baseline, which 
occur after signing the informed consent through the final Follow-up Visit (Day 29 ± 3 days) 
must be recorded. Adverse events that are ongoing at the time of treatment discontinuation 
should be followed up to 30 days after the last dose of study treatment. All SAEs will be 
followed up to 30 days after the last dose of study treatment, or until the SAE has resolved.  


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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). 
• The date of onset of the event. 
• The date of resolution of the event. 
• Whether the event is serious or not. 
• Intensity of the event (see below for definitions). 
• Relationship of the event to study treatment (see below for definitions). 
• Action taken: none; change in the study drug administration (eg, temporary interruption 
in dosing); drug treatment required; nondrug treatment required; hospitalization or 
prolongation of hospitalization required (complete SAE page); diagnostic procedure 
performed; subject discontinued from the study (complete End of Study Visit). 
• Outcome: subject recovered without sequelae; subject recovered with sequelae; event 
ongoing; subject died (notify the Medical Monitor immediately, and complete the SAE 
form). 
Intensity of all AEs will be graded according to CTCAE v4.03 (Appendix 3). 
Relationship to study drug administration will be determined by the Investigator according to 
the following criteria: 
•  Not Related:  Exposure to the study treatment did not occur, or the occurrence of the AE 
is not reasonably related in time, or the AE is considered unlikely to be related to the 
study treatment. 
•  Possibly Related:  The study treatment and the AE were reasonably related in time, and 
the AE could be explained equally well by causes other than exposure to the study 
treatment. An attribution of possibly related means that there are facts in evidence to 
suggest a possible relationship. 
•  Probably Related:  The study treatment and the AE were reasonably related in time, and 
the AE was more likely explained by exposure to the study treatment than by other 
causes, or the study treatment was the most likely cause of the AE. An attribution of 
probably related means that there are facts in evidence to suggest a probable relationship. 
For the purpose of safety analyses related to final database review, all AEs that are classified as 
Possibly Related or Probably Related will be considered treatment-related AEs. 
11.3. 
Pregnancy Reporting 
Pregnancy is neither an AE nor an SAE, unless a complication relating to the pregnancy occurs 
(eg, spontaneous abortion, which may qualify as an SAE). However, any pregnancy in a 
participating female subject or a female partner of a participating male subject that occurs during 
this study or within 30 days following the last dose of AG-348 must be reported to the Sponsor 
or Medical Monitor within 30 days of being notified of the pregnancy.  


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The Investigator must follow up and document the course and outcome of all pregnancies even if 
the subject was discontinued from the study or if the study has finished. The female subject or 
partner of a male subject should receive any necessary counseling regarding the risks of 
continuing the pregnancy and the possible effects on the fetus. Monitoring should continue until 
conclusion of the pregnancy. 
All outcomes of pregnancy must be reported by the Investigator to the Sponsor or Sponsor’s 
designee on a Pregnancy Outcome Report form within 30 days after he/she has gained 
knowledge of the delivery or elective abortion. 
Any SAE that occurs during pregnancy must be recorded on the SAE report form (eg, maternal 
serious complications, spontaneous or therapeutic abortion, ectopic pregnancy, stillbirth, 
neonatal death, congenital anomaly, or birth defect) and reported within 24 hours in accordance 
with the procedure for reporting SAEs. 
All subjects, male and female, must agree to use effective contraception during the entire study 
and for 30 days following the last dose of AG-348. Abstinence is an acceptable method only 
when this is in line with the normal life style of the subject, meaning that the subject plans to 
remain abstinent continuously throughout the duration of the study and for at least 30 days after 
the last dose of study drug. Periodic abstinence (eg, calendar, symptothermal, and postovulation 
methods) and withdrawal are not acceptable methods of contraception. 


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12. 
STATISTICAL METHODS 
12.1. 
Sample Size and Power  
Due to the rare disease setting, the minimal sample size in each dose arm may be determined by 
feasibility. Depending on possible early termination of 1 or both of the initial 2 arms or the 
addition of a 3rd dose arm, the study could enroll up to a maximum of 75 subjects.  
In order to evaluate the primary objective of safety and tolerability of AG-348 in Arm 1 and 
Arm 2, up to a maximum of 25 subjects may be randomized onto each arm. The actual number 
of subjects enrolled into Arms 1 and 2 will depend on the safety reviews and decisions made by 
the DRT. In addition, up to 25 additional subjects may be enrolled to evaluate an additional dose 
arm (Arm 3; see Section 7.1).  
As for Arms 1 and 2, the actual enrollment in a potential Arm 3 will depend on the safety 
reviews and decisions made by the DRT. Therefore, up to approximately 75 total subjects may 
be enrolled in this study across 2 to 3 dose arms. 
Table 6 provides the probability within a dose arm of detecting 1 or more AEs with varying 
sample size and the true underlying AE rates. 
Table 6: 
Probability of Observing at Least 1 Specific AE Given Different Underlying 
AE Incidence Rates 
Sample Size 
True Underlying AE Rate 
15% 
10% 
5% 
5 
56% 
41% 
23% 
10 
80% 
65% 
40% 
15 
91% 
79% 
54% 
25 
98% 
93% 
72% 
50 
> 99% 
99% 
92% 
Abbreviations: AE = adverse event. 
 
 


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12.2. 
Analysis Sets 
The following subject populations (ie, analysis sets) will be evaluated and used for presentation 
of the data: 
• Safety Analysis Set: All subjects who receive at least 1 dose of study treatment. The 
Safety Analysis Set will be the primary set for the analysis of safety data. Subjects will be 
classified according to the initial treatment group, defined as the assigned treatment if it 
is received at least once, or as the first treatment received if assigned treatment is never 
received. 
 
Unless otherwise stated, the Safety Analysis Set will be the default analysis set for all 
data analyses. Additional analysis by actual dose group may be conducted. Details will be 
provide in the Statistical Analysis Plan (SAP). 
• Efficacy Analysis Set: All subjects who are enrolled and received any study drug for at 
least 3 weeks. The Efficacy Analysis Set will be the primary set for the analysis of 
preliminary clinical activity data. Subjects will be classified by randomized treatment. 
Additional analysis by actual dose group may be conducted. Details will be provided in 
the SAP. 
12.3. 
Analysis Periods 
Analyses of safety and of indicators of clinical activity will be conducted for the Core Period, 
and for the Cumulative Period (Core Period and Extension Period), if applicable. Unless 
specified otherwise, safety analysis will be based on the treatment-emergent period, which is 
defined as from the first dose to 30 days after the last dose of the corresponding period.  
 
Efficacy analysis will be based on the efficacy window defined as from the first dose to 1 day 
after the last dose of the corresponding period.  
12.4. 
Statistical Analysis 
This section presents a summary of the planned statistical analyses of efficacy and safety for this 
study. Statistical analysis and presentation details will be provided in the SAP for the study, 
which will be finalized before the database lock after all subjects have completed the Core 
Period. The results of this analysis will be presented in a clinical study report (CSR). All 
deviations from the most recent approved SAP before the database lock will be provided in the 
final CSR.  
Additional data collected during the Extension Period after the Core Period database lock will be 
analyzed for inclusion in a subsequent CSR addendum. 
  
General Methods 
12.4.1.
The primary objective for the Core Period of this Phase 2 study is to evaluate the safety and 
tolerability of up to 24 weeks of AG-348 in subjects with PK deficiency. Therefore, the analysis 
of this study will be primarily descriptive in nature; there will be no formal hypothesis testing. 
Summaries will be produced for disposition, baseline disease characteristics and demographic 
data.  


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Categorical variables will be summarized by frequency distributions (number and percentages) 
and continuous variables will be summarized by descriptive statistics (mean, standard deviation 
[SD], median, minimum, and maximum). 
No imputation will be performed for missing data elements. When summarizing AE data, partial 
dates will be imputed as described in the SAP. Additional rules addressing the handling of 
missing data will be detailed in the SAP. 
  
Disposition 
12.4.2.
A summary of the disposition of subjects will be presented, including the number enrolled, the 
number treated, the number discontinued in the Core Period and the reasons, the number rolled 
to the Extension period and completed extension, the number discontinued in the Extension 
period and the reason for discontinuation. Entry criteria and protocol deviations will be listed. 
  
Exposure and Safety Analyses 
12.4.3.
The actual dose and duration in days of AG-348, and the compliance (computed as the ratio of 
actual dose and the planned dose) will be listed and summarized using descriptive statistics. 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) 
and the incidence of AEs occurring in the treatment-emergent period (new or worsening from 
baseline) will be summarized by primary MedDRA System Organ Class (SOC) and Preferred 
Term, severity, outcome, action taken with study drug, and relationship to the study drug. 
Separate summaries will be produced for all TEAEs, treatment-related AEs (those considered by 
the Investigator as at least possibly study drug-related), SAEs, AESIs, AEs leading to 
discontinuation, and AEs ≥ Grade 3 severity. Individual subject listings will be provided for 
deaths, SAEs, and TEAEs leading to treatment modification, interruption, or discontinuation.  
Descriptive statistics will be provided for clinical laboratory values (eg, hematology, serum 
chemistry, coagulation studies, urinalysis) and VS data, presented as both actual values and 
changes from baseline relative to each on-study evaluation. Shift analyses will be conducted for 
selected laboratory parameters based on the baseline CTCAE grade to maximum CTCAE grade. 
Where applicable CTCAE terms do not exist, a grading system based on the upper and/or lower 
limits of normal will be used to classify the results. 
Electrocardiogram analyses will include individual subject listings and summaries of abnormal 
and clinically significant ECG results. Actual values and changes from baseline in PR, QRS, and 
heart-rate corrected QT interval (QTc) intervals will be summarized by visit.  
Data collected from the menstrual diaries such as the start and stop dates of the menses and the 
subject reported characteristics of the menses will be presented in a by-subject listing. Additional 
descriptions of the data may also be performed. 
Concomitant medications will be coded using the World Health Organization Drug Dictionary. 
Concomitant medications prior to and after the start of the study drug will be listed by subject, 
and summarized by Anatomical Therapeutic Chemical Classification System term and dose arm. 


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Sex Hormone Analysis  
12.4.4.
Hormone data, including the actual values and their changes from baseline at each visit, will be 
summarized by sex using descriptive statistics (mean, SD, median, min and max). Spaghetti plots 
will be provided by sex.  
  
Clinical Activity Analyses 
12.4.5.
Details on analyses to evaluate indicators of potential clinical activity of AG-348 in subjects with 
PK deficiency will be described in the SAP. These will include changes in Hb, HCT, reticulocyte 
count, Hp, COHb, LDH, total and indirect bilirubin, EPO, hepcidin, ferritin, and transferrin 
saturation (serum iron/iron-binding capacity).  
  
Analysis Specific to the Extension Period 
12.4.6.
Number and percentage of subjects at individual optimal maintenance dose will be summarized. 
If necessary, additional analyses related to the dose-taper regimen in the Extension Period, 
focusing on the long-term treatment effect in the Cumulative Period, may be conducted and will 
be described in a separate analysis plan after the Core Period database lock, but before the 
database lock for the Extension Period. 
12.5. 
PK/PD Analysis 
 
 
. If such analyses are performed, they will be 
described in a separate analysis plan and may be reported separately in a standalone report. 
  
Pharmacokinetic Analysis 
12.5.1.
Descriptive statistics will be used to summarize PK parameters for the parent compound AG-348 
and the metabolite AGI-8702 for each dose group, and where appropriate, for the entire 
population. Pharmacokinetic parameters will be summarized using the following descriptive 
statistics: n, mean, SD, coefficient of variation Additional PK analyses, if conducted, may be 
described in a separate analysis plan. 
  
Pharmacodynamic Analysis 
12.5.2.
Descriptive statistics will be used to summarize PD parameters for 2,3-DPG and ATP for each 
dose group, and where appropriate for the entire population. Pharmacodynamic parameters will 
be summarized using the following descriptive statistics: n, mean, SD, coefficient of variation %, 
median, minimum, and maximum, geometric mean, and geometric coefficient of variation %. 
Additional PD analyses, if conducted, may be described in a separate analysis plan. 
12.7. 
Interim Analysis 
No formal statistical interim analysis will be conducted.  


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Safety data will be reviewed on an ongoing basis by the DRT, who will meet to review safety, 
PK, PD, and preliminary clinical activity data at regular intervals (approximately every 6 weeks 
during the Core Period and approximately every 4 to 6 months during the Extension Period once 
all subjects have completed the Core Period) throughout the duration of the study.  
The DRT’s decisions to suspend, terminate, or open a potential third dosing arm, or re-assign 
subjects’ dosing in a terminated arm to match the dose and schedule of another arm of the study 
will be based on the totality of the data including, safety, PK, PD, and preliminary clinical 
activity (eg, changes in Hb levels). When all the subjects are in the Extension Period, PK/PD 
data will no longer be reviewed by the DRT. 
Additional interim reviews of data may be conducted to support decision making concerning the 
current clinical study, the Sponsor’s development programs in general, or in case of any safety 
concerns. 


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13. 
ADMINSTRATIVE REQUIREMENTS 
13.1. 
Good Clinical Practice 
The study will be conducted in accordance with the ICH for Good Clinical Practice (GCP) and 
the appropriate regulatory requirement(s). The Investigator will be thoroughly familiar with the 
appropriate use of the study drug as described in the protocol and Investigator’s Brochure. 
Essential clinical documents will be maintained to demonstrate the validity of the study and the 
integrity of the data collected. Master files should be established at the beginning of the study, 
maintained for the duration of the study, and retained according to the appropriate regulations. 
13.2. 
Ethical Considerations 
The study will be conducted in accordance with ethical principles founded in the Declaration of 
Helsinki (Appendix 5). 
The Investigator must obtain IRB approval for the investigation and must submit written 
documentation of the approval to the Sponsor before he or she can enroll any subject into the 
study. The IRB will review all appropriate study documentation in order to safeguard the rights, 
safety, and well-being of the subjects. The study will only be conducted at sites where IRB 
approval has been obtained. The protocol, Investigator’s Brochure, informed consent, 
advertisements (if applicable), written information given to the subjects, annual progress reports, 
and any revisions to these documents will be provided to the IRB.  
The IRB is to be notified of any amendment to the protocol in accordance with local 
requirements. Progress reports and notifications of serious unexpected adverse drug reactions are 
to be provided to the IRB according to local regulations and guidelines. 
13.3. 
Subject Information and Informed Consent 
The Investigator or trained designee will ensure that the subject is given full and adequate oral 
and written information about the nature, purpose, possible risk, and benefit of the study. 
Subjects must also be notified that they are free to discontinue from the study at any time. The 
subject should be given the opportunity to ask questions and allowed time to consider the 
information provided. 
After the study has been fully explained, written informed consent will be obtained from the 
subject prior to study participation.  
The subject’s signed and dated informed consent must be obtained before conducting any study-
related procedures. The Investigator must maintain the original, signed consent form. A copy of 
the signed form must be given to the subject. 
The method of obtaining and documenting the informed consent and the contents of the consent 
will comply with ICH-GCP and all applicable regulatory requirement(s). 
13.4. 
Subject Confidentiality 
In order to maintain subject privacy, all source documents, study drug accountability records, 
study reports and communications will identify the subject by the assigned subject number. The 
Investigator will grant monitor(s) and auditor(s) from the Sponsor or its designee and regulatory 
authority(ies) access to the subject’s original medical records for verification of data gathered on 


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the source documents and to audit the data collection process. The subject’s confidentiality will 
be maintained and will not be made publicly available to the extent permitted by the applicable 
laws and regulations. 
13.5. 
Protocol Compliance 
The Investigator will conduct the study in compliance with the protocol. Modifications to the 
protocol should not be made without agreement of both the Investigator and the Sponsor. 
Changes to the protocol will require written IRB approval/favorable opinion prior to 
implementation, except when the modification is needed to eliminate an immediate hazard(s) to 
subjects. The IRB may provide, if applicable, where regulatory authority(ies) permit, expedited 
review and approval/favorable opinion for minor change(s) in ongoing studies that have the 
approval/favorable opinion of the IRB. The Sponsor or designee will submit all protocol 
modifications to the regulatory authority(ies) in accordance with the governing regulations.  
When immediate deviation from the protocol is required to eliminate an immediate hazard(s) to 
subjects, the Investigator will contact the Sponsor’s Medical Monitor (or Responsible Medical 
Officer), if circumstances permit, to discuss the planned course of action. Any departures from 
the protocol must be fully documented in the source documents/database. 
13.6. 
Data Management 
A paper or eCRF will be completed for each subject or an electronic data capture (EDC) system 
will be used. The EDC system (Medidata Rave®) is a software tool designed to ensure quality 
assurance and facilitate data capture during clinical trials. Through a system regulated workflow 
that includes barcode scanning and interfaces to medical equipment to avoid manual data entry, 
study operations performance is controlled and captured in real time. The system is fully Code of 
Federal Regulations (CFR) 21 Part 11-compliant.  
Source documentation supporting the data should indicate participation in the study and should 
document the dates and details of study procedures, AEs, and subject status. The Investigator, or 
trained designee should complete and the Investigator should verify the source documents as the 
information is collected. Any outstanding entries must be completed immediately after the final 
examination. An explanation should be given for all missing data.  
The Investigator will ensure the accuracy, completeness, and timeliness of the data reported to 
the Sponsor. Data collection processes and procedures will be reviewed and validated to ensure 
completeness, accuracy, reliability, and consistency. A complete audit trail will be maintained of 
all data changes. The Investigator or designee will cooperate with the Sponsor’s representative(s) 
for the periodic review of study documents to ensure the accuracy and completeness of the data 
capture system at each scheduled monitoring visit.  
 
Electronic consistency checks and manual review will be used to identify any errors or 
inconsistencies in the data. This information will be provided to the respective study sites by 
means of electronic or manual queries. The Investigator or designee will prepare and maintain 
adequate and accurate study documents (medical records, ECGs, AE and concomitant 
medication reporting, raw data collection forms, et cetera) designed to record all observations 
and other pertinent data for each subject receiving study treatment.  


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The Investigator will allow Sponsor representatives, contract designees, authorized regulatory 
authority inspectors, and the IRB to have direct access to all documents pertaining to the study. 
13.7. 
Source Document/Case Report Form Completion 
Source documents will be completed for each study subject. It is the Investigator’s responsibility 
to ensure the accuracy, completeness, and timeliness of the data reported in the subject’s source 
document. 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, preferably on the same day that a study subject is seen for 
an examination, treatment, or any other study procedure. Any outstanding entries must be 
completed immediately after the final examination. An explanation should be given for all 
missing data. 
The Investigator will sign and date each required assessment for all study subjects. The 
Investigator will retain all completed source documents. 
13.8. 
Direct Access to Source Data 
The study will be monitored by the Sponsor or its designee. Monitoring will be done by personal 
visits from a representative of the Sponsor (site monitor) and will include on-site review of the 
source documents for completeness and clarity, cross-checking with source documents, and 
clarification of administrative matters will be performed. The review of medical records will be 
performed in a manner to ensure that subject confidentiality is maintained. 
The site monitor will ensure that the investigation is conducted according to protocol design and 
regulatory requirements by frequent communications (letter, telephone, e-mail, and fax). 
All unused study drug and other study materials are to be returned to the Sponsor or designee 
after the study has been completed and the database has been locked. 
Regulatory authorities, the IRB, and/or the Sponsor’s clinical quality assurance group or 
designee may request access to all source documents, database, and any other applicable study 
documentation for an on-site audit or inspection. Direct access to these documents must be 
guaranteed by the Investigator, who must provide support at all times for these activities. 
13.9. 
Record Retention 
The Investigator will maintain all study records according to ICH-GCP and applicable regulatory 
requirement(s). Records will be retained for at least 2 years after the last marketing application 
approval or 2 years after formal discontinuation of the clinical development of the investigational 
product or according to applicable regulatory requirement(s). If the Investigator withdraws from 
the responsibility of keeping the study records, custody must be transferred to a person willing to 
accept the responsibility. The Sponsor must be notified in writing if a custodial change occurs. 
13.10. 
Liability and Insurance 
The Sponsor has subscribed to an insurance policy covering, in its terms and provisions, its legal 
liability for injuries caused to participating persons and arising out of this research performed 


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Amendment #5, Version 6.0 
30 June 2017  
Page 87  
strictly in accordance with the scientific protocol as well as with applicable law and professional 
standards.  
13.11. 
Publication of Study Findings and Use of Information 
All information regarding AG-348 supplied by the Sponsor or designee to the Investigator is 
privileged and confidential information. The Investigator agrees to use this information to 
accomplish the study and will not use it for other purposes without consent from the Sponsor. It 
is understood that there is an obligation to provide the Sponsor with complete data obtained 
during the study. The information obtained from the clinical study will be used towards the 
development of AG-348 and may be disclosed to regulatory authority(ies), other Investigators, 
corporate partners, or consultants as required. 


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Amendment #5, Version 6.0 
30 June 2017  
Page 88  
14. 
LIST OF REFERENCES 
Beutler E, and Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene 
frequency in the general white population. Blood. 2000;95(11):3585-8. 
Black JA, Rittenberg MB, Bigley RH, and Koler RD. Hemolytic anemia due to pyruvate kinase 
deficiency: characterization of the enzymatic activity from eight subjects. Am J Hum Genet. 
1979;31(3):300-10. 
Christensen RD, Lambert DK, Henry E, Yaish HM, and Prchal JT. End-tidal carbon monoxide 
as an indicator of the hemolytic rate. Blood Cells Mol Dis. 2015;54(3):292-6. 
Christensen RD, Malleske DT, Lambert DK, Baer VL, Prchal JT, Denson LE, et al. Measuring 
End-Tidal Carbon Monoxide of Jaundiced Neonates in the Birth Hospital to Identify Those with 
Hemolysis. Neonatology. 2016;109(1):1-5. 
Christensen RD, Yaish HM, Johnson CB, Bianchi P, and Zanella A. Six children with pyruvate 
kinase deficiency from one small town: molecular characterization of the PK-LR gene. J Pediatr. 
2011;159(4):695-7. 
FDA. Guidance for Industry: Drug-Induced Liver Injury: Premarketing Clinical Evaluation. 
2009. 
Ghidini A, Sirtori M, Romero R, Yarkoni S, Solomon L, and Hobbins JC. Hepatosplenomegaly 
as the only prenatal finding in a fetus with pyruvate kinase deficiency anemia. Am J Perinatol. 
1991;8(1):44-6. 
Hennekam RC, Beemer FA, Cats BP, Jansen G, and Staal GE. Hydrops fetalis associated with 
red cell pyruvate kinase deficiency. Genet Couns. 1990;1(1):75-9. 
Kahn A, Marie J, Galand C, and Boivin P. Molecular mechanism of erythrocyte pyruvate kinase 
deficiency. Humangenetik. 1975;29(4):271-80. 
Mentzer WC, Jr., Baehner RL, Schmidt-Schonbein H, Robinson SH, and Nathan DG. Selective 
reticulocyte destruction in erythrocyte pyruvate kinase deficiency. J Clin Invest. 1971;50(3):688-
99. 
Rider NL, Strauss KA, Brown K, Finkenstedt A, Puffenberger EG, Hendrickson CL, et al. 
Erythrocyte pyruvate kinase deficiency in an old-order Amish cohort: longitudinal risk and 
disease management. Am J Hematol. 2011;86(10):827-34. 
Rijksen G, Veerman AJ, Schipper-Kester GP, and Staal GE. Diagnosis of pyruvate kinase 
deficiency in a transfusion-dependent subject with severe hemolytic anemia. Am J Hematol. 
1990;35(3):187-93. 
Schwartz JC. The histamine H3 receptor: from discovery to clinical trials with pitolisant. Br J 
Pharmacol. 2011;163(4):713-21. 
Steer K, Stavnichuk M, Morris M, Komarova SV. Bone health in subjects with hematopoietic 
disorders of bone marrow origin: systematic review and meta-analysis. J Bone Miner Res. 2017 
Apr; 32(4):731-42. 


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Amendment #5, Version 6.0 
30 June 2017  
Page 89  
Strauss KA, Robinson DL, Vreman HJ, Puffenberger EG, Hart G, and Morton DH. Management 
of hyperbilirubinemia and prevention of kernicterus in 20 subjects with Crigler-Najjar disease. 
Eur J Pediatr. 2006;165(5):306-19. 
Tanphaichitr VS, Suvatte V, Issaragrisil S, Mahasandana C, Veerakul G, Chongkolwatana V, et 
al. Successful bone marrow transplantation in a child with red blood cell pyruvate kinase 
deficiency. Bone Marrow Transplant. 2000;26(6):689-90. 
van Wijk R, and van Solinge WW. The energy-less red blood cell is lost: erythrocyte enzyme 
abnormalities of glycolysis. Blood. 2005;106(13):4034-42. 
Wong P, Fuller PJ, Gillespie MT, Kartsogiannis V, Kerr PG, Doery JC, et al. Thalassemia bone 
disease: a 19-year longitudinal analysis. J Bone Miner Res. 2014 Nov;29(11):2468-73. 
Zanella A, Fermo E, Bianchi P, and Valentini G. Red cell pyruvate kinase deficiency: molecular 
and clinical aspects. Br J Haematol. 2005;130(1):11-25. 


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Amendment #5, Version 6.0 
30 June 2017  
Page 90  
15. 
APPENDICES


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Agios Pharmaceuticals, Inc. 
Amendment #5, Version 6.0 
30 June 2017 
Page 91  
APPENDIX 1: 
SCHEDULES OF ASSESSMENTS 
Appendix 1.1. 
Schedule of Assessments: Core Period 
Timing: 
Assessment(s) 
Pre-treatment 
Month 1 
Months 2 and 3 
Months 4, 5, 6 
Follow-up1 
Visit 
Screening 
Baseline 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day(s) 
-42 to -
1
-40 to
-12
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window (days) 
-- 
-- 
-- 
± 2 
± 2 
± 2 
± 7 
± 7 
± 7 
± 7 
± 7 
± 7 
± 7 
Written informed consent 
X 
PK enzyme assay4 
(confirmation of PK 
deficiency) 
X 
PKR genotype 
(for randomization) 
X 
Demographics 
X 
Medical/surgical history5 
(general and PK deficiency-
specific) 
X 
Medication history 
X 
Transfusion history 
X 
Confirmation of 
vaccinations 
(splenectomized subjects) 
X 
Physical examination6/ 
Height and weight6 
X 
X 
X 
X 
X 
X 
X 
X 
X 
ECOG Performance Status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs7 
(BP, HR, RR, T) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG8
X 
X 
X 
X 
X 
X 
DXA scan 
X9 
X10 
Laboratory evaluations11 
HBsAg, HCV Ab, HIV1 
and 2 Ab 
X 
RBC antibody screen 
X 
Hematology (CBC) 
X12 
X12,13 
X12 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin14 
X 
X 
X 
X 
X 


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Amendment #5, Version 6.0 
30 June 2017 
Page 92  
Timing: 
Assessment(s) 
Pre-treatment 
Month 1 
Months 2 and 3 
Months 4, 5, 6 
Follow-up1 
Visit 
Screening 
Baseline 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day(s) 
-42 to -
1
-40 to
-12
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window (days) 
-- 
-- 
-- 
± 2 
± 2 
± 2 
± 7 
± 7 
± 7 
± 7 
± 7 
± 7 
± 7 
EPO levels15 
X 
X 
X 
X 
X 
G6PD screen 
X 
Hepcidin 
X 
X 
X 
X 
X 
Serum chemistry16 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Iron panel17 
X 
X 
X 
COHb18 
X 
X 
X 
X 
X 
X 
X 
X 
Coagulation studies19 
X 
X 
X 
X 
X 
X 
Urinalysis20 
X 
X 
X 
X 
X 
X 
Serum or urine 
pregnancy21 
X 
X 
Lipids22 
X 
X 
X 
X 
X 
Hormonal testing23 
X 
X24 
X 
X 
X 
X 
Serum osteocalcin-N- 
mid and CTX25 
X 
X 
X 
25-hydroxy
vitamins D2 and D3
X 
X 
X 
Randomization26 
X 
Study drug administration 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X27 
Study drug dispensed28 
X 
X 
X 
X 
X 
X 
X 
X 
X 
PK blood sampling29 
X 
X 
X 
X 
X 
X 
X 
X 
X 
PD assessments29 
2,3-DPG/ATP 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Dispense/collect menstrual 
cycle diary31 
X 
X 
X 
X 
X 
X 
X 
Adverse events32 
Continuous 
X 
Transfusion record33 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 


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Agios Pharmaceuticals, Inc. 
Amendment #5, Version 6.0 
30 June 2017 
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Timing: 
Assessment(s) 
Pre-treatment 
Month 1 
Months 2 and 3 
Months 4, 5, 6 
Follow-up1 
Visit 
Screening 
Baseline 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day(s) 
-42 to -
1
-40 to
-12
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window (days) 
-- 
-- 
-- 
± 2 
± 2 
± 2 
± 7 
± 7 
± 7 
± 7 
± 7 
± 7 
± 7 
Concomitant 
medications/procedures 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Rollover to Extension 
Period34 
X 
Abbreviations: 2,3-DPG = 2,3 diphosphoglycerate; Ab = antibody; ATP = adenosine triphosphate; BP = blood pressure; CBC= complete blood count; COHb = carboxyhemoglobin; 
CTX = C-terminal telopeptide; DPG = diphosphoglycerate; DXA = dual-energy x-ray absorptiometry; ECG = electrocardiogram; ECOG = Eastern Cooperative Oncology Group; EPO = 
erythropoietin; FSH = follicle-stimulating hormone; G6PD = glucose-6-phosphate-dehydrogenase; HBsAg = hepatitis B surface antigen; HCV Ab = hepatitis C virus antibody; HDL-C = 
high-density lipoprotein-cholesterol; HIV = human immunodeficiency virus; HR = heart rate; PD = pharmacodynamic; PK = pharmacokinetic; PK deficiency = pyruvate kinase deficiency; 
PKR = pyruvate kinase isoform R; RBC = red blood cell; RR = resting rate; T = temperature; W = week. 
Note: Whenever more than 1 assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive assessment (eg, VS, 
ECG, then blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time. The order of procedures may be revised if agreed upon in prior 
discussion between Sponsor and study site. 
1.
The  Week 28 Follow-up Visit will only be conducted for subjects who do not enter the Extension Period.
2.
To be performed at least 2 days after the first Screening Visit.
3.
In-clinic visits on Day 8 and Day 22 may be performed by the subject’s primary care physician if necessary and must be approved by the Sponsor on a case-by-case basis. In
these instances, PK/PD sampling would not be required and dispensing of study medication would not be performed.
For the Day 8 visit performed by the subject’s primary care physician, the primary care medical office will collect a blood sample for hematology using the blood sample collection
and shipping supplies from the kit prepared by the designated central laboratory. The kit will be sent to the primary care physician’s office. No other testing or procedures will be
asked of the primary care physician on Day 8 (VS and serum chemistry will not be required). The 12-lead ECG scheduled for Day 8 will instead be performed at the main study
center on Day 15.
For the Day 22 visit performed by the subject’s primary care physician, the primary care medical office will collect a blood sample for hematology using the blood sample
collection and shipping supplies from the kit prepared by the designated central laboratory. The kit will be sent to the primary care physician’s office. No other testing or
procedures will be asked of the primary care physician on Day 22.  (VS, serum chemistry, coagulation, haptoglobin, EPO level, carboxyhemoglobin, and PK/PD samples will not
be required.) The 12-lead electrocardiogram scheduled for Day 22 will instead be performed at the main study center on Day 43 (Week 6).
For subjects having their Day 8 and/or Day 22 visits performed by the primary care physician, the Principal Investigator will himself/herself or have a qualified research nurse or
other designated site staff member make telephone contact with the subject to inquire about any adverse events. These must be recorded as if the subject appeared in the main study
center.


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Amendment #5, Version 6.0 
30 June 2017 
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3.
(continued) The Principal Investigator will exercise prudent clinical judgment in determining any clinical course of action to take based on any adverse events discovered.
These telephone contacts on Day 8 and Day 22 must be explained to the subject in advance and scheduled in advance to maximize the likelihood of successfully making
contact.
4.
May be performed either by a designated central laboratory or any participating investigative site’s local hematology laboratory.
5.
Medical history, including all relevant prior medical history, current medical conditions, and hematology profile (CBCs) over prior 6 months, will be obtained at the
Screening assessment.
6.
A complete physical examination (including neurological examination; genital and rectal examinations will be performed at the discretion of the Investigator) will be
obtained at Screening, Baseline, Week 12, and the Follow-up Visit (Week 28), or at Week 24 for subjects rolling over to the Extension Period. The neurological examination
must include an assessment of general orientation and mental status including level of alertness (assess as normal or abnormal and specify any abnormality). Limited focused
physical examinations will be performed at all other specified visits. Height to be collected at Screening only.
7.
Vital signs will include systolic and diastolic blood pressure, heart rate, respiratory rate, and temperature.
8.
12-lead ECGs are to be conducted after 5 minutes of recumbency.
9.
If a DXA scan of the hip and spine with T and Z scores has been performed within 3 months preceding the first day of dosing, this may be used to meet this requirement.
10. Week 24 DXA scan may be performed any time between Weeks 24 and 28, and must be performed at the same imaging center on the same DXA machine as the original Screening
DXA scan.
11. Laboratory evaluations (hematology, serum chemistry, coagulation studies, and urinalysis) are to be collected in the morning. These should be collected following an overnight
fast on Baseline Day 1 Week 6 (Day 43), Week 12 (Day 85), Week 24 (Day 169), and Follow-up Week 28 (Day 197), when the lipid samples are also included.
12. Three Screening/Baseline samples will be collected for complete blood count (CBC). Samples will be collected in the AM on 3 different days; the samples collected on Baseline/
Day 1 may comprise one of these; samples may be taken at the same time as Screening/Baseline hormone assessments (any 2 time points during Screening at least 2 days apart).
CBC will include HCT, Hb, red blood cell (RBC) count, absolute reticulocyte count, percent reticulocyte count, mean corpuscular volume (MCV), mean corpuscular Hb (MCH),
mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW), white blood cell (WBC) count with automated (or manual, as indicated) differential, absolute
neutrophil count (ANC), absolute lymphocyte count (ALC), and platelet count.
In the event that the designated central laboratory for the study is unable to provide a valid result for any specific component of the defined CBC for a specific subject, the site
may be asked to have the test performed at their local laboratory. The result of the local CBC will be entered into the study database along with the local normal reference range.
13. The second Screening hematology (CBC) should be drawn in the morning (does not have to be fasting), and may be drawn at the same time the subject returns for the second
estradiol and free and total testosterone sample.
14. Haptoglobin will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28.
15. Erythropoietin (EPO) levels will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28.


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Agios Pharmaceuticals, Inc. 
Amendment #5, Version 6.0 
30 June 2017 
Page 95  
16. Alkaline phosphatase, sodium, potassium, chloride, calcium, magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, albumin, total protein, glucose, blood urea
nitrogen (BUN), creatinine, uric acid, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin and indirect
bilirubin (and estimated creatinine clearance or glomerular filtration rate for Screening only, as appropriate).
17. Iron, total iron-binding capacity (TIBC), transferrin saturation, and ferritin will be performed prior to dosing on Day 1, at the end of Week 12 and at the end of
Week 24.
18. To be collected before the AG-348 morning dose is administered.
19. Fibrinogen, activated partial thromboplastin time (aPTT), and international normalized ratio (INR) will be performed at Screening, prior to dosing on Day 1, at the end
of Week 2, the end of Week 12, the end of Week 24, and the end of Week 28.
20. Color, appearance, pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite, urobilinogen, and occult blood. Microscopic inspection of sediment should only be
performed for cause or to investigate an abnormal dipstick finding per the Investigator’s discretion. Urinalysis will be performed at Screening, prior to dosing on Day 1,
at the end of Week 2, the end of Week 12, the end of Week 24, and the end of Week 28.
21. Must be repeated at any point throughout the study period if pregnancy is clinically suspected.
22. Total cholesterol, HDL-C, and triglyceride samples will be collected in the morning following an overnight fast.
23. Three Screening/Baseline samples will be collected for estradiol and testosterone (total and free). Samples will be collected in the AM on 3 different days; the samples collected on
Baseline/Day 1 may comprise one of these; the Screening samples may be collected at any 2 time points during Screening at least 2 days apart. Serum estrone, estradiol, and free
and total testosterone will then follow the schedule indicated on Day 1 and Weeks 12, 24, and 28. FSH will only be performed at Screening for female subjects only for
confirmation of postmenopausal status.
24. The second Screening hormone testing will consist of estradiol and testosterone (free and total) only. Samples should be drawn in the AM (does not need to be fasting), and may
be drawn at the same time the subject returns for the second CBC sample.
25. Serum osteocalcin-N-mid and CTX will be drawn in the AM each time, approximately between 8-10 AM, and after an overnight fast of 10-12 hours.
26. Randomization will be performed following PKR genotyping and prior to and as close as feasible to dosing on Day 1.
27. Study drug administration is not required on W24/D169 for subjects not continuing into the Extension Period.
28. Study drug will be dispensed on a 28-day schedule, or on an alternate schedule (< 28 days) as needed to accommodate subject visit schedule and dose modifications. The amount
of study drug dispensed should be sufficient to provide an adequate reserve supply of AG-348 to ensure uninterrupted dosing in the event of an unexpected delay for the next
scheduled study visit (7 extra days of dosing supply is recommended during the Core Period).
29. For the first 10 subjects treated, extensive PK/PD sampling will be conducted on Days 1 and 15 (see for details), followed by limited PK/PD sampling from Week 3 to Week 24
(see Appendix 1.3). Limited PK/PD sampling will be conducted on the remainder of subjects treated (Appendix 1.4). See Section 10.6 and Section 10.7 for details of blood
sampling for PK and PD assessments, respectively, and refer to Section 10.9 for sample processing and storage guidelines.
31. Menstruating female subjects will record their menstrual cycles (start, stop, characteristics) monthly. Paper-based menstrual cycle diaries will be dispensed at study visits
approximately every month. The previous month’s diary will be collected at these visits as well.
32. Randomized subjects will be evaluated for AEs from the time they sign informed consent until they complete all scheduled study follow-up visits or withdraw consent, whichever
occurs first.
33. All transfusions must be recorded in the eCRF.
34. Subject must have completed 24 weeks of treatment and tolerated AG-348 (may have had dose modifications). Investigator and Medical Monitor or Responsible Medical Officer
must agree with the subject continuing on treatment and subject must sign a separate ICF for the Extension Period.


Clinical Study Protocol AG348-C-003 
Agios Pharmaceuticals, Inc. 
Amendment #5, Version 6.0 
30 June 2017 
Appendix 1.2. 
Schedule of Assessments: Extension Period 
Visit: 
Assessment(s) 
Month 9 
Month 12 
Month 15 
Month 18 
Month 21 
Month 24 
Month 27 
Month 30 
Follow-up 
Approximate 
Study Day 
259 
349 
439 
529 
619 
709 
799 
889 
919 
Visit Window 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
Physical examination/weight1 
X 
X 
X 
X 
X 
X 
X 
X 
X 
ECOG Performance Status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs  
(BP, HR, RR, T)2 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG3
X 
X 
X 
X 
X 
DXA scan 
X 
X 
X 
X 
Laboratory evaluations4 
Hematology (CBC)5 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin 
X 
X 
X 
X 
X 
EPO levels6 
X 
X 
X 
X 
X 
Hepcidin 
X 
X 
X 
X 
Serum chemistry7 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Iron panel8 
X 
X 
X 
X 
COHb9 
X 
X 
X 
X 
Coagulation studies10 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urinalysis11 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum or urine pregnancy12 
Lipids13 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Hormonal testing14 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum osteocalcin-N-mid 
 and CTX15 
X 
X 
Study drug administration 
X 
X 
X 
X 
X 
X 
X 
X 
Study drug dispensed16 
X 
X 
X 
X 
X 
X 
X 
PK blood sampling17 
X 
X 
X 
X 
X 
X 
X 
X 
PD assessments 
(2,3-DPG/ATP, 
)18 
X 
X 
X 
X 
X 
X 
X 
X 
Dispense/collect menstrual cycle diary19 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse events20 
Continuous 
X 
Transfusion record21 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant medications/procedures 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  2,3-DPG = 2,3 diphosphoglycerate; ATP = adenosine triphosphate; BP = blood pressure; CBC= complete blood count; COHb = carboxyhemoglobin; 
CTX = C-terminal telopeptide;  DPG = diphosphoglycerate; DXA = dual-energy x-ray absorptiometry; ECG = electrocardiogram; ECOG = Eastern Cooperative Oncology Group; EPO = 
erythropoietin; HDL-C = high-density lipoprotein-cholesterol; HIV = human immunodeficiency virus; HR = heart rate; PD = pharmacodynamic; PK = pharmacokinetic; PK deficiency = 
pyruvate kinase deficiency; PKR = pyruvate kinase isoform R; RBC = red blood cell; RR = resting rate; T = temperature; W = week. 
Page 96  


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Amendment #5, Version 6.0 
30 June 2017 
Page 97  
Note: Whenever more than 1 assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive assessment (eg, VS, 
ECG, then blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time. The order of procedures may be revised with prior discussion 
between Sponsor and site. 
1. A focused physical examination (including neurological examination; genital and rectal examinations will be performed at the discretion of the Investigator) will be
obtained every 3 months; a complete physical examination will be performed at the Month 30 visit. The neurological examination must include an assessment of general
orientation and mental status including level of  alertness (assess as normal or abnormal and specify any abnormality).
2. Vital signs will include systolic and diastolic blood pressure, heart rate, respiratory rate, and temperature.
3. 12-lead ECGs are to be conducted after 5 minutes of recumbency.
4. Laboratory evaluations (hematology, serum chemistry, coagulation studies, and urinalysis) are to be collected in the morning. These should be collected following an
overnight fast.
5. CBC will include HCT, Hb, red blood cell (RBC) count, absolute reticulocyte count, percent reticulocyte count, mean corpuscular volume (MCV), mean corpuscular Hb
(MCH), mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW),  white blood cell (WBC) count with automated (or manual, as indicated)
differential, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and platelet count. In the event that the designated central laboratory for the study is unable
to provide a valid result for any specific component of the defined CBC for a specific subject, the site may be asked to have the test performed at their local laboratory. The
result of the local CBC will be entered into the study database along with the local normal reference range.
6. Erythropoietin (EPO) levels will be performed prior to dosing at Month 12, Month 18, Month 24, and Month 30.
7. Alkaline phosphatase, sodium, potassium, chloride, calcium, magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, albumin, total protein, glucose, blood urea
nitrogen (BUN), creatinine, uric acid, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin and indirect bilirubin.
8. Iron, total iron-binding capacity (TIBC), transferrin saturation, and ferritin will be performed prior to dosing at Month 12, Month 18, Month 24, and Month 30.
9. To be collected before the AG-348 morning dose is administered.
10. Fibrinogen, activated partial thromboplastin time (aPTT), and international normalized ratio (INR) will be performed at each study visit.
11. Color, appearance, pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite, urobilinogen, and occult blood. Microscopic inspection of sediment should only be
 performed for cause or to investigate an abnormal dipstick finding per the Investigator’s discretion. Urinalysis will be performed prior to dosing at each study visit.
12. Must be obtained at any point throughout the study period if pregnancy is clinically suspected. Effective contraception must be continued throughout the Extension Period in
all subjects and any pregnancies must be reported (see Section 11.3).
13. Total cholesterol, HDL-C, and triglyceride samples will be collected in the morning following an overnight fast.
14. Serum estrone, estradiol, and free and total testosterone.
15. Serum osteocalcin-N-mid and CTX will be drawn in the AM each time, approximately between 8-10 AM, and after an overnight fast of 10-12 hours.
16. Study drug will be dispensed on a 3-month schedule, or on an alternate schedule (< 3 months) as needed to accommodate subject visit schedule and dose modifications. The
amount of study drug dispensed should be sufficient to provide an adequate reserve supply of AG-348 to ensure uninterrupted dosing in the event of an unexpected delay for
the next scheduled study visit (14 extra days of dosing supply is recommended during the Extension Period).
17. Predose; PK sampling will only include AG-348 and AGI-8702 concentrations.


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18.  Predose.
19.  Menstruating female subjects will record their menstrual cycles (start, stop, characteristics) monthly. Paper-based menstrual cycle  diaries will be dispensed and collected at 
      each study visit.
20.  All randomized subjects will be evaluated for AEs from the time they sign informed consent until they complete all scheduled study follow-up visits or withdraw consent, 
       whichever occurs first.
21.  All transfusions must be recorded in the eCRF.    


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Appendix 1.3. 
Schedule of Assessments: Extensive PK/PD Sampling during the Core Period 
Sample 
Timing/Interval 
Month 1 
Months 2 and 3 
Months 4, 5, 6 
Visit(s) 
Baseline/D1 
W2/D15 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
Study Day(s) 
1/15 
22 
43 
64 
85 
113 
141 
169 
Visit Window 
(days) 
± 2 (D15) 
± 2 
± 7 
± 7 
± 7 
± 7 
± 7 
± 7 
Timing: 
Assessment(s) 
Predose1 
30 
min2 
1 
hr2 
2 
hr2 
4 
hr3 
8 
hr3,6 
12 
hr3,6 
Predose1 
Predose1 
Predose1 
Predose1 
Predose1 
Predose1 
Predose1 
PK blood sample 
X 
X 
X 
X 
X 
X 
X4 
X 
X 
X 
X 
X 
X 
X 
2,3 DPG/ATP 
X 
X 
X 
X 
X 
X 
X4 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  2,3 DPG = 2,3 diphosphoglycerate; ATP = adenosine triphosphate; D = day; DPG = diphosphoglycerate; hr = hours; min = minutes; PD = pharmacodynamic; PK = 
pharmacokinetic; PKM2 = pyruvate kinase isoform M2; PKR = pyruvate kinase isoform R; W = week. 
1.
The acceptable time window will be within 60 minutes prior to study treatment dose administration for the predose PK/PD sample. Study drug administration is not required on W24/
D169 for subjects not continuing into the Extension Period.
2.
The acceptable time window will be within ± 5 minutes of the scheduled collection time for the 30-minute, 1-hour, and 2-hour PK/PD samples.
3.
The acceptable time window will be within ± 30 minutes of the scheduled collection time for the 4, 8, and 12-hour PK/PD samples.
4.
To be collected on Day 1 only.
6.
If the 12-hour time point sample cannot be collected at site on Day 1, an 8-hour time point sample may be collected instead.
 


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Appendix 1.4. 
Schedule of Assessments: Limited PK/PD Sampling during the Core Period 
Sample Timing/ 
Interval 
Month 1 
Months 2 and 3 
Months 4, 5, 6 
Visit(s) 
Baseline/ 
D1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
Study Day 
1 
15 
22 
43 
64 
85 
113 
141 
169 
Visit Window (days) 
-- 
± 2 
± 2 
± 2 
± 7 
± 7 
± 7 
± 7 
± 7 
Timing: 
Assessment(s) 
Predose1 
Predose1 
Predose1 
Predose1 
Predose1 
Predose1 
Predose1 
Predose1 
Predose1 
PK blood sample 
X 
X 
X 
X 
X 
X 
X 
X 
X 
2,3 DPG/ATP 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Abbreviations: 2,3 DPG = 2,3 diphosphoglycerate; ATP = adenosine triphosphate; D = day; DPG = diphosphoglycerate; hr = hours; min = minutes; PD = pharmacodynamic; PK = 
pharmacokinetic; PKM2 = pyruvate kinase isoform M2; PKR = pyruvate kinase isoform R; W = week. 
1.
The predose blood sample for plasma PK/PD analysis should be collected within 60 minutes prior to study treatment dose administration.
 


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APPENDIX 2: 
EASTERN COOPERATIVE ONCOLOGY GROUP 
PERFORMANCE STATUS SCORING 
Grade 
Symptomatology 
0 
Fully active, able to carry on all predisease performance without restriction. 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or 
sedentary nature, eg, light house work, office work. 
2 
Ambulatory and capable of all self-care but unable to carry out any work activities. Up and about 
more than 50% of waking hours. 
3 
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours. 
4 
Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair. 
5 
Dead. 
Source: Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, et al. Toxicity and Response Criteria of the 
    Eastern Cooperative Oncology Group. Am J Clin Oncol 1982;5:649-655. 


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APPENDIX 3: 
NATIONAL CANCER INSTITUTE COMMON 
TERMINOLOGY CRITERIA FOR ADVERSE EVENTS 
The NCI CTCAE, Version 4.03, can be accessed using the following link:   
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_8.5x11.pdf 


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APPENDIX 4: 
CONCOMITANT THERAPY TO BE AVOIDED 
(PROHIBITED) OR REQUIRING CAREFUL 
MONITORING (USE WITH CAUTION) 
Appendix 4.1. 
Strong and Moderate CYP3A4 Inhibitors 
Strong CYP3A4 Inhibitors: Prohibited 
Moderate CYP3A4 Inhibitors: Substitute or 
Use with Caution 
Boceprevir 
Amprenavir 
Clarithromycin 
Aprepitant 
Conivaptan 
Atazanavir 
Grapefruit and grapefruit juice2 
Ciprofloxacin 
Indinavir 
Darunavir 
Itraconazole 
Diltiazem1
Ketoconazole 
Erythromycin1 
Ritonavir/Lopinavir 
Fluconazole 
Mibefradil (withdrawn from US market) 
Fosamprenavir 
Nefazodone 
Verapamil1 
Nelfinavir 
Posaconazole, 
Saquinavir 
Suboxone 
Telaprevir, 
Telithromycin 
Voriconazole 
Note: Strong Inhibitor = > 5-fold increase in area under the curve (AUC); Moderate Inhibitor = > 2-fold, < 5-fold increase in 
AUC. 
1.
Erythromycin, verapamil, and diltiazem are contraindicated because they are strong P-gp inhibitors (see Appendix 4.3).
2.
Although grapefruit is classified as a moderate CYP3A4 inhibitor, it is prohibited, as is grapefruit juice.


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Appendix 4.2. 
Strong CYP3A4 Inducers (Prohibited) 
Carbamazepine 
Oxcarbazepine 
Phenobarbital 
Phenytoin 
Rifabutin 
Rifampin 
St. John’s Wort 
Note: Corticosteroids may induce CYP3A4. Although the use of corticosteroids is not prohibited, their use should be minimized 
as much as is medically feasible. 
 
 


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Appendix 4.3. 
Strong P-glycoprotein Inhibitors (Prohibited) 
Amiodarone 
Felodipine 
Azithromycin 
Itraconazole 
Captopril 
Ketoconazole 
Carvedilol 
Lopinavir 
Clarithromycin 
Ritonavir 
Conivaptan 
Quercetin 
Cyclosporine 
Quinidine 
Diltiazem 
Ranolazine 
Dronedarone 
Ticagrelor 
Erythromycin 
Verapamil 
 


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Appendix 4.4. 
Sensitive CYP3A4 Substrates (Substitute or Use with Caution) 
Benzodiazepines: 
Antihistamines: 
Miscellaneous: 
Chlorpheniramine 
Alfentanil 
Finasteride 
Salmeterol 
Aprepitant 
Gleevec 
Sildenafil 
Calcium Channel 
Blockers: 
Aripiprazole 
Haloperidol 
Sirolimus 
Alprazolam 
Amlodipine 
Boceprevir 
Irinotecan 
Sorafenib 
Diazepam→3OH 
Lercanidipine 
Buspirone 
LAAM 
Sunitinib 
Midazolam 
Nifedipine 
Cafergot 
Lidocaine 
Tamoxifen 
Triazolam 
Nisoldipine 
Caffeine→TMU 
Methadone 
Taxol 
Nitrendipine 
Cilostazol 
Nateglinide 
Telaprevir 
Immune Modulators: 
Cocaine 
Nevirapine 
Terfenadine 
Tacrolimus (FK506) 
HMG CoA 
Reductase 
Inhibitors: 
Codeine-N-
demethylation 
Ondansetron 
Torisel 
Atorvastatin 
Dapsone 
Pimozide 
Trazodone 
Steroid 6beta-OH: 
Cerivastatin 
Dextromethorphan 
Propranolol 
Vemurafenib 
Estradiol 
Lovastatin 
Docetaxel 
Quetiapine 
Vincristine 
Hydrocortisone (and 
other glucocorticoids) 
Simvastatin 
Domperidone 
Quinine 
Zaleplon 
Progesterone 
Eplerenone 
Risperidone 
Ziprasidone 
Testosterone 
Fentanyl 
Romidepsin 
Zolpidem 
 
 
 


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Appendix 4.5. 
Sensitive CYP2B6 Substrates* (Substitute or Use with Caution) 
Bupropion 
Efavirenz 
  * The areas under the curve of these substrates were not increased by 5-fold or more with a CYP2B6 inhibitor, but they 
     represent the most sensitive substrates studied with available inhibitors evaluated to date. 
 
 


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Appendix 4.6. 
Proton-Pump Inhibitors and H2-Receptor Antagonists   
 
 
 
 
 
 
(Substitute or Use with Caution) 
Proton-pump inhibitors: 
Dexlansoprazole 
Omeprazole 
Esomeprazole 
Rabeprazole 
Lansoprazole 
Pantoprazole 
H2-receptor antagonists: 
Cimetidine 
Nizatidine 
Famotidine 
Ranitidine 
 
 
 


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APPENDIX 5:  
DECLARATION OF HELSINKI 
WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI: 
Recommendations Guiding Medical Doctors in Biomedical Research Involving Human Subjects 
Adopted by the 18th World Medical Association (WMA) General Assembly, Helsinki, Finland, 
June 1964 and amended by the 29th WMA General Assembly, Tokyo, Japan, October 1975, 35th 
WMA General Assembly, Venice, Italy, October 1983, and the 41st WMA General Assembly, 
Hong Kong, September 1989, the 48th WMA General Assembly, Somerset West, Republic of 
South Africa, October 1996; 52nd WMA General Assembly, Edinburgh, Scotland, October 2000; 
53rd WMA General Assembly, Washington 2002 (Note of Clarification on paragraph 29 added); 
55th WMA General Assembly, Tokyo 2004 (Note of Clarification on Paragraph 30 added); and 
59th WMA General Assembly, Seoul, October 2008. 
A. INTRODUCTION  
1. The World Medical Association (WMA) has developed the Declaration of Helsinki as a 
statement of ethical principles for medical research involving human subjects, including 
research on identifiable human material and data.  
The Declaration is intended to be read as a whole and each of its constituent paragraphs 
should not be applied without consideration of all other relevant paragraphs.  
2. Although the Declaration is addressed primarily to physicians, the WMA encourages 
other participants in medical research involving human subjects to adopt these principles.  
3. It is the duty of the physician to promote and safeguard the health of subjects, including 
those who are involved in medical research. The physician's knowledge and conscience 
are dedicated to the fulfilment of this duty.  
4. The Declaration of Geneva of the WMA binds the physician with the words, "The health 
of my subject will be my first consideration," and the International Code of Medical 
Ethics declares that, "A physician shall act in the subject's best interest when providing 
medical care."  
5. Medical progress is based on research that ultimately must include studies involving 
human subjects. Populations that are underrepresented in medical research should be 
provided appropriate access to participation in research.  
6. In medical research involving human subjects, the well-being of the individual research 
subject must take precedence over all other interests.  
7. The primary purpose of medical research involving human subjects is to understand the 
causes, development and effects of diseases and improve preventive, diagnostic and 
therapeutic interventions (methods, procedures and treatments). Even the best current 
interventions must be evaluated continually through research for their safety, 
effectiveness, efficiency, accessibility and quality.  
8. In medical practice and in medical research, most interventions involve risks and 
burdens.  


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9. Medical research is subject to ethical standards that promote respect for all human 
subjects and protect their health and rights. Some research populations are particularly 
vulnerable and need special protection. These include those who cannot give or refuse 
consent for themselves and those who may be vulnerable to coercion or undue influence.  
10. Physicians should consider the ethical, legal and regulatory norms and standards for 
research involving human subjects in their own countries as well as applicable 
international norms and standards. No national or international ethical, legal or regulatory 
requirement should reduce or eliminate any of the protections for research subjects set 
forth in this Declaration.  
B. BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH  
1. It is the duty of physicians who participate in medical research to protect the life, health, 
dignity, integrity, right to self-determination, privacy, and confidentiality of personal 
information of research subjects.  
2. Medical research involving human subjects must conform to generally accepted scientific 
principles, be based on a thorough knowledge of the scientific literature, other relevant 
sources of information, and adequate laboratory and, as appropriate, animal 
experimentation. The welfare of animals used for research must be respected.  
3. Appropriate caution must be exercised in the conduct of medical research that may harm 
the environment.  
4. The design and performance of each research study involving human subjects must be 
clearly described in a research protocol. The protocol should contain a statement of the 
ethical considerations involved and should indicate how the principles in this Declaration 
have been addressed. The protocol should include information regarding funding, 
Sponsors, institutional affiliations, other potential conflicts of interest, incentives for 
subjects and provisions for treating and/or compensating subjects who are harmed as a 
consequence of participation in the research study. The protocol should describe 
arrangements for poststudy access by study subjects to interventions identified as 
beneficial in the study or access to other appropriate care or benefits.  
5. The research protocol must be submitted for consideration, comment, guidance and 
approval to a research ethics committee before the study begins. This committee must be 
independent of the researcher, the Sponsor and any other undue influence. It must take 
into consideration the laws and regulations of the country or countries in which the 
research is to be performed as well as applicable international norms and standards but 
these must not be allowed to reduce or eliminate any of the protections for research 
subjects set forth in this Declaration. The committee must have the right to monitor 
ongoing studies. The researcher must provide monitoring information to the committee, 
especially information about any SAEs. No change to the protocol may be made without 
consideration and approval by the committee.  
6. Medical research involving human subjects must be conducted only by individuals with 
the appropriate scientific training and qualifications. Research in subjects with a 
particular diagnosis or healthy adult subjects requires the supervision of a competent and 
appropriately qualified physician or other health care professional. The responsibility for 


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the protection of research subjects must always rest with the physician or other health 
care professional and never the research subjects, even though they have given consent.  
7. Medical research involving a disadvantaged or vulnerable population or community is 
only justified if the research is responsive to the health needs and priorities of this 
population or community and if there is a reasonable likelihood that this population or 
community stands to benefit from the results of the research.  
8. Every medical research study involving human subjects must be preceded by careful 
assessment of predictable risks and burdens to the individuals and communities involved 
in the research in comparison with foreseeable benefits to them and to other individuals 
or communities affected by the condition under investigation.  
9. Every clinical trial must be registered in a publicly accessible database before recruitment 
of the first subject.  
10. Physicians may not participate in a research study involving human subjects unless they 
are confident that the risks involved have been adequately assessed and can be 
satisfactorily managed. Physicians must immediately stop a study when the risks are 
found to outweigh the potential benefits or when there is conclusive proof of positive and 
beneficial results.  
11. Medical research involving human subjects may only be conducted if the importance of 
the objective outweighs the inherent risks and burdens to the research subjects.  
12. Participation by competent individuals as subjects in medical research must be voluntary. 
Although it may be appropriate to consult family members or community leaders, no 
competent individual may be enrolled in a research study unless he or she freely agrees.  
13. Every precaution must be taken to protect the privacy of research subjects and the 
confidentiality of their personal information and to minimize the impact of the study on 
their physical, mental and social integrity.  
14. In medical research involving competent human subjects, each potential subject must be 
adequately informed of the aims, methods, sources of funding, any possible conflicts of 
interest, institutional affiliations of the researcher, the anticipated benefits and potential 
risks of the study and the discomfort it may entail, and any other relevant aspects of the 
study. The potential subject must be informed of the right to refuse to participate in the 
study or to withdraw consent to participate at any time without reprisal. Special attention 
should be given to the specific information needs of individual potential subjects as well 
as to the methods used to deliver the information. After ensuring that the potential subject 
has understood the information, the physician or another appropriately qualified 
individual must then seek the potential subject's freely-given informed consent, 
preferably in writing. If the consent cannot be expressed in writing, the nonwritten 
consent must be formally documented and witnessed.  
15. For medical research using identifiable human material or data, physicians must normally 
seek consent for the collection, analysis, storage and/or reuse. There may be situations 
where consent would be impossible or impractical to obtain for such research or would 
pose a threat to the validity of the research. In such situations the research may be done 
only after consideration and approval of a research ethics committee.  


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16. When seeking informed consent for participation in a research study the physician should 
be particularly cautious if the potential subject is in a dependent relationship with the 
physician or may consent under duress. In such situations the informed consent should be 
sought by an appropriately qualified individual who is completely independent of this 
relationship.  
17. For a potential research subject who is incompetent, the physician must seek informed 
consent from the legally authorized representative. These individuals must not be 
included in a research study that has no likelihood of benefit for them unless it is intended 
to promote the health of the population represented by the potential subject, the research 
cannot instead be performed with competent persons, and the research entails only 
minimal risk and minimal burden.  
18. When a potential research subject who is deemed incompetent is able to give assent to 
decisions about participation in research, the physician must seek that assent in addition 
to the consent of the legally authorized representative. The potential subject's dissent 
should be respected.  
19. Research involving subjects who are physically or mentally incapable of giving consent, 
for example, unconscious subjects, may be done only if the physical or mental condition 
that prevents giving informed consent is a necessary characteristic of the research 
population. In such circumstances the physician should seek informed consent from the 
legally authorized representative. If no such representative is available and if the research 
cannot be delayed, the study may proceed without informed consent provided that the 
specific reasons for involving subjects with a condition that renders them unable to give 
informed consent have been stated in the research protocol and the study has been 
approved by a research ethics committee. Consent to remain in the research should be 
obtained as soon as possible from the subject or a legally authorized representative.  
20. Authors, editors and publishers all have ethical obligations with regard to the publication 
of the results of research. Authors have a duty to make publicly available the results of 
their research on human subjects and are accountable for the completeness and accuracy 
of their reports. They should adhere to accepted guidelines for ethical reporting. Negative 
and inconclusive as well as positive results should be published or otherwise made 
publicly available. Sources of funding, institutional affiliations and conflicts of interest 
should be declared in the publication. Reports of research not in accordance with the 
principles of this Declaration should not be accepted for publication.  
C. ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH COMBINED WITH 
MEDICAL CARE  
1. The physician may combine medical research with medical care only to the extent that 
the research is justified by its potential preventive, diagnostic or therapeutic value and if 
the physician has good reason to believe that participation in the research study will not 
adversely affect the health of the subjects who serve as research subjects.  


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2. The benefits, risks, burdens and effectiveness of a new intervention must be tested 
against those of the best current proven intervention, except in the following 
circumstances: 
• The use of placebo, or no treatment, is acceptable in studies where no current 
proven intervention exists.  
• Where for compelling and scientifically sound methodological reasons the use of 
placebo is necessary to determine the efficacy or safety of an intervention and the 
subjects who receive placebo or no treatment will not be subject to any risk of 
serious or irreversible harm. Extreme care must be taken to avoid abuse of this 
option.  
3. At the conclusion of the study, subjects entered into the study are entitled to be informed 
about the outcome of the study and to share any benefits that result from it, for example, 
access to interventions identified as beneficial in the study or to other appropriate care or 
benefits.  
4. The physician must fully inform the subject which aspects of the care are related to the 
research. The refusal of a subject to participate in a study or the subject's decision to 
withdraw from the study must never interfere with the subject-physician relationship.  
5. In the treatment of a subject, where proven interventions do not exist or have been 
ineffective, the physician, after seeking expert advice, with informed consent from the 
subject or a legally authorized representative, may use an unproven intervention if, in the 
physician's judgment, it offers hope of saving life, re-establishing health or alleviating 
suffering. Where possible, this intervention should be made the object of research, 
designed to evaluate its safety and efficacy. In all cases, new information should be 
recorded and, where appropriate, made publicly available. 
 


 
1. 
TITLE PAGE 
Clinical Study Protocol AG348-C-003 
EudraCT No. 2015-000484-13 
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, 
Pharmacokinetic and Pharmacodynamic Study of AG-348 in Adult Patients 
with Pyruvate Kinase Deficiency 
Study Sponsor: 
Agios Pharmaceuticals, Inc. 
88 Sidney Street 
Cambridge, MA 02139-4169 
Phone:  617-649-8600 
Fax:  617-649-8618 
Responsible Medical Officer: 
, MD, PhD 
 
 
Agios Pharmaceuticals, Inc. 
Phone: 
 
Email:  
 
Study Medical Monitor 
, MD 
 
On behalf of Agios Pharmaceuticals, Inc. 
Mobile Phone: 
 
Office Phone: 
 
Email: 
  
Document Version (Date): 
Revised 
Version 1.0 (05 January 2015) 
Amendment 1, Protocol Version 2.0 (02 February 2015) Final 
Amendment 2, Protocol Version 3.0 (05 August 2015) Final 
Amendment 3, Protocol Version 4.0 (10 November 2015) 
Final 
Amendment 4, Protocol Version 5.0 (30 March 2016) Final 
This study will be conducted according to the protocol and in compliance with Good Clinical Practice, the 
ethical principles stated in the Declaration of Helsinki, and other applicable regulatory requirements. 
CONFIDENTIALITY NOTE: 
The information contained in this document is confidential and proprietary to Agios Pharmaceuticals, Inc. Any 
distribution, copying, or disclosure is strictly prohibited unless such disclosure is required by federal regulations or 
state law. Persons to whom the information is disclosed must know that it is confidential and that it may not be 
further disclosed by them. 
1 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
INVESTIGATOR’S AGREEMENT 
 
I understand that all documentation provided to me by Agios 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). No changes will be made to the study protocol without the 
prior written approval of Agios and the IRB, except where necessary to eliminate an immediate 
hazard to the patient. 
I have read, understood, and agree to conduct this study as outlined in the protocol and in 
accordance with the guidelines and all applicable government regulations. 
 
 
Investigator Name (printed) 
 
Investigator Signature 
 
Date 
 
 
 
 
Investigational site or name of institution and location (printed) 
2 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
2. 
SYNOPSIS 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Title of Study:  
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, Pharmacokinetic and 
Pharmacodynamic Study of AG-348 in Adult Patients with Pyruvate Kinase Deficiency 
Study Center(s): 
This study will be conducted at multiple study centers. 
Phase of Development: 2 
Objectives: 
Core Period 
Primary: 
• 
Evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in patients with 
pyruvate kinase deficiency (PK deficiency). 
Secondary: 
• 
Evaluate the pharmacokinetics (PK) of AG-348 and the metabolite AGI-8702. 
• 
Evaluate the pharmacodynamic (PD) response of adenosine triphosphate (ATP) and 
2,3-diphosphoglycerate (2,3-DPG) after administration of AG-348. 
• 
Evaluate indicators of clinical activity of AG-348 in patients with PK deficiency, including changes 
in hemoglobin (Hb), hematocrit (HCT), reticulocyte count, haptoglobin (Hp), carboxyhemoglobin 
(COHb), end tidal carbon monoxide (ETCO), lactate dehydrogenase (LDH), total and indirect 
bilirubin, erythropoietin (EPO), hepcidin, ferritin, and transferrin saturation (serum iron/iron binding 
capacity). 
Extension Period 
Primary: 
• 
Evaluate the safety and tolerability of up to 30 months of AG-348 administration in patients with PK 
deficiency. 
Secondary: 
• 
Evaluate the PK of AG-348 and the metabolite AGI-8702. 
• 
Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348. 
3 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
• 
Evaluate indicators of clinical activity of AG-348 in patients with PK deficiency, including changes 
in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, hepcidin, 
ferritin, and transferrin saturation (serum iron/iron binding capacity). 
Methodology: 
Study AG348-C-003 is a Phase 2, open label, two arm, multicenter, randomized, dose-ranging study in 
adult patients with PK deficiency; the study will be divided in to a Core Period and an Extension Period. 
During the Core Period, patients will receive multiple doses of AG-348 for up to 24 weeks; patients who 
are eligible can enter the Extension Period to receive AG-348 for up to 2 years following the end of the 
Core Period. Patients with PK deficiency confirmed by red blood cell (RBC) PK enzymatic assay 
performed at Screening will be eligible to participate in this study. At Week 25, patients who safely 
tolerate AG-348 and demonstrate clinical activity of AG-348 may be eligible to immediately roll over to 
the Extension Period for continued treatment. Patients who complete treatment at the end of the Core 
Period (24 weeks) will undergo follow-up assessment 4 weeks after the last dose of study drug. If a 
patient discontinues at any other time (including early discontinuation or discontinuation during the Core 
or Extension Period), the follow-up assessments will be conducted 4 weeks after discontinuation. 
Patients with toxicity suspected to be related to study drug will continue follow-up until the adverse 
event (AE) resolves, is declared chronic by the Investigator, or the patient is lost to follow-up.  
For the Core Period, up to 25 patients will be initially randomized on an open-label 1:1 basis to each of 
two twice-daily (BID) doses of AG-348 (up to 50 patients total; see Figure 1, Study Schema). The dose 
of Arm 1 is 300 mg of AG-348 administered orally (PO) every 12 hours (q12h, BID). The dose of Arm 2 
is 50 mg of AG-348 administered PO q12h (BID). Randomization will be stratified by PKR mutation in 
order to maintain balance as much as possible across the dose arms for the specific mutations expected 
to be most frequently enrolled. The PKR mutation stratification factor will consist of 4 levels (R510Q, 
R486W, and R479H) and all other mutations (“other”). Mutation status is defined by the presence of at 
least one of the indicated mutations; patients with more than one stratified mutation will be assigned 
based on Sponsor’s discretion.  
The doses for each arm of the Core Period have been selected from the AG348-C-001 single ascending 
dose (SAD) and AG348-C-002 multiple ascending dose (MAD) studies in healthy adult volunteers to 
represent the range of doses/exposures that were safely tolerated and resulted in maximal or near-
maximal PD effects on 2,3-DPG and ATP.  
Because PK deficiency is a rare disease with a limited eligible patient population and because the 
underlying pathophysiology and clinical phenotype of affected patients is heterogeneous due to the wide 
variety of mutations in PKR that cause the disease, it is important to focus closely on dose findings in 
this first-in-patient study. Therefore, in addition to initiating this study with 2 different doses of AG-348 
administered q12h, a Data Review Team (DRT) will be established to review study data on a regular 
basis and adapt the study design, dose and schedule of AG-348. 
4 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
The DRT will monitor safety on an on-going basis and meet at regular intervals of approximately every 
6 weeks, or ad hoc, as necessary, for as long as any patients are still in the Core Period to review AEs, 
vital signs (VS), clinical laboratory assessments (hematology, clinical chemistry, coagulation, and 
urinalysis), and electrocardiograms (ECGs). The DRT will also review available PK/PD data and 
indicators of clinical activity (e.g., changes from baseline in Hb). These DRT meetings will also include 
data review for all patients that may be under treatment in the Extension Period. If there are no patients 
still being treated in the Core Period, and the only patients being treated are those in the Extension 
Period, then the frequency of the DRT meetings will reduce to approximately every 3 months in order to 
match the frequency of patient visits (and new data collection) in the Extension Period. The DRT will be 
comprised of the study Coordinating Investigator, treating Investigators, Medical Monitor, Clinical 
Pharmacologist, Statistician, and Sponsor’s Responsible Medical Officer.  
Beginning 6 weeks after the first patient is dosed in the Core Period or ad hoc as necessary, and 
proceeding according to the schedule indicated above (approximately every 6 weeks during the Core 
Period, and approximately every 3 months during the Extension Period once all patients have completed 
the Core Period), the DRT will review cumulative safety data, available PK/PD data, and clinical 
activity data. Based on the DRT’s recurring reviews, the DRT may exercise one or more of the 
following options during the Core Period: 
• 
Continue treatment and enrollment in existing arms without change. 
• 
Add 1 new dose arm (Arm 3) to enroll up to 25 patients at a dose to be determined; the dose for 
Arm 3 may be lower or higher than Arm 1 and Arm 2 doses, but will not exceed 360 mg q12h; 
and the dose regimen may be less frequent than q12h. 
• 
Terminate or suspend enrollment to allow further review of clinical data in Arm 1 and/or Arm 2 
(and/or potential Arm 3). Enrollment in an arm could be terminated or suspended to allow 
further review, for example, for unacceptable safety/tolerability, poor PD response, or lack of 
signs of clinical activity. 
• 
Re-assign patients’ doses and schedule in a terminated arm to match the dose and schedule of 
another arm of the study. In this case, the patients in the terminated arm will remain in their 
original arm, i.e., they will not count towards the enrollment quota of the arm whose dose and 
schedule is being adopted. 
• 
Implement specific genotype restrictions to enrollment in one or more arms to ensure 
representation of patients with genotypes of greatest clinical relevance. 
The DRT may exercise one or more of the following options during the Extension Period: 
• 
Continue treatment without change; 
• 
Re-assign patients’ doses and schedule to an existing dose and schedule that has been 
determined to be safer, and/or have a better PD response, or produce signs of clinical activity; 
• 
Terminate or suspend treatment to allow further review of clinical data (eg, for unacceptable 
safety/tolerability, poor PD response, or lack of signs of clinical activity). 
The data that the DRT will review to make these decisions is expected to include, but are not necessarily 
limited to, the following:  
5 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
• 
Safety Observations: all AEs; VS, clinical laboratory (hematology, clinical chemistry, 
coagulation, and urinalysis), and ECG;  
• 
PK and PD Observations: including changes in 2,3-DPG and ATP; 
• 
Indicators of Clinical Activity: including changes in Hb, HCT, reticulocyte count, Hp, COHb, 
ETCO (Core Period only), LDH, EPO, hepcidin, total and indirect bilirubin, ferritin, and 
transferrin saturation.  
If a third dose arm is implemented, the dose of AG-348 selected will not exceed 360 mg BID, as this 
was the highest dose that demonstrated acceptable safety and tolerance in the 14-day multiple BID 
dosing study in healthy volunteers. The PK/PD sampling schedule in a potential third dosing arm will be 
determined by Sponsor’s discretion and may follow either the extensive or limited PK/PD sampling 
schedules as specified. 
The DRT will monitor the data in an ongoing manner as described and may make a decision to terminate 
enrollment in an arm if unacceptable AEs are observed, PD activity is limited, and/or markers of clinical 
activity indicate no effect. Unacceptable AEs are defined as ≥ Grade 3 AEs (using National Cancer 
Institute Common Terminology Criteria for Adverse Events [NCI CTCAEv4.03]) that are assessed as at 
least possibly related to AG-348, with the exception of Grade 3 headache, hot flash/flushing, nausea, 
vomiting, and/or diarrhea that is transient in duration (< 24 hours) or able to be medically managed to 
≤ Grade 2 within 24 hours. The DRT may also exercise discretion to terminate enrollment in an arm if it 
observes a frequency of Grade 2 AEs that would make it difficult to support long-term dosing. 
Patients in the Extension Period will continue on the dose they were randomized to in the Core Period, 
unless the DRT had reason to establish a different dose/schedule during the course of the Core Period 
(the DRT will not propose a dose higher than 360 mg q12h), or unless the treating Investigator exercises 
the option for intra-patient dose escalation (see Section 9.7.2).  
Due to the potential for AG-348-mediated aromatase inhibition, dual-energy x-ray absorptiometry 
(DXA) scan (hip and spine) will be performed at Screening (if patient has not had prior DXA scan 
within 3 months of Day 1) to obtain T and Z scores. These data are intended to serve as a baseline 
measure of bone mineral density for all enrolling patients, and are deemed of particular importance for 
those who enter the longer term Extension Period after completing 24 weeks of treatment (Core Period). 
All patients will have a second DXA scan in the interval between Weeks 24 and 28 for the Core Period. 
Patients in the Extension Period will have additional DXA scans at Months 18 and 30. 
As the number of enrolling arms changes in the study (for example, from 2 to 3), the randomization 
scheme will adjust to enable balanced randomization into each actively accruing arm. Randomization 
and stratification will cease in the event that only a single arm is left enrolling. 
Depending on possible early termination of 1 or both of the initial 2 arms, or the addition of a third arm, 
the study could enroll up to a maximum of 75 patients. 
6 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Figure 1:  Study Schema:  Core Period 
 
Abbreviations:  BID (q12h) = twice-daily (every 12 hours); DRT = data review team; PKR = pyruvate kinase red blood cell 
isoform; TBD= to be determined; w = week(s). 
 
Visit Schedule 
Screening assessments will occur within 42 days prior to the first dose of study treatment. During the 
Core Treatment Period, patients will attend visits at baseline (Day 1), weekly through Week 3 (Days 8, 
15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12), and monthly through 
Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate AG-348 of AG-348 through Week 24 
(Core Period) may be eligible to immediately enter the Extension Period for continued treatment for up 
to 2 years after completion of the Core Period upon agreement of the treating Investigator and the 
Medical Monitor or Responsible Medical Officer. Study visits for safety and clinical activity 
assessments will occur approximately every 3 months during the Extension Period. All patients will 
undergo a follow-up assessment 4 weeks after the last dose of AG-348, regardless of whether this was 
due to early discontinuation, the last dose in the Core Period for a patient who chooses not to continue in 
the Extension Period, or the last dose of the Extension Period.  
 
7 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Dose Modifications for Safety and/or Increase in Hb Level 
The Investigator will monitor all patients for safety and tolerability. Modification of an individual 
patient’s dose of AG-348 will be based on AEs and/or observed changes in Hb level as described in 
Section 9.7.1 and Section 9.7.2. The same criteria for dose modifications will apply in the Extension 
Period as in the Core Period. 
Number of patients (planned): Up to approximately 75 patients. 
Diagnosis and main criteria for inclusion: 
Inclusion criteria: 
For entry into the Core Period, patients must meet all of the following criteria during the Screening or 
other specified period: 
1. Signed written informed consent obtained prior to performing any study procedure, including 
screening procedures.  
2. Male or female, aged 18 years and older. 
3. Known medical history of PK deficiency.  
4. All patients must have documented clinical laboratory confirmation of PK deficiency by RBC 
pyruvate kinase enzymatic assay performed at Screening either by a designated central 
laboratory or by any participating investigative site’s local hematology laboratory. Patients with 
prior documentation of PK deficiency by RBC enzymatic assay will have a reconfirmation of 
this result during Screening as a condition of enrollment. 
i. 
In the event that a patient’s screening pyruvate kinase enzymatic assay is negative (i.e., 
shows normal pyruvate kinase activity), the patient will be eligible for enrollment if the 
genotyping shows a mutant genotype that has been previously documented in the 
literature to be associated with PK deficiency. If the genotyping shows a previously 
undescribed mutation in the PKR gene, then the eligibility for enrollment will be 
determined on a case-by-case basis by the Coordinating Investigator and Medical 
Monitor in discussion with the Investigator. If no mutation is defined, then the patient 
will not be eligible. 
5. ALL patients must have a blood sample for genotypic characterization of the mutant PKR gene 
performed by the designated central laboratory at Screening. The designated central laboratory-
determined genotype will generally serve as the basis for genotyping for enrollment. However, 
patients whose genotype has already been determined by another laboratory may be enrolled on 
the basis of that report, with the approval of the Medical Monitor, in case of unexpected delay in 
return of the designated central laboratory result during the Screening Period. Enrollment on the 
basis of a result from a laboratory other than the designated central genotyping laboratory does 
not relieve the inclusion requirement that ALL patients must have a sample sent to the 
8 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
designated central genotyping laboratory. 
6. All patients must have genotypic characterization of the UGT1A1 gene performed by a 
designated central laboratory to document whether they may have underlying Gilbert's Disease. 
Patients with Gilbert’s Disease are eligible to enroll. 
7. Males must have Hb ≤ 12.0 g/dL; females must have Hb ≤ 11.0 g/dL.  
8. All patients must be considered transfusion independent as defined by: no greater than 3 units of 
RBCs transfused in the 12-month period up to the first day of study dosing and no transfusions 
within 4 months of first day of study dosing. Patients who have received more transfusion 
support than described above will be evaluated for eligibility on a case-by-case basis by the 
Medical Monitor. 
9. Eligible patients may still have their spleens in place, or may have undergone prior splenectomy. 
For splenectomized patients: 
i. 
Must have undergone their procedure at least 6 months prior to Screening. 
ii. 
Must be current in their vaccinations for Pneumococcal Conjugate (PCV13), 
Pneumococcal Polysaccharide (PPSV23), Quadrivalent Meningococcal vaccine, and 
Haemophilus influenzae type b as recommended by Centers for Disease Control and 
Prevention's (CDC) Advisory Committee on Immunization Practices (ACIP) or 
immunization advisory groups in Canada and the European Union (for patients enrolled 
in Canada and the EU). [http://www.cdc.gov/vaccines/schedules/downloads/adult/adult-
schedule.pdf] [Any missing vaccinations may be administered starting with the 
Screening Period and during the trial following the initiation of AG-348 dosing as 
necessary according to recommended vaccination guidance.] 
10. Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2. (Appendix 15.2) 
11. Patients must be taking at least 1 mg of folic acid daily for at least 21 days prior to first dose and 
continue daily during study participation. 
12. Adequate organ function, defined as: 
i. 
Serum aspartate aminotransferase (AST) ≤ 2.5 × upper limit of normal (ULN; unless the 
increased AST is assessed by the Investigator as due to hemolysis and/or hepatic iron 
deposition); and alanine aminotransferase (ALT) ≤ 2.5 × ULN (unless the increased 
ALT is assessed by the Investigator as due to hepatic iron deposition). 
ii. 
Normal or elevated levels of serum bilirubin. In patients with serum bilirubin > ULN, 
the elevation must be attributed to hemolysis with or without Gilbert's syndrome and 
must not be choledocholithiasis, cholecystitis, biliary obstruction, or hepatocellular 
disease. 
iii. 
Serum creatinine ≤ 1.25 × ULN. If serum creatinine > 1.25 × ULN, then 24-hour 
measured or calculated (Cockcroft-Gault) glomerular filtration rate (GFR) ≥ 60 mL/min. 
9 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
iv. 
Absolute neutrophil count (ANC) ≥ 1.0 × 109/L. 
v. 
Platelet count ≥ 100 × 109/L. 
vi. 
Activated partial thromboplastin time (aPTT) and international normalized ratio (INR) 
≤ 1.25 × ULN, unless the patient is receiving therapeutic anticoagulants. 
13. Women of childbearing potential (WOCBP) must agree to abstain from sexual intercourse or to 
use an acceptable/effective method of contraception (i.e., condom plus spermicide, condom plus 
oral contraceptive, condom plus intrauterine device [IUD], condom plus diaphragm with 
spermicide) from as soon as feasible during the Screening period until 30 days following the last 
dose of AG-348. Abstinence is an acceptable method only when this is in line with the normal 
life style of the patient, meaning that the patient plans to remain abstinent continuously 
throughout the duration of the study and for at least 30 days after the last dose of study drug. 
Periodic abstinence, e.g., calendar, sympathothermal and post-ovulation methods, and 
withdrawal are not acceptable methods of contraception. 
i. 
WOCBP include any female who has experienced menarche and who has not undergone 
successful surgical sterilization (hysterectomy, bilateral tubal ligation, and/or bilateral 
oophorectomy) or is not post-menopausal. Post-menopausal is defined as: 
ii. 
Amenorrhea ≥ 12 consecutive months without another cause, and a documented serum 
follicle stimulating hormone (FSH) level > 35 mIU/mL; 
iii. 
Amenorrhea ≥ 12 consecutive months in women ≥ 62 years old (FSH testing is not 
required). 
14. WOCBP must have a negative serum or urine pregnancy test within 72 hours before start of 
AG-348 dosing. 
15. Women must not be breastfeeding.  
16. Male patients, with the exception of those who have undergone vasectomy at least 6 months 
prior to Screening, must agree to abstain from sexual intercourse or, if sexually active, to use a 
condom with spermicide as contraception (regardless of their female partner's childbearing 
potential or their partner's use of their own contraception) from Day 1 of dosing until 30 days 
following the last dose of AG-348. Abstinence is an acceptable method only when this is in line 
with the normal life style of the patient, meaning that the patient plans to remain abstinent 
continuously throughout the duration of the study and for at least 30 days after the last dose of 
study drug. Periodic abstinence, e.g., selective timing of intercourse based on partner’s calendar, 
sympathothermal and post-ovulation methods, and withdrawal are not acceptable methods of 
contraception. 
For entry into the Extension Period, patients must meet the following criteria: 
17. Signed written informed consent obtained prior to performing any study procedure during the 
Extension Period.  
10 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
18. Patient must have completed 24 weeks of treatment during the Core Period and tolerated 
AG-348 (defined as having completed 24 weeks with or without permitted dose modifications) 
19. The patient’s treating Investigator agrees that there is a potential for clinical benefit to continued 
treatment and recommends participation in the Extension Period 
20. The Sponsor's designated Medical Monitor or Responsible Medical Officer approves the 
patient’s participation in the Extension Period  
21. As applicable, the patient must agree to continue to follow the same sexual 
abstinence/contraception rules as stated in Inclusion Criteria 13 and 16. 
Exclusion criteria: 
Patients who meet any of the following criteria at Screening or prior to dosing on Day 1 will not be 
enrolled in the Core Period: 
1. Hemoglobin level > 12.0 g/dL if male; Hb > 11.0 g/dL if female. 
2. Additional diagnosis of any other congenital or acquired blood disorder, including 
glucose-6-phosphate-dehydrogenase (G6PD) deficiency, or any other hemolytic anemia process 
except for mild allo-immunization as a consequence of transfusion therapy. 
3. Iron overload (hemosiderosis or concurrent hemochromatosis) sufficiently severe to result in a 
clinical diagnosis by the Investigator of cardiac, hepatic, or pancreatic insufficiency. 
4. Prior bone marrow or stem cell transplant. 
5. Clinically symptomatic cholelithiasis or cholecystitis. (Prior cholecystectomy is not 
exclusionary. Patients with symptomatic cholelithiasis or cholecystitis may be rescreened once 
the disorder has been treated and clinical symptoms have resolved.) 
6. Currently enrolled in another therapeutic clinical trial involving on-going therapy with any 
investigational or marketed product or placebo. Concurrent participation in the Pyruvate Kinase 
Deficiency Natural History Study (NCT02053480) is permitted. 
7. Exposure to any investigational drug, device, or procedure within 28 days prior to Screening or 
during trial participation. 
8. Concurrent medical condition that could compromise participation in the study such as: 
a. Poorly controlled hypertension (defined as systolic blood pressure (BP) > 150 mm Hg 
or diastolic BP > 90 mm Hg) refractory to medical management. 
b. History of recent (within < 6 months from Screening date) congestive heart failure; 
myocardial infarction or unstable angina pectoris; or hemorrhagic, embolic, or 
11 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
thrombotic stroke; deep venous thrombosis; or pulmonary or arterial embolism.  
c. Currently active infection requiring the use of parenteral anti-microbial agents or that is 
greater than Grade 3 (CTCAEv4.03) within 6 months of first dose. 
d. A pattern or frequency of post-splenectomy sepsis that in the assessment of the 
Investigator could reasonably be expected to interfere with the ability of the patient to 
complete the 24 week Core Period study participation. 
e. Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) 
antibody with signs of active Hepatitis B or C virus infection.  
f. Positive test for human immunodeficiency virus (HIV) 1 or 2 antibody. 
g. Diabetes mellitus judged to be in poor control by the Investigator or requiring > 3 anti-
diabetic agents counting insulin (all insulins are considered one agent); use of insulin 
per se is not exclusionary.  
h. History of any primary malignancy with the exception of: curatively treated non-
melanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; or 
other primary tumor treated with curative intent and no known active disease present 
and no treatment administered during the last 3 years. 
9. Undergone major surgery within 6 months of first dose.  
10. Current or recent history of psychiatric disorder that in the opinion of the Investigator or 
Medical Monitor could compromise the ability of the patient to cooperate with study visits and 
procedures. 
11. Use of any of the restricted list of products known to strongly inhibit cytochrome P450 
(CYP) 3A4 drug metabolism (Appendix 15.4, Table 9) within 5 days prior to Day 1 dosing; or 
to strongly induce CYP3A4 metabolism (Appendix 15.4, Table 10) within 28 days prior to Day 
1 dosing; or to strongly inhibit P-glycoprotein (P-gp) transporter (Appendix 15.4, Table 11) 
within 5 days prior to Day 1 dosing; or digoxin within 5 days prior to Day 1 dosing.  
12. Serum bilirubin > ULN attributable to factors other than hemolysis and/or Gilbert's syndrome. 
13. Male patients with heart-rate corrected QT interval -Fridericia's method (QTcF) interval 
> 450 msec, or female patients with QTcF interval > 470 msec with the exception of patients 
with a left bundle branch block (LBBB). Medical Monitor approval needed in patients with a 
LBBB. 
14. Cardiac dysrhythmias judged as clinically significant by the Investigator or requiring therapy 
with drugs that are primarily substrates of CYP3A4. 
15. History of allergy to sulfonamides if characterized by acute hemolytic anemia, anaphylaxis, or 
12 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
rash of erythema multiforme type or Stevens-Johnson syndrome. 
16. Any other medical or psychological condition deemed by the Investigator to be likely to 
interfere with a patient's ability to understand and sign informed consent; cooperate with study 
visits, tests, and procedures; or otherwise safely and reliably participate in the study. 
Patients will not be permitted to enter the Extension Period if: 
17. The patient experienced AEs during the Core Period that are considered by the treating 
Investigator or the Sponsor’s designated Medical Monitor or Responsible Medical Officer to 
pose a significant safety risk to the patient if treatment were to be extended. 
Investigational product, dosage, and mode of administration: 
AG-348 sulfate hydrate capsules will be provided as 5 mg, 25 mg, or 100 mg (free-base equivalent) of 
AG-348 sulfate hydrate without excipients in dark green opaque (5 mg), Swedish orange (25 mg), or 
white opaque (100 mg) gelatin capsules (size 2 capsules for all dose strengths). AG-348 will be 
administered PO BID. The number of capsules per dose will vary by assigned dose group. AG-348 will 
be administered with water and may be administered with or without food. 
Reference therapy, dosage and mode of administration:  
Not applicable. 
Duration of treatment:  
The duration of treatment for all patients in the Core Period will be up to 24 weeks. Patients who safely 
tolerate AG-348 and for whom the Investigator agrees with continuation of AG-348 treatment may be 
eligible to immediately roll over to the Extension Period for continued treatment (up to 2 years following 
completion of the Core Period). 
Criteria for evaluation: 
Safety: 
Monitoring of AEs in randomized patients, including determination of serious AEs (SAEs) and AEs 
leading to discontinuation; safety laboratory parameters (hematology, chemistry, urinalysis, 
coagulation); physical examination findings (including neurological examination); VS; 12-lead ECGs, 
and DXA scans. Adverse events will be graded using CTCAE, Version 4.03. Serum sex hormone levels 
(testosterone [total and free], estrone, and estradiol), bone turnover markers (serum osteocalcin-N-mid 
and serum C-terminal telopeptide [CTX]), 25-hydroxy vitamin D2 and D3, total cholesterol, high-
density lipoprotein-cholesterol (HDL-C), and triglycerides will be monitored for evidence of potential 
inhibition of aromatase by AG-348. Menstruating female patients will also keep a paper-based menstrual 
cycle diary throughout the study. 
Indicators of Clinical Activity: 
Monitoring of potential indicators of clinical activity will include evaluating changes in Hb, HCT, 
reticulocyte count, Hp, COHb, ETCO (Core Period only), LDH, total and indirect bilirubin, EPO, 
hepcidin, ferritin, and transferrin saturation.  
13 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Pharmacokinetics: 
Approximately the first 10 patients treated in the Core Period, contingent on clinical site feasibility, will 
undergo extensive PK sampling as detailed in Appendix 15.1, Table 7. The remainder of treated patients 
will undergo limited PK sampling as detailed in Appendix 15.1, Table 8. During the Core Period, serial 
blood sampling for determination of concentration-time profiles of AG-348 and its metabolite AGI-8702 
will be conducted following the first dose and the morning Day 15 dose, and additional trough levels of 
AG-348 and AGI-8702 will be obtained. During the Extension Period, predose PK samples will be 
drawn for the measurement of trough levels of AG-348 and AGI-8702 at each study visit (every 
3 months; see Appendix 15.1, Table 6). AG-348 and AGI-8702 will be analyzed using qualified assays 
to determine concentrations in plasma. Pharmacokinetic parameters on Day 1 and Day 15 will be 
computed using standard non-compartmental methods based on observed plasma AG-348 and AGI-8702 
concentrations. 
Pharmacodynamics: 
Pharmacodynamic assessments during the Core Period will include 2,3-DPG, ATP (secondary 
objectives), 
 
 
 
Approximately the first 10 patients treated during the Core Period will undergo extensive PD sampling 
as detailed in Appendix 15.1, Table 7. The remainder of treated patients will undergo limited PD 
sampling as detailed in Appendix 15.1, Table 8. During the Core Period, serial blood sampling for 
determination of levels of ATP and, 2,3-DPG will be conducted following the first dose and the morning 
Day 15 dose, and additional trough levels of ATP and 2,3-DPG will be obtained. During the Extension 
Period, predose PD samples will be drawn for the measurement of trough levels of 2,3-DPG, ATP, 
 
 at each study visit (every 3 months; see Appendix 15.1, Table 6). Adenosine triphosphate 
and 2,3 DPG will be analyzed using qualified assays to determine concentrations in whole blood. 
Pharmacodynamic parameters on Day 1 and Day 15 will be computed based on observed whole blood 
ATP and 2,3-DPG concentrations.  
Statistical methods: 
The primary objective during the Core Period of this Phase 2 study is to evaluate the safety and 
tolerability of up to 24 weeks of AG-348 in patients with PK deficiency. Therefore, analyses will be 
primarily descriptive in nature; no formal hypothesis testing will be conducted. All analyses will be 
14 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
conducted separately within each dose arm, or pooled when appropriate Analyses of safety and of 
indicators of clinical activity will be conducted for the Core Period, Extension Period, and overall, if 
applicable. For the Core Period, the data to be analyzed will include all collected data through 24 weeks 
of treatment for patients who directly enter the Extension period. For patients who do not enter the 
Extension Period, the analyses will include all collected data through the duration of treatment 
(24 weeks or less) plus 4 week follow up data. For patients who move directly from the Core to the 
Extension Period, the 4 week follow up data will be analyzed as part of the Extension Period. 
Summaries will be produced for disposition, baseline disease characteristics and demographic data 
including genotype, safety measurements, PK, PD parameters and indicators of clinical activity. Data 
from each AG-348 dose group will be analyzed separately, and pooled across all dose groups where 
appropriate. All data will also be listed by individual patient. Categorical variables will be summarized 
by frequency distributions (number and percentages) and continuous variables will be summarized by 
descriptive statistics (mean, standard deviation [SD], median, minimum, and maximum). 
Populations for analysis will include a Safety Analysis Set, a PK Analysis Set, and an Efficacy Analysis 
Set. The Safety Analysis set will include all patients who are enrolled and receive any dose of study 
treatment; this population will be the primary set for the analysis of safety data and the default analysis 
set for all data analyses unless otherwise stated. Patients will be classified according to treatment 
received, where treatment received is defined as the assigned treatment if it is received at least once, or 
as the first treatment received if assigned treatment is never received. The PK Analysis Set will include 
all patients in the Safety Analysis Set with sufficient plasma sample data to assess PK parameters. 
Results of the potential PD activity of AG-348 will also be based on the PK analysis set. The Efficacy 
Analysis Set will include all patients who are enrolled and achieve at least 50% compliance with their 
assigned dose intensity for at least 4 weeks of continued dosing. Additional efficacy analyses may be 
performed on subsets of patients with other degrees of compliance with their assigned dose intensity 
and/or duration of dosing. The Efficacy Analysis Set will be the primary set for the analysis of 
preliminary clinical activity data. Patients will be classified according to assigned treatment. 
 
 
 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) and the 
incidence of treatment-emergent AEs (TEAEs) (new or worsening from baseline) will be summarized by 
primary system organ class (SOC), preferred term, CTCAE Version 4.03 severity, outcome, action taken 
with study drug, and relationship to study drug by dose group. Separate summaries will be produced for 
all TEAEs, treatment-related AEs (those considered by the Investigator as at least possibly drug related), 
SAEs, discontinuations due to AEs, and AEs ≥ Grade 3 severity. Individual patient listings will be 
provided for any deaths, SAEs, AEs leading to interruption and/or reduction of study drug dose, and 
AEs leading to discontinuation of treatment. Analyses of AEs will be performed for those events that are 
considered treatment-emergent, where treatment-emergent is defined as any AE with onset beginning at 
the day of first administration of study drug, throughout the treatment period until 30 days after the last 
dose of study drug, or any event that was present at baseline but worsened in intensity or was 
subsequently considered drug-related by the Investigator through the end of the study. 
15 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Descriptive statistics will be provided for clinical laboratory values (e.g., hematology, serum chemistry, 
coagulation studies, urinalysis) and VS data, presented as both actual values and changes from baseline 
relative to each on-study evaluation. Shift analyses will be conducted for selected laboratory parameters 
based on the baseline CTCAE grade to maximum CTCAE grade. Where applicable CTCAE terms do 
not exist, a grading system based on the upper and/or lower limits of normal will be used to classify the 
results. 
Electrocardiogram analyses will include individual patient listings and summaries of abnormal and 
clinically significant ECG results. Actual values and changes from baseline in the portion of the ECG 
wave from the beginning of the P wave to the beginning of the QRS complex (PR), QRS, heart rate-
corrected QT intervals (QTc) will be summarized by visit and dose arm.  
Data collected from the menstrual diaries such as the start and stop dates of the menses and the patient 
reported characteristics of the menses will be presented in a by-patient listing. Additional descriptions of 
the data may also be performed. 
Concomitant medications will be coded using the WHO Drug Dictionary. Concomitant medications 
prior to and after the start of the study drug will be listed by patient, and summarized by ATC term and 
dose arm. 
Descriptive statistics will be used to summarize PK parameters for the parent compound AG-348 and the 
metabolite AGI-8702 for each dose group, and where appropriate, for the entire population. 
Pharmacokinetic parameters will be summarized using the following descriptive statistics: n, mean, SD, 
coefficient of variation %, median, minimum, and maximum, geometric mean, and geometric coefficient 
of variation.  
Descriptive statistics will be used to summarize PD parameters for 2,3-DPG and ATP for each dose 
group, and where appropriate for the entire population. Pharmacodynamic parameters will be 
summarized using the following descriptive statistics: n, mean, SD, coefficient of variation %, median, 
minimum, and maximum, geometric mean, and geometric coefficient of variation %. 
Analyses of serum sex hormones will use appropriate graphic displays and statistical analyses to 
evaluate patient change from baseline for each parameter. These analyses will include summaries of 
actual values and change from baseline using appropriate descriptive statistics (mean, SD, median, min 
and max) as described above.  
Analyses evaluating indicators of potential clinical activity of AG-348 in patients with PK deficiency 
will include changes in Hb, HCT, reticulocyte count, Hp, COHb, ETCO (Core Period only), LDH, total 
and indirect bilirubin, EPO, hepcidin, ferritin, and transferrin saturation (serum iron/iron binding 
capacity). Characterization of Hb response (e.g., % of patients whose Hb increases by a certain amount), 
as well as time to Hb response, and duration of Hb response will be explored, among others. 
The study database will be locked and statistical analysis will be performed after all patients have 
completed the Core Period. The results of this analysis will be presented in a clinical study report (CSR). 
Additional data collected during the Extension Period after the CSR database lock will be analyzed for 
inclusion in a subsequent CSR addendum. 
Interim Review 
No formal statistical analysis will be conducted. Safety data will be reviewed on an ongoing basis by the 
16 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
DRT, who will meet to review safety, PK, PD, and preliminary clinical activity data at regular intervals 
(approximately every 6 weeks during the Core Period and approximately every 3 months during the 
Extension Period once all patients have completed the Core Period) throughout the duration of the study. 
The composition of the DRT, its meeting schedule, the data to be reviewed, and the decisions it is 
empowered to make have been described previously. 
Additional interim reviews of data may be conducted to support decision making concerning the current 
clinical study, the sponsor’s development programs in general, or in case of any safety concerns.  
 
Sample Size 
Due to the rare disease setting, the minimal sample size may be determined by feasibility. Depending on 
possible early termination of 1 or both of the initial 2 arms or the addition of a 3rd dose arm, the study 
could enroll up to a maximum of 75 patients. 
17 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF 
FIGURES 
TABLE OF CONTENTS 
1. 
TITLE PAGE ................................................................................................................1 
2. 
SYNOPSIS ...................................................................................................................3 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES ...............18 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS.............................23 
5. 
INTRODUCTION ......................................................................................................27 
5.1. 
Pyruvate Kinase Deficiency .......................................................................................27 
5.2. 
AG-348 .......................................................................................................................28 
5.2.1. 
Summary of Nonclinical Data ....................................................................................28 
5.2.1.1. 
Pharmacology .............................................................................................................29 
5.2.1.2. 
Safety Pharmacology ..................................................................................................29 
5.2.1.3. 
Pharmacokinetics ........................................................................................................29 
5.2.1.4. 
Toxicology ..................................................................................................................30 
5.2.2. 
Summary of Clinical Data ..........................................................................................32 
5.2.2.1. 
Pharmacokinetics ........................................................................................................32 
5.2.2.2. 
Pharmacodynamics .....................................................................................................33 
5.2.2.3. 
Safety ..........................................................................................................................33 
5.3. 
Study Rationale ...........................................................................................................34 
5.3.1. 
Summary of Overall Safety Management Plan ..........................................................35 
6. 
TRIAL OBJECTIVES AND ENDPOINTS ...............................................................37 
6.1. 
Core Period .................................................................................................................37 
6.1.1. 
Primary Objective .......................................................................................................37 
6.1.2. 
Secondary Objectives .................................................................................................37 
 
37 
6.2. 
Extension Period .........................................................................................................37 
6.2.1. 
Primary Objective .......................................................................................................37 
6.2.2. 
Secondary Objectives .................................................................................................37 
 
38 
6.3. 
Study Measures and Endpoints ...................................................................................38 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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6.3.1. 
Safety Measures and Endpoints ..................................................................................38 
6.3.2. 
Clinical Activity Measures and Endpoints .................................................................38 
6.3.3. 
Pharmacokinetic and Pharmacodynamic Measures and Endpoints ............................38 
 
39 
7. 
INVESTIGATIONAL PLAN .....................................................................................40 
7.1. 
Overall Study Design ..................................................................................................40 
7.2. 
Justification of the Study Design ................................................................................44 
7.3. 
Rationale for the Starting Dose, Dose Range, and Duration of Dosing .....................45 
7.4. 
Criteria for Study Termination ...................................................................................46 
8. 
STUDY POPULATION .............................................................................................48 
8.1. 
Number of Patients .....................................................................................................48 
8.2. 
Inclusion Criteria ........................................................................................................48 
8.3. 
Exclusion Criteria .......................................................................................................51 
8.4. 
Patient Identification and Registration .......................................................................52 
8.5. 
Patient Randomization ................................................................................................52 
8.6. 
Patient Withdrawal Criteria ........................................................................................53 
8.7. 
Replacement of Patients .............................................................................................53 
9. 
STUDY TREATMENT ..............................................................................................54 
9.1. 
Description of Study Drug ..........................................................................................54 
9.2. 
Study Drug Packaging and Labeling ..........................................................................54 
9.3. 
Study Drug Storage .....................................................................................................54 
9.4. 
Method of Assigning Patients to Treatment ...............................................................54 
9.5. 
Blinding ......................................................................................................................54 
9.6. 
Study Drug Preparation and Administration ..............................................................55 
9.7. 
Criteria for Dose Escalation, Dose Modification, or Discontinuation of Study 
Drug ............................................................................................................................56 
9.7.1. 
Dose Modification for Safety .....................................................................................56 
9.7.2. 
Dose Modification for Increase in Hemoglobin Level ...............................................57 
9.7.3. 
Stopping Criteria .........................................................................................................58 
9.8. 
Duration of Patient Participation ................................................................................58 
9.9. 
Treatment Compliance ................................................................................................58 
9.10. 
Study Drug Accountability .........................................................................................59 
9.11. 
Prior and Concomitant Medications and Treatments .................................................59 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
9.11.1. 
Prior Medications and Procedures ..............................................................................59 
9.11.2. 
Prohibited Concomitant Therapy ................................................................................59 
9.11.3. 
Allowed Concomitant Therapy ...................................................................................61 
9.11.4. 
Potential for Phototoxicity ..........................................................................................61 
9.11.5. 
Management of Nausea, Vomiting, and Diarrhea ......................................................61 
9.11.6. 
Other Restrictions and Precautions .............................................................................62 
10. 
STUDY ASSESSMENTS ..........................................................................................63 
10.1. 
Schedule of Assessments ............................................................................................63 
10.2. 
Informed Consent and Confirmation of Eligibility ....................................................64 
10.3. 
Demographic Data, Medical and Medication History ................................................64 
10.4. 
PKR Enzymatic Assay and PKR Genotyping ............................................................65 
10.5. 
Safety Assessments .....................................................................................................65 
10.5.1. 
Physical Examination, Height, and Weight ................................................................65 
10.5.2. 
Vital Signs ..................................................................................................................65 
10.5.3. 
Electrocardiogram .......................................................................................................65 
10.5.4. 
DXA Scans .................................................................................................................65 
10.5.5. 
Safety Laboratory Assessments ..................................................................................66 
10.5.5.1. 
Hematology, Serum Chemistry, Coagulation Studies, and Urinalysis .......................66 
10.5.5.2. 
Screening Serology .....................................................................................................67 
10.5.6. 
Menstrual Cycle Diary ................................................................................................67 
10.5.7. 
Adverse Events ...........................................................................................................68 
10.6. 
Pharmacokinetic Assessments ....................................................................................68 
10.7. 
Pharmacodynamic Assessments .................................................................................68 
10.8. 
Ordering of Blood Sample Collection ........................................................................70 
10.9. 
Sample Processing, Storage, and Shipment ................................................................70 
11. 
ADVERSE EVENTS..................................................................................................71 
11.1. 
Definition of Adverse Events .....................................................................................71 
11.1.1. 
Adverse Event .............................................................................................................71 
11.1.2. 
Suspected Adverse Reaction .......................................................................................71 
11.1.3. 
Unexpected Adverse Event .........................................................................................71 
11.1.4. 
Serious Adverse Event ................................................................................................71 
11.1.4.1. 
Potential Severe Drug-Induced Liver Injury ..............................................................72 
11.2. 
Procedures for Reporting Adverse Events and Serious Adverse Events ....................72 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
11.3. 
Pregnancy Reporting ..................................................................................................75 
12. 
STATISTICAL METHODS .......................................................................................76 
12.1. 
Sample Size Estimation ..............................................................................................76 
12.2. 
Populations for Analysis .............................................................................................76 
12.3. 
Procedures for Handling Missing, Unused, and Spurious Data .................................77 
12.4. 
Interim Analysis ..........................................................................................................77 
12.5. 
Statistical Methodology ..............................................................................................78 
12.5.1. 
General Methods .........................................................................................................78 
12.5.2. 
Disposition ..................................................................................................................78 
12.5.3. 
Exposure and Safety Analyses ....................................................................................78 
12.5.4. 
Pharmacokinetic Analyses ..........................................................................................79 
12.5.5. 
Pharmacodynamic Analyses .......................................................................................79 
12.5.6. 
Aromatase Hormone Analysis ....................................................................................79 
12.5.7. 
Clinical Activity ..........................................................................................................79 
 
80 
12.6. 
Procedures for Reporting Deviations to Original Statistical Analysis Plan ...............80 
13. 
ADMINSTRATIVE REQUIREMENTS ....................................................................81 
13.1. 
Good Clinical Practice ................................................................................................81 
13.2. 
Ethical Considerations ................................................................................................81 
13.3. 
Patient Information and Informed Consent ................................................................81 
13.4. 
Patient Confidentiality ................................................................................................81 
13.5. 
Protocol Compliance ..................................................................................................82 
13.6. 
Data Management .......................................................................................................82 
13.7. 
Source Document/Case Report Form Completion .....................................................83 
13.8. 
Direct Access to Source Data .....................................................................................83 
13.9. 
Record Retention ........................................................................................................83 
13.10. 
Liability and Insurance ...............................................................................................83 
13.11. 
Publication of Study Findings and Use of Information ..............................................84 
14. 
LIST OF REFERENCES ............................................................................................85 
15. 
APPENDICES ............................................................................................................87 
15.1. 
Schedules of Assessments ..........................................................................................88 
15.2. 
Eastern Cooperative Oncology Group Performance Status Scoring ..........................99 
15.3. 
National Cancer Institute Common Terminology Criteria for Adverse Events .........99 
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30 March 2016 
 
15.4. 
Potential Drug Interactions .......................................................................................100 
15.5. 
Declaration of Helsinki .............................................................................................103 
 
LIST OF TABLES 
Table 1: 
Dose Modification for Adverse Events Deemed at Least Possibly Related to 
AG-348 .......................................................................................................................57 
Table 2: 
Dose Reduction Table (by Dosing Arm) ....................................................................58 
Table 3: 
Summary of Assessments When Day 8 and/or Day 22 In-Clinic Visits Are 
Performed By Primary Care Physician (Core Period) ................................................64 
Table 4: 
Sample Size Estimation ..............................................................................................76 
Table 5: 
Schedule of Assessments: Core Period .......................................................................88 
Table 6: 
Schedule of Assessments: Extension Period ..............................................................94 
Table 7: 
Schedule of Assessments: Extensive PK/PD Sampling during the Core 
Period ..........................................................................................................................97 
Table 8: 
Schedule of Assessments: Limited PK/PD Sampling during the Core Period ...........98 
Table 9: 
Strong and Moderate CYP3A4 Inhibitors ................................................................100 
Table 10: Strong CYP3A4 Inducers .........................................................................................101 
Table 11: Strong P-glycoprotein Inhibitors ..............................................................................101 
Table 12: Sensitive CYP3A4 Substrates...................................................................................102 
 
LIST OF FIGURES 
Figure 1:  Study Schema:  Core Period .........................................................................................7 
Figure 2: 
Red Cell Glycolysis ....................................................................................................27 
Figure 3: 
Study Schema:  Core Period .......................................................................................43 
Figure 4: 
PKR Enzymatic Reaction ...........................................................................................69 
 
22 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation or Specialist Term 
Explanation 
2,3-DPG 
2,3-diphosphoglycerate 
ACIP 
Advisory Committee on Immunization Practices 
ADP 
Adenosine diphosphate 
AE 
Adverse event 
ALC 
Absolute lymphocyte count 
ALP 
Alkaline phosphatase 
ALT 
Alanine aminotransferase 
ANC 
Absolute neutrophil count 
aPTT 
Activated partial thromboplastin time 
AST 
Aspartate aminotransferase 
ATP 
Adenosine triphosphate 
AUC 
Area under the curve 
AUC0-12hr 
Area under the plasma concentration versus time curve from 0 to 12 
hours 
AUC0 24hr 
Area under the plasma concentration versus time curve from 0 to 
24 hours 
AUC0-∞ 
Area under the plasma concentration versus time curve from 0 to 
infinity 
BCRP 
Breast cancer resistance protein 
BID 
Twice daily 
BP 
Blood pressure 
BUN 
Blood urea nitrogen 
CBC 
Complete blood count 
CDC 
Centers for Disease Control and Prevention 
Cmax 
Maximum plasma concentration 
CO2 
Carbon dioxide 
COHb 
Carboxyhemoglobin 
CSR 
Clinical study report 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTX 
Serum C-terminal telopeptide 
CV 
Cardiovascular 
23 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Abbreviation or Specialist Term 
Explanation 
DDI 
Drug-drug interaction 
CYP 
Cytochrome P450 
DILI 
Drug-induced liver injury 
DLT 
Dose-limiting toxicity 
DRT 
Data review team 
DXA 
Dual-energy x-ray absorptiometry 
ECG 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic case report form 
EDC 
Electronic Data Capture 
EPO 
Erythropoietin 
ETCO 
End tidal carbon monoxide 
FDA 
Food and Drug Administration 
FSH 
Follicle stimulating hormone 
G6PD 
Glucose-6-phosphate-dehydrogenase 
GCP 
Good Clinical Practice 
GFR 
Glomerular filtration rate 
GLP 
Good Laboratory Practice 
Hb 
Hemoglobin 
HBsAg 
Hepatitis B surface antigen 
HCT 
Hematocrit 
HCV 
Hepatitis C virus 
HDL-C 
High-density lipoprotein-C 
HIV 
Human immunodeficiency virus 
Hp 
Haptoglobin 
IC50 
Concentration of drug that achieved half-maximal inhibition 
ICH 
International Conference on Harmonization 
IEC 
Independent Ethics Committee 
INR 
International normalized ratio 
IRB 
Institutional Review Board 
IUD 
Intrauterine device 
LBBB 
Left bundle branch block 
24 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Abbreviation or Specialist Term 
Explanation 
LDH 
Lactate dehydrogenase 
MAD 
Multiple ascending dose 
MCH 
Mean corpuscular hemoglobin 
MCHC 
Mean corpuscular hemoglobin concentration 
MCV 
Mean corpuscular volume 
MedDRA 
Medical Dictionary for Regulatory Activities 
mPKR 
PKR mutants 
MTD 
Maximum tolerated dose 
NCI 
National Cancer Institute 
NOEL 
No-observed-effect-level 
NOAEL 
No-observed-adverse-effect-level 
P-gp 
P-glycoprotein 
PCV13 
Pneumococcal Conjugate 
PD 
Pharmacodynamic 
PEP 
Phosphoenolpyruvate 
PK 
Pharmacokinetic 
PK deficiency 
Pyruvate kinase deficiency 
PKM2 
Pyruvate kinase isoform M2 
PKR 
Pyruvate kinase isoform R 
PO 
Oral(ly) 
PPSV23 
Pneumococcal polysaccharide 
PR 
The portion of the ECG wave from the beginning of the P wave to 
the beginning of the QRS complex 
q12h 
Every 12 hours 
q24h 
Every 24 hours 
QD 
Once-daily 
QTc 
Heart-rate corrected QT interval 
QTcB 
Corrected QT interval - Bazett correction formula 
QTcF 
Corrected QT interval - Fridericia’s method 
RBC 
Red blood cell (count) 
RDW 
Red cell distribution width 
SAD 
Single ascending dose 
SAE 
Serious adverse event 
25 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Abbreviation or Specialist Term 
Explanation 
SAP 
Statistical analysis plan 
SD 
Standard deviation 
SOC 
System organ class 
t½ 
Apparent terminal half-life 
TIBC 
Total iron-binding capacity 
Tmax 
Time to maximum plasma concentration 
TEAE 
Treatment-emergent adverse event 
ULN 
Upper limit of normal 
VS 
Vital signs 
Vss 
Volume of distribution at steady-state 
Vz/F 
Mean apparent volume of distribution 
WBC 
White blood cell  
WMA 
World Medical Association 
WOCBP 
Women of childbearing potential 
WT 
Wild type 
26 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
5. 
INTRODUCTION 
5.1. 
Pyruvate Kinase Deficiency 
Pyruvate kinase deficiency (PK deficiency) is a glycolytic enzymopathy that results in non-
spherocytic hemolytic anemia with a variable clinical presentation, ranging from mild or fully 
compensated forms to life-threatening neonatal anemia and life-long chronic hemolytic anemia 
associated with severe, debilitating co-morbidities. Pyruvate kinase deficiency is caused by 
mutations in the PKLR gene, which results in defective pyruvate kinase isoform R (PKR) in the 
red blood cell (RBC). As shown in the diagram below, PKR catalyzes the final and irreversible 
step in glycolysis, converting phosphoenolpyruvate (PEP) to pyruvate, and formation of 
adenosine triphosphate (ATP) from adenosine diphosphate (ADP). Mature RBCs rely almost 
exclusively on the process of glycolysis to generate the energy carrier molecule ATP. PKR is 
thus a key enzyme for maintaining energy homeostasis in erythrocytes, and it has been proposed 
that ATP levels are critical for optimally maintaining RBC membrane integrity (van Wijk and 
van Solinge 2005). 
Figure 2: 
Red Cell Glycolysis 
 
Abbreviations:  1,3 DPG = 1,3 diphosphoglycerate; 2,3 DPG = 2,3 diphosphoglycerate; 3-PG = 3-phosphoglycerate; 
ADP = adenosine diphosphate; ATP = adenosine triphosphate; FBP = fructose 1,6-bisphosphate; PEP = 
phosphoenolpyruvate; PKR = red blood cell-specific form of pyruvate kinase 
Note: Not all steps in glycolysis are shown. 
The majority of mutations in PKR that have been described have a deleterious effect on PKR 
catalytic activity, protein stability, and/or protein expression. Pyruvate kinase deficiency is 
associated with reduced RBC survival as well as impaired red cell maturation. Mature 
erythrocytes lack mitochondria relying predominantly on glycolysis to generate ATP. It is 
hypothesized that insufficient energy production promotes erythrocyte hemolysis due to impaired 
maintenance of cellular membrane homeostasis. 
Pyruvate kinase deficiency is an autosomal recessive disease, in which both homozygotes and 
compound heterozygotes develop hemolytic anemia. More than 200 different mutations in the 
PKLR gene have been identified in patients with PK deficiency to date, and the majority of these 
mutations are single nucleotide missense mutations (Zanella, et al. 2005). The prevalence of PK 
deficiency, based on the most common pyruvate kinase mutation in Southern Europe, is 
estimated at 51 cases per million in the Caucasian population (Beutler and Gelbart 2000). 
Some patients with PK deficiency have a clinically severe disease course that can present as a 
severe hemolytic anemia in early infancy, requiring immediate care which typically includes 
blood transfusion support (Christensen, et al. 2011; Ghidini, et al. 1991; Hennekam, et al. 1990). 
Infants with PK deficiency may have erythroblastosis fetalis with hepatosplenomegaly, 
extramedullary hematopoiesis, and severe indirect hyperbilirubinemia, which is associated with a 
Glucose
Pyruvate
PKR
`
ATP
FBP
1,3-DPG
2,3-DPG
3-PG
PEP
ADP
27 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
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high risk of kernicterus. Hyperbilirubinemia associated with the hemolytic state often requires 
supportive treatment with intensive phototherapy. For other infants, the hyperbilirubinemia 
caused by hemolysis can require exchange transfusion, as well as supportive transfusion to keep 
their hematocrit (HCT) levels >60%. 
Adults with PK deficiency are reported to have life-long hemolysis and the subsequent 
associated co-morbidities including anemia and transfusion dependence. Other co-morbidities 
include frequent miscarriages, aplastic crises, as well as symptoms associated with an acute on 
chronic hemolytic anemia (Rider, et al. 2011). Since unconjugated bilirubin is often chronically 
elevated, pigmented gallstones are common in children and adults. Severe and sometimes life-
threatening iron deposition occurs and is typically progressive. 
The current therapeutic options for patients with PK deficiency are supportive. Most require life-
long treatment, including blood transfusions at a frequency depending on the disease state. Long-
term surveillance for systemic iron overload, even in transfusion-independent patients, is 
standard as is the use of chelation therapy. Case reports of cure by allogeneic bone marrow 
transplant have been published (Tanphaichitr, et al. 2000) but have been infrequently performed. 
Splenectomy, while not curative, can reduce the need for supportive blood transfusions and 
decrease bilirubin levels in some patients. However, this intervention is associated with 
significant morbidity including infection risk, and possible increased risk of thrombosis. 
5.2. 
AG-348 
AG-348 is an orally available, allosteric activator of PKR. It is hypothesized that drug 
intervention with AG-348 restores glycolytic pathway activity and normalizes red cell 
metabolism in vivo. Biochemical experiments demonstrate that AG-348 is a potent, pan-activator 
of many PKR alleles associated with PK deficiency. Pyruvate kinsase deficiency red cells and 
their progenitors are characterized by changes in metabolism associated with defective 
glycolysis, including a build-up of PEP and 2,3-diphosphoglycerate (2,3-DPG), and lowered 
ATP levels. Treatment of PK deficiency patient red cells ex vivo with AG-348 results in 
increased ATP levels, and reductions in PEP and 2,3-DPG, consistent with pharmacological 
activation of PKR enzyme activity. In vitro studies against wild type (WT) PKR enzymes and ex 
vivo studies in blood from mice, monkeys and humans indicate that AG-348 activates WT PKR 
with a similar potency and efficacy as it does with the mutant versions. In vivo studies in mice 
confirm the potency of AG-348 in increasing WT PKR enzyme activity and modulating the 
levels of downstream markers such as ATP and 2,3-DPG. This therapeutic approach may be an 
effective way to correct the underlying pathology of PK deficiency and, importantly, provide 
clinical benefit to patients. 
5.2.1. 
Summary of Nonclinical Data 
A series of exploratory pharmacology studies were conducted to characterize the ability of 
AG-348 to activate WT PKR and anemia-associated PKR mutants (mPKR) in vitro, ex vivo, and 
in vivo. Additional details regarding nonclinical data can be found in the AG-348 Investigator’s 
Brochure 
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5.2.1.1. 
Pharmacology 
AG-348 is a potent, broad-spectrum activator of PKR with low nM potency against both WT and 
mutant enzymes. AG-348 is also able to effectively stabilize mPKR enzymes that are 
hypersensitive to thermal denaturation, suggesting that AG-348 may be able to increase PKR 
pathway activity by preventing destabilization and degradation of mPKR enzymes. Its major 
metabolite, AGI-8702, has low potency mixed activity against PKR and mutant isoforms and can 
increase the thermostability of some mPKR, albeit with significantly weaker potency than that of 
AG-348. 
The effect of AG-348 and AGI-8702 on PKR activity and a number of downstream pathway 
markers was evaluated in human RBCs. AG-348 dose-response curves in human whole blood 
showed potent activation of PKR, while AGI-8702 is a weak activator of the PKR enzyme. In 
whole blood from mice treated with AG-348, 13C-glucose labeling through the PKR reaction was 
increased by 80% demonstrating AG-348-mediated activation of the WT PKR enzyme. AG-348 
treatment of RBCs from healthy human donors potently increased levels of the PKR reaction 
product ATP consistent with activation of PKR.  
The effects of AG-348 on PKR activity and RBC metabolism also were assessed in blood 
samples from patients with PK deficiency. AG-348 activated PKR and induced metabolic 
changes (increased ATP levels and decreased 2,3-DPG levels) consistent with increased 
glycolytic pathway activity in RBCs from PK deficiency patients with different mutations in the 
PKR enzyme.  
Assessments of binding inhibition potential and enzymatic activity were evaluated in a panel of 
89 receptors, ion channels, and enzymes. Although AG-348 is a histamine H3 antagonist/inverse 
agonist and an aromatase inhibitor, no effects of histamine H3 modulation have been observed in 
safety pharmacology or toxicology studies. Effects consistent with aromatase inhibition were 
seen in toxicology studies (Section 5.2.1.4). 
5.2.1.2. 
Safety Pharmacology 
Safety pharmacology studies demonstrated that neither AG-348 nor AGI-8702 exhibited 
significant inhibition of the human ether-à-go-go related gene (hERG) current (concentration of 
drug that achieved half-maximal inhibition [IC50] 29.4 μM and > 10 μM, respectively). 
Additionally, in the monkey cardiovascular (CV) study, no CV effects were seen at any dose 
level. No effects were seen in AG-348 Good Laboratory Practice (GLP)-compliant Irwin and 
respiratory safety pharmacology studies in Sprague-Dawley rats. AG-348 administered via oral 
gavage had a dose-dependent emetic activity in the ferret at ≥ 30 mg/kg. 
5.2.1.3. 
Pharmacokinetics 
Absorption, distribution, metabolism, and excretion (ADME) studies of AG-348 were performed 
in Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys. Both oral (PO) and intravenous 
(IV) routes were evaluated at doses ranging from 1 to 50 mg/kg. A dose of 200 mg (50 mg/kg) 
was administered PO in the food-effect evaluation in monkeys. The PK of AG-348 in animal 
species is characterized by rapid oral absorption, medium to high total body plasma clearance 
(CLp), and high volume of distribution at steady-state (Vss) in rats, dogs, and monkeys. 
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AG-348 has a medium to long apparent terminal half-life (t1/2) in the rat, dog, and monkey. 
Medium to high oral bioavailability was observed for AG-348 in animals. A high-fat diet led to 
lower oral absorption of AG-348 in monkeys. 
AG-348 showed low brain penetration in rats (brain/plasma ratio of 5.7% to 9.1%) based on area 
under the plasma concentration versus time curve from 0 to 24 hours (AUC0 24hr) following 
single and repeated-dose administration, respectively. Metabolism appears to be the major 
elimination pathway for AG-348 with minimal biliary or urinary excretion.  
Both in vitro and in vivo, the predominant metabolite was the N-dealkylated product, AGI-8702. 
There were no metabolites unique to human liver microsomes and no direct Phase 2 metabolites. 
The plasma metabolite profile of AG-348 did not change with repeat or ascending dosing, 
suggesting that AG-348 is not susceptible to metabolic switching with increasing or prolonged 
dose administration. 
AG-348 is a weak direct inhibitor of cytochrome P450 (CYP) 2C8, CYP2C9, CYP2C19, 
CYP2D6, and CYP3A4/5 (testosterone 6β-hydroxylation). There was no direct inhibition of 
CYP1A2, CYP2B6, or CYP3A4/5 (midazolam 1′-hydroxylation) by AG-348. There was 
evidence of metabolism-dependent inhibition of CYP2C19 (largely reversible) and CYP3A4 
(largely irreversible) by AG-348. AG-348 is considered a weak time-dependent CYP3A4 
inhibitor. AGI-8702 is a weak direct inhibitor of CYP2C9 and CYP2C19. AG-348 has the 
potential to cause an induction-related drug-drug interaction (DDI) with sensitive CYP2B6 and 
CYP3A4 substrates. 
The routes of metabolism for AG-348 are via multiple CYPs with CYP3A4 contributing > 70% 
of the total. CYP1A2, CYP2C9, and CYP2C8 contribute approximately 6%, 10%, and 7% to the 
remaining metabolism of AG-348; other isoforms contribute < 4% each. 
AG-348 is a substrate for P-glycoprotein (P-gp), but not breast cancer resistance protein (BCRP). 
AG-348 is an inhibitor of P-gp (91% and 99% inhibition at 41 and 411 µM, respectively), but 
does not inhibit BCRP. AGI-8702 is not an inhibitor of P-gp or BCRP under the concentrations 
tested (5 and 100 μM). 
5.2.1.4. 
Toxicology 
In rats, the no-observed-effect-level/no-observed-adverse-effect-level (NOEL/NOAEL), 
determined as 2000 mg/kg, was associated with area under the plasma concentration versus time 
curve from 0 to 12 hours (AUC0-12hr) values 223- to 526-fold the projected human efficacious 
AUC0-12hr value. In dogs, clinical observations consistent with anaphylactoid reactions were seen, 
and the maximum tolerated dose (MTD) was 62.5 mg/kg, which was associated with an 
AUC0-12hr value 5.7-fold the projected efficacious AUC0-12hr value. The NOEL/NOAEL in dogs 
was 10 mg/kg, which was associated with an AUC0-12hr value 0.8-fold the projected efficacious 
AUC0-12hr value. In monkeys, the NOAEL was 1000 mg/kg, with only non-adverse emesis and 
body weight loss seen; this dose was associated with an AUC0-12hr value 70-fold the projected 
efficacious AUC0 12hr value. Based on the results of single-dose studies, the rat and the monkey 
were chosen as the most appropriate species for further evaluation in toxicology studies.  
Dose-limiting toxicity (DLT) in cynomolgus monkeys was defined in non-GLP 5-day and 
14-day repeat-dose studies as emesis, inappetence, and weight loss. These toxicities became dose 
limiting at AUC0-12hr values 27- to 34-fold the projected efficacious AUC0-12hr value, and 
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precluded meaningful evaluation of other toxicities at this exposure level. Potential other effects 
at this exposure level were observed in a number hematology and serum chemistry parameters as 
well as in lymphoid organs. Additionally, minimal potential effects in kidneys (renal 
tubulointerstitial nephritis) and heart (myocardial degeneration), which could not be 
differentiated from spontaneous background lesions, were seen when monkeys were exposed to 
AG-348 AUC0-12hr values ≥ 27- to 34-fold the projected human efficacious AUC0-12hr value.  
In GLP-compliant 28-day monkey study, the dose of 150 mg/kg/day (75 mg/kg/dose twice daily 
[BID]) was the NOAEL. Effects were limited to increased liver weights without serum chemistry 
or microscopic correlate. At this dosage level, the Day 27 AUC0-12hr values were 8.9- and 
8.5-fold the projected efficacious AUC0-12hr value in males and females respectively. In the same 
study, the low dosage of 20 mg/kg/day (10 mg/kg/dose) resulted in AUC0-12hr values that 
approximated the efficacious AUC0-12hr value, and there were no test article-related effects seen. 
The next highest dose of 50 mg/kg/day (25 mg/kg/dose) was the NOEL and was associated with 
AUC0-12hr values 3.1- and 2.6-fold the projected efficacious AUC0-12hr value in males and females 
respectively.  
In the GLP-compliant 28-day rat study, the NOAEL in males was the highest dose tested, 
600 mg/kg/day (300 mg/kg/dose), and the NOAEL in females was the lowest dose tested, 
20 mg/kg/day (10 mg/kg/dose). At the 600 mg/kg/day dosage level in males, AG-348-related 
findings were limited to mild effects on hematology, serum chemistry, and urinalysis parameters, 
and microscopic findings in the adrenal gland (minimal to mild vacuolation of the adrenal zona 
glomerulosa and decreased thickness of the zona fasciculate), liver (minimal to mild 
hepatocellular hypertrophy), kidney (minimal tubular vacuolation), pancreas (minimal to 
moderate decreased zymogen granules), heart (minimal myocardial vacuolation), and prostate 
(minimal to mild decreased secretion). All findings were fully reversible over the 14-day 
recovery period with the exception of decreased serum glucose levels and decreased prostate 
secretion. In females, the highest dosage tested was 200 mg/kg/day (100 mg/kg/dose); adverse 
effects observed were similar to those observed in the 600 mg/kg/day males, with the exception 
that in females, fewer effects in hematology and serum chemistry parameters were seen, and also 
in females, adverse effects in the reproductive organs consistent with aromatase inhibition were 
observed.  
In the 13-week repeat-dose rat study, adverse effects in males were identified in the testes and 
consisted of seminiferous tubular degeneration, spermatid retention, and Leydig cell 
hypertrophy. These effects were defined as adverse at dose levels at which they are expected to 
impair fertility. These effects were either not present or present with incidence and severity 
similar to that of the vehicle group in lower dose levels. Adverse effects in females included 
uterine atrophy and increased folding of the luminal surface; these effects were defined as 
adverse at the dose level at which they are expected to impair fertility  
In the 13-week repeat-dose study in monkeys, no adverse effects were identified, and no new 
effects were identified when compared to the 4-week repeat-dose study. Similar to what occurred 
on the 4-week study, inappetence and emesis during the initial 1-2 weeks of dosing occurred, 
precluding evaluation of higher doses.  
AG-348 inhibits human aromatase activity with an IC50 of 2050 nM (based on human placental 
microsomes) and rat aromatase with an IC50 of 493 nM (based on rat ovarian microsomes). In the 
GLP-compliant 28-day rat study, histologic effects consistent with aromatase inhibition were 
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seen in the female reproductive tract at the mid- and high-dosage levels (100 and 200 mg/kg/day) 
and included incomplete corpora lutea; ovarian follicular cysts; ovarian cystic, luteinized 
follicles; uterine atrophy; vaginal mucification; and altered cyclicity. Although these findings 
were minimal to mild and were fully reversible (over 14 days), they were considered adverse and 
the next lower dosage evaluated, 20 mg/kg/day (10 mg/kg/dose BID) was the NOAEL in 
females. The Day 27 AUC0-12hr value associated with this dosage level was 6.9-fold the projected 
human efficacious AUC0-12hr value. The potential for aromatase inhibition effects occurring in 
female rats at AUC0-12hr values > 6.9-fold and < 53-fold the projected efficacious AUC0-12hr value 
has been addressed in a 13-week rat study. In this study using doses between the NOAEL and 
LOAEL in the 28-day study, the NOAEL for histologic lesions of the uterus that may be 
associated with aromatase inhibition resulted in an AUC0-12hr that was 26-fold the projected 
efficacious value. Notably, due to the potency difference of AG-348 against rat versus human 
aromatase inhibition, there is potential for a 4-fold wider margin for aromatase inhibition in 
humans versus rats. AGI-8702 is not an aromatase inhibitor. 
5.2.2. 
Summary of Clinical Data 
To date, 72 healthy adult volunteers have been exposed to AG-348 in 2 clinical studies, a single 
ascending dose (SAD) study and a multiple ascending dose (MAD) study, with 31 of these 
subjects exposed to a single AG-348 dose under fasted conditions; 5 exposed to 2 doses of 
AG-348 under fasted and fed conditions; and 36 exposed to repeated doses of AG-348 for up to 
14 days. Except as otherwise specified, the following discussion of clinical data refers only to 
healthy adult volunteer subjects, as this is the first clinical trial in which patients with PK 
deficiency will be treated with AG-348. 
5.2.2.1. 
Pharmacokinetics 
The pharmacokinetics (PK) of AG-348 showed low to moderate variability between subjects. 
After single doses of AG-348 from 30 to 2500 mg, AG-348 was rapidly absorbed as indicated by 
the short time to maximum plasma concentration (Tmax). There appeared to be some lengthening 
of the period of absorption at higher doses as indicated by higher Tmax and a less than 
proportional increase in maximum plasma concentration (Cmax). Dose-normalized area under the 
curve (AUC) generally remained constant over the dose range studied, suggesting that AG-348 
total exposure increased in a dose-proportional manner. The mean t1/2 ranged from 17.8 to 20.4 
hours when samples were collected through 72 hours and from 50.3 to 79.3 when samples were 
collected through 120 hours. However, this terminal elimination phase contributed little to 
overall exposure of AG-348, as indicated by the small difference between AUC0-12 and area 
under the plasma concentration versus time curve from 0 to infinity (AUC0-∞), suggesting a 
shorter effective half-life of approximately 3 to 6 hours. AG-348 was extensively distributed 
(mean apparent volume of distribution [Vz/F] range of 271 to 1148 L) and had a moderate rate of 
clearance (geometric mean clearance [CL/F] range of 10.3 to 13.9 L/hr). The fraction of AG-348 
excreted in urine ranged from 0.0145 to 0.0209 across the dose levels suggesting that renal 
excretion plays a minor role in the systemic elimination of AG-348. 
The preliminary repeat-dose PK of AG-348 at doses ranging from 15 mg every 12 hours (q12h) 
to 700 mg q12h also showed an increase in AG-348 exposure in a dose-proportional manner 
after the first dose. AG-348 exposure observed after repeated dosing for 14 days at doses of 
120 mg every 24 hours (q24h) to 700 mg q12h were lower than observed on Day 1 after the first 
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dose. The magnitude of the effect was greater at higher doses; lower doses of 15 mg q12h and 
60 mg q12h did not show this effect. This observation may be related to the nonclinical finding 
that AG-348 is a potential inducer of human CYP3A4, an enzyme which biotransforms AG-348. 
It is possible that multiple doses of AG-348 leads to an increased rate of its own metabolism. 
Evaluation of the effect of food on the PK of a single 700 mg dose of AG-348 in 5 subjects who 
were administered the drug fasting and then, after an appropriate wash-out period, re-
administered the drug following ingestion of a standard US Food and Drug Administration 
(FDA) high fat meal, showed that food likely has a minimal effect on the PK of AG-348. 
5.2.2.2. 
Pharmacodynamics 
After a single dose of AG-348, a decrease in the concentration of 2,3-DPG was observed at 
3 hours post-dose, decreased in a dose-dependent manner to a minimum at 24-hour post-dose, 
and then returned to values similar to baseline by 72 to 120 hours post-dose. The mean decrease 
at 24 hours was approximately 300 µg/mL at the 700 through 2500 mg dose levels. Similar 
decreases were observed after the first dose of multiple doses and prior to doses in the MAD 
study. After the final dose of multiple doses, the concentration of 2,3-DPG returned to values 
similar to baseline between 72 and 120 hours post- dose. 
After a single dose of AG-348, a minimal increase in the concentration of ATP was observed at 
24 to 120 hours post-dose. In the MAD study, no increase was observed at 12 hours after the first 
dose; the concentration of ATP was increased on Day 8 to concentrations greater than in the 
single dose study, continued to trend upward on Day 11, and remained at a similar level through 
Day 14. The concentration of ATP remained elevated through 120 hours after the last dose on 
Day 14. The magnitude of the increase in ATP was similar across the dose range from 60 mg 
BID to 700 mg BID. 
5.2.2.3. 
Safety 
Overall, AG-348 has been well-tolerated among healthy volunteers at doses that produced strong 
pharmacodynamic (PD) effects on 2,3-DPG and ATP. 
After a single AG-348 dose, treatment-emergent adverse events (TEAEs) reported by > 1 subject 
at any time on study (either under fasted or fed conditions) included headache (22%), nausea 
(14%), and contact dermatitis and vomiting (each 6%). After repeated dosing of AG-348 for 
14 days, TEAEs that occurred in > 5% of all AG-348-treated subjects across all cohorts included 
headache (13.9%), nausea (13.9%), vomiting (8.3%), decreased appetite (8.3%), feeling hot 
(8.3%), restlessness (8.3%), and dizziness, fatigue, vessel puncture site bruise, hyperhidrosis, 
dermatitis allergic, and drug eruption (5.6% each). 
A dose-relationship was apparent with regard to the incidence of gastrointestinal events, 
primarily nausea and vomiting, with the incidence of such events increasing with increasing 
dose. Nausea and/or vomiting were observed only at doses ≥ 1400 mg in the single dose study 
and only at doses ≥ 700 BID in the MAD study. Nausea and vomiting were not observed at any 
dose ≤ 360 mg in either the single or multiple dose studies. 
All but 1 TEAE reported to date has been mild or moderate (Grade 1 or 2) in intensity. The only 
Grade 3 TEAE was elevated liver transaminases (alanine aminotransferase [ALT]; aspartate 
aminotransferase [AST]) in a single subject in the MAD study treated with 700 mg AG-348 BID. 
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The event was considered to be possibly related to AG-348, was declared a DLT, and led to 
study drug discontinuation, following which the elevated liver transaminases resolved. 
Altogether, 3 of 6 subjects treated with AG-348 at 700 mg BID discontinued study drug in the 
MAD study: the subject described above with Grade 3 elevated transaminases, and 2 others who 
withdrew themselves for nausea and vomiting. A fourth subject treated with AG-348 at 60 mg 
BID was discontinued from the study for a drug eruption. No AG-348-treated subject 
discontinued in the SAD study due to an adverse event (AE). 
No deaths or other serious AEs (SAEs) have been reported in any clinical study of AG-348. 
Furthermore, no DLTs were observed after a single administration of AG-348 at doses of 30 mg 
to 2500 mg or after repeat administration of AG-348 for 14 days at daily doses of 15 mg to 
700 mg BID except for the event of Grade 3 elevated liver function tests described above. 
Due to preclinical observations pertaining to the potential for inhibition of the aromatase enzyme 
(see Section 5.2.1.4), the AG348-C-002 multiple dose study included assessment of baseline and 
serial measures of free and total serum testosterone and serum estradiol and estrone.  
In males treated with AG-348, compared with placebo-treated males, the aromatase-dependent 
hormone assessments demonstrated an increase in total and free testosterone mean serum 
concentrations and decreased concentrations of estradiol and estrone at all doses of AG-348, 
including the lowest dose of 15 mg q12h. Most of the increases in total and free testosterone 
remained within the reference range, except at the 360 mg q12h dose where the dose group 
means on Day 8 and Day 14 exceeded the ULN. Most of the estradiol concentrations observed in 
males remained within the reference range, but the mean male estrone concentrations dropped to 
the lower limit of quantification in all dose groups except 15 mg q12h. These changes in 
aromatase-dependent hormone levels in the male subjects treated with AG-348 are consistent 
with inhibition of human aromatase, and were reversible within 14 days upon cessation of 
dosing. The study did not enroll enough female subjects to draw any definitive conclusions 
regarding serial changes in aromatase-dependent hormones over time. 
As of as this protocol amendment (Study AG348-C-003, Version 5.0), 1 patient with PK 
deficiency (
 year-old 
 1151C>T [T384M]/1529G>A [R510Q]) treated with 300 mg 
AG-348 BID experienced an increase in hemoglobin (Hb) from baseline 
 g/dL (
 g/L) to 
 g/dL (
 g/L) and a decrease in serum bilirubin at Day 17. The patient’s treatment was 
suspended for 2 days, according to the dose modification guidance in Amendment 2, and then 
resumed at 100 mg BID on Day 19. The Hb level decreased to 
 g/dL (
 g/L) by Day 21 
(before increasing again upon resuming AG-348) and was associated with an increase in serum 
bilirubin and symptoms of nausea and headache that were similar to symptoms the patient had 
previously noted during hemolytic episodes. This patient’s course suggests a robust Hb response 
to AG-348 followed by hemolysis upon suspension of the drug. The patient tolerated these 
symptoms and did not require hospitalization. See Section 9.7.2 for further guidance on dose 
modification for increases in Hb level. 
5.3. 
Study Rationale 
Study AG348-C-003 is the first study that will be conducted in patients with PK deficiency. This 
study is primarily intended to evaluate the safety and tolerability and potential indicators of 
clinical activity of AG-348 administered for up to 24 weeks. This study will also evaluate the PK 
profile of AG-348 and its metabolite AGI-8702, the PD responses in ATP and 2,3-DPG 
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following administration of AG-348, and the clinical activity of AG-348 in PK deficiency 
patients. Two previous double-blind, placebo-controlled clinical trials of AG-348 conducted in 
healthy adult male and female volunteers (AG348-C-001, a SAD study; and AG348-C-002, a 
MAD study) have established an acceptable safety and tolerability profile for AG-348 for up to 
14 days of both once-daily (QD) and BID dosing at exposures that result in significant PD 
changes in whole blood levels of the glycolytic metabolites 2,3-DPG and ATP. Due to the 
potential for AG-348-mediated aromatase inhibition, dual-energy x-ray absorptiometry (DXA) 
scan (hip and spine) will be performed at Screening (if patient has not had prior DXA scan 
within 3 months of Day 1) to obtain T and Z scores. These data will serve as baseline measures 
of bone mineral density for all enrolling patients (see Section 7.1 for more details). 
The target population of this study consists of adult males and females with a diagnosis of PK 
deficiency, who are anemic but non-transfusion dependent. Non-transfusion dependent patients 
are preferred for this study in order to reduce any potential confounding effect of transfusion 
therapies on evaluation of potential indicators of clinical activity and PD responses. The safety, 
tolerability, and PK/PD findings in this study will form the basis for subsequent clinical 
development of AG-348. 
The Extension Period is offered in order to allow for longer study of AG-348 in patients who 
experience a benefit and tolerate the drug. 
5.3.1. 
Summary of Overall Safety Management Plan 
Measures to minimize the risks to patients enrolled in the Core and Extension Periods have been 
taken with respect to the following study design elements: 
• The initial doses for Arms 1 and 2 have been selected on the basis of safety and 
tolerability observations already carefully made in two predecessor clinical trials in 
adult healthy male and female volunteers;  
• The specified inclusion/exclusion criteria have been carefully considered to avoid 
enrollment of subjects for whom exposure to the study drug might pose a hazard; 
• A designated Data Review Team (DRT) consisting of the overall study Coordinating 
Investigator, treating Investigators, Medical Monitor, Clinical Pharmacologist, 
Statistician, and Sponsor’s Responsible Medical Officer has been established to meet 
regularly at approximately 6-week intervals throughout the duration of the Core 
Period, and approximately every 3 months during the Extension Period once all 
patients have completed the Core Period to review the accumulating study data and 
will exercise options to suspend enrollment to one or both of the initial two study 
dose arms, discontinue enrollment to one or both of the initial two study dose arms, 
adjust the dose of patients in one or both of the initial two study arms, and/or 
implement one new study dose arm. If one new dosing arm is implemented in the 
Core Period, the dose selected will not exceed 360 mg BID, the highest dose that 
demonstrated acceptable safety and tolerance in the 14-day multiple BID dosing 
study in healthy volunteers. Group cohort stopping rules for terminating enrollment 
into an arm based on the severity (Common Terminology Criteria for Adverse Events 
[CTCAE]v4.03 grade) and frequency of AEs are defined;  
• Dose modification and stopping rules are defined for individual patients; 
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• Guidance for permitted, prohibited, and cautionary concomitant medications is 
specified based on the estimated potential for DDIs from hepatic cytochrome enzyme 
interactions with AG-348. 
• Due to the potential for AG-348-mediated aromatase inhibition, bone mineral density 
will be monitored using DXA scans (hip and spine) at Baseline (if patient has not had 
prior DXA scan within 3 months of Day 1) and between Week 24 and Week 28. 
In the event that any clear and unequivocal, previously unidentified/unexpected toxicities occur 
in pre-clinical toxicology studies, the Sponsor will notify the Investigators, Institutional Review 
Boards (IRBs)/Independent Ethics Committees (IECs), and regulatory authorities. The Sponsor 
will also report these to the DRT for review and discussion of appropriate actions, which may 
include, but are not necessarily limited to:  
• Amending the protocol to adjust the inclusion/exclusion criteria (e.g., to exclude 
patients with certain at-risk concurrent conditions); and/or  
• Amending the protocol to adjust safety monitoring procedures (e.g., to require 
additional monitoring of specified AEs, physical examinations, clinical laboratory 
testing, ECG monitoring, or other testing as appropriate); and/or  
• Adjusting the dose of an arm of the study as appropriate; and/or 
• Adjusting the dose modification and/or stopping rules (Section 9.7); and/or 
• Adjusting the patient withdrawal criteria (Section 8.6); or 
• Terminating the trial. 
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6. 
TRIAL OBJECTIVES AND ENDPOINTS 
6.1. 
Core Period 
6.1.1. 
Primary Objective 
The primary objective of the study is to: 
• Evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in 
patients with PK deficiency. 
6.1.2. 
Secondary Objectives 
The secondary objectives of the study are to: 
• Evaluate the PK of AG-348 and the metabolite AGI-8702. 
• Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348. 
• Evaluate indicators of clinical activity of AG-348 in patients with PK deficiency, 
including changes in Hb, HCT, reticulocyte count, haptoglobin (Hp), 
carboxyhemoglobin (COHb), end tidal carbon monoxide (ETCO), lactate 
dehydrogenase (LDH), total and indirect bilirubin, erythropoietin (EPO), hepcidin, 
ferritin, and transferrin saturation (serum iron/iron binding capacity). 
6.2. 
Extension Period 
6.2.1. 
Primary Objective 
The primary objective of the study is to: 
• Evaluate the safety and tolerability of up to 30 months of AG-348 administration in 
patients with PK deficiency. 
6.2.2. 
Secondary Objectives 
The secondary objectives of the study are to: 
• Evaluate the PK of AG-348 and the metabolite AGI-8702. 
• Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348. 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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• Evaluate indicators of clinical activity of AG-348 in patients with PK deficiency, 
including changes in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and 
indirect bilirubin, EPO, hepcidin, ferritin, and transferrin saturation (serum iron/iron 
binding capacity). 
6.3. 
Study Measures and Endpoints 
6.3.1. 
Safety Measures and Endpoints 
Safety will be evaluated by: 
• Monitoring of AEs, including determination of SAEs and AEs leading to 
discontinuation; safety laboratory parameters (hematology, chemistry, urinalysis, 
coagulation); physical examination findings; vital signs (VS); 12 lead 
electrocardiograms (ECGs); and DXA scans. Adverse events will be graded using 
CTCAE, Version 4.03. Serum sex hormone levels (testosterone [total and free], 
estrone, and estradiol), bone turnover markers (serum osteocalcin-N-mid and serum 
C-terminal telopeptide [CTX]), 25-hydroxy vitamin D2 and D3, total cholesterol, 
high-density lipoprotein-cholesterol (HDL-C), and triglycerides will be monitored for 
evidence of potential inhibition of aromatase by AG-348. Menstruating female 
patients will also keep a paper-based menstrual cycle diary throughout the Core and 
Extension Periods. 
6.3.2. 
Clinical Activity Measures and Endpoints 
• Monitoring of potential indicators of clinical activity will include evaluating changes 
in Hb, HCT, reticulocyte count, Hp, COHb, ETCO (Core Period only), LDH, total 
and indirect bilirubin, EPO, hepcidin, ferritin, and transferrin saturation. 
6.3.3. 
Pharmacokinetic and Pharmacodynamic Measures and Endpoints 
The PK and PD profile of AG-348 will be evaluated by: 
• Approximately the first 10 patients treated during the Core Period, contingent on 
clinical site feasibility, will undergo extensive PK sampling as detailed in 
Appendix 15.1, Table 7. The remainder of treated patients will undergo limited PK 
sampling as detailed in Appendix 15.1, Table 8. During the Core Period, serial blood 
sampling for determination of concentration-time profiles of AG-348 and its 
metabolite AGI-8702 will be conducted following the first dose and the morning 
Day 15 dose, and additional trough levels of AG-348 and AGI-8702 will be obtained. 
During the Extension Period, predose PK samples will be drawn for the measurement 
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of trough levels of AG-348 and AGI-8702 at each study visit (every 3 months; 
Appendix 15.1, Table 6). AG-348 and AGI-8702 will be analyzed using qualified 
assays to determine concentrations in plasma. Pharmacokinetic parameters on Day 1 
and Day 15 will be computed using standard non-compartmental methods based on 
observed plasma AG-348 and AGI-8702 concentrations. 
• Pharmacodynamic assessments during the Core Period will include 2,3-DPG, ATP 
(secondary objectives), 
 
 
 
Approximately the first 10 patients treated during the Core Period will undergo 
extensive PD sampling as detailed in Appendix 15.1, Table 7. The remainder of 
treated patients will undergo limited PD sampling as detailed in Appendix 15.1, 
Table 8. During the Core Period, serial blood sampling for determination of levels of 
ATP and, 2,3-DPG will be conducted following the first dose and the morning Day15 
dose, and additional trough levels of ATP and 2,3-DPG will be obtained. During the 
Extension Period, predose PD samples will be drawn for the measurement of trough 
levels of 2,3-DPG, ATP, 
 at each study visit (every 3 months; 
Appendix 15.1, Table 6). Adenosine triphosphate and 2,3-DPG will be analyzed 
using qualified assays to determine concentrations in whole blood. PD parameters on 
Day 1 and Day 15 will be computed based on observed whole blood ATP and 2,3-
DPG concentrations.  
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7. 
INVESTIGATIONAL PLAN 
7.1. 
Overall Study Design 
Study AG348-C-003 is a Phase 2, open-label, two-arm, multicenter, randomized, dose-ranging 
study in adult patients with PK deficiency; the study will be divided in to a Core Period and an 
Extension Period. During the Core Period, patients will receive multiple doses of AG-348 for up 
to 24 weeks; patients who are eligible can enter the Extension Period to receive AG-348 for up to 
2 years following the end of the Core Period. Patients with PK deficiency confirmed by RBC PK 
enzymatic assay performed at Screening will be eligible to participate in this study. At Week 25, 
patients who safely tolerate AG-348 and demonstrate clinical activity of AG-348 may be eligible 
to immediately roll over to the Extension Period for continued treatment. Patients who complete 
treatment at the end of the Core Period (24 weeks) will undergo follow-up assessment 4 weeks 
after the last dose of study drug. If a patient discontinues at any other time (including early 
discontinuation or discontinuation during the Core or Extension Period), follow-up assessments 
will be conducted 4 weeks after discontinuation. Patients with toxicity suspected to be related to 
study drug will continue follow-up until the AE resolves, is declared chronic by the Investigator, 
or the patient is lost to follow-up. 
For the Core Period, up to 25 patients will be initially randomized on an open-label, 1:1 basis to 
each of two BID doses of AG-348 (up to 50 patients; see Figure 3, Study Schema). The dose of 
Arm 1 is 300 mg of AG-348 administered orally q12h (BID). The dose of Arm 2 is 50 mg of 
AG-348 administered orally q12h (BID). Randomization will be stratified by PKR mutation in 
order to maintain balance as much as possible across the dose arms for the specific mutations 
expected to be most frequently enrolled. The PKR mutation stratification factor will consist of 
4 levels (R510Q, R486W, and R479H) and all other mutations (“other”). Mutation status is 
defined by the presence of at least one of the indicated mutations; patients with more than one 
stratified mutation will be assigned based on Sponsor’s discretion. 
The doses for each arm of the Core Period have been selected from the AG348-C-001 SAD 
study and AG348-C-002 MAD studies in healthy adult volunteers to represent the range of 
doses/exposures that were safely tolerated and resulted in maximal or near maximal PD effects 
on 2,3-DPG and ATP. 
Because PK deficiency is a rare disease with a limited eligible patient population and because the 
underlying pathophysiology and clinical phenotype of affected patients is heterogeneous due to 
the wide variety of mutations in PKR that cause the disease, it is deemed important to focus 
closely on dose findings in this first-in-patient study. Therefore, in addition to initiating this 
study with 2 different doses of AG-348 administered q12h, a DRT will be established to review 
study data on a frequent basis and adapt the study design, dose and schedule of AG-348 as 
indicated.  
The DRT will monitor safety on an on-going basis and meet at regular intervals of approximately 
every 6 weeks, or ad hoc as necessary, for as long as any patients are still in the Core Period to 
review AEs, VS, clinical laboratory (hematology, clinical chemistry, coagulation, and 
urinalysis), and ECGs. The DRT will also review available PK/PD data and indicators of clinical 
activity (e.g., changes from baseline in Hb). These DRT meetings will also include data review 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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for all patients that may be under treatment in the Extension Period. If there are no patients still 
being treated in the Core Period, and the only patients on treatment are those in the Extension 
Period, then the frequency of the DRT meetings will reduce to approximately every 3 months in 
order to match the frequency of patient visits (and new data collection) in the Extension Period. 
The DRT will be comprised of the study Coordinating Investigator, treating Investigators, 
Medical Monitor, Clinical Pharmacologist, Statistician, and Sponsor’s Responsible Medical 
Officer. 
Beginning 6 weeks after the first patient is dosed in the Core Period or ad hoc as necessary, and 
proceeding according the schedule indicated above (approximately every 6 weeks during the 
Core Period, approximately every 3 months during the Extension Period once all patients have 
completed the Core Period), the DRT will review cumulative safety data, available PK/PD data, 
and clinical activity data. Based on the DRT’s recurring, the DRT may exercise one or more of 
the following options during the Core Period: 
• Continue treatment and enrollment in existing arms without change. 
• Add 1 new dose arm (Arm 3) to enroll up to 25 patients at a dose to be determined; 
the dose for Arm 3 may be lower or higher than Arm 1 and Arm 2 doses, but will not 
exceed 360 mg q12h; and the dose regimen may be less frequent than q12h. 
• Terminate or suspend enrollment to allow further review of clinical data in Arm 1 
and/or Arm 2 (and/or potential Arm 3). Enrollment in an arm could be terminated or 
suspended to allow further review, for example, for unacceptable safety/tolerability, 
poor PD response, or lack of signs of clinical activity. 
• Re-assign patient’s doses and schedule in a terminated arm to match the dose and 
schedule of another arm of the study. In this case, the patients in the terminated arm 
will remain in their original arm, i.e., they will not count towards the enrollment 
quota of the arm whose dose and schedule is being adopted. 
• Implement specific genotype restrictions to enrollment in one or more arms to ensure 
representation of patients with genotypes of greatest clinical relevance. 
The DRT may exercise one or more of the following options during the Extension Period: 
• Continue treatment without change; 
• Re-assign patients’ doses and schedule to an existing dose and schedule that has been 
determined to be safer, and/or have a better PD response, or produce signs of clinical 
activity; 
• Terminate or suspend treatment to allow further review of clinical data (eg, for 
unacceptable safety/tolerability, poor PD response, or lack of signs of clinical 
activity). 
The data that the DRT will review to make these decisions is expected to include, but are not 
necessarily limited to, the following:  
• Safety Observations: all AEs; VS, clinical laboratory (hematology, clinical chemistry, 
coagulation, and urinalysis), and ECGs;  
• PK and PD Observations: including changes in 2,3-DPG and ATP; 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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• Indicators of Clinical Activity: including changes in Hb, HCT, reticulocyte count, Hp, 
COHb, ETCO (Core Period only), LDH, EPO, hepcidin, total and indirect bilirubin, 
ferritin, and transferrin saturation.  
If a third dose arm is implemented, the dose of AG-348 selected will not exceed 360 mg BID, as 
this was the highest dose that demonstrated acceptable safety and tolerance in the 14-day 
multiple BID dosing study in healthy volunteers. The PK/PD sampling schedule in a potential 
third dosing arm will be determined by Sponsor’s discretion and may follow either the extensive 
or limited PK/PD sampling schedules as specified. 
The DRT will monitor the safety data in an ongoing manner as described and may make a 
decision to terminate enrollment in an arm if unacceptable AEs are observed, PD activity is 
limited, and/or markers of clinical activity indicate no effect. Unacceptable AEs are defined as 
≥ Grade 3 AEs (using National Cancer Institute (NCI) CTCAEv4.03) that are assessed as at least 
possibly related to AG-348, with the exception of Grade 3 headache, hot flash/flushing, nausea, 
vomiting, and/or diarrhea that is transient in duration (< 24 hours) or able to be medically 
managed to ≤ Grade 2 within 24 hours. The DRT may also exercise discretion to terminate 
enrollment in an arm if it observes a frequency of Grade 2 AEs that would make it difficult to 
support long-term dosing. 
Patients in the Extension Period will continue on the dose they were randomized to in the Core 
Period, unless the DRT had reason to establish a different dose/schedule during the course of the 
Core Period (the DRT will not propose a dose higher than 360 mg q12h), or unless the treating 
Investigator exercises the option for intra-patient dose escalation (see Section 9.7.2). 
Due to the potential for AG-348-mediated aromatase inhibition, DXA scan (hip and spine) will 
be performed at Screening (if patient has not had prior DXA scan within 3 months of Day 1) to 
obtain T and Z scores. These data are intended to serve as a baseline measure of bone mineral 
density for all enrolling patients, and are deemed of particular importance for those who may 
enter the longer term Extension Period after completing 24 weeks of treatment (Core Period). All 
patients will have a second DXA scan in the interval between Weeks 24 and 28 for the Core 
Period. Patients in the Extension Period will have additional DXA scans at Months 18 and 30. 
As the number of enrolling arms changes in the study (for example, from 2 to 3), the 
randomization scheme will adjust to enable balanced randomization into each actively accruing 
arm. Randomization and stratification will cease in the event that only a single arm is left 
enrolling. 
Depending on possible early termination of 1 or both of the initial 2 arms, or the addition of a 
third arm, the study could enroll up to a maximum of 75 patients. 
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Figure 3: 
Study Schema:  Core Period 
 
Abbreviations:  BID (q12h) = twice-daily (every 12 hours); DRT = data review team; PKR = pyruvate kinase red 
blood cell isoform; TBD = to be determined; w = weeks.  
The Investigator will monitor all patients for safety and tolerability. Modification of an 
individual patient’s dose of AG-348 will be based on AEs and observed changes in Hb levels as 
detailed in Section 9.7.1 and Section 9.7.2. 
Screening assessments will occur within 42 days prior to the first dose of study treatment. During 
the Core Treatment period, patients will attend visits at baseline (Day 1), weekly through Week 3 
(Days 8, 15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12), and 
monthly through Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate AG 348 through 
Week 24 (Core Period) may be eligible to immediately enter the Extension Period for continued 
treatment. Study visits for safety and clinical activity assessments will occur approximately 
every 3 months during the Extension Period for up to 2 years after the end of the Core Period. 
All patients will undergo a follow-up assessment 4 weeks after the last dose of AG-348, 
regardless of whether this was due to early discontinuation, the last dose in the Core Period for a 
patient who chooses not to continue in the Extension Period, or the last dose of the Extension 
Period. 
Safety assessments will include monitoring of AEs, including determination of SAEs and AEs 
leading to discontinuation; safety laboratory parameters (e.g., hematology, serum chemistry, 
coagulation studies, and urinalysis); physical examination findings; VS; 12 lead ECGs, and DXA 
scans. Additional safety assessments will include monitoring of sex hormone levels (testosterone 
[total and free], estrone, and estradiol), bone turnover markers (osteocalcin-N-mid and CTX), 
25-hydroxy vitamin D2 and D3 levels, total cholesterol, HDL-C, and triglycerides. Follow-up 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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assessments will be conducted on Day 197 (Week 28) for patients who do not enter the 
Extension Period and will include physical examination, weight, performance status, VS, 12-lead 
ECGs, laboratory evaluations (hematology, Hp, EPO levels, serum chemistry, coagulation 
studies, urinalysis; lipids, hormonal testing), AEs, and transfusion record. These follow-up 
assessments will be performed approximately 4 weeks after discontinuation of AG-348 for 
patients who discontinue prior to completion of the Core Period and for those who discontinue in 
the Extension Period. Menstruating female patients will also be required to keep a paper-based 
menstrual cycle diary throughout the study.  
Pharmacokinetic assessments will include serial blood sampling for PK profiles of AG-348 and 
its metabolite AGI-8702. Pharmacodynamic evaluations will include serial blood sampling for 
determination of levels of ATP and 2,3 DPG. Extensive PK/PD sampling will be conducted on 
the first approximately 10 patients total treated in Arms 1 and 2 of the Core Period (see 
Appendix 15.1, Table 7) while limited PK/PD sampling will be conducted on the remainder of 
treated patients (see Appendix 15.1, Table 8). Limited trough sampling will be conducted every 
3 months during the Extension Period (Appendix 15.1, Table 6). 
 
 
 
 
7.2. 
Justification of the Study Design 
The primary and secondary objectives of this study are to evaluate the safety, tolerability, PK 
and PD, and indicators of clinical activity of AG-348 in patients with PK deficiency. The choice 
of dose and schedule of administration of AG -348 for Arms 1 and 2 was based on the highest 
safely tolerated dose (Arm 1: 300 mg BID) and the lowest dose with potentially relevant PD 
activity (Arm 2: 50 mg BID) from the forerunner AG348-C-002 MAD study in healthy 
volunteers. Decisions regarding continuing enrollment and treatment in these initial dose arms 
and/or implementation of an additional dose arm will be based on DRT review of safety, PK and 
PD data, and indicators of clinical activity collected from all patients treated in Arm 1 and 
Arm 2. This design was chosen to minimize risk to patients while allowing evaluation of safe 
and pharmacologically active dose levels of AG-348, and to allow the necessary flexibility to 
adjust dose and schedule should the safety, tolerability, PK, and/or PD be different in patients 
with PK deficiency compared with healthy volunteers.  
Additional safety measures intended to minimize risk to patients include monitoring of AEs by 
the DRT and specified provisions for individual patient dose modification as needed for safety 
and (potentially) large increases in Hb level (Section 9.7.1 and Section 9.7.2). Measures intended 
to maximize the opportunity for patients with demonstrated safety and tolerability to continue to 
derive benefit from any observed clinical activity of AG-348 include the option for continued 
treatment in the Extension Period.  
A comprehensive series of safety evaluations, including laboratory parameters, physical 
examinations, VS, 12-lead ECGs, and monitoring for SAEs and AEs, will be conducted to 
evaluate the safety profile of AG-348 and to aid in the determination of the recommended dose 
for continued development. 
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The study includes serial blood sampling across at least two different doses of the study drug to 
assess its PK and PD profiles.  
Consistent with the design of many Phase 2 studies, preliminary evaluation of the potential PD 
and clinical activity of AG-348 are secondary objectives of this study. The latter will include 
assessments of the PK/PD relationship between AG-348 and the biomarkers ATP and 2,3-DPG, 
 
7.3. 
Rationale for the Starting Dose, Dose Range, and Duration of Dosing 
Prior to execution of this study, Agios conducted two clinical studies of AG-348 in healthy 
volunteers, including a SAD study (AG348-C-001) and a MAD (14 day q12h) study 
(AG348-C-002). Available details of these studies are discussed in the current Investigator’s 
Brochure (IB). Between these two studies, 72 healthy human subjects have been dosed with 
AG-348. In vitro investigations, also reported in the IB, had previously demonstrated that 
AG-348 increased the activity of wild-type PKR approximately to the same extent as it did a 
series a recombinant mPKRs. Therefore it was deemed reasonable to study the safety, 
tolerability, PK, and PD of AG-348 in healthy subjects in a controlled Phase 1 setting as a more 
efficient means of obtaining information than in the actual rare disease population of patients 
with PK deficiency.  
The MAD study demonstrated that the exposures produced by AG-348 doses from 60 mg q12h 
to 360 mg q12h (including 120 mg q24h) resulted in maximal changes from baseline for the PD 
markers 2,3-DPG (reduction from baseline) and ATP (increase from baseline). The exposures 
resulting from doses less than 60 mg q12h were of lesser magnitude and the exposures resulting 
from doses greater than 360 mg q12h were of no greater magnitude than the aforementioned 
range. Therefore the starting doses for this first dose ranging study in patients with PK deficiency 
were selected to be 300 mg q12h (Arm 1) and 50 mg q12h (Arm 2). These doses were 
demonstrated to be safe and tolerable in the healthy volunteer studies. The availability of ATP is 
proposed as being critical for optimally maintaining RBC membrane integrity (see Section 5.1). 
The dose ranges from 50 mg q12h to 300 mg q12h may result in clinically effective modulation 
of PKR in PK deficiency patients if the mutated enzyme is responsive to AG-348 in a similar 
manner to the wild-type enzyme in healthy subjects. However, there are many different 
mutations in PKR that result in PK deficiency, and these mutations produce variable effects on 
the enzyme in terms of catalytic activity and thermal stability. It is not known if different 
mutations will respond clinically in a similar manner to the same exposure to AG-348. 
Therefore, it is prudent to study the range of safe and pharmacodynamically relevant doses as 
specified in this study, and to allow flexibility for the DRT to analyze the evolving study data to 
adapt the dose and schedule of administration of AG-348 to produce the optimal combination of 
safety, tolerability, and PD, and, potentially, clinical response. 
Justification for Duration of Dosing  
The initial treatment duration of 24 weeks (6 months) for the Core Period was chosen for this 
proof-of-concept trial for 2 principal reasons: 1) to begin establishing a safety database 
addressing the chronic administration of AG-348; and 2) to allow sufficient time for clinical 
response to treatment to appear.  
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It is anticipated that this treatment, if successful, may be taken for life, as PK deficiency is a 
genetically determined inborn error of metabolism. Therefore, it is important to begin to 
investigate the long-term safety of the treatment. As will be discussed below, the safety package 
supports the treatment duration of 6 months.  
Red blood cell turnover is typically 120 days (4 months), although it may be shorter in some 
populations of pyruvate kinase deficient RBCs (Mentzer, et al. 1971). Different PKR mutations 
produce a variety of physiologic consequences. In mutations where normal or nearly normal 
levels of PKR protein persist, but the protein’s catalytic function is impaired, an improvement in 
PKR functional activity might be seen relatively quickly upon exposure to AG-348. However, in 
cases where the PKR mutation results in an unstable mutant, PKR protein levels may be low and 
additional time may be required for stabilization of the newly synthesized mutant enzyme in 
developing bone marrow erythroblasts so that eventually more mature RBCs may be produced 
with more nearly normal levels of functional PKR protein.   
This study plans up to 24 weeks of dosing with AG-348 during the Core Period with the 
possibility of continued dosing beyond 24 weeks during the Extension Period in patients for 
whom AG-348 is safely tolerated and demonstrates clinical activity. The International 
Conference on Harmonisation (ICH) Guideline M3(R2) on Nonclinical Safety Studies for the 
Conduct of Human Clinical Trials and Marketing Authorisation for Pharmaceuticals, 
recommends that clinical trials of a duration between 2 weeks and 6 months should be supported 
by repeat-dose toxicity studies of at least equivalent duration, and clinical trials lasting beyond 
6 months should be supported by 6-month rodent and 9-month non-rodent studies. This guidance 
allows for clinical trials of longer duration to be initiated based on 3 months of nonclinical data, 
and for clinical dosing in serious or life-threatening indications to be extended based on complete 
chronic rodent data and in-life and necropsy data in the non-rodent (and complete histopathology 
in the non-rodent provided within an additional 3 months).  
For the current investigational product (AG-348), 13-week, repeat dose toxicology studies in the 
rat and monkey have been completed and are summarized in Section 5.2.1.4 of this protocol and 
in the current Investigator Brochure prepared to support initiation of this clinical study. 
Considering that PK deficiency is a serious condition with an unmet medical need, it is 
appropriate to initiate this clinical study with the available nonclinical data. Six-month rodent 
and nine-month monkey toxicology studies were initiated in January 2015, and the Sponsor will 
report the results of these studies in each applicable regulatory region as required before any 
patients will be treated for greater than 6 months (i.e., enter the Extension Period).  
7.4. 
Criteria for Study Termination 
This study may be prematurely terminated if, in the opinion of the Sponsor, there is sufficient 
reasonable cause. In the event of such action, written notification documenting the reason for 
study termination will be provided to each Investigator. 
Circumstances that may warrant termination include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to patients; 
• Insufficient adherence to protocol requirements; 
• Plans to modify, suspend, or discontinue the development of the study drug; 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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• Other administrative reasons. 
Should the study be closed prematurely, all study materials must be returned to the Sponsor or 
the Sponsor’s designee. Patients who have participated in the Extension Period may be able to 
screen for eligibility in a study for a second generation allosteric PKR activator, if such a 
compound is available at that time, and after a suitable washout period has been observed. 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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8. 
STUDY POPULATION 
8.1. 
Number of Patients 
Up to approximately 75 patients may be enrolled in this study. 
8.2. 
Inclusion Criteria 
For entry into the Core Period, patients must meet all of the following criteria during the 
Screening or other specified period: 
1. Signed written informed consent obtained prior to performing any study procedure, 
including screening procedures.  
2. Male or female, aged 18 years and older. 
3. Known medical history of PK deficiency. 
4. All patients must have documented clinical laboratory confirmation of PK deficiency by 
RBC pyruvate kinase enzymatic assay performed at Screening either by a designated 
central laboratory or by any participating investigative site’s local hematology laboratory. 
Patients with prior documentation of PK deficiency by RBC enzymatic assay will have a 
reconfirmation of this result during Screening as a condition of enrollment. 
a. In the event that a patient’s screening pyruvate kinase enzymatic assay is negative 
(i.e., shows normal pyruvate kinase activity), the patient will be eligible for 
enrollment if the genotyping shows a mutant genotype that has been previously 
documented in the literature to be associated with PK deficiency. If the 
genotyping shows a previously undescribed mutation in the PKR gene, then the 
eligibility for enrollment will be determined on a case-by-case basis by the 
Coordinating Investigator and Medical Monitor in discussion with the 
Investigator. If no mutation is defined, then the patient will not be eligible. 
5. ALL patients must have a blood sample for genotypic characterization of the mutant PKR 
gene performed by the designated central laboratory at Screening. The designated central 
laboratory-determined genotype will generally serve as the basis for genotyping for 
enrollment. However, patients whose genotype has already been determined by another 
laboratory may be enrolled on the basis of that report, with the approval of the Medical 
Monitor, in case of unexpected delay in return of the designated central laboratory result 
during the Screening Period. Enrollment on the basis of a result from a laboratory other 
than the designated central genotyping laboratory does not relieve the inclusion 
requirement that ALL patients must have a sample sent to the designated central 
genotyping laboratory. 
6. All patients must have genotypic characterization of the UGT1A1 gene performed by a 
designated central laboratory to document whether they may have underlying Gilbert's 
Disease. Patients with Gilbert’s Disease are eligible to enroll. 
7. Males must have Hb ≤ 12.0 g/dL; females must have Hb ≤ 11.0 g/dL. 
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8. All patients must be considered transfusion independent as defined by: no greater than 
3 units of RBCs transfused in the 12-month period up to the first day of study dosing and 
no transfusions within 4 months of first day of study dosing. Patients who have received 
more transfusion support than described above will be evaluated for eligibility on a case-
by-case basis by the Medical Monitor. 
9. Eligible patients may still have their spleens in place, or may have undergone prior 
splenectomy. For splenectomized patients: 
a. Must have undergone their procedure at least 6 months prior to Screening. 
b. Must be current in their vaccinations for Pneumococcal Conjugate (PCV13), 
Pneumococcal Polysaccharide (PPSV23), Quadrivalent Meningococcal vaccine, 
and Haemophilus influenzae type b  as recommended by Centers for Disease 
Control and Prevention's (CDC) Advisory Committee on Immunization Practices 
(ACIP) or immunization advisory groups in Canada and the European Union (for 
patients enrolled in Canada and the EU). 
[http://www.cdc.gov/vaccines/schedules/downloads/adult/adult-schedule.pdf] 
[Any missing vaccinations may be administered starting with the Screening 
Period and during the trial following the initiation of AG-348 dosing as necessary 
according to recommended vaccination guidance.] 
10. Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2 (Appendix 15.2). 
11. Patients must be taking at least 1 mg of folic acid daily for at least 21 days prior to first 
dose and continued daily during study participation. 
12. Adequate organ function, defined as: 
a. Serum AST ≤ 2.5 × ULN (unless the increased AST is assessed by the 
Investigator as due to hemolysis and/or hepatic iron deposition); and ALT 
≤ 2.5 × ULN (unless the increased ALT is assessed by the Investigator as due to 
hepatic iron deposition). 
b. Normal or elevated levels of serum bilirubin. In patients with serum bilirubin 
> ULN, the elevation must be attributed to hemolysis with or without Gilbert's 
syndrome and must not be choledocholithiasis, cholecystitis, biliary obstruction, 
or hepatocellular disease. 
c. Serum creatinine ≤ 1.25 × ULN. If serum creatinine > 1.25 × ULN, then 24-hour 
measured or calculated (Cockcroft-Gault) glomerular filtration rate 
(GFR) ≥ 60 mL/min. 
d. Absolute neutrophil count (ANC) ≥ 1.0 × 109/L. 
e. Platelet count ≥ 100 × 109/L. 
f. Activated partial thromboplastin time (aPTT) and international normalized ratio 
(INR) ≤ 1.25 × ULN, unless the patient is receiving therapeutic anticoagulants. 
13. Women of childbearing potential (WOCBP) must agree to abstain from sexual 
intercourse or to use an acceptable/effective method of contraception (i.e., condom plus 
spermicide, condom plus oral contraceptive, condom plus intrauterine device [IUD], 
condom plus diaphragm with spermicide) from as soon as feasible during the Screening 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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period until 30 days following the last dose of AG-348. Abstinence is an acceptable 
method only when this is in line with the normal life style of the patient, meaning that the 
patient plans to remain abstinent continuously throughout the duration of the study and 
for at least 30 days after the last dose of study drug. Periodic abstinence, e.g. calendar, 
sympathothermal and post-ovulation methods, and withdrawal are not acceptable 
methods of contraception. 
a. WOCBP include any female who has experienced menarche and who has not 
undergone successful surgical sterilization (hysterectomy, bilateral tubal ligation, 
and/or bilateral oophorectomy) or is not post-menopausal. Post-menopausal is 
defined as: 
i. Amenorrhea ≥ 12 consecutive months without another cause, and a 
documented serum follicle stimulating hormone (FSH) level 
> 35 mIU/mL; 
ii. Amenorrhea≥ 12 consecutive months in women ≥ 62 years old (FSH 
testing is not required). 
14. WOCBP must have a negative serum or urine pregnancy test within 72 hours before start 
of AG-348 dosing. 
15. Women must not be breastfeeding.  
16. Male patients, with the exception of those who have undergone vasectomy at least 
6 months prior to Screening, must agree to abstain from sexual intercourse or, if sexually 
active, to use a condom with spermicide as contraception (regardless of their female 
partner's childbearing potential or their partner's use of their own contraception) from 
Day 1 of dosing until 30 days following the last dose of AG-348. Abstinence is an 
acceptable method only when this is in line with the normal life style of the patient, 
meaning that the patient plans to remain abstinent continuously throughout the duration 
of the study and for at least 30 days after the last dose of study drug. Periodic abstinence, 
e.g. selective timing of intercourse based on partner's calendar, sympathothermal and 
post-ovulation methods, and withdrawal are not acceptable methods of contraception. 
For entry into the Extension Period, patients must meet the following criteria: 
17. Signed written informed consent obtained prior to performing any study procedure during 
the Extension Period. 
18. Patient must have completed 24 weeks of treatment during the Core Study and tolerated 
AG-348 (defined as having completed 24 weeks with or without permitted dose 
modifications). 
19. The patient’s treating Investigator agrees that there is a potential for clinical benefit to 
continued treatment and recommends participation in the Extension Period 
20. The Sponsor's designated Medical Monitor or Responsible Medical Officer approves the 
patient’s participation in the Extension Period  
21. As applicable, the patient must agree to continue to follow the same sexual 
abstinence/contraception rules as stated in Inclusion Criteria 13 and 16. 
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8.3. 
Exclusion Criteria 
Patients who meet any of the following criteria at Screening or prior to dosing on Day 1 will not 
be enrolled in Core Period: 
1. Hemoglobin level > 12.0 g/dL if male; Hb > 11.0 g/dL if female. 
2. Additional diagnosis of any other congenital or acquired blood disorder, including 
glucose-6-phosphate-dehydrogenase (G6PD) deficiency, or any other hemolytic anemia 
process except for mild allo-immunization as a consequence of transfusion therapy. 
3. Iron overload (hemosiderosis or concurrent hemochromatosis) sufficiently severe to 
result in a clinical diagnosis by the Investigator of cardiac, hepatic, or pancreatic 
insufficiency. 
4. Prior bone marrow or stem cell transplant. 
5. Clinically symptomatic cholelithiasis or cholecystitis. (Prior cholecystectomy is not 
exclusionary. Patients with symptomatic cholelithiasis or cholecystitis may be rescreened 
once the disorder has been treated and clinical symptoms have resolved.) 
6. Currently enrolled in another therapeutic clinical trial involving on-going therapy with 
any investigational or marketed product or placebo. Concurrent participation in the 
Pyruvate Kinase Deficiency Natural History Study (NCT02053480) is permitted. 
7. Exposure to any investigational drug, device, or procedure within 28 days prior to 
Screening or during trial participation. 
8. Concurrent medical condition that could compromise participation in the study such as: 
a. Poorly controlled hypertension (defined as systolic blood pressure (BP) 
> 150 mm Hg or diastolic BP > 90 mm Hg) refractory to medical management. 
b. History of recent (within < 6 months from Screening date) congestive heart 
failure; myocardial infarction or unstable angina pectoris; or hemorrhagic, 
embolic, or thrombotic stroke; deep venous thrombosis; or pulmonary or arterial 
embolism. 
c. Currently active infection requiring the use of parenteral anti-microbial agents or 
that is ≥ Grade 3 (CTCAEv4.03) within 6 months of first dose. 
d. A pattern or frequency of post-splenectomy sepsis that in the assessment of the 
Investigator could reasonably be expected to interfere with the ability of the 
patient to complete the 24 week Core Period study participation. 
e. Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) 
antibody with signs of active Hepatitis B or C virus infection.  
f. Positive test for human immunodeficiency virus (HIV) 1 or 2 antibody. 
g. Diabetes mellitus judged to be in poor control by the Investigator or requiring 
> 3 anti-diabetic agents counting insulin; use of insulin per se is not exclusionary.  
h. History of any primary malignancy with the exception of: curatively treated non-
melanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; 
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or other primary tumor treated with curative intent and no known active disease 
present and no treatment administered during the last 3 years. 
9. Undergone major surgery within 6 months of first dose.  
10. Current or recent history of psychiatric disorder that in the opinion of the Investigator or 
Medical Monitor could compromise the ability of the patient to cooperate with study 
visits and procedures. 
11. Use of any of the restricted list of products known to strongly inhibit CYP3A4 
metabolism (Appendix 15.4, Table 9) within 5 days prior to Day 1 dosing; or to strongly 
induce CYP3A4 metabolism (Appendix 15.4, Table 10) within 28 days prior to Day 1 
dosing; or to strongly inhibit P-gp transporter (Appendix 15.4, Table 11) within 5 days 
prior to Day 1 dosing; or digoxin within 5 days prior to Day 1 dosing.  
12. Serum bilirubin > ULN attributable to factors other than hemolysis and/or Gilbert's 
syndrome. 
13. Male patients with heart-rate corrected QT interval -Fridericia's method (QTcF) 
interval > 450 msec, or female patients with QTcF interval > 470 msec with the 
exception of patients with a left bundle branch block (LBBB). Medical Monitor approval 
needed in patients with a LBBB. 
14. Cardiac dysrhythmias judged as clinically significant by the Investigator or requiring 
therapy with drugs that are primarily substrates of CYP3A4. 
15. History of allergy to sulfonamides if characterized by acute hemolytic anemia, 
anaphylaxis, or rash of erythema multiforme type or Stevens-Johnson syndrome. 
16. Any other medical or psychological condition deemed by the Investigator to be likely to 
interfere with a patient’s ability to understand and sign informed consent; cooperate with 
study visits, tests, and procedures; or otherwise safely and reliably participate in the 
study. 
Patients will not be allowed to enter the Extension Period if: 
17. The patient experienced AEs during the Core Period that are considered by the treating 
Investigator or the Sponsor’s designated Medical Monitor or Responsible Medical 
Officer to pose a significant safety risk to the patient if treatment were to be extended. 
8.4. 
Patient Identification and Registration 
Patients who are candidates for enrollment into the study will be evaluated for eligibility by the 
Investigator to ensure that the inclusion and exclusion criteria (see Section 8.2 and Section 8.3, 
respectively) have been satisfied and that the patient is eligible for participation in this clinical 
study. The site will submit to the Sponsor an Eligibility form for each eligible patient and the 
Medical Monitor will confirm eligibility for all patients prior to receipt of the first dose of 
AG-348. 
8.5. 
Patient Randomization 
Patients who have been confirmed as eligible will be randomized in an equal ratio to a treatment 
arm (e.g., 1:1 or 1:1:1 depending on which arms are open). The site will provide a request for 
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randomization form (including the patient’s confirmed genotype) to the study Medical Monitor. 
The randomization will be stratified by PKR mutation in order to maintain balance across the 
dose arms for the specific mutations expected to be most frequently enrolled. The PKR mutation 
stratification factor will consist of 4 levels (R510Q, R486W, and R479H) and all other mutations 
(“other”). Since this is an open label study, randomization will not be blinded. 
Please refer to the study manual for the randomization procedure. 
8.6. 
Patient Withdrawal Criteria 
Patients have the right to withdraw from the study at any time for any reason. Patients will be 
withdrawn from study-related procedures and treatments under the following conditions: 
• Withdrawal of consent; 
• Experiences unacceptable toxicity; 
• Development of an intercurrent medical condition that precludes further participation 
in the trial; 
• Patient requires use of a prohibited concomitant medication (Section 9.11.2); 
• Investigator decision; 
• Protocol violation: non-adherence to protocol requirements; 
• Pregnancy; 
• Lost to follow-up. 
Should a patient decide to withdraw, all efforts will be made to complete and report the protocol-
defined study observations up to the time of the patient’s withdrawal as completely as possible 
and to determine the reason for withdrawal. 
In the event a patient is withdrawn from the study, the Medical Monitor must be informed. If 
there is a medical reason for withdrawal, the patient will remain under the supervision of the 
Investigator until satisfactory health is returned. 
When a patient withdraws from the study, the primary reason for discontinuation must be 
recorded in the appropriate section of the electronic case report form (eCRF) and all efforts will 
be made to complete and report final study observations as thoroughly as possible. 
All AEs should be followed until resolution or for a period of 30 days from the last dose of study 
drug, whichever is shorter. Patients with toxicity suspected to be related to study drug will 
continue follow-up until the AE resolves, is declared chronic by the Investigator, or the patient is 
lost to follow-up. 
8.7. 
Replacement of Patients 
Patients who drop out of the Core Period prior to completing the first 12 weeks of assigned 
dosing for reasons other than AEs may be replaced at the Sponsor’s discretion. 
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9. 
STUDY TREATMENT 
9.1. 
Description of Study Drug 
AG-348 sulfate hydrate capsules will be provided as 5 mg, 25 mg, or 100 mg (free-base 
equivalent) of AG-348 sulfate hydrate without excipients in dark green opaque (5 mg), Swedish 
orange (25 mg), or white opaque (100 mg) gelatin capsules (size 2 capsules for all dose 
strengths). 
All study drugs are for investigational use only and are to be used only within the context of this 
study. All study drug products will be supplied by the Sponsor. The Sponsor reserves the right to 
discontinue the supply of any specific capsule strength should the evolving trial experience 
demonstrate that the specific capsule strength fills no additional need beyond the other capsule 
strengths available. Please see the Investigator’s Brochure for further details regarding study 
drug. 
9.2. 
Study Drug Packaging and Labeling 
AG-348 sulfate hydrate capsules are packaged in white, high-density polyethylene (HDPE) 
induction sealed bottles with a child-resistant screw cap. 
Packaging and labeling will be prepared to meet all regulatory requirements. 
9.3. 
Study Drug Storage 
AG-348 sulfate hydrate drug capsules must be stored at room temperature of 15 to 30°C 
(59 - 86°F). 
All study drug products must be stored in a secure, limited-access location and may be dispensed 
only by the Investigator or by a member of the staff specifically authorized by the Investigator. 
9.4. 
Method of Assigning Patients to Treatment 
Up to a maximum of 25 patients will be randomized to any one of the dosing arms in this study. 
Randomization will be balanced 1:1 or 1:1:1, depending on whether 2 or potentially 3 arms are 
open, and will be stratified by PKR mutation (see Section 8.5). The dose and schedule of 
AG-348 each patient receives will be dependent upon which dose arm is open for enrollment 
when the patient qualifies for and is randomized into the study. Patients in the Extension Period 
will continue on the dose they were randomized to in the Core Period, unless the DRT had 
reason to establish a different dose/schedule during the course of the Core Period (the DRT will 
not propose a dose higher than 360 mg q12h).  
9.5. 
Blinding 
This is an open-label study; no blinding methods will be used. 
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9.6. 
Study Drug Preparation and Administration 
For the initial two treatment arms, (Arm 1 and Arm 2) in the Core Period, AG-348 will be 
administered orally BID (approximately q12h with a minimum of 10 hours between doses) over 
a 24 week treatment period. Starting with Day 1, dosing will be continuous; there will be no rest 
periods. Patients who do not meet any of the treatment withdrawal criteria (see Section 8.5) may 
continue treatment for the entire 24-week treatment period. 
Patients will be dispensed the appropriate number of Sponsor-packaged, labeled bottles to allow 
for 28 days of dosing until the next scheduled visit. The amount of study drug dispensed should 
be sufficient to provide an adequate reserve supply of AG-348 to ensure uninterrupted dosing in 
the event of an unexpected delay for the next scheduled study visit (7 extra days of dosing supply 
is recommended during the Core Period; 14 extra days is recommended during the Extension 
Period). 
Patients will be given a dosing diary to be used for each 28-day dosing period. They should 
record relevant information regarding their study drug in the diary (e.g., confirmation that each 
daily dose was taken, reasons for missed doses). 
Treatment compliance will be assessed based on return of unused drug and the dosing diary (see 
Section 9.9). 
Patients should be instructed to take their daily dose at approximately the same times each day 
except for dosing on in-clinic visiting days.  
Patients who undergo extensive PK/PD sampling during the Core Period (see Appendix 15.1, 
Table 7) should be instructed from Week 3 on to bring the AM dose with them for in-clinic visits 
and to ingest the dose following PK/PD blood draws.  
Patients receiving limited PK/PD sampling during the Core Period (see Appendix 15.1, Table 8) 
should be instructed to bring the AM dose with them for all in-clinic visits and to take the AM 
dose following PK/PD blood draws.  
Patients receiving extensive PK/PD sampling on Day 1 and 15 will also have limited PK/PD on 
other visit days. As a general rule, regardless of extensive or limited schedule, patients will bring 
in the AM dose for all visits and take this dose following PK/PD blood draws. Patients not 
continuing into the Extension Period are not required to take the Week 24/Day 169 morning dose 
of AG-348 after the required PK/PD blood samples are collected, as these patients will be 
discontinuing the study. 
A minimum of 10 hours between the AM and PM dose will be required on those dosing days. 
Each dose should be taken with a glass of water and consumed over as short a time as possible. 
AG-348 may be taken with or without food. Patients should be instructed to swallow capsules 
whole and to not chew the capsules. For patients who have difficulty swallowing tablet(s), the 
Medical Monitor should be contacted to discuss administration. 
Patients will receive their first dose of AG-348 in the clinic on Day 1 and then may take the 
remaining doses on an outpatient basis (except for in-clinic visit days, as described above). 
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9.7. 
Criteria for Dose Escalation, Dose Modification, or Discontinuation 
of Study Drug 
Intra-patient dose escalations will be permitted in this study under 2 circumstances. First, the 
DRT may decide to re-assign patients’ doses and schedule in a terminated arm to match the dose 
and schedule of another arm of the study. In this case, the patients in the terminated arm will 
remain in their originally assigned arm; i.e., they will not count towards the enrollment quota of 
the arm whose dose and schedule is being adopted. Second, a treating Investigator, with Medical 
Monitor approval, may elect to increase the dose for a patient in Arm 2 (50 mg BID) or a 
potential third arm of the study (if < 300 mg BID) to 300 mg BID if the patient is adequately 
tolerating his/her current dose and if their Hb has not reached at least the lower limit of the 
normal gender-adjusted reference range as specified by the designated central laboratory (male: 
13.0 g/dL; female: 11.6 g/dL) after at least 12 weeks of treatment in the Core Period. The patient 
must have completed the 12-week visit during the Core Period and had all assigned 
tests/procedures for that visit before an intra-patient dose escalation will be allowed. An intra-
patient dose escalation may also be made later than the 12-week visit in the Core Period or 
anytime during the Extension Period. 
All dosing modifications, as outlined below, will be implemented following discussions with the 
Medical Monitor. The same criteria for dose modifications or discontinuation of study drug 
apply in the Extension Period as in the Core Period. 
Patients should be advised not to discontinue dosing without first speaking with the treating 
Investigator—abrupt discontinuation of AG-348 dosing in a patient who experience a substantial 
increase in Hb may result in withdrawal hemolysis. 
9.7.1. 
Dose Modification for Safety 
The Investigator will monitor all patients for safety and tolerability. Modification of the patient’s 
dose of AG-348 will be based on AEs and observed changes in Hb levels (see Section 9.7.2). 
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Table 1: 
Dose Modification for Adverse Events Deemed at Least Possibly Related to 
AG-348 
Adverse Events(s) 
AG-348 Dose Adjustment 
Grade 1 
None required. 
Grade 2 
None required; Investigator and Medical Monitor judgment to manage as for 
Grade 3. 
Grade 3 
Suspend dosing1;  
If event resolves to Grade 1 or baseline within approximately 14 days of 
suspension, resume dosing with at least a 1 dose level reduction and discuss the 
advisability of additional dose modification with the Medical Monitor (see 
Table 2 below).  
If event does not resolve to Grade 1 or baseline within approximately 14 days of 
suspension, permanently discontinue dosing, unless the benefits outweigh the 
risks of resuming treatment and are approved by the Medical Monitor. 
Grade 4 
Permanently discontinue dosing, unless the benefits outweigh the risks of 
resuming treatment and are approved by the Medical Monitor. 
1 A related AE of Grade 3 severity would generally be expected to require suspension of dosing with AG-348. 
However, in some circumstances in which the Hb concentration has normalized prior to the onset of the related 
AE, the Investigator may elect to discuss with the Medical Monitor tapering the dose of AG-348 in order to 
mitigate potential withdrawal hemolysis (see Section 5.2.2.3 and Section 9.7.2). 
Dose modifications for Grade 2 and 3 AEs should be maintained long enough for the 
Investigator to be confident that the reduced dose is being well tolerated before considering a 
request of the Medical Monitor to permit subsequent re-escalation to the former dose level. 
Dosing for an individual patient will be discontinued permanently for Grade 3 and Grade 4 AEs 
that do not resolve to Grade 1 or baseline within approximately 14 days of suspension of dosing, 
unless the benefits outweigh the risks of resuming treatment and are approved by the Medical 
Monitor. No patients may be re-escalated to their former dose level after a dose modification 
without discussion with the Medical Monitor. If following the first dose reduction for a Grade 3 
AE the patient experiences a second occurrence of the same Grade 3 AE, then treatment with 
AG-348 must be immediately and permanently discontinued.  
It should be noted that if the initial dose of 300 mg BID selected for Arm 1 demonstrates an 
unacceptable safety profile resulting in multiple patients undergoing dose modifications, the 
DRT may exercise its option to re-assign these patients’ dose and schedule to match the dose and 
schedule of another study arm (for example, Arm 2 of the study, or to match the dose and 
schedule of a [potential] Arm 3, if implemented). 
9.7.2. 
Dose Modification for Increase in Hemoglobin Level 
It is presently unknown to what magnitude, how rapidly, or even whether, AG-348 will result in 
increased Hb levels in patients with PK deficiency across the variety of potential PKR mutations 
that may be encountered during this study. Moreover, it is unknown whether any potential 
increases in Hb experienced by patients in this study may be a safety risk. As a conservative 
measure, this study incorporates guidance for dose modification based on potential large 
increases in Hb levels that exceed the midpoint of the typical normal range by gender. The intent 
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of this guidance is to strike a balance between abruptly stopping AG-348 treatment, thereby 
potentially risking withdrawal hemolysis, versus reducing the dose sufficiently to allow an 
opportunity for Hb to settle more gradually to a level less than the midpoint of the typical normal 
range by gender. The Investigator will monitor all patients for changes in Hb levels and should 
adjust the dose of AG-348 as outlined in Table 2 per the following guidelines and with Medical 
Monitor approval. 
• Males: If Hb > 15.0 g/dL and confirmed with a second test, reduce dose by at least 
1 dose level (Table 2) and discuss the advisability of additional dose modification or 
suspension with the Medical Monitor. 
• Females: If Hb > 13.5 g/dL and confirmed with a second test, reduce dose by at least 
1 dose level (Table 2) and discuss the advisability of additional dose modification or 
suspension with the Medical Monitor. 
• The treating Investigator will discuss with the Medical Monitor questions relating to 
additional dose modifications and the need for additional unscheduled Hb monitoring 
on an as needed basis.  
Table 2: 
Dose Reduction Table (by Dosing Arm) 
Dose Group 
Starting Dose 
1st Dose Reduction 
2nd Dose Reduction 
Arm 1 
300 mg BID 
200 mg BID 
100 mg BID 
Arm 2 
50 mg BID 
25 mg BID 
TBD1 
Potential Arm 3 
TBD 
To approximately 
50-66% of initial dose 
To approximately 
25-33% of initial dose 
1 Dose to be determined by Medical Monitor. 
Hemoglobin levels above the ULN by gender should be reported as an AE, graded per the 
CTCAEv4.03, according to the guidance provided in Section 11.2. 
9.7.3. 
Stopping Criteria 
Dosing for an individual patient will be discontinued permanently for Grade 3 AEs that do not 
resolve to Grade 1 or baseline within approximately 14 days of suspension of dosing, and for 
Grade 4 AEs, unless the benefits outweigh the risks of resuming treatment and are approved by 
the Medical Monitor (Section 9.7.1). Other reasons for treatment termination are provided in 
Section 8.5. 
9.8. 
Duration of Patient Participation 
The duration of treatment for all patients will be up to 24 weeks in the Core Period. Patients who 
safely tolerate AG-348 and for whom the Investigator agrees with continuation of AG-348 
treatment may be eligible to immediately roll over to the Extension Period for continued 
treatment (up to 2 years following completion of the Core Period). 
9.9. 
Treatment Compliance 
During in-clinic visits, doses of AG-348 will be ingested by the patient under the supervision of 
clinical facility personnel. For at-home dosing, patients will be given a dosing diary to be used 
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for the duration of the 24-week Core Treatment Period; the diary will also be used by patients 
who roll over to the Extension Period. Patients should record relevant information regarding their 
study drug in the diary (e.g., confirmation that each daily dose was taken, reasons for missed 
doses) and return the diary at each study visit. 
9.10. 
Study Drug Accountability 
Accountability for the study drug at the clinical facility is the responsibility of the Investigator. 
The Investigator will ensure that the study drug is used only in accordance with this protocol. 
Where allowed, the Investigator may choose to assign drug accountability responsibilities to a 
pharmacist or other appropriate individual. 
The Investigator or delegate will maintain accurate drug accountability records indicating the 
drug’s delivery date to the site, inventory at the site, use by each patient, and return to Agios or 
its designee (or disposal of the drug, if approved by Agios). These records will adequately 
document that the patients were provided the doses as specified in the protocol and should 
reconcile all study drug received from Agios. Accountability records will include dates, 
quantities, batch/serial numbers, expiration dates (if applicable), and patient numbers. An 
unblinded monitor will review drug accountability at the site on a schedule agreed to by the 
Sponsor. 
Study drug must not be used for any purpose other than the present study.  
All unused and used study drug will be retained at the site until it is inventoried by the Study 
Monitor after database lock has occurred. All used, unused or expired study drug will be returned 
to Agios or its designee or, if authorized, disposed of at the study site per the site’s Standard 
Operating Procedures (SOPs) and documented. All material containing AG-348 will be treated 
and disposed of as hazardous waste in accordance with governing regulations. 
9.11. 
Prior and Concomitant Medications and Treatments 
9.11.1. 
Prior Medications and Procedures 
All medications administered and procedures conducted within 28 days prior to, or from 
Screening Visit #1 until the first day of study drug administration, whichever interval is longer, 
are to be recorded on the source documentation and included in the eCRF. 
9.11.2. 
Prohibited Concomitant Therapy 
All concomitant medications and procedures administered from 28 days prior to, or from 
Screening Visit #1 until the first day of study drug administration, whichever interval is longer, 
through the last Follow-up Visit must be recorded in the appropriate section of the source 
documentation and eCRF along with dosage information, dates of administration, and reason for 
use.  
The following are prohibited at all times during participation in this study: 
• Investigational drugs must be discontinued no less than 28 days prior to the first dose 
of study drug; 
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• Products known to strongly inhibit CYP3A4 metabolism (listed in Appendix 15.4, 
Table 9) must be discontinued within 5 days prior to Day 1 dosing; 
• Products known to strongly induce CYP3A4 metabolism (listed in Appendix 15.4, 
Table 10) must be discontinued within 28 days prior to Day 1 dosing; 
• Products known to strongly inhibit P-gp transporter (listed in Appendix 15.4, 
Table 11) must be discontinued within 5 days prior to Day 1 dosing; 
• Digoxin must be discontinued within 5 days prior to Day 1 dosing; 
• Hematopoietic stimulating agents (EPOs, granulocyte colony stimulating factors, 
thrombopoietins, etc) must be discontinued no less than 28 days prior to the first dose 
of study drug. [Folic acid 1 mg orally per day is required for all patients. B12 
injections are permitted for patients with a prior diagnosis of B12 deficiency 
syndromes, but must be repleted to stability of the Hb and mean corpuscular volume 
(MCV) prior to enrollment in the study]; 
• Anabolic steroids, including testosterone preparations, administered for anemia must 
be discontinued no less than 28 days prior to the first dose of study drug;  
• As the target population for this study consists of transfusion independent patients 
and transfusion of blood products could confound key endpoints of the study, blood 
transfusions of any type must be strictly avoided except in cases of compelling 
medical need. If medical circumstances permit, the Medical Monitor should be 
contacted for discussion before any transfusions are administered. 
Drugs that displace unconjugated bilirubin from albumin (including some common sulfa 
antibiotics [including sulfamethoxazole/trimethoprim], cephalosporins, salicylates, and 
aminophylline) should be used with caution with the understanding that patients with elevated 
levels of unconjugated bilirubin may potentially be at risk for kernicterus syndrome (Strauss, et 
al. 2006). 
Potential Drug Interactions 
Strong inhibitors of CYP3A4 (listed in Appendix 15.4, Table 9) are not permitted for use with 
AG-348. Based on modeling of AG-348 metabolism and tolerability data from the Phase 1 MAD 
study, moderate inhibitors of CYP3A4 do not appear to pose a risk to patients dosed with 
AG-348. 
In-vivo data from the Phase 1 MAD show AG-348 induces CYP3A4 and its own metabolism, 
presumably via CYP3A4. The use of strong CYP3A4 inducers in combination with AG-348 is 
expected to reduce AG-348’s efficacy. Therefore, administration of strong CYP3A4 inducers 
(listed in Appendix 15.4, Table 10) is not permitted with AG-348. Corticosteroids may induce 
CYP3A4. Although the use of corticosteroids is not prohibited, their use should be minimized as 
much as medically feasible. 
Strong inhibitors of drug transport (listed in Appendix 15.4, Table 11) are not permitted for use 
with AG-348. 
Digoxin is not permitted for use with AG-348. 
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Induction of CYP3A4 by AG-348 is expected to reduce the efficacy of certain sensitive co-
medications. Co-administration of AG-348 with the drugs in Appendix 15.4, Table 12 should be 
done with caution, as their efficacy may be reduced.  
Of note, women in the trial utilizing oral contraception must utilize barrier methods as per the 
Inclusion Criteria 14 (Section 8.2) while taking AG-348. 
The expected patient co-medications deferoxamine, deferasirox, deferiprone, and oral penicillin 
are not expected to interact with AG-348. 
9.11.3. 
Allowed Concomitant Therapy 
Medications and treatments other than those specified above are permitted during the study. All 
intercurrent medical conditions will be treated at the discretion of the Investigator according to 
acceptable local standards of medical care. Subjects may receive analgesics, antiemetics, anti-
infectives (including penicillins), and antipyretics as medically indicated and consistent with the 
guidance in the previous two sections. Patients may continue iron chelation therapy with 
deferoxamine, deferasirox, or deferiprone. Patients must continue taking at least 1 mg of folic 
acid for the duration of the study. 
All concomitant medications, including transfusions of blood products, procedures performed 
during the study, including those used to treat AEs, will be recorded on the eCRF.  
9.11.4. 
Potential for Phototoxicity 
AG-348 may cause sensitivity to direct and indirect sunlight. Patients should be warned to avoid 
direct sun exposure. When exposure to sunlight is anticipated for longer than 15 minutes, the 
patient should be instructed to apply factor 30 or higher sunscreen to exposed areas and wear 
protective clothing and sunglasses. 
9.11.5. 
Management of Nausea, Vomiting, and Diarrhea 
As the primary objective of this study is to assess the safety and tolerability of up to 24 weeks of 
AG-348, routine use of prophylactic anti-emetic and antidiarrheal or other classes of medications 
is prohibited. However, if patients experience AEs relating to nausea, vomiting, or diarrhea these 
may be treated at the Investigator's clinical discretion with recommended medications as follows: 
• Nausea/vomiting: allowed agents include standard clinical dosing with palonosetron 
(Aloxi), dolasetron (Anzemet), or promethazine (Phenergen). Promethazine is a 
substrate for CYP2B6, and it is presently unknown if the potential for 2B6 induction 
after AG-348 dosing could be sufficient to reduce the therapeutic effect of 
promethazine. Not recommended are aprepitant (Emend; CYP3A4 inhibitor), 
ondansetron (Zofran; CYP3A4 inducer), chlorpromazine (Thorazine; CYP3A4 
inducer), prochlorperazine (Compazine; CYP3A4 substrate, QT prolongation has 
been reported), and granisetron (Kytril; CYP3A4 substrate, QT prolongation has been 
reported);  
• Diarrhea: recommended management includes standard clinical dosing with 
Kaopectate or other non-absorbable anti-diarrheals, diphenoxylate/atropine (Lomotil), 
or loperamide (Imodium). Loperamide is the least preferred choice because it is both 
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a substrate and inhibitor for CYP3A4, a substrate for CYP2B6, and a substrate for 
P-gp. 
• For the use of any medications not specifically mentioned above the Investigator may 
confer with the Sponsor’s Medical Monitor. 
9.11.6. 
Other Restrictions and Precautions 
Patients should be advised to refrain from altering their normal exercise routine for the first 
28 days of treatment with AG-348 as symptoms of anemia could theoretically temporarily 
worsen before the eventual potential beneficial effect of AG-348 on the Hb level becomes 
evident. The elevated blood levels of 2,3-DPG commonly seen in patients with PK deficiency 
may produce a right shift in the Hb-O2 dissociation curve. The effect of this is to enhance 
oxygen delivery at the tissue level, thus counteracting to some extent the physiologic 
consequences of the anemia. AG-348 was shown to produce rapid decreases (within the first 
12 hours following a single dose) in 2,3-DPG in two prior clinical trials with healthy adult male 
and female volunteers. In patients with PK deficiency who have elevated 2,3-DPG levels, it is 
theoretically possible that decreases in 2,3-DPG that precede correction of the anemia could 
result in a temporary decrease of oxygen delivery at the tissue level resulting in clinical 
symptoms (e.g., increased fatigue). 
As discussed in Section 5.2.1.1 of this protocol and in the Investigator Brochure, AG-348 has 
been identified as a histamine H3 receptor antagonist/inverse agonist. No effects of histamine H3 
modulation have been observed in safety pharmacology or toxicology studies. Nonetheless, 
patients should be monitored for potential AEs related to wakefulness and insomnia (Schwartz 
2011). 
Patients should be advised not to discontinue dosing without first speaking with the treating 
Investigator—abrupt discontinuation of AG-348 dosing in a patient who experience a substantial 
increase in Hb may result in withdrawal hemolysis. 
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10. 
STUDY ASSESSMENTS 
10.1. 
Schedule of Assessments 
The Schedules of Assessments for this study are provided in Appendix 15.1.  
After obtaining written informed consent, patients will undergo screening evaluations. The 
Screening visit is to be conducted within approximately 42 days prior to first dose of study 
treatment.  
During the Core Treatment Period, patients will attend visits at baseline (Day 1), weekly through 
Week 3 (Days 8, 15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 
12) and monthly through Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate AG-348 
through Week 24 (Core Period) and for whom the Investigator agrees with continuation of 
AG-348 treatment may be eligible to immediately enter the Extension Period for continued 
treatment upon agreement of the treating Investigator and the Medical Monitor or Responsible 
Medical Officer. Study visits for safety and clinical activity assessments will occur 
approximately every 3 months during the Extension Period. All patients will undergo a follow-up 
assessment 4 weeks after the last dose of AG-348, regardless of whether this was due to early 
discontinuation, the last dose in the Core Period for a patient who chooses not to continue in the 
Extension Period, or the last dose of the Extension Period. 
Although not encouraged, as a convenience for patients who travel long distances to the study 
site, in-clinic visits on Day 8 and Day 22 may be performed by the patient’s primary care 
physician if necessary and must be approved by the Sponsor on a case by case basis. For details, 
please refer to Table 5 in Appendix 15.1 : Schedule of Assessments. For patients having their 
Day 8 and/or Day 22 visits performed by the primary care physician, the principal investigator 
will him or herself, or have a qualified research nurse or other designated site staff member, 
make telephone contact with the patient to inquire about any AEs. These must be recorded as if 
the patient appeared in the main study center. The Principal Investigator will exercise prudent 
clinical judgment in determining any clinical course of action to take based on any AEs 
discovered. These telephone contacts on Day 8 and Day 22 must be explained to the patient in 
advance and scheduled in advance to maximize the likelihood of successfully making contact. 
Having in-clinic visits on Days 8 and/or Day 22 performed by the primary care physician will 
necessitate re-scheduling certain assessments that the primary care physician’s office may not be 
reasonably expected to perform. Table 3 summarizes the details of the re-scheduling of these 
assessments as described in Table 5 in Appendix 15.1.  
During the Extension Period, all scheduled visits must be conducted by the Investigator and at 
the participating authorized investigative site; local primary care visits will not be allowed. 
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Table 3: 
Summary of Assessments When Day 8 and/or Day 22 In-Clinic Visits Are 
Performed By Primary Care Physician (Core Period) 
Day 8 Visit by Primary Care Physician 
Day 22 Visit by Primary Care Physician 
Primary 
Care Office 
Main 
Study 
Site 
Assessment 
to Move to 
Day 15 Main 
Study Site 
Assessments 
Not Required  
Primary Care 
Office 
Main 
Study 
Site 
Assessment 
to Move to 
Day 43 Main 
Study Site 
Assessments Not 
Required 
Hematology 
to central lab 
Phone 
contact 
with 
patient 
12-lead ECG 
VS; serum 
chemistry; 
ETCO 
Hematology 
sample to 
central lab 
Phone 
contact 
with 
patient 
12-lead ECG 
VS; serum chemistry; 
coagulation; 
haptoglobin; EPO level; 
carboxyhemoglobin; 
ETCO; PK/PD 
Abbreviations: ECG = electrocardiogram; EPO = erythropoietin; PK/PD = pharmacokinetics/pharmacodynamics; 
VS = vital signs 
Whenever more than one assessment is scheduled for the same nominal time, the assessments 
should be performed in the order of least invasive to most invasive assessment (e.g., VS first, 
ECG, blood draw). The timing of these assessments should allow the PK blood draw to occur at 
the exact nominal time. The order of procedures may be revised with prior discussion between 
Sponsor and Site. 
Minor adjustments to the timing, number of planned safety monitoring procedures (e.g., VS, 
ECG, blood draw), and PK/PD assessments may be made during the course of the study based on 
collected data to ensure appropriate safety monitoring and will not require a protocol 
amendment. These minor changes will require prior approval from the Sponsor’s Medical 
Monitor (or Responsible Medical Officer) as well as appropriate documentation in the study 
records. The addition of new safety monitoring procedures or other assessments will require a 
protocol amendment. 
10.2. 
Informed Consent and Confirmation of Eligibility 
A complete description of the study is to be presented to each potential patient and a signed and 
dated informed consent is to be obtained before any study specific procedures are performed. 
Separate informed consent forms will be used for the Core and Extension Periods. 
Patient’s eligibility will be confirmed at Screening and within 24 hours prior to study treatment 
dose. If a patient is determined to be ineligible, the patient will be excluded from participation. 
10.3. 
Demographic Data, Medical and Medication History 
Patient demographic data, including gender, date of birth, age, race, and ethnicity, will be 
obtained at Screening. Collection of demographic data will be modified by country regulatory 
requirements, as appropriate.  
Medication history, including all relevant prior medical history and current medical conditions, 
will be obtained at the Screening assessment and on Day -1; all concomitant medications 
administered and procedures conducted within 28 days prior to, or from Screening Visit #1 until 
the first day of study drug administration, whichever interval is longer, should be reported in the 
source documentation and eCRF. Investigators will be asked to provide information on the 
patient’s history of any medical diagnoses (e.g., iron overload) and surgical procedures (e.g., 
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splenectomy, cholecystectomy) pertaining to their diagnosis of PK deficiency and prior available 
complete blood counts (CBCs) over the preceding 6 months and transfusion history over the 
preceding 12 months prior to the date of signing informed consent.  
10.4. 
PKR Enzymatic Assay and PKR Genotyping 
Assessments for PKR enzymatic activity and PKR genotyping will be performed at Screening 
only for confirmation of study eligibility. PKR enzymatic assays will be conducted at 
 
 or any participating investigative site’s local hematology 
laboratory. PKR genotyping will be conducted at 
 
10.5. 
Safety Assessments 
10.5.1. 
Physical Examination, Height, and Weight 
A complete physical examination (including neurological examination; genital and rectal 
examinations will be performed at the discretion of the Investigator) will be obtained at 
Screening, Baseline, Week 12, and the Follow up Visit (Week 28). The neurological examination 
must include an assessment of general orientation and mental status including level of alertness 
(assess as normal or abnormal and specify any abnormality). Limited focused physical 
examinations will be performed at all other visits during the Core Period. Focused physical 
examinations, including neurological examination, will continue every 3 months during the 
Extension Period; a complete physical examination will be performed at Month 30. Any findings 
will be recorded on the eCRF. Height will be collected at Screening only. 
10.5.2. 
Vital Signs 
Vital signs, including systolic and diastolic BP, heart rate, respiratory rate, and temperature, will 
be obtained according to the according to the Schedules of Assessments (Appendix 15.1). Vital 
sign measurements will be repeated if clinically significant or machine/equipment errors occur. 
Out of range BP or heart rate measurements will be repeated at the Investigator’s discretion. Any 
confirmed, clinically significant vital sign measurements will be recorded as AEs. 
10.5.3. 
Electrocardiogram  
A 12-lead ECG will be obtained according to the Schedules of Assessments (Appendix 15.1). 
The ECGs will be measured using an ECG machine that reports the heart rate and PR, QRS, QT, 
QTcB (Bazett correction formula; may also be calculated), and QTcF intervals (may also be 
calculated). Only QTcF (not QTcB) will be used for determination of eligibility. 
The 12-lead ECGs should be obtained following 5 minutes of recumbency. ECGs will be 
repeated if clinically significant abnormalities are observed, if artifacts are present, or if 
machine/equipment errors occur. Any confirmed, clinically significant ECG findings will be 
recorded as AEs. 
10.5.4. 
DXA Scans 
DXA scans (hip and spine) will be performed at Screening to obtain T and Z scores and bone 
mineral density that will serve as a baseline measure for all enrolling patients. An additional 
DXA scan for the Core Period will be conducted in the interval between Week 24 and Week 28, 
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and scans will be conducted at Months 18 and 30 during the Extension Period as indicated in the 
Schedules of Assessments (Appendix 15.1). All redacted DXA scan reports must be held at the 
study site and will be collected by the Sponsor. 
10.5.5. 
Safety Laboratory Assessments 
10.5.5.1. Hematology, Serum Chemistry, Coagulation Studies, and Urinalysis 
Laboratory values obtained prior to Screening and RBC antibodies obtained at Screening will be 
performed at a local laboratory. On-study clinical laboratory evaluations are to be performed by 
a central laboratory. If Investigators believe that it is clinically indicated to obtain safety 
laboratory results from their own local laboratories on the day of the subject's visit, and before 
the results are returned from the designated central laboratory, they are free to exercise their 
discretion to do so. Investigators should be aware that since the Hb level is a secondary endpoint 
of the study, it behooves them to minimize blood volumes drawn, and samples for the central lab 
must still be collected, since it will serve as the official lab result for this study. 
Clinical laboratory evaluations are to be collected according to the Schedules of Assessments 
(Appendix 15.1). In addition, all clinically significant laboratory abnormalities noted on testing 
will be followed by repeat testing and further investigated according to the judgment of the 
Investigator. Please note that serum estradiol, free and total testosterone, and CBC will be 
collected in the AM at any 2 time points during Screening at least 2 days apart in addition to 
Baseline/Day 1 (total of 3 time points prior to Day 1 dosing).  
The following safety laboratory parameters are to be determined: 
Hematology: 
HCT, Hb, RBC count, absolute reticulocyte count, percent 
reticulocyte count, MCV, mean corpuscular Hb (MCH), mean 
corpuscular Hb concentration (MCHC), red cell distribution width 
(RDW), white blood cell (WBC) count with automated (or manual, 
as indicated) differential, ANC, and absolute lymphocyte count 
(ALC), and platelet count. In the event that the designated central 
laboratory for the study is unable to provide a valid result for any 
specific component of the defined CBC for a specific patient, the site 
may be asked to have the test performed at their local laboratory. 
The result of the local CBC will be entered into the study database 
along with the local normal reference range. G6PD (may be 
conducted at Mayo Medical Laboratories [Rochester, MN] or any 
participating investigative site’s local hematology laboratory) and 
RBC antibody screen will be performed at Screening only 
Other 
EPO, Hp, COHb, ETCO (at a subset of sites during the Core Period), 
hepcidin, 25-hydroxy vitamin D2 and D3. 
Measurement of ETCO by CoSense® Monitor 
Assessment of ETCO as measured by the CoSense® Monitor device 
will be performed at a limited number of participating study sites on 
consenting patients during the Core Period. The patient will be fitted 
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with small single-use, disposable nasal prongs attached to a cannula 
that is connected to the CoSense® monitor. The patient breathes 
normally and the CoSense® monitor collects and analyzes the 
patient’s exhaled air within approximately 5 minutes, generating a 
measurement of end tidal carbon monoxide in parts per million 
(ppm). This value is entered into the clinical database. The 
assessment will be performed according to the schedule detailed in 
Section 15.1, Table 5. Carbon monoxide is produced in a 1:1 ratio 
from the breakdown of heme and has been correlated with the rate of 
hemolysis (Christensen, et al. 2015; Christensen, et al. 2016). 
Serum Chemistry: 
alkaline phosphatase (ALP), sodium, potassium, chloride, calcium, 
magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, 
albumin, total protein, glucose, blood urea nitrogen (BUN), 
creatinine, uric acid, LDH, ALT, AST, total bilirubin, indirect 
bilirubin, (and estimated creatinine clearance or GFR for screening 
only, as appropriate). 
Sex Hormones: 
testosterone (total and free), estrone, and estradiol. FSH will only be 
performed at Screening for female patients only for confirmation of 
post-menopausal status. 
Bone Turnover 
serum osteocalcin-N-mid and CTX. 
Lipids 
total cholesterol, HDL-C, triglycerides. 
Iron Panel 
iron, total iron-binding capacity (TIBC), transferrin saturation, 
ferritin. 
Coagulation Studies: 
fibrinogen, aPTT, INR. 
Urinalysis: 
color and appearance, pH, specific gravity, ketones, protein, glucose, 
bilirubin, nitrite, urobilinogen, pregnancy screen (dipstick), occult 
blood. Microscopic inspection of sediment will only be performed 
for cause or to investigate an abnormal dipstick finding per the 
Investigator’s discretion. 
 
10.5.5.2. Screening Serology 
A blood sample for serology, including HBsAg, HCV Ab screen, and HIV1 and HIV2 Ab, is to 
be collected from all patients at Screening.  
10.5.6. 
Menstrual Cycle Diary 
Menstruating female patients will be required to fill out a paper-based menstrual cycle diary each 
month in order to monitor any changes. Diaries will be dispensed and collected as indicated in 
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the Schedules of Assessments (Appendix 15.1). Patients will record the start date, stop date, and 
any notable characteristics of each menstrual cycle. 
10.5.7. 
Adverse Events 
Each patient will be carefully monitored for the development of any AEs throughout the study 
from signing of the informed consent through all scheduled study follow-up visits, or withdrawal 
of consent, whichever occurs first. In addition, SAEs that are assessed as possibly or probably 
related to study treatment that occur > 30 days post-treatment also are to be reported. 
Adverse events will be evaluated by the Investigator and recorded as described in the Schedules 
of Assessments. On dosing visits, all AEs (elicited and spontaneously reported) will be 
continuously evaluated by the Investigator and recorded. At any non-dosing day visit, AEs will 
be evaluated by the Investigator and recorded. Any AEs already documented at a previous 
assessment and designated as ongoing will be reviewed at subsequent visits or assessment time 
points as necessary. If these AEs have resolved, this will be documented. 
All AEs will be graded using the CTCAE grading system (Appendix 15.3 ). 
Complete details on AE monitoring are provided in Section 11. 
10.6. 
Pharmacokinetic Assessments 
The first approximately 10 patients treated in the Core Period, contingent on clinical site 
feasibility, will undergo extensive PK sampling as detailed in Appendix 15.1, Table 7. The 
remainder of treated patients will undergo limited PK sampling as detailed in Appendix 15.1, 
Table 8. The in-clinic visit on Day 22 may be performed by the patient’s primary care physician 
if necessary and must be approved by the Sponsor on a case by case basis. In this instance, PK 
sampling will not be required on Day 22. (Additional details regarding Day 8 and Day 22 visits 
performed by the patient’s primary care physician can be found in Table 5 in Appendix 15.1: 
Schedule of Assessments: Core Period.) During the Extension Period, predose PK samples will 
be drawn for the measurement of trough levels of AG-348 and AGI-8702 at each study visit 
(every 3 months; see Appendix 15.1, Table 6). 
The collection times for post-dose PK samples will start from the time that dosing is completed. 
(For example, a PK draw at 30 minutes will be collected 30 minutes after the last capsule has 
been ingested.) The completion time of each dose will be recorded. 
Procedures for sample collection and processing will be provided in a separate study manual. 
The actual time point of each sample collection will be recorded. 
Samples for PK and PD assessments may be retained for up to 2 years from collection.  
10.7. 
Pharmacodynamic Assessments 
The first approximately 10 patients treated in the Core Period, contingent on clinical site 
feasibility, will undergo extensive PD sampling for 2,3-DPG and ATP as detailed in 
Appendix 15.1, Table 7. The remainder of treated patients will undergo limited PD for 2,3-DPG 
and ATP sampling as detailed in Appendix 15.1, Table 8. During the Extension Period, predose 
PD samples will be drawn for the measurement of trough levels of 2,3-DPG, ATP, 
 
 at each study visit (every 3 months; see Appendix 15.1, Table 6). 
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The collection times for post-dose PD samples will start from the time that dosing is completed. 
(For example, a PD draw at 30 minutes will be collected 30 minutes after the last capsule has 
been ingested.) The completion time of each dose will be recorded. 
Procedures for sample collection and processing will be provided in a separate study manual. 
The actual time point of each sample collection will be recorded. 
Pharmacodynamic assessments during the Core Period will include 2,3-DPG, ATP,
 
 
 The in-clinic visit on Day 22 may be performed by the patient’s primary 
care physician if necessary and must be approved by the Sponsor on a case by case basis. In this 
instance, PD sampling will not be required on Day 22. (Additional details regarding Day 8 and 
Day 22 visits performed by the patient’s primary care physician are stated in Table 5 in 
Appendix 15.1: Schedule of Assessments.) 
Figure 4 provides a brief schematic outlining the PKR reaction and how each of these PD 
assessments fits into a complete mechanistic understanding of the action of AG-348. 
Figure 4: 
PKR Enzymatic Reaction 
 
The PKR enzyme catalyzes the PEP to pyruvate reaction, with concomitant formation of ATP. 
• Binding of AG-348 to the PKR tetramer can be assessed through an ex-vivo 
biochemical assay of cell lysates from AG-348 treated patients. Because WBCs 
contain a high level of pyruvate kinase from a non-PKR pyruvate kinase isoform, 
WBCs are first removed by filtration before the purified red cells are frozen.
 
 
It has been reported in the literature that there may be 
compensatory expression of PKM2 in the RBCs of some patients with PKR 
deficiency (Black, et al. 1979; Kahn, et al. 1975; Rijksen, et al. 1990).  Therefore, 
levels of PKM2 and appropriate reference proteins (e.g. actin, GAPDH) may be 
evaluated in these whole blood samples. 
• 
 
 
 
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• AG-348 target engagement 
 has been 
shown in preclinical models and healthy volunteer clinical studies to result in 
accumulation of ATP and depletion of the upstream metabolite 2,3-DPG. Therefore, 
levels of these metabolites will be measured by mass spectrometry in frozen whole 
blood samples. 
The first approximately 10 patients treated during the Core Period, contingent on clinical site 
feasibility, will undergo extensive PD sampling as detailed in Appendix 15.1, Table 7. The 
remainder of treated patients will undergo limited PD sampling as detailed in Appendix 15.1, 
Table 8. The collection times for post-dose PD samples will start from the time that dosing is 
completed. (For example, a PD draw at 30 minutes will be collected 30 minutes after the last 
capsule has been ingested.) The completion time of each dose will be recorded. 
Blood samples will be stored at the site and regularly transported at -80°C ±10 C to the 
bioanalytical laboratory for analysis. Procedures for sample collection and processing will be 
provided in a separate study manual. 
10.8. 
Ordering of Blood Sample Collection 
When more than 1 blood sample is collected at the same nominal time, the samples will be 
collected in the following order: 
1. Safety laboratory assessments 
2. PD (2,3 DPG, ATP) 
 
4. PK  
 
 
10.9. 
Sample Processing, Storage, and Shipment 
Instructions for the processing, storage and shipment of all study samples for central analysis 
will be provided in a separate study manual. 
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11. 
ADVERSE EVENTS 
Monitoring of AEs will be conducted throughout the study. Adverse events and SAEs will be 
recorded in the source documentation and eCRF from time of the signing informed consent 
through the Follow-up Visits for randomized patients. All AEs should be monitored until they 
are resolved or are clearly determined to be due to a patient’s stable or chronic condition or 
intercurrent illness(es). 
All SAEs will be followed through 30 days after the last dose of study treatment or until the SAE 
has resolved. Any SAEs that are assessed as possibly or probably related to study treatment that 
occur > 30 days post-treatment also are to be reported. 
11.1. 
Definition of Adverse Events 
11.1.1. 
Adverse Event 
An AE is any untoward medical occurrence associated with the use of a drug in humans, whether 
or not considered drug related. An AE (also referred to as an adverse experience) can be any 
unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a drug, without any judgment about causality. An AE can 
arise from any use of the drug (e.g., off-label use, use in combination with another drug) and 
from any route of administration, formulation, or dose, including an overdose. 
11.1.2. 
Suspected Adverse Reaction 
A suspected adverse reaction is any AE for which there is a reasonable possibility that the drug 
caused the AE. For the purposes of expedited safety reporting, ‘reasonable possibility’ means 
that there is evidence to suggest a causal relationship between the drug and the AE.  
11.1.3. 
Unexpected Adverse Event 
An unexpected AE is one for which the nature or severity of the event is not consistent with the 
applicable product information, e.g., the Investigator’s Brochure. 
11.1.4. 
Serious Adverse Event 
An AE or suspected adverse reaction is considered serious (SAE) if, in the view of either the 
Investigator or Sponsor, it results in any of the following outcomes: 
• Death; 
• Life-threatening. Life-threatening means that the patient was at immediate risk of 
death from the reaction as it occurred, i.e., it does not include a reaction which 
hypothetically might have caused death had it occurred in a more severe form; 
• In-patient hospitalization or prolongation of existing hospitalization. Hospitalization 
admissions and/or surgical operations scheduled to occur during the study period, but 
planned prior to study entry are not considered AEs if the illness or disease existed 
before the patient was enrolled in the study, provided that it did not deteriorate in an 
unexpected manner during the study (e.g., surgery performed earlier than planned); 
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• A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions; 
• Congenital anomaly/birth defect; 
• Important medical event. An important medical event is an event that may not result 
in death, be life-threatening, or require hospitalization but may be considered an SAE 
when, based upon appropriate medical judgment, it may jeopardize the patient or 
patient and 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 convulsions that do not result in in-patient hospitalization, 
or the development of drug dependency or drug abuse. 
11.1.4.1. Potential Severe Drug-Induced Liver Injury 
The document entitled FDA Guidance for Industry, Drug-Induced Liver Injury: Premarketing 
Clinical Evaluation (FDA 2009) provides guidance on how the measurement of various 
laboratory parameters may be used to assess a given drug’s potential to cause severe liver injury 
(i.e., irreversible liver failure that is fatal or requires liver transplantation). Such cases are 
suggested by the following three components: 
1. The drug causes hepatocellular injury, generally shown by a higher incidence of 3-fold or 
greater elevations above the ULN of ALT or AST than the (non-hepatotoxic) control 
drug or placebo; 
2. Among trial patients showing such aminotransferase elevations, often with 
aminotransferases much greater than 3×ULN, one or more also show elevation of serum 
total bilirubin to > 2×ULN, without initial findings of cholestasis (elevated serum ALP); 
3. No other reason can be found to explain the combination of increased aminotransferases 
and total bilirubin, such as viral hepatitis A, B, or C; pre-existing acute liver disease; or 
another drug capable of causing the observed injury.  
Clinical safety laboratory results compatible with the definition of drug-induced liver injury 
(DILI) stated above must be repeated for confirmation as soon as possible, and if confirmed, will 
be scored as an unacceptable AE and reported to FDA as a serious unexpected AE. 
11.2. 
Procedures for Reporting Adverse Events and Serious Adverse 
Events  
Each patient must be carefully monitored for the development of any AEs. This information 
should be obtained in the form of non-leading questions (e.g., “How are you feeling?”) and from 
signs and symptoms detected during each examination, observations of study personnel, and 
spontaneous reports from patients. 
All AEs (serious and non-serious) spontaneously reported by the patient and/or in response to an 
open question from study personnel or revealed by observation, physical examination, or other 
diagnostic procedures will be recorded in the source documentation and eCRF. Any clinically 
relevant deterioration in laboratory assessments or other clinical findings is considered an AE 
and must be recorded in the appropriate source documentation and eCRF. Treatment-emergent 
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abnormal clinical laboratory results should generally be reported as AEs if there are 
accompanying symptoms; if additional diagnostic evaluations or medical (including drug 
therapy) or surgical interventions are undertaken; if a change in study drug dosing or study drug 
discontinuation is required; or, if the laboratory result is considered clinically significant by the 
Investigator. Although it is an objective of this study to determine if treatment with AG-348 
results in increased Hb levels in patients with PK deficiency, overshoot of the Hb level above the 
ULN by gender is not recommended and should be reported as an AE, graded per the 
CTCAEv4.03. When possible, signs and symptoms indicating a common underlying pathology 
should be noted as one comprehensive event. 
All SAEs that occur during the course of the study must be promptly reported by the Investigator 
to 
 Global Safety and Pharmacovigilance (see below). Deaths and AEs assessed 
as life-threatening are to be reported immediately and SAEs that meet other criteria are to be 
reported within 24 hours from the point in time when the Investigator becomes aware of the 
SAE. All SAEs must be reported whether or not they are considered causally related to AG-348. 
Serious AE forms will be completed and the information collected will include subject number, a 
narrative description of the event, and an assessment by the Investigator as to the severity of the 
event and relatedness to study drug. Follow-up information on the SAE may be requested by the 
Sponsor or Medical Monitor. 
Contact Information: 
 Safety Management 
SAE Hotline (United States): 
 
SAE Fax (United States): 
 
SAE Hotline (France): 
 
SAE Fax (France): 
  
SAE Hotline (Netherlands): 
 
SAE Fax (Netherlands): 
 
SAE Hotline (Italy): 
 
SAE Fax (Italy): 
 
SAE Hotline (United Kingdom): 
 
SAE Fax (United Kingdom): 
 
E-mail: 
 
If there are serious, unexpected adverse drug reactions associated with the use of AG-348, the 
Sponsor will notify the appropriate regulatory agency(ies) and all participating Investigators on 
an expedited basis. The local IRB/IEC will be promptly notified based on local regulations 
where required by the IRB/IEC of all serious, unexpected adverse drug reactions involving risk 
to human subjects. 
All AEs, whether serious or not, will be described in the source documents in the database. All 
new events, as well as those that worsen in intensity or frequency relative to baseline, which 
occur after signing the informed consent through the final Follow-up Visit (Day 29 ± 3 days) 
must be recorded. Adverse events that are ongoing at the time of treatment discontinuation 
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should be followed up to 30 days after the last dose of study treatment. All SAEs will be 
followed up to 30 days after the last dose of study treatment, or until the SAE has resolved.  
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); 
• The date of onset of the event; 
• The date of resolution of the event; 
• Whether the event is serious or not; 
• Intensity of the event (see below for definitions); 
• Relationship of the event to study treatment (see below for definitions); 
• Action taken: none; change in the study drug administration (e.g., temporary 
interruption in dosing); drug treatment required; non-drug treatment required; 
hospitalization or prolongation of hospitalization required (complete SAE page); 
diagnostic procedure performed; patient discontinued from the study (complete End 
of Study visit); 
• Outcome: patient recovered without sequelae; patient recovered with sequelae; event 
ongoing; patient died (notify the Medical Monitor immediately, and complete the 
SAE form). 
Intensity of all AEs will be graded according to the NCI CTCAE Version 4.03 (Appendix 15.3). 
Relationship to study drug administration will be determined by the Investigator according to 
the following criteria: 
• Not Related:  Exposure to the study treatment did not occur, or the occurrence of the 
AE is not reasonably related in time, or the AE is considered unlikely to be related to 
the study treatment. 
• Possibly Related:  The study treatment and the AE were reasonably related in time, 
and the AE could be explained equally well by causes other than exposure to the 
study treatment. An attribution of possibly related means that there are facts in 
evidence to suggest a possible relationship. 
• Probably Related:  The study treatment and the AE were reasonably related in time, 
and the AE was more likely explained by exposure to the study treatment than by 
other causes, or the study treatment was the most likely cause of the AE. An 
attribution of probably related means that there are facts in evidence to suggest a 
probable relationship. 
For the purpose of safety analyses related to final database review, all AEs that are classified as 
possible or probable will be considered treatment-related AEs. 
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11.3. 
Pregnancy Reporting 
Pregnancy is neither an AE nor an SAE, unless a complication relating to the pregnancy occurs 
(e.g., spontaneous abortion, which may qualify as an SAE). However, any pregnancy in a 
participating female patient or a female partner of a participating male patient that occurs during 
this study or within 30 days following the last dose of AG-348 must be reported to the Sponsor 
or Medical Monitor within 30 days of being notified of the pregnancy. The Investigator must 
follow up and document the course and outcome of all pregnancies even if the patient was 
discontinued from the study or if the study has finished. The female patient or partner of a male 
patient should receive any necessary counseling regarding the risks of continuing the pregnancy 
and the possible effects on the fetus. Monitoring should continue until conclusion of the 
pregnancy. 
All outcomes of pregnancy must be reported by the Investigator to the Sponsor or Sponsor’s 
designee on a Pregnancy Outcome Report form within 30 days after he/she has gained 
knowledge of the delivery or elective abortion. 
Any SAE that occurs during pregnancy must be recorded on the SAE report form (e.g., maternal 
serious complications, spontaneous or therapeutic abortion, ectopic pregnancy, stillbirth, 
neonatal death, congenital anomaly, or birth defect) and reported within 24 hours in accordance 
with the procedure for reporting SAEs. 
All patients, male and female, must agree to use effective contraception during the entire study 
and for 30 days following the last dose of AG-348. Abstinence is an acceptable method only 
when this is in line with the normal life style of the patient, meaning that the patient plans to 
remain abstinent continuously throughout the duration of the study and for at least 30 days after 
the last dose of study drug. Periodic abstinence, e.g. calendar, sympathothermal and post-
ovulation methods, and withdrawal are not acceptable methods of contraception. 
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12. 
STATISTICAL METHODS 
The primary objective of this Phase 2 study is to evaluate the safety and tolerability of up to 
24 weeks of AG-348 in patients with PK deficiency. Therefore, analyses will be primarily 
descriptive in nature; no formal hypothesis testing will be conducted. All analyses will be 
conducted separately within each dose arm, or pooled where appropriate. Analyses of safety and 
of indicators of clinical activity will be conducted for the Core Period, Extension Period, and 
overall, if applicable. For the Core Period, the data to be analyzed will include all collected data 
through 24 weeks of treatment for patients who directly enter the Extension period. For patients 
who do not enter the Extension Period, the analyses will include all collected data through the 
duration of treatment (24 weeks or less) plus 4 week follow up data. For patients who move 
directly from the Core to the Extension Period, the 4 week follow up data will be analyzed as 
part of the Extension Period. 
12.1. 
Sample Size Estimation 
Due to the rare disease setting, the minimal sample size in each dose arm may be determined by 
feasibility. In order to evaluate the primary objective of safety and tolerability of AG-348 in 
Arm 1 and Arm 2, up to a maximum of 25 patients may be randomized onto each arm. The 
actual number of patients enrolled into Arms 1 and 2 will depend on the safety reviews and 
decisions made by the DRT. In addition, up to 25 additional patients may be enrolled to evaluate 
an additional dose arm (Arm 3; see Section 7.1). As for Arms 1 and 2, the actual enrollment in a 
potential Arm 3 will depend on the safety reviews and decisions made by the DRT. Therefore, up 
to approximately 75 total patients may be enrolled in this study across 2 to 3 dose arms. 
Table 4 provides the probability within a dose arm of detecting 1 or more AEs with varying 
sample size and the true underlying AE rates. 
Table 4: 
Sample Size Estimation 
Sample Size 
True Underlying AE Rate 
15% 
10% 
5% 
5 
56% 
41% 
23% 
10 
80% 
65% 
40% 
15 
91% 
79% 
54% 
25 
98% 
93% 
72% 
 
12.2. 
Populations for Analysis 
The following patient populations (i.e., analysis sets) will be evaluated and used for presentation 
of the data: 
• Safety Analysis Set: All patients who are enrolled and receive any dose of study 
treatment. The Safety Analysis Set will be the primary set for the analysis of safety 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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data. Patients will be classified according to the actual treatment received and 
whether they underwent an intra-patient dose escalation, where treatment received is 
defined as the assigned treatment if it is received at least once, or as the first treatment 
received if assigned treatment is never received. Unless otherwise stated, the Safety 
Analysis Set will be the default analysis set for all data analyses. 
• Pharmacokinetic Analysis Set: All patients in the Safety Analysis Set with sufficient 
plasma sample data to assess PK parameters. Results of the potential PD activity of 
AG-348 will also be based on the PK analysis set.  
• Efficacy Analysis Set: All patients who are enrolled and achieve at least 50% 
compliance with their assigned dose intensity for at least 4 weeks of continuous 
dosing. Additional efficacy analyses may be performed on subsets of patients with 
other degrees of compliance with their assigned dose intensity and/or duration of 
dosing. The Efficacy Analysis Set will be the primary set for the analysis of 
preliminary clinical activity data. Patients will be classified according to the actual 
treatment received and whether they underwent an intra-patient dose escalation, 
where treatment received is defined as the assigned treatment if it is received at least 
once, or as the first treatment received if assigned treatment is never received. 
 
 
 If such analyses are performed, they will be 
described in a separate PK Statistical Analysis Plan (SAP) and may be reported separately in a 
stand-alone report. 
12.3. 
Procedures for Handling Missing, Unused, and Spurious Data 
No imputation will be performed for missing data elements. When summarizing AE data, partial 
dates will be imputed as described in the SAP. Additional rules addressing the handling of 
missing data will be detailed in the SAP. 
12.4. 
Interim Analysis 
No formal statistical interim analysis will be conducted. Safety data will be reviewed on an 
ongoing basis by the DRT, who will meet to review safety, PK, PD, and preliminary clinical 
activity data at regular intervals (approximately every 6 weeks during the Core Period and 
approximately every 3 months during the Extension Period once all patients have completed the 
Core Period) throughout the duration of the Core Period. The DRT’s decisions to suspend, 
terminate, or open a potential third dosing arm, or re-assign patients’ dosing in a terminated arm 
to match the dose and schedule of another arm of the study will be based on the totality of the 
data including, safety, PK, PD, and preliminary clinical activity (e.g., changes in Hb levels). 
Additional interim reviews of data may be conducted to support decision making concerning the 
current clinical study, the sponsor’s development programs in general, or in case of any safety 
concerns. 
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12.5. 
Statistical Methodology 
12.5.1. 
General Methods 
This study will be primarily descriptive in nature; therefore, there will be no formal hypothesis 
testing. Summaries will be produced for disposition, baseline disease characteristics and 
demographic data including genotype, safety measurements, PK, PD parameters and indicators 
of clinical activity. Data from each AG-348 dose group will be analyzed separately, and pooled 
across all dose groups where appropriate. All data will also be listed by individual patient. 
Categorical variables will be summarized by frequency distributions (number and percentages) 
and continuous variables will be summarized by descriptive statistics (mean, standard deviation 
[SD], median, minimum, and maximum). 
The study database will be locked and statistical analysis will be performed after all patients 
have completed the Core Period. The results of this analysis will be presented in a clinical study 
report (CSR). Additional data collected during the Extension Period after the CSR database lock 
will be analyzed for inclusion in a subsequent CSR addendum. 
12.5.2. 
Disposition 
A summary of the disposition of patients will be presented, including the number enrolled, the 
number treated, and the reasons for study discontinuation. Entry criteria and protocol deviations 
will be listed. 
12.5.3. 
Exposure and Safety Analyses 
Patients will receive multiple PO doses of AG-348 over a 24-week treatment period. The actual 
dose and duration in days of AG-348, as well as the dose intensity (computed as the ratio of 
actual dose received and actual duration) and the relative dose intensity (computed as the ratio of 
dose intensity and planned dose received/planned duration) will be listed and summarized using 
descriptive statistics by dose arm. 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) 
and the incidence of TEAEs (new or worsening from baseline) will be summarized by primary 
system organ class (SOC), preferred term, CTCAE Version 4.03 severity, outcome, action taken 
with study drug, and relationship to the study drug by dose group. Separate summaries will be 
produced for all TEAEs, treatment-related AEs (those considered by the Investigator as at least 
possibly drug related), SAEs, discontinuations due to AEs, and AEs ≥ Grade 3 severity. 
Individual patient listings will be provided for deaths, SAEs, AEs leading to interruption and/or 
reduction of study drug dose, and AEs leading to discontinuation of treatment. 
Analyses of AEs will be performed for those events that are considered treatment-emergent, 
where treatment-emergent is defined as any AE with onset beginning at the day of first 
administration of study drug, throughout the treatment period until 30 days after the last dose of 
study drug, or any event that was present at baseline but worsened in intensity or was 
subsequently considered drug-related by the Investigator through the end of the study. 
Descriptive statistics will be provided for clinical laboratory values (e.g., hematology, serum 
chemistry, coagulation studies, urinalysis) and VS data, presented as both actual values and 
changes from baseline relative to each on-study evaluation. Shift analyses will be conducted for 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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selected laboratory parameters based on the baseline CTCAE grade to maximum CTCAE grade. 
Where applicable CTCAE terms do not exist, a grading system based on the upper and/or lower 
limits of normal will be used to classify the results. 
No formal hypothesis-testing analysis of AE incidence rates will be performed. 
Electrocardiogram analyses will include individual patient listings and summaries of abnormal 
and clinically significant ECG results. Actual values and changes from baseline in PR, QRS, and 
heart-rate corrected QT interval (QTc) intervals will be summarized by visit and dose arm. Full 
details of the QTc analysis including correction methods used will be described in the SAP. 
Data collected from the menstrual diaries such as the start and stop dates of the menses and the 
patient reported characteristics of the menses will be presented in a by-patient listing. Additional 
descriptions of the data may also be performed. 
Concomitant medications will be coded using the WHO Drug Dictionary. Concomitant 
medications prior to and after the start of the study drug will be listed by patient, and 
summarized by ATC term and dose arm. 
12.5.4. 
Pharmacokinetic Analyses 
Descriptive statistics will be used to summarize PK parameters for the parent compound AG-348 
and the metabolite AGI-8702 for each dose group, and where appropriate, for the entire 
population. Pharmacokinetic parameters will be summarized using the following descriptive 
statistics: n, mean, SD, coefficient of variation %, median, minimum and maximum, geometric 
mean, and geometric coefficient of variation. PK analyses will be described in a separate PK 
SAP. 
12.5.5. 
Pharmacodynamic Analyses 
Descriptive statistics will be used to summarize PD parameters for 2,3-DPG and ATP for each 
dose group, and where appropriate for the entire population. Pharmacodynamic parameters will 
be summarized using the following descriptive statistics: n, mean, SD, coefficient of variation %, 
median, minimum, and maximum, geometric mean, and geometric coefficient of variation %. PD 
analyses will be described in a separate PD SAP. 
12.5.6. 
Aromatase Hormone Analysis  
The analyses of serum sex hormones will use appropriate graphic displays and statistical 
analyses to evaluate patient change from baseline for each parameter. These analyses will 
include summaries of actual values and change from baseline using appropriate descriptive 
statistics (mean, SD, median, min and max) as described above.  
 
These analyses will present information by each dose arm, and analyses of a pooled 
AG-348 cohort. Additional details regarding these analyses will be provided in the SAP. 
12.5.7. 
Clinical Activity 
Details on analyses to evaluate indicators of potential clinical activity of AG-348 in patients with 
PK deficiency will be described in the SAP. These will include changes in Hb, HCT, reticulocyte 
count, Hp, COHb, ETCO (Core Period only), LDH, total and indirect bilirubin, EPO, hepcidin, 
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ferritin, and transferrin saturation (serum iron/iron binding capacity). Characterization of Hb 
response which will include, but is not limited to percent of patients with increase in Hb, time to 
Hb response, and duration of Hb response will be explored. 
12.6. 
Procedures for Reporting Deviations to Original Statistical Analysis 
Plan 
All deviations from the original SAP will be provided in the final CSR. 
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13. 
ADMINSTRATIVE REQUIREMENTS 
13.1. 
Good Clinical Practice 
The study will be conducted in accordance with the ICH for Good Clinical Practice (GCP) and 
the appropriate regulatory requirement(s). The Investigator will be thoroughly familiar with the 
appropriate use of the study drug as described in the protocol and Investigator’s Brochure. 
Essential clinical documents will be maintained to demonstrate the validity of the study and the 
integrity of the data collected. Master files should be established at the beginning of the study, 
maintained for the duration of the study, and retained according to the appropriate regulations. 
13.2. 
Ethical Considerations 
The study will be conducted in accordance with ethical principles founded in the Declaration of 
Helsinki (Appendix 15.5). 
The Investigator must obtain IRB approval for the investigation and must submit written 
documentation of the approval to the Sponsor before he or she can enroll any patient into the 
study. The IRB will review all appropriate study documentation in order to safeguard the rights, 
safety, and well-being of the patients. The study will only be conducted at sites where IRB 
approval has been obtained. The protocol, Investigator’s Brochure, informed consent, 
advertisements (if applicable), written information given to the patients, annual progress reports, 
and any revisions to these documents will be provided to the IRB. The IRB is to be notified of 
any amendment to the protocol in accordance with local requirements. Progress reports and 
notifications of serious unexpected adverse drug reactions are to be provided to the IRB 
according to local regulations and guidelines. 
13.3. 
Patient Information and Informed Consent 
The Investigator or trained designee will ensure that the patient is given full and adequate oral 
and written information about the nature, purpose, possible risk, and benefit of the study. Patients 
must also be notified that they are free to discontinue from the study at any time. The patient 
should be given the opportunity to ask questions and allowed time to consider the information 
provided. 
After the study has been fully explained, written informed consent will be obtained from the 
patient prior to study participation.  
The patient’s signed and dated informed consent must be obtained before conducting any study-
related procedures. The Investigator must maintain the original, signed consent form. A copy of 
the signed form must be given to the patient. 
The method of obtaining and documenting the informed consent and the contents of the consent 
will comply with ICH-GCP and all applicable regulatory requirement(s). 
13.4. 
Patient Confidentiality 
In order to maintain patient privacy, all source documents, study drug accountability records, 
study reports and communications will identify the patient by the assigned patient number. The 
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Investigator will grant monitor(s) and auditor(s) from the Sponsor or its designee and regulatory 
authority(ies) access to the patient’s original medical records for verification of data gathered on 
the source documents and to audit the data collection process. The patient’s confidentiality will 
be maintained and will not be made publicly available to the extent permitted by the applicable 
laws and regulations. 
13.5. 
Protocol Compliance 
The Investigator will conduct the study in compliance with the protocol. Modifications to the 
protocol should not be made without agreement of both the Investigator and the Sponsor. 
Changes to the protocol will require written IRB approval/favorable opinion prior to 
implementation, except when the modification is needed to eliminate an immediate hazard(s) to 
patients. The IRB may provide, if applicable, where regulatory authority(ies) permit, expedited 
review and approval/favorable opinion for minor change(s) in ongoing studies that have the 
approval/favorable opinion of the IRB. The Sponsor or designee will submit all protocol 
modifications to the regulatory authority(ies) in accordance with the governing regulations.  
When immediate deviation from the protocol is required to eliminate an immediate hazard(s) to 
patients, the Investigator will contact the Sponsor’s Medical Monitor (or Responsible Medical 
Officer), if circumstances permit, to discuss the planned course of action. Any departures from 
the protocol must be fully documented in the source documents/database. 
13.6. 
Data Management 
A paper or eCRF will be completed for each patient or an electronic data capture (EDC) system 
will be used. The EDC system (Medidata Rave®) is a software tool designed to ensure quality 
assurance and facilitate data capture during clinical trials. Through a system regulated workflow 
that includes barcode scanning and interfaces to medical equipment to avoid manual data entry, 
study operations performance is controlled and captured in real time. The system is fully Code of 
Federal Regulations (CFR) 21 Part 11 compliant. Source documentation supporting the data 
should indicate participation in the study and should document the dates and details of study 
procedures, AEs, and patient status. The Investigator, or trained designee should complete and 
the Investigator should verify the source documents as the information is collected. Any 
outstanding entries must be completed immediately after the final examination. An explanation 
should be given for all missing data. The Investigator will ensure the accuracy, completeness, 
and timeliness of the data reported to the Sponsor. Data collection processes and procedures will 
be reviewed and validated to ensure completeness, accuracy, reliability, and consistency. A 
complete audit trail will be maintained of all data changes. The Investigator or designee will 
cooperate with the Sponsor’s representative(s) for the periodic review of study documents to 
ensure the accuracy and completeness of the data capture system at each scheduled monitoring 
visit. Electronic consistency checks and manual review will be used to identify any errors or 
inconsistencies in the data. This information will be provided to the respective study sites by 
means of electronic or manual queries. The Investigator or designee will prepare and maintain 
adequate and accurate study documents (medical records, ECGs, AE, and concomitant 
medication reporting, raw data collection forms, etc.) designed to record all observations and 
other pertinent data for each patient receiving study treatment. The Investigator will allow 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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Sponsor representatives, contract designees, authorized regulatory authority inspectors, and the 
IRB to have direct access to all documents pertaining to the study. 
13.7. 
Source Document/Case Report Form Completion 
Source documents will be completed for each study patient. It is the Investigator’s responsibility 
to ensure the accuracy, completeness, and timeliness of the data reported in the patient’s source 
document. The source document should indicate the patient’s participation in the study and 
should document the dates and details of study procedures, AEs and patient status. 
The Investigator, or designated representative, should complete the source document as soon as 
possible after information is collected, preferably on the same day that a study patient is seen for 
an examination, treatment, or any other study procedure. Any outstanding entries must be 
completed immediately after the final examination. An explanation should be given for all 
missing data. 
The Investigator will sign and date each required assessment for all study patients. The 
Investigator will retain all completed source documents. 
13.8. 
Direct Access to Source Data 
The study will be monitored by the Sponsor or its designee. Monitoring will be done by personal 
visits from a representative of the Sponsor (site monitor) and will include on-site review of the 
source documents for completeness and clarity, cross-checking with source documents, and 
clarification of administrative matters will be performed. The review of medical records will be 
performed in a manner to ensure that patient confidentiality is maintained. 
The site monitor will ensure that the investigation is conducted according to protocol design and 
regulatory requirements by frequent communications (letter, telephone, e-mail, and fax). 
All unused study drug and other study materials are to be returned to the Sponsor or designee 
after the study has been completed and the database has been locked. 
Regulatory authorities, the IRB, and/or the Sponsor’s clinical quality assurance group or 
designee may request access to all source documents, database, and any other applicable study 
documentation for an on-site audit or inspection. Direct access to these documents must be 
guaranteed by the Investigator, who must provide support at all times for these activities. 
13.9. 
Record Retention 
The Investigator will maintain all study records according to ICH-GCP and applicable regulatory 
requirement(s). Records will be retained for at least 2 years after the last marketing application 
approval or 2 years after formal discontinuation of the clinical development of the investigational 
product or according to applicable regulatory requirement(s). If the Investigator withdraws from 
the responsibility of keeping the study records, custody must be transferred to a person willing to 
accept the responsibility. The Sponsor must be notified in writing if a custodial change occurs. 
13.10. 
Liability and Insurance 
The Sponsor has subscribed to an insurance policy covering, in its terms and provisions, its legal 
liability for injuries caused to participating persons and arising out of this research performed 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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strictly in accordance with the scientific protocol as well as with applicable law and professional 
standards.  
13.11. 
Publication of Study Findings and Use of Information 
All information regarding AG-348 supplied by the Sponsor or designee to the Investigator is 
privileged and confidential information. The Investigator agrees to use this information to 
accomplish the study and will not use it for other purposes without consent from the Sponsor. It 
is understood that there is an obligation to provide the Sponsor with complete data obtained 
during the study. The information obtained from the clinical study will be used towards the 
development of AG-348 and may be disclosed to regulatory authority(ies), other Investigators, 
corporate partners, or consultants as required. 
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14. 
LIST OF REFERENCES 
 
Beutler E, and Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene 
frequency in the general white population. Blood. 2000;95(11):3585-8. 
Black JA, Rittenberg MB, Bigley RH, and Koler RD. Hemolytic anemia due to pyruvate kinase 
deficiency: characterization of the enzymatic activity from eight patients. Am J Hum Genet. 
1979;31(3):300-10. 
Christensen RD, Lambert DK, Henry E, Yaish HM, and Prchal JT. End-tidal carbon monoxide 
as an indicator of the hemolytic rate. Blood Cells Mol Dis. 2015;54(3):292-6. 
Christensen RD, Malleske DT, Lambert DK, Baer VL, Prchal JT, Denson LE, et al. Measuring 
End-Tidal Carbon Monoxide of Jaundiced Neonates in the Birth Hospital to Identify Those with 
Hemolysis. Neonatology. 2016;109(1):1-5. 
Christensen RD, Yaish HM, Johnson CB, Bianchi P, and Zanella A. Six children with pyruvate 
kinase deficiency from one small town: molecular characterization of the PK-LR gene. J Pediatr. 
2011;159(4):695-7. 
FDA. Guidance for Industry: Drug-Induced Liver Injury: Premarketing Clinical Evaluation. 
2009. 
Ghidini A, Sirtori M, Romero R, Yarkoni S, Solomon L, and Hobbins JC. Hepatosplenomegaly 
as the only prenatal finding in a fetus with pyruvate kinase deficiency anemia. Am J Perinatol. 
1991;8(1):44-6. 
Hennekam RC, Beemer FA, Cats BP, Jansen G, and Staal GE. Hydrops fetalis associated with 
red cell pyruvate kinase deficiency. Genet Couns. 1990;1(1):75-9. 
Kahn A, Marie J, Galand C, and Boivin P. Molecular mechanism of erythrocyte pyruvate kinase 
deficiency. Humangenetik. 1975;29(4):271-80. 
Mentzer WC, Jr., Baehner RL, Schmidt-Schonbein H, Robinson SH, and Nathan DG. Selective 
reticulocyte destruction in erythrocyte pyruvate kinase deficiency. J Clin Invest. 1971;50(3):688-
99. 
Rider NL, Strauss KA, Brown K, Finkenstedt A, Puffenberger EG, Hendrickson CL, et al. 
Erythrocyte pyruvate kinase deficiency in an old-order Amish cohort: longitudinal risk and 
disease management. Am J Hematol. 2011;86(10):827-34. 
Rijksen G, Veerman AJ, Schipper-Kester GP, and Staal GE. Diagnosis of pyruvate kinase 
deficiency in a transfusion-dependent patient with severe hemolytic anemia. Am J Hematol. 
1990;35(3):187-93. 
Schwartz JC. The histamine H3 receptor: from discovery to clinical trials with pitolisant. Br J 
Pharmacol. 2011;163(4):713-21. 
Strauss KA, Robinson DL, Vreman HJ, Puffenberger EG, Hart G, and Morton DH. Management 
of hyperbilirubinemia and prevention of kernicterus in 20 patients with Crigler-Najjar disease. 
Eur J Pediatr. 2006;165(5):306-19. 
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Tanphaichitr VS, Suvatte V, Issaragrisil S, Mahasandana C, Veerakul G, Chongkolwatana V, et 
al. Successful bone marrow transplantation in a child with red blood cell pyruvate kinase 
deficiency. Bone Marrow Transplant. 2000;26(6):689-90. 
van Wijk R, and van Solinge WW. The energy-less red blood cell is lost: erythrocyte enzyme 
abnormalities of glycolysis. Blood. 2005;106(13):4034-42. 
Zanella A, Fermo E, Bianchi P, and Valentini G. Red cell pyruvate kinase deficiency: molecular 
and clinical aspects. Br J Haematol. 2005;130(1):11-25. 
 
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15. 
APPENDICES 
 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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15.1. 
Schedules of Assessments 
Table 5: 
Schedule of Assessments: Core Period 
Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up1 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-42 to -1 
-40 to -12 
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Written Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
PK enzyme assay 
(confirmation of PK 
deficiency)4 
X 
 
 
 
 
 
 
 
 
 
 
 
 
PKR Genotype 
(for randomization) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
UGT1A1 Genotype 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medical/Surgical 
History (General and 
PK deficiency-specific)
5 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medication History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Transfusion History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Confirmation of 
Vaccinations 
(Splenectomized 
Patients) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Examination6/ 
Height6 and Weight 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
X 
X 
Performance Status 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
X 
X 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up1 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-42 to -1 
-40 to -12 
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Vital signs  
(BP, HR, RR, T)7  
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG8 
X 
 
X 
X 
 
X 
 
 
 
 
 
X 
X 
DXA Scan9 
X 
 
 
 
 
 
 
 
 
 
 
X10 
 
Laboratory Evaluations
11 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBsAg, HCV Ab, 
HIV1 and 2 Ab 
X 
 
 
 
 
 
 
 
 
 
 
 
 
RBC antibody 
Screen  
X 
 
 
 
 
 
 
 
 
 
 
 
 
Hematology (CBC)12 
X 
X13 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin14 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
EPO levels15 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
G6PD screen 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Hepcidin 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Serum Chemistry16 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Iron Panel17 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Carboxyhemoglobin 
(COHb)18 
 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
 
ETCO19 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Coagulation Studies
20 
X 
 
X 
 
X 
 
 
 
X 
 
 
X 
X 
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Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up1 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-42 to -1 
-40 to -12 
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Urinalysis21 
X 
 
X 
 
X 
 
 
 
X 
 
 
X 
X 
Serum or Urine 
Pregnancy22 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
Lipids23 
 
 
X 
 
 
 
X 
 
X 
 
 
X 
X 
Hormonal Testing24 
X 
X25 
X 
 
 
 
 
 
X 
 
 
X 
X 
Serum 
osteocalcin-N-mid 
and CTX26 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
25-hydroxy Vitamin 
D2 and D3 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Randomization27 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Study Drug 
Administration 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X28 
 
Dispense Study Drug29 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
PK blood sampling30 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
PD Assessments30 
 
 
 
 
 
 
 
 
 
 
 
 
 
2,3-DPG/ATP 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
Dispense/Collect 
Menstrual Cycle Diary
32 
 
 
X 
 
 
 
X 
 
X 
X 
X 
X 
X 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up1 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-42 to -1 
-40 to -12 
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Adverse Events33 
 
 
Continuous 
X 
Transfusion Record34 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
Medications/Procedures 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Rollover to Extension 
Period35 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Abbreviations:  Ab = antibody; ATP = adenosine triphosphate; BP = blood pressure; CBC= complete blood count; COHb = carboxyhemoglobin; 
CTX = C-terminal telopeptide; D = day; DPG = diphosphoglycerate; DXA = Dual-energy x-ray absorptiometry; ECG = electrocardiogram; 
EPO = erythropoietin; ETCO = end tidal carbon monoxide; FSH = follicle stimulating hormone; G6PD = glucose-6-phosphate-dehydrogenase; HBsAg = 
hepatitis B surface antigen; HCV Ab = hepatitis C virus antibody; HDL-C = high-density lipoprotein-cholesterol; HIV = human immunodeficiency virus; HR 
= heart rate; PD = pharmacodynamic; PK = pharmacokinetic; PK deficiency = pyruvate kinase deficiency; PKR = pyruvate kinase isoform R; RR = resting 
rate; W = week. 
Whenever more than one assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive 
assessment (e.g., VS, ECG, blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time. The order of 
procedures may be revised with prior discussion between Sponsor and site. 
 
1 The Week 28 Follow-up Visit will only be conducted for patients who do not enter the Extension Period. 
2 To be performed at least 2 days after the first Screening Visit. 
3 In-clinic visits on Day 8 and Day 22 may be performed by the patient’s primary care physician if necessary and must be approved by the Sponsor on a case by 
case basis; in these instances PK/PD sampling would not be required and dispensing of study medication would not be performed. For the Day 8 visit 
performed by the patient’s primary care physician, the primary care medical office will collect a blood sample for hematology using the blood sample 
collection and shipping supplies from the kit prepared by the designated central laboratory. The kit will be sent to the primary care physician’s office. No 
other testing or procedures will be asked of the primary care physician on Day 8 [VS and serum chemistry will not be required]. The 12-lead ECG scheduled 
for Day 8 will instead be performed at the main study center on Day 15. For the Day 22 visit performed by the patient’s primary care physician, the primary 
care medical office will collect a blood sample for hematology using the blood sample collection and shipping supplies from the kit prepared by the 
designated central laboratory. The kit will be sent to the primary care physician’s office. No other testing or procedures will be asked of the primary care 
physician on Day 22. [VS, serum chemistry, coagulation, haptoglobin, EPO level, carboxyhemoglobin, and PK/PD samples will not be required.] The 12-lead 
electrocardiogram scheduled for Day 22 will instead be performed at the main study center on Day 43 (Week 6). For patients having their Day 8 and/or 
Day 22 visits performed by the primary care physician, the Principal Investigator will him or herself, or have a qualified research nurse or other designated 
91 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
site staff member, make telephone contact with the patient to inquire about any adverse events. These must be recorded as if the patient appeared in the main 
study center. The Principal Investigator will exercise prudent clinical judgment in determining any clinical course of action to take based on any adverse 
events discovered. These telephone contacts on Day 8 and Day 22 must be explained to the patient in advance and scheduled in advance to maximize the 
likelihood of successfully making contact. 
4 May be performed either by a designated central laboratory or any participating investigative site’s local hematology laboratory. 
5 Medical history, including all relevant prior medical history, current medical conditions, and hematology profile (CBCs) over prior 6 months, will be obtained 
at the Screening assessment. 
6 A complete physical examination (including neurological examination; genital and rectal examinations will be performed at the discretion of the Investigator) 
will be obtained at Screening, Baseline, Week 12, and the Follow up Visit (Week 28), or Week 24 for patients rolling over to the Extension Period. The 
neurological examination must include an assessment of general orientation and mental status including level of alertness (assess as normal or abnormal and 
specify any abnormality). Limited focused physical examinations will be performed at all other specified visits. Height to be collected at Screening only. 
7 Vital signs will include systolic and diastolic blood pressure, heart rate, respiratory rate, and temperature.  
8 12-lead ECGs are to be conducted after 5 minutes of recumbency.  
9 If a DXA scan of the hip and spine with T and Z scores has been performed within 3 months preceding the first day of dosing, this may be used to meet this 
requirement.  
10 Week 24 DXA scan may be performed anytime between Weeks 24 and 28 and must be performed at the same imaging center on the same DXA machine as 
the original Screening DXA scan. 
11 Laboratory evaluations (hematology, serum chemistry, coagulation studies, and urinalysis) are to be collected in the morning. These should be collected 
following an overnight fast on Baseline Day 1 Week 6 (Day 43), Week 12 (Day 85), Week 24 (Day 169), and Follow-up Week 28 (Day 197), when the lipid 
samples are also included. 
12 Three Screening/Baseline samples will be collected for complete blood count (CBC). Samples will be collected in the AM on 3 different days; the samples 
collected on Baseline/Day 1 may comprise one of these; samples may be taken at the same time as Screening/Baseline hormone assessments (any 2 time 
points during Screening at least 2 days apart). CBC will include HCT, Hb, red blood cell (RBC) count, absolute reticulocyte count, percent reticulocyte count, 
mean corpuscular volume (MCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW), white 
blood cell (WBC) count with automated (or manual, as indicated) differential, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and 
platelet count. In the event that the designated central laboratory for the study is unable to provide a valid result for any specific component of the defined 
CBC for a specific patient, the site may be asked to have the test performed at their local laboratory. The result of the local CBC will be entered into the study 
database along with the local normal reference range. 
13 The second Screening hematology (CBC) should be drawn in the morning (does not have to be fasting), and may be drawn at the same time the patient returns 
for the second estradiol and free and total testosterone sample. 
14 Haptoglobin will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28. 
15 Erythropoietin (EPO) levels will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 
28. 
16 Alkaline phosphatase, sodium, potassium, chloride, calcium, magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, albumin, total protein, glucose, 
blood urea nitrogen (BUN), creatinine, uric acid, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total 
bilirubin and indirect bilirubin, (and estimated creatinine clearance or glomerular filtration rate for screening only, as appropriate). 
17 Iron, total iron-binding capacity (TIBC), transferrin saturation, and ferritin will be performed prior to dosing on Day 1, at the end of Week 12 and at the end 
of Week 24. 
18 To be collected before the AG-348 morning dose is administered. 
92 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
19 End tidal carbon monoxide (ETCO) assessment by CoSense® End Tidal Carbon Monoxide Monitor to be performed only at a subset of investigative sites that 
elect to participate in this assessment and only during the Core Period. The screening ETCO may be performed at either Screening 1 or Screening 2. ETCO 
measurement should performed before administration of AG-348 on Baseline/Day 1 and on W2, W3, W6, W9 W12, W16, W20, W24, and W28 (W28 only if 
not going into Extension Period). 
20 Fibrinogen, activated partial thromboplastin time (aPTT), and international normalized ratio (INR) will be performed at Screening, prior to dosing on Day 1, 
at the end of Week 2, the end of Week 12, the end of Week 24, and the end of Week 28. 
21 Color, appearance, pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite, urobilinogen, and occult blood. Microscopic inspection of sediment 
should only be performed for cause or to investigate an abnormal dipstick finding per the Investigator’s discretion. Urinalysis will be performed at Screening, 
prior to dosing on Day 1, at the end of Week 2, the end of Week 12, the end of Week 24, and the end of Week 28. 
22 Must be repeated at any point throughout the study period if pregnancy is clinically suspected. 
23 Total cholesterol, HDL-C, and triglyceride samples will be collected in the morning following an overnight fast. 
24 Three Screening/Baseline samples will be collected for estradiol and testosterone (total and free). Samples will be collected in the AM on 3 different days; the 
samples collected on Baseline/Day 1 may comprise one of these; the Screening samples may be collected at any 2 time points during Screening at least 2 days 
apart. Serum estrone, estradiol, and free and total testosterone will then follow the schedule indicated on Day 1 and Weeks 12, 24, and 28. FSH will only be 
performed at Screening for female patients only for confirmation of post-menopausal status. 
25 The second Screening hormone testing will consist of estradiol and testosterone (free and total) only. Samples should be drawn in the AM (does not need to 
be fasting), and may be drawn at the same time the patient returns for the second CBC sample. 
26 Serum osteocalcin-N-mid and CTX will be drawn in the AM each time, approximately between 8-10 AM, and after an overnight fast of 10-12 hours. 
27 Randomization will be performed following PKR genotyping and prior to and as close as feasible to dosing on Day 1. 
28 Study drug administration is not required on W24/D169 for patients not continuing into the Extension Period. 
29 Study drug will be dispensed on a 28-day schedule, or on an alternate schedule (< 28 days) as needed to accommodate patient visit schedule and dose 
modifications. The amount of study drug dispensed should be sufficient to provide an adequate reserve supply of AG-348 to ensure uninterrupted dosing in 
the event of an unexpected delay for the next scheduled study visit (7 extra days of dosing supply is recommended during the Core Period). 
30 For the first 10 patients treated, extensive PK/PD sampling will be conducted on Days 1 and 15 (see Appendix 15.1, Table 7 for details), followed by limited 
PK/PD sampling from Week 3 to Week 24 (see Appendix 15.1, Table 8 for details). Limited PK/PD sampling will be conducted on the remainder of patients 
treated (see Appendix 15.1, Table 8). See Section 10.6, Section 10.7, and Section 10.9 for details on blood sampling for PK and PD assessments, respectively, 
and guidelines on sample processing and storage. 
 
 
32 Menstruating female patients will record their menstrual cycles (start, stop, characteristics) monthly. Paper-based menstrual cycle diaries will be dispensed at 
study visits approximately every month. The previous month’s diary will be collected at these visits as well. 
33 All randomized patients will be evaluated for AEs from the time they sign informed consent until they complete all scheduled study follow-up visits or 
withdraw consent, whichever occurs first.  
34 All transfusions must be recorded in the eCRF 
35 Patient must have completed 24 weeks of treatment and tolerated AG-348 (may have had dose modifications). Investigator and Medical Monitor or 
Responsible Medical Officer must agree with the patient continuing on treatment and patient must sign a separate ICF for the Extension Period. 
93 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Table 6: 
Schedule of Assessments: Extension Period 
Visit 
Month 9 
Month 12 
Month 15 
Month 18 
Month 21 
Month 24 
Month 27 
Month 30 
Follow-up 
Approximate Study Day 
259 
349 
439 
529 
619 
709 
799 
889 
919 
Visit Window 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
Physical Examination/Weight1 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Performance Status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs  
(BP, HR, RR, T)2 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG3 
 
X 
 
X 
 
X 
 
X 
X 
DXA Scan 
 
 
 
X 
 
 
 
X 
 
Laboratory Evaluations4 
 
 
 
 
 
 
 
 
 
Hematology (CBC)5 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin 
 
X 
 
X 
 
X 
 
X 
X 
EPO levels6 
 
X 
 
X 
 
X 
 
X 
X 
Hepcidin 
 
X 
 
X 
 
X 
 
X 
 
Serum Chemistry7 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Iron Panel8 
 
X 
 
X 
 
X 
 
X 
 
Carboxyhemoglobin (COHb)
9 
 
X 
 
X 
 
X 
 
X 
 
Coagulation Studies10 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urinalysis11 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum or Urine Pregnancy12 
 
 
 
 
 
 
 
 
 
Lipids13 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Hormonal Testing14 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum osteocalcin-N-mid and 
CTX15 
 
 
 
X 
 
 
 
X 
 
94 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Visit 
Month 9 
Month 12 
Month 15 
Month 18 
Month 21 
Month 24 
Month 27 
Month 30 
Follow-up 
Approximate Study Day 
259 
349 
439 
529 
619 
709 
799 
889 
919 
Visit Window 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
Study Drug Administration 
X 
X 
X 
X 
X 
X 
X 
X 
 
Dispense Study Drug16 
X 
X 
X 
X 
X 
X 
X 
 
 
PK blood sampling17 
X 
X 
X 
X 
X 
X 
X 
X 
 
PD Assessments18 
(2,3-DPG/ATP, 
) 
X 
X 
X 
X 
X 
X 
X 
X 
 
Dispense/Collect Menstrual 
Cycle Diary19 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Events20 
Continuous 
X 
Transfusion Record21 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
Medications/Procedures 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; BP = blood pressure; CBC= complete blood count; COHb = carboxyhemoglobin; CTX = C-terminal telopeptide; 
DPG = diphosphoglycerate; DXA = Dual-energy x-ray absorptiometry; ECG = electrocardiogram; EPO = erythropoietin; HDL-C = high-density lipoprotein-
cholesterol; HIV = human immunodeficiency virus; HR = heart rate; PD = pharmacodynamic; PK = pharmacokinetic; PK deficiency = pyruvate kinase 
deficiency; PKR = pyruvate kinase isoform R; RR = resting rate; W = week. 
Whenever more than one assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive 
assessment (e.g., VS, ECG, blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time. The order of 
procedures may be revised with prior discussion between Sponsor and site. 
 
1 A focused physical examination (including neurological examination; genital and rectal examinations will be performed at the discretion of the Investigator) 
will be obtained every 3 months; a complete physical examination will be performed at the Month 30 visit. The neurological examination must include an 
assessment of general orientation and mental status including level of alertness (assess as normal or abnormal and specify any abnormality). 
2 Vital signs will include systolic and diastolic blood pressure, heart rate, respiratory rate, and temperature 
3 12-lead ECGs are to be conducted after 5 minutes of recumbency.  
4 Laboratory evaluations (hematology, serum chemistry, coagulation studies, and urinalysis) are to be collected in the morning. These should be collected 
following an overnight fast. 
5 CBC will include HCT, Hb, red blood cell (RBC) count, absolute reticulocyte count, percent reticulocyte count, mean corpuscular volume (MCV), mean 
corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW), white blood cell (WBC) count with automated (or 
manual, as indicated) differential, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and platelet count. In the event that the designated 
central laboratory for the study is unable to provide a valid result for any specific component of the defined CBC for a specific patient, the site may be asked 
95 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
to have the test performed at their local laboratory. The result of the local CBC will be entered into the study database along with the local normal reference 
range. 
6 Erythropoietin (EPO) levels will be performed prior to dosing at Month 12, Month 18, Month 24, and Month 30. 
7 Alkaline phosphatase, sodium, potassium, chloride, calcium, magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, albumin, total protein, glucose, 
blood urea nitrogen (BUN), creatinine, uric acid, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total 
bilirubin and indirect bilirubin. 
8 Iron, total iron-binding capacity (TIBC), transferrin saturation, and ferritin will be performed prior to dosing at Month 12, Month 18, Month 24, and 
Month 30. 
9 To be collected before the AG-348 morning dose is administered. 
10 Fibrinogen, activated partial thromboplastin time (aPTT), and international normalized ratio (INR) will be performed at each study visit. 
11 Color, appearance, pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite, urobilinogen, and occult blood. Microscopic inspection of sediment 
should only be performed for cause or to investigate an abnormal dipstick finding per the Investigator’s discretion. Urinalysis will be performed prior to 
dosing at each study visit. 
12 Must be obtained at any point throughout the study period if pregnancy is clinically suspected. Effective contraception must be continued throughout the 
Extension Period in all patients and any pregnancies must be reported (see Section 11.3). 
13 Total cholesterol, HDL-C, and triglyceride samples will be collected in the morning following an overnight fast. 
14 Serum estrone, estradiol, and free and total testosterone.  
15 Serum osteocalcin-N-mid and CTX will be drawn in the AM each time, approximately between 8-10 AM, and after an overnight fast of 10-12 hours. 
16 Study drug will be dispensed on a 3-month schedule, or on an alternate schedule (< 3 months) as needed to accommodate patient visit schedule and dose 
modifications. The amount of study drug dispensed should be sufficient to provide an adequate reserve supply of AG-348 to ensure uninterrupted dosing in 
the event of an unexpected delay for the next scheduled study visit (14 extra days of dosing supply is recommended during the Extension Period). 
17 Predose; PK sampling will only include AG-348 and AGI-8702 concentrations. 
18 Predose. 
19 Menstruating female patients will record their menstrual cycles (start, stop, characteristics) monthly. Paper-based menstrual cycle diaries will be dispensed 
and collected at each study visit. 
20 All randomized patients will be evaluated for AEs from the time they sign informed consent until they complete all scheduled study follow-up visits or 
withdraw consent, whichever occurs first.  
21 All transfusions must be recorded in the eCRF. 
96 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Table 7: 
Schedule of Assessments: Extensive PK/PD Sampling during the Core Period 
Sample Timing/Interval 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Visit 
Baseline / D1 
W2 / D15 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
Study Day 
1/15 
22 
43 
64 
85 
113 
141 
169 
Visit Window 
± 2 D (D15) 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Timing 
Pre-
dose1 
30 
min2 
1 hr2 
2 hr2 
4 hr3 
8 hr3 
12 
hr3 
Pre-
dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
PK blood sample 
X 
X 
X 
X 
X 
X 
X4 
X 
X 
X 
X 
X 
X 
X 
2,3 DPG/ATP 
X 
X 
X 
X 
X 
X 
X4 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; D = day; DPG = diphosphoglycerate; PD = pharmacodynamic; PK = pharmacokinetic; PKR = pyruvate kinase 
isoform R; W = week. 
1 The acceptable time window will be within 60 minutes prior to study treatment dose administration for the pre-dose PK/PD sample. Study drug administration 
is not required on W24/D169 for patients not continuing into the Extension Period. 
2 The acceptable time window will be within ± 5 minutes of the scheduled collection time for the 30 minute, 1 and 2 hour PK/PD samples. 
3 The acceptable time window will be within ± 30 minutes of the scheduled collection time for the 4, 8, and 12 hour PK/PD samples. 
4 To be collected on Day 1 only. 
 
  
6 If the 12 hour time point cannot be collected at site on Day 1, an 8 hour time point may be collected instead. 
 
 
97 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Table 8: 
Schedule of Assessments: Limited PK/PD Sampling during the Core Period 
Sample Timing/Interval 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Visit 
Baseline / 
D1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
Study Day 
1 
15 
22 
43 
64 
85 
113 
141 
169 
Visit Window 
- 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Timing 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
PK blood sample 
X 
X 
X 
X 
X 
X 
X 
X 
X 
2,3 DPG/ATP 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; D = day; DPG = diphosphoglycerate; PD = pharmacodynamic; PK = pharmacokinetic; PKR = pyruvate kinase 
isoform R; W = week. 
 
1 The predose blood sample for plasma PK/PD analysis should be collected within 60 minutes prior to study treatment dose administration. Study drug 
administration is not required on W24/D169 for patients not continuing into the Extension Period. 
 
 
 
98 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
15.2. 
Eastern Cooperative Oncology Group Performance Status Scoring 
 
Grade 
Symptomatology 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a 
light or sedentary nature, e.g., light house work, office work 
2 
Ambulatory and capable of all self-care but unable to carry out any work activities. Up and 
about more than 50% of waking hours 
3 
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours 
4 
Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair 
5 
Dead 
Source: Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, et al. Toxicity and Response 
Criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol 1982;5:649-655. 
 
15.3. 
National Cancer Institute Common Terminology Criteria for 
Adverse Events 
 
The NCI CTCAE, Version 4.03, can be accessed using the following link:   
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_8.5x11.pdf 
99 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
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15.4. 
Potential Drug Interactions 
Strong inhibitors of CYP3A4 (listed in Table 9) are not permitted for use with AG-348. Based on 
modeling of AG-348 metabolism and tolerability data from the Phase 1 MAD study, it is thought 
that moderate inhibitors of CYP3A4 do not pose a risk to patients dosed with AG-348. 
In-vivo data from the Phase 1 MAD show AG-348 induces CYP3A4 and induces its own 
metabolism, presumably via CYP3A4. The use of strong CYP3A4 inducers in combination with 
AG-348 is expected to reduce AG-348’s efficacy. Therefore, administration of strong CYP3A4 
inducers (listed in Table 10) with AG-348 is not permitted. Strong inhibitors of drug transport 
(listed in Table 11) are not permitted for use with AG-348. 
Digoxin is not permitted for use with AG-348. 
Induction of CYP3A4 by AG-348 is expected to reduce the efficacy of certain sensitive co-
medications. Co-administration of AG-348 with the drugs in Table 12 should be done with 
caution, as their efficacy may be reduced.  
Of note, in accordance with Inclusion Criteria 14, women in the trial utilizing oral contraception 
must utilize barrier methods while taking AG-348. 
The expected patient co-medications deferoxamine, deferasirox, deferiprone, and oral penicillin 
are not expected to interact with AG-348. 
Table 9: 
Strong and Moderate CYP3A4 Inhibitors 
Strong CYP3A4 Inhibitors: Contraindicated 
Moderate CYP3A4 Inhibitors: No Action 
Indinavir 
Aprepitant 
Nelfinavir 
Erythromycin1 
Ritonavir 
Fluconazole 
Clarithromycin 
Verapamil1 
Itraconazole 
Diltiazem1 
Ketoconazole 
 
Nefazodone 
 
Saquinavir 
 
Suboxone 
 
Telithromycin 
 
Grapefruit juice2 
 
Strong Inhibitor; > 5 fold increase in AUC 
Moderate Inhibitor; > 2 fold, < 5 fold increase in AUC 
1 Erythromycin, verapamil and diltiazem are contraindicated because they are strong P-gp inhibitors (see Table 11) 
2 Although classified as a moderate CYP3A4 inhibitor, grapefruit and grapefruit juice are prohibited 
 
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Protocol AG348-C-003, Amendment 4, Version 5.0 
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Table 10: 
Strong CYP3A4 Inducers 
Strong CYP3A4 Inducers:  Contraindicated 
Efavirenz 
Phenytoin 
Nevirapine 
Pioglitazone 
Carbamazepine 
Rifabutin 
Modafinil 
Rifampin 
Oxcarbazepine 
St. John's Wort 
Phenobarbital 
Troglitazone 
Note: Corticosteroids may induce CYP3A4. Although the use of corticosteroids is not prohibited, their use should be 
minimized as much as medically feasible. 
 
Table 11: 
Strong P-glycoprotein Inhibitors 
Strong P-gp Inhibitors:  Contraindicated 
Amiodarone 
Felodipine 
Azithromycin 
Itraconazole 
Captopril 
Ketoconazole 
Carvedilol 
Lopinavir 
Clarithromycin 
Ritonavir 
Conivaptan 
Quercetin 
Cyclosporine 
Quinidine 
Diltiazem 
Ranolazine 
Dronedarone 
Ticagrelor 
Erythromycin 
Verapamil 
 
101 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
Table 12: 
Sensitive CYP3A4 Substrates 
Sensitive CYP3A4 Substrates:  Substitute or Use with Caution 
 
Antihistamines: 
Miscellaneous: 
 
 
 
Chlorpheniramine 
Alfentanil 
Finasteride 
Salmeterol 
 
 
Aprepitant 
Gleevec 
Sildenafil 
Benzodiazepines: 
Calcium Channel 
Blockers: 
Aripiprazole 
Haloperidol 
Sirolimus 
Alprazolam 
Amlodipine 
Boceprevir 
Irinotecan 
Sorafenib 
Diazepam→3OH 
Lercanidipine 
Buspirone 
LAAM 
Sunitinib 
Midazolam 
Nifedipine 
Cafergot 
Lidocaine 
Tamoxifen 
Triazolam 
Nisoldipine 
Caffeine→TMU 
Methadone 
Taxol 
Nitrendipine 
Cilostazol 
Nateglinide 
Telaprevir 
Immune Modulators: 
 
Cocaine 
Nevirapine 
Terfenadine 
Tacrolimus (FK506) 
HMG CoA 
Reductase 
Inhibitors: 
Codeine-N-
demethylation 
Ondansetron 
Torisel 
 
Atorvastatin 
Dapsone 
Pimozide 
Trazodone 
Steroid 6beta-OH: 
Cerivastatin  
Dextromethorphan 
Propranolol 
Vemurafenib 
Estradiol 
Lovastatin 
Docetaxel 
Quetiapine 
Vincristine 
Hydrocortisone (and 
other glucocorticoids) 
Simvastatin 
Domperidone 
Quinine 
Zaleplon 
Progesterone 
 
Eplerenone 
Risperidone 
Ziprasidone 
Testosterone 
 
Fentanyl 
Romidepsin 
Zolpidem 
102 
 


Protocol AG348-C-003, Amendment 4, Version 5.0 
30 March 2016 
 
15.5. 
Declaration of Helsinki 
WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI: 
Recommendations Guiding Medical Doctors in Biomedical Research Involving Human Subjects 
Adopted by the 18th World Medical Association (WMA) General Assembly, Helsinki, Finland, 
June 1964 and amended by the 29th WMA General Assembly, Tokyo, Japan, October 1975, 35th 
WMA General Assembly, Venice, Italy, October 1983, and the 41st WMA General Assembly, 
Hong Kong, September 1989, the 48th WMA General Assembly, Somerset West, Republic of 
South Africa, October 1996; 52nd WMA General Assembly, Edinburgh, Scotland, October 2000; 
53rd WMA General Assembly, Washington 2002 (Note of Clarification on paragraph 29 added); 
55th WMA General Assembly, Tokyo 2004 (Note of Clarification on Paragraph 30 added); and 
59th WMA General Assembly, Seoul, October 2008. 
A. INTRODUCTION  
1. The World Medical Association (WMA) has developed the Declaration of Helsinki as a 
statement of ethical principles for medical research involving human subjects, including 
research on identifiable human material and data.  
The Declaration is intended to be read as a whole and each of its constituent paragraphs 
should not be applied without consideration of all other relevant paragraphs.  
2. Although the Declaration is addressed primarily to physicians, the WMA encourages 
other participants in medical research involving human subjects to adopt these principles.  
3. It is the duty of the physician to promote and safeguard the health of subjects, including 
those who are involved in medical research. The physician's knowledge and conscience 
are dedicated to the fulfilment of this duty.  
4. The Declaration of Geneva of the WMA binds the physician with the words, "The health 
of my subject will be my first consideration," and the International Code of Medical 
Ethics declares that, "A physician shall act in the subject's best interest when providing 
medical care."  
5. Medical progress is based on research that ultimately must include studies involving 
human subjects. Populations that are underrepresented in medical research should be 
provided appropriate access to participation in research.  
6. In medical research involving human subjects, the well-being of the individual research 
subject must take precedence over all other interests.  
7. The primary purpose of medical research involving human subjects is to understand the 
causes, development and effects of diseases and improve preventive, diagnostic and 
therapeutic interventions (methods, procedures and treatments). Even the best current 
interventions must be evaluated continually through research for their safety, 
effectiveness, efficiency, accessibility and quality.  
8. In medical practice and in medical research, most interventions involve risks and 
burdens.  
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9. Medical research is subject to ethical standards that promote respect for all human 
subjects and protect their health and rights. Some research populations are particularly 
vulnerable and need special protection. These include those who cannot give or refuse 
consent for themselves and those who may be vulnerable to coercion or undue influence.  
10. Physicians should consider the ethical, legal and regulatory norms and standards for 
research involving human subjects in their own countries as well as applicable 
international norms and standards. No national or international ethical, legal or regulatory 
requirement should reduce or eliminate any of the protections for research subjects set 
forth in this Declaration.  
B. BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH  
11. It is the duty of physicians who participate in medical research to protect the life, health, 
dignity, integrity, right to self-determination, privacy, and confidentiality of personal 
information of research subjects.  
12. Medical research involving human subjects must conform to generally accepted scientific 
principles, be based on a thorough knowledge of the scientific literature, other relevant 
sources of information, and adequate laboratory and, as appropriate, animal 
experimentation. The welfare of animals used for research must be respected.  
13. Appropriate caution must be exercised in the conduct of medical research that may harm 
the environment.  
14. The design and performance of each research study involving human subjects must be 
clearly described in a research protocol. The protocol should contain a statement of the 
ethical considerations involved and should indicate how the principles in this Declaration 
have been addressed. The protocol should include information regarding funding, 
Sponsors, institutional affiliations, other potential conflicts of interest, incentives for 
subjects and provisions for treating and/or compensating subjects who are harmed as a 
consequence of participation in the research study. The protocol should describe 
arrangements for post-study access by study subjects to interventions identified as 
beneficial in the study or access to other appropriate care or benefits.  
15. The research protocol must be submitted for consideration, comment, guidance and 
approval to a research ethics committee before the study begins. This committee must be 
independent of the researcher, the Sponsor and any other undue influence. It must take 
into consideration the laws and regulations of the country or countries in which the 
research is to be performed as well as applicable international norms and standards but 
these must not be allowed to reduce or eliminate any of the protections for research 
subjects set forth in this Declaration. The committee must have the right to monitor 
ongoing studies. The researcher must provide monitoring information to the committee, 
especially information about any SAEs. No change to the protocol may be made without 
consideration and approval by the committee.  
16. Medical research involving human subjects must be conducted only by individuals with 
the appropriate scientific training and qualifications. Research on subjects or healthy 
volunteers requires the supervision of a competent and appropriately qualified physician 
or other health care professional. The responsibility for the protection of research subjects 
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must always rest with the physician or other health care professional and never the 
research subjects, even though they have given consent.  
17. Medical research involving a disadvantaged or vulnerable population or community is 
only justified if the research is responsive to the health needs and priorities of this 
population or community and if there is a reasonable likelihood that this population or 
community stands to benefit from the results of the research.  
18. Every medical research study involving human subjects must be preceded by careful 
assessment of predictable risks and burdens to the individuals and communities involved 
in the research in comparison with foreseeable benefits to them and to other individuals 
or communities affected by the condition under investigation.  
19. Every clinical trial must be registered in a publicly accessible database before recruitment 
of the first subject.  
20. Physicians may not participate in a research study involving human subjects unless they 
are confident that the risks involved have been adequately assessed and can be 
satisfactorily managed. Physicians must immediately stop a study when the risks are 
found to outweigh the potential benefits or when there is conclusive proof of positive and 
beneficial results.  
21. Medical research involving human subjects may only be conducted if the importance of 
the objective outweighs the inherent risks and burdens to the research subjects.  
22. Participation by competent individuals as subjects in medical research must be voluntary. 
Although it may be appropriate to consult family members or community leaders, no 
competent individual may be enrolled in a research study unless he or she freely agrees.  
23. Every precaution must be taken to protect the privacy of research subjects and the 
confidentiality of their personal information and to minimize the impact of the study on 
their physical, mental and social integrity.  
24. In medical research involving competent human subjects, each potential subject must be 
adequately informed of the aims, methods, sources of funding, any possible conflicts of 
interest, institutional affiliations of the researcher, the anticipated benefits and potential 
risks of the study and the discomfort it may entail, and any other relevant aspects of the 
study. The potential subject must be informed of the right to refuse to participate in the 
study or to withdraw consent to participate at any time without reprisal. Special attention 
should be given to the specific information needs of individual potential subjects as well 
as to the methods used to deliver the information. After ensuring that the potential subject 
has understood the information, the physician or another appropriately qualified 
individual must then seek the potential subject's freely-given informed consent, 
preferably in writing. If the consent cannot be expressed in writing, the non-written 
consent must be formally documented and witnessed.  
25. For medical research using identifiable human material or data, physicians must normally 
seek consent for the collection, analysis, storage and/or reuse. There may be situations 
where consent would be impossible or impractical to obtain for such research or would 
pose a threat to the validity of the research. In such situations the research may be done 
only after consideration and approval of a research ethics committee.  
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26. When seeking informed consent for participation in a research study the physician should 
be particularly cautious if the potential subject is in a dependent relationship with the 
physician or may consent under duress. In such situations the informed consent should be 
sought by an appropriately qualified individual who is completely independent of this 
relationship.  
27. For a potential research subject who is incompetent, the physician must seek informed 
consent from the legally authorized representative. These individuals must not be 
included in a research study that has no likelihood of benefit for them unless it is intended 
to promote the health of the population represented by the potential subject, the research 
cannot instead be performed with competent persons, and the research entails only 
minimal risk and minimal burden.  
28. When a potential research subject who is deemed incompetent is able to give assent to 
decisions about participation in research, the physician must seek that assent in addition 
to the consent of the legally authorized representative. The potential subject's dissent 
should be respected.  
29. Research involving subjects who are physically or mentally incapable of giving consent, 
for example, unconscious subjects, may be done only if the physical or mental condition 
that prevents giving informed consent is a necessary characteristic of the research 
population. In such circumstances the physician should seek informed consent from the 
legally authorized representative. If no such representative is available and if the research 
cannot be delayed, the study may proceed without informed consent provided that the 
specific reasons for involving subjects with a condition that renders them unable to give 
informed consent have been stated in the research protocol and the study has been 
approved by a research ethics committee. Consent to remain in the research should be 
obtained as soon as possible from the subject or a legally authorized representative.  
30. Authors, editors and publishers all have ethical obligations with regard to the publication 
of the results of research. Authors have a duty to make publicly available the results of 
their research on human subjects and are accountable for the completeness and accuracy 
of their reports. They should adhere to accepted guidelines for ethical reporting. Negative 
and inconclusive as well as positive results should be published or otherwise made 
publicly available. Sources of funding, institutional affiliations and conflicts of interest 
should be declared in the publication. Reports of research not in accordance with the 
principles of this Declaration should not be accepted for publication.  
C. ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH COMBINED WITH 
MEDICAL CARE  
31. The physician may combine medical research with medical care only to the extent that 
the research is justified by its potential preventive, diagnostic or therapeutic value and if 
the physician has good reason to believe that participation in the research study will not 
adversely affect the health of the subjects who serve as research subjects.  
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32. The benefits, risks, burdens and effectiveness of a new intervention must be tested 
against those of the best current proven intervention, except in the following 
circumstances:  
• The use of placebo, or no treatment, is acceptable in studies where no current proven 
intervention exists; or  
• Where for compelling and scientifically sound methodological reasons the use of 
placebo is necessary to determine the efficacy or safety of an intervention and the 
subjects who receive placebo or no treatment will not be subject to any risk of serious 
or irreversible harm. Extreme care must be taken to avoid abuse of this option.  
33. At the conclusion of the study, subjects entered into the study are entitled to be informed 
about the outcome of the study and to share any benefits that result from it, for example, 
access to interventions identified as beneficial in the study or to other appropriate care or 
benefits.  
34. The physician must fully inform the subject which aspects of the care are related to the 
research. The refusal of a subject to participate in a study or the subject's decision to 
withdraw from the study must never interfere with the subject-physician relationship.  
35. In the treatment of a subject, where proven interventions do not exist or have been 
ineffective, the physician, after seeking expert advice, with informed consent from the 
subject or a legally authorized representative, may use an unproven intervention if in the 
physician's judgement it offers hope of saving life, re-establishing health or alleviating 
suffering. Where possible, this intervention should be made the object of research, 
designed to evaluate its safety and efficacy. In all cases, new information should be 
recorded and, where appropriate, made publicly available. 
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1. 
TITLE PAGE 
Clinical Study Protocol AG348-C-003 
EudraCT No. 2015-000484-13 
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, 
Pharmacokinetic and Pharmacodynamic Study of AG-348 in Adult Patients 
with Pyruvate Kinase Deficiency 
Study Sponsor: 
Agios Pharmaceuticals, Inc. 
88 Sidney Street 
Cambridge, MA 02139-4169 
Phone:  617-649-8600 
Fax:  617-649-8618 
Responsible Medical Officer: 
, MD, PhD 
 
 
Agios Pharmaceuticals, Inc. 
Phone: 
 
Email:  
 
Study Medical Monitor 
, MD 
 
On behalf of Agios Pharmaceuticals, Inc. 
Mobile Phone: 
 
Office Phone: 
 
Email: 
  
Document Version (Date): 
Revised 
Version 1.0 (05 January 2015) 
Amendment 1, Protocol Version 2.0 (02 February 2015) Final 
Amendment 2, Protocol Version 3.0 (05 August 2015) Final 
Amendment 3, Protocol Version 4.0 (10 November 2015) 
Final 
This study will be conducted according to the protocol and in compliance with Good Clinical 
Practice, the ethical principles stated in the Declaration of Helsinki, and other applicable 
regulatory requirements. 
CONFIDENTIALITY NOTE: 
The information contained in this document is confidential and proprietary to Agios Pharmaceuticals, Inc. Any 
distribution, copying, or disclosure is strictly prohibited unless such disclosure is required by federal regulations or 
state law. Persons to whom the information is disclosed must know that it is confidential and that it may not be 
further disclosed by them. 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
INVESTIGATOR’S AGREEMENT 
 
I understand that all documentation provided to me by Agios 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). No changes will be made to the study protocol without the 
prior written approval of Agios and the IRB, except where necessary to eliminate an immediate 
hazard to the patient. 
I have read, understood, and agree to conduct this study as outlined in the protocol and in 
accordance with the guidelines and all applicable government regulations. 
 
 
Investigator Name (printed) 
 
Investigator Signature 
 
Date 
 
 
 
 
Investigational site or name of institution and location (printed) 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
2. 
SYNOPSIS 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Title of Study:  
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, Pharmacokinetic and 
Pharmacodynamic Study of AG-348 in Adult Patients with Pyruvate Kinase Deficiency 
Study Center(s): 
This study will be conducted at multiple study centers. 
Phase of Development: 2 
Objectives: 
Core Period 
Primary: 
• 
Evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in patients with 
pyruvate kinase deficiency (PK deficiency). 
Secondary: 
• 
Evaluate the pharmacokinetics (PK) of AG-348 and the metabolite AGI-8702. 
• 
Evaluate the pharmacodynamic (PD) response of adenosine triphosphate (ATP) and 
2,3-diphosphoglycerate (2,3-DPG) after administration of AG-348. 
• 
Evaluate indicators of clinical activity of AG-348 in patients with PK deficiency, including changes 
in hemoglobin (Hb), hematocrit (HCT), reticulocyte count, haptoglobin (Hp), carboxyhemoglobin 
(COHb), lactate dehydrogenase (LDH), total and indirect bilirubin, erythropoietin (EPO), ferritin, 
and transferrin saturation (serum iron/iron binding capacity). 
Extension Period 
Primary: 
• 
Evaluate the safety and tolerability of up to 30 months of AG-348 administration in patients with PK 
deficiency. 
Secondary: 
• 
Evaluate the PK of AG-348 and the metabolite AGI-8702. 
• 
Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348. 
• 
Evaluate indicators of clinical activity of AG-348 in patients with PK deficiency, including changes 
3 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, ferritin, and 
transferrin saturation (serum iron/iron binding capacity). 
Methodology: 
Study AG348-C-003 is a Phase 2, open label, two arm, multicenter, randomized, dose-ranging study in 
adult patients with PK deficiency; the study will be divided in to a Core Period and an Extension Period. 
During the Core Period, patients will receive multiple doses of AG-348 for up to 24 weeks; patients who 
are eligible can enter the Extension Period to receive AG-348 for up to 2 years following the end of the 
Core Period. Patients with PK deficiency confirmed by red blood cell (RBC) PK enzymatic assay 
performed at Screening will be eligible to participate in this study. At Week 25, patients who safely 
tolerate AG-348 and demonstrate clinical activity of AG-348 may be eligible to immediately roll over to 
the Extension Period for continued treatment. Patients who complete treatment at the end of the Core 
Period (24 weeks) will undergo follow-up assessment 4 weeks after the last dose of study drug. If a 
patient discontinues at any other time (including early discontinuation or discontinuation during the Core 
or Extension Period), the follow-up assessments will be conducted 4 weeks after discontinuation. 
Patients with toxicity suspected to be related to study drug will continue follow-up until the adverse 
event (AE) resolves, is declared chronic by the Investigator, or the patient is lost to follow-up.  
For the Core Period, up to 25 patients will be initially randomized on an open-label 1:1 basis to each of 
two twice-daily (BID) doses of AG-348 (up to 50 patients total; see Figure 1, Study Schema). The dose 
of Arm 1 is 300 mg of AG-348 administered orally (PO) every 12 hours (q12h, BID). The dose of Arm 2 
is 50 mg of AG-348 administered PO q12h (BID). Randomization will be stratified by PKR mutation in 
order to maintain balance as much as possible across the dose arms for the specific mutations expected 
to be most frequently enrolled. The PKR mutation stratification factor will consist of 4 levels (R510Q, 
R486W, and R479H) and all other mutations (“other”). Mutation status is defined by the presence of at 
least one of the indicated mutations; patients with more than one stratified mutation will be assigned 
based on Sponsor’s discretion.  
The doses for each arm of the Core Period have been selected from the AG348-C-001 single ascending 
dose (SAD) and AG348-C-002 multiple ascending dose (MAD) studies in healthy adult volunteers to 
represent the range of doses/exposures that were safely tolerated and resulted in maximal or near-
maximal PD effects on 2,3-DPG and ATP.  
Because PK deficiency is a rare disease with a limited eligible patient population and because the 
underlying pathophysiology and clinical phenotype of affected patients is heterogeneous due to the wide 
variety of mutations in PKR that cause the disease, it is important to focus closely on dose findings in 
this first-in-patient study. Therefore, in addition to initiating this study with 2 different doses of AG-348 
administered q12h, a Data Review Team (DRT) will be established to review study data on a regular 
basis and adapt the study design, dose and schedule of AG-348. 
The DRT will monitor safety on an on-going basis and meet at regular intervals of approximately every 
4 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
6 weeks, or ad hoc, as necessary, for as long as any patients are still in the Core Period to review AEs, 
vital signs (VS), clinical laboratory assessments (hematology, clinical chemistry, coagulation, and 
urinalysis), and electrocardiograms (ECGs). The DRT will also review available PK/PD data and 
indicators of clinical activity (e.g., changes from baseline in Hb). These DRT meetings will also include 
data review for all patients that may be under treatment in the Extension Period. If there are no patients 
still being treated in the Core Period, and the only patients being treated are those in the Extension 
Period, then the frequency of the DRT meetings will reduce to approximately every 3 months in order to 
match the frequency of patient visits (and new data collection) in the Extension Period. The DRT will be 
comprised of the study Coordinating Investigator, treating Investigators, Medical Monitor, Clinical 
Pharmacologist, Statistician, and Sponsor’s Responsible Medical Officer.  
Beginning 6 weeks after the first patient is dosed in the Core Period or ad hoc as necessary, and 
proceeding according to the schedule indicated above (approximately every 6 weeks during the Core 
Period, and approximately every 3 months during the Extension Period once all patients have completed 
the Core Period), the DRT will review cumulative safety data, available PK/PD data, and clinical 
activity data. Based on the DRT’s recurring reviews, the DRT may exercise one or more of the 
following options during the Core Period: 
• 
Continue treatment and enrollment in existing arms without change. 
• 
Add 1 new dose arm (Arm 3) to enroll up to 25 patients at a dose to be determined; the dose for 
Arm 3 may be lower or higher than Arm 1 and Arm 2 doses, but will not exceed 360 mg q12h; 
and the dose regimen may be less frequent than q12h. 
• 
Terminate or suspend enrollment to allow further review of clinical data in Arm 1 and/or Arm 2 
(and/or potential Arm 3). Enrollment in an arm could be terminated or suspended to allow 
further review, for example, for unacceptable safety/tolerability, poor PD response, or lack of 
signs of clinical activity. 
• 
Re-assign patients’ doses and schedule in a terminated arm to match the dose and schedule of 
another arm of the study. In this case, the patients in the terminated arm will remain in their 
original arm, i.e., they will not count towards the enrollment quota of the arm whose dose and 
schedule is being adopted. 
• 
Implement specific genotype restrictions to enrollment in one or more arms to ensure 
representation of patients with genotypes of greatest clinical relevance. 
The DRT may exercise one or more of the following options during the Extension Period: 
• 
Continue treatment without change; 
• 
Re-assign patients’ doses and schedule to an existing dose and schedule that has been 
determined to be safer, and/or have a better PD response, or produce signs of clinical activity; 
• 
Terminate or suspend treatment to allow further review of clinical data (eg, for unacceptable 
safety/tolerability, poor PD response, or lack of signs of clinical activity). 
The data that the DRT will review to make these decisions is expected to include, but are not necessarily 
limited to, the following:  
• 
Safety Observations: all AEs; VS, clinical laboratory (hematology, clinical chemistry, 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
coagulation, and urinalysis), and ECG;  
• 
PK and PD Observations: including changes in 2,3-DPG and ATP; 
• 
Indicators of Clinical Activity: including changes in Hb, HCT, reticulocyte count, Hp, COHb, 
LDH, EPO, total and indirect bilirubin, ferritin, and transferrin saturation.  
If a third dose arm is implemented, the dose of AG-348 selected will not exceed 360 mg BID, as this 
was the highest dose that demonstrated acceptable safety and tolerance in the 14-day multiple BID 
dosing study in healthy volunteers. The PK/PD sampling schedule in a potential third dosing arm will be 
determined by Sponsor’s discretion and may follow either the extensive or limited PK/PD sampling 
schedules as specified. 
The DRT will monitor the data in an ongoing manner as described and may make a decision to terminate 
enrollment in an arm if unacceptable AEs are observed, PD activity is limited, and/or markers of clinical 
activity indicate no effect. Unacceptable AEs are defined as ≥ Grade 3 AEs (using National Cancer 
Institute Common Terminology Criteria for Adverse Events [NCI CTCAEv4.03]) that are assessed as at 
least possibly related to AG-348, with the exception of Grade 3 headache, hot flash/flushing, nausea, 
vomiting, and/or diarrhea that is transient in duration (< 24 hours) or able to be medically managed to 
≤ Grade 2 within 24 hours. The DRT may also exercise discretion to terminate enrollment in an arm if it 
observes a frequency of Grade 2 AEs that would make it difficult to support long-term dosing. 
Patients in the Extension Period will continue on the dose they were randomized to in the Core Period, 
unless the DRT had reason to establish a different dose/schedule during the course of the Core Period 
(the DRT will not propose a dose higher than 360 mg q12h).  
Due to the potential for AG-348-mediated aromatase inhibition, dual-energy x-ray absorptiometry 
(DXA) scan (hip and spine) will be performed at Screening (if patient has not had prior DXA scan 
within 3 months of Day 1) to obtain T and Z scores. These data are intended to serve as a baseline 
measure of bone mineral density for all enrolling patients, and are deemed of particular importance for 
those who enter the longer term Extension Period after completing 24 weeks of treatment (Core Period). 
All patients will have a second DXA scan in the interval between Weeks 24 and 28 for the Core Period. 
Patients in the Extension Period will have additional DXA scans at Months 18 and 30. 
As the number of enrolling arms changes in the study (for example, from 2 to 3), the randomization 
scheme will adjust to enable balanced randomization into each actively accruing arm. Randomization 
and stratification will cease in the event that only a single arm is left enrolling. 
Depending on possible early termination of 1 or both of the initial 2 arms, or the addition of a third arm, 
the study could enroll up to a maximum of 75 patients. 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Figure 1:  Study Schema:  Core Period 
 
Abbreviations:  BID (q12h) = twice-daily (every 12 hours); DRT = data review team; PKR = pyruvate kinase red blood cell 
isoform; TBD= to be determined; w = week(s). 
 
Visit Schedule 
Screening assessments will occur within 28 days prior to the first dose of study treatment. During the 
Core Treatment Period, patients will attend visits at baseline (Day 1), weekly through Week 3 (Days 8, 
15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12), and monthly through 
Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate AG-348 of AG-348 through Week 24 
(Core Period) may be eligible to immediately enter the Extension Period for continued treatment for up 
to 2 years after completion of the Core Period upon agreement of the treating Investigator and the 
Medical Monitor or Responsible Medical Officer. Study visits for safety and clinical activity 
assessments will occur approximately every 3 months during the Extension Period. All patients will 
undergo a follow-up assessment 4 weeks after the last dose of AG-348, regardless of whether this was 
due to early discontinuation, the last dose in the Core Period for a patient who chooses not to continue in 
the Extension Period, or the last dose of the Extension Period.  
 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Dose Modifications for Safety and/or Increase in Hb Level 
The Investigator will monitor all patients for safety and tolerability. Modification of an individual 
patient’s dose of AG-348 will be based on AEs and/or observed changes in Hb level as described in 
Section 9.7.1 and Section 9.7.2. The same criteria for dose modifications will apply in the Extension 
Period as in the Core Period. 
Number of patients (planned): Up to approximately 75 patients. 
Diagnosis and main criteria for inclusion: 
Inclusion criteria: 
For entry into the Core Period, patients must meet all of the following criteria during the Screening or 
other specified period: 
1. Signed written informed consent obtained prior to performing any study procedure, including 
screening procedures.  
2. Male or female, aged 18 years and older. 
3. Known medical history of PK deficiency.  
4. All patients must have documented clinical laboratory confirmation of PK deficiency by RBC 
pyruvate kinase enzymatic assay performed at Screening by a designated central laboratory. 
Patients with prior documentation of PK deficiency by RBC enzymatic assay will have a 
reconfirmation of this result during Screening as a condition of enrollment. 
i. 
In the event that a patient’s screening pyruvate kinase enzymatic assay is negative (i.e., 
shows normal pyruvate kinase activity), the patient will be eligible for enrollment if the 
genotyping shows a mutant genotype that has been previously documented in the 
literature to be associated with PK deficiency. If the genotyping shows a previously 
undescribed mutation in the PKR gene, then the eligibility for enrollment will be 
determined on a case-by-case basis by the Coordinating Investigator and Medical 
Monitor in discussion with the Investigator. If no mutation is defined, then the patient 
will not be eligible. 
5. All patients must have genotypic characterization of the mutant PKR gene performed by the 
designated central laboratory at Screening. 
6. All patients must have genotypic characterization of the UGT1A1 gene performed by a 
designated central laboratory to document whether they may have underlying Gilbert's Disease. 
Patients with Gilbert’s Disease are eligible to enroll. 
7. Males must have Hb ≤ 12.0 g/dL; females must have Hb ≤ 11.0 g/dL.  
8. All patients must be considered transfusion independent as defined by: no greater than 3 units of 
RBCs transfused in the 12-month period up to the first day of study dosing and no transfusions 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
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Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
within 4 months of first day of study dosing. Patients who have received more transfusion 
support than described above will be evaluated for eligibility on a case-by-case basis by the 
Medical Monitor. 
9. Eligible patients may still have their spleens in place, or may have undergone prior splenectomy. 
For splenectomized patients: 
i. 
Must have undergone their procedure at least 6 months prior to Screening. 
ii. 
Must be current in their vaccinations for Pneumococcal Conjugate (PCV13), 
Pneumococcal Polysaccharide (PPSV23), Quadrivalent Meningococcal vaccine, and 
Haemophilus influenzae type b as recommended by Centers for Disease Control and 
Prevention's (CDC) Advisory Committee on Immunization Practices (ACIP) or 
immunization advisory groups in Canada and the European Union (for patients enrolled 
in Canada and the EU). [http://www.cdc.gov/vaccines/schedules/downloads/adult/adult-
schedule.pdf] [Any missing vaccinations may be administered during the Screening 
period. If the patient requires both PCV13 and PPSV23, PCV13 must be given before 
PPSV23, if possible. Administration of PPSV23 should follow PCV13 by at least 
8 weeks; it is permissible to give PCV13 during Screening followed by PPSV23 
following the initiation of AG-348 treatment.] 
10. Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2. (Appendix 15.2) 
11. Patients must be taking at least 1 mg of folic acid daily for at least 21 days prior to first dose and 
continue daily during study participation. 
12. Adequate organ function, defined as: 
i. 
Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) 
≤ 1.5 × upper limit of normal (ULN) (unless the increased AST is assessed by the 
Investigator as due to hemolysis). 
ii. 
Normal or elevated levels of serum bilirubin. In patients with serum bilirubin > ULN, 
the elevation must be attributed to hemolysis with or without Gilbert's syndrome and 
must not be choledocholithiasis, cholecystitis, biliary obstruction, or hepatocellular 
disease. 
iii. 
Serum creatinine ≤ 1.25 × ULN. If serum creatinine > 1.25 × ULN, then 24-hour 
measured or calculated (Cockcroft-Gault) glomerular filtration rate (GFR) ≥ 60 mL/min. 
iv. 
Absolute neutrophil count (ANC) > 1.0 × 109/L. 
v. 
Platelet count ≥ 100 × 109/L. 
vi. 
Activated partial thromboplastin time (aPTT) and international normalized ratio (INR) 
≤ 1.25 × ULN, unless the patient is receiving therapeutic anticoagulants. 
13. Women of childbearing potential (WOCBP) must agree to abstain from sexual intercourse or to 
use an acceptable/effective method of contraception (i.e., condom plus spermicide, condom plus 
oral contraceptive, condom plus intrauterine device [IUD], condom plus diaphragm with 
spermicide) from as soon as feasible during the Screening period until 30 days following the last 
9 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
dose of AG-348. Abstinence is an acceptable method only when this is in line with the normal 
life style of the patient, meaning that the patient plans to remain abstinent continuously 
throughout the duration of the study and for at least 30 days after the last dose of study drug. 
Periodic abstinence, e.g., calendar, sympathothermal and post-ovulation methods, and 
withdrawal are not acceptable methods of contraception. 
i. 
WOCBP include any female who has experienced menarche and who has not undergone 
successful surgical sterilization (hysterectomy, bilateral tubal ligation, and/or bilateral 
oophorectomy) or is not post-menopausal. Post-menopausal is defined as: 
ii. 
Amenorrhea ≥ 12 consecutive months without another cause, and a documented serum 
follicle stimulating hormone (FSH) level > 35 mIU/mL; 
iii. 
Amenorrhea ≥ 12 consecutive months in women ≥ 62 years old (FSH testing is not 
required). 
14. WOCBP must have a negative serum or urine pregnancy test within 72 hours before start of 
AG-348 dosing. 
15. Women must not be breastfeeding.  
16. Male patients, with the exception of those who have undergone vasectomy at least 6 months 
prior to Screening, must agree to abstain from sexual intercourse or, if sexually active, to use a 
condom with spermicide as contraception (regardless of their female partner's childbearing 
potential or their partner's use of their own contraception) from Day 1 of dosing until 30 days 
following the last dose of AG-348. Abstinence is an acceptable method only when this is in line 
with the normal life style of the patient, meaning that the patient plans to remain abstinent 
continuously throughout the duration of the study and for at least 30 days after the last dose of 
study drug. Periodic abstinence, e.g., selective timing of intercourse based on partner’s calendar, 
sympathothermal and post-ovulation methods, and withdrawal are not acceptable methods of 
contraception. 
For entry into the Extension Period, patients must meet the following criteria: 
17. Signed written informed consent obtained prior to performing any study procedure during the 
Extension Period.  
18. Patient must have completed 24 weeks of treatment during the Core Period and tolerated 
AG-348 (defined as having completed 24 weeks with or without permitted dose modifications) 
19. The patient’s treating Investigator agrees that there is a potential for clinical benefit to continued 
treatment and recommends participation in the Extension Period 
20. The Sponsor's designated Medical Monitor or Responsible Medical Officer approves the 
patient’s participation in the Extension Period  
21. As applicable, the patient must agree to continue to follow the same sexual 
10 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
abstinence/contraception rules as stated in Inclusion Criteria 13 and 16. 
Exclusion criteria: 
Patients who meet any of the following criteria at Screening or prior to dosing on Day 1 will not be 
enrolled in the Core Period: 
1. Hemoglobin level > 12.0 g/dL if male; Hb > 11.0 g/dL if female. 
2. Additional diagnosis of any other congenital or acquired blood disorder, including 
glucose-6-phosphate-dehydrogenase (G6PD) deficiency, or any other hemolytic anemia 
process except for mild allo-immunization as a consequence of transfusion therapy. 
3. Iron overload (hemosiderosis or concurrent hemochromatosis) sufficiently severe to result in a 
clinical diagnosis by the Investigator of cardiac, hepatic, or pancreatic insufficiency. 
4. Prior bone marrow or stem cell transplant. 
5. Clinically symptomatic cholelithiasis or cholecystitis. (Prior cholecystectomy is not 
exclusionary. Patients with symptomatic cholelithiasis or cholecystitis may be rescreened once 
the disorder has been treated and clinical symptoms have resolved.) 
6. Currently enrolled in another therapeutic clinical trial involving on-going therapy with any 
investigational or marketed product or placebo. Concurrent participation in the Pyruvate Kinase 
Deficiency Natural History Study (NCT02053480) is permitted. 
7. Exposure to any investigational drug, device, or procedure within 28 days prior to Screening. 
8. Concurrent medical condition that could compromise participation in the study such as: 
a. Poorly controlled hypertension (defined as systolic blood pressure (BP) > 150 mm Hg 
or diastolic BP > 90 mm Hg) refractory to medical management. 
b. History of recent (within < 6 months from Screening date) congestive heart failure; 
myocardial infarction or unstable angina pectoris; or hemorrhagic, embolic, or 
thrombotic stroke; deep venous thrombosis; or pulmonary or arterial embolism.  
c. Currently active infection requiring the use of parenteral anti-microbial agents or that is 
greater than Grade 3 (CTCAEv4.03) within 6 months of first dose. 
d. A pattern or frequency of post-splenectomy sepsis that in the assessment of the 
Investigator could reasonably be expected to interfere with the ability of the patient to 
complete the 24 week study participation. 
e. Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) 
antibody with signs of active Hepatitis B or C virus infection.  
11 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
f. Positive test for human immunodeficiency virus (HIV) 1 or 2 antibody. 
g. Diabetes mellitus judged to be in poor control by the Investigator or requiring > 3 anti-
diabetic agents counting insulin (all insulins are considered one agent); use of insulin 
per se is not exclusionary.  
h. History of any primary malignancy with the exception of: curatively treated non-
melanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; or 
other primary tumor treated with curative intent and no known active disease present 
and no treatment administered during the last 3 years. 
9. Undergone major surgery within 6 months of first dose.  
10. Current or recent history of psychiatric disorder that in the opinion of the Investigator or 
Medical Monitor could compromise the ability of the patient to cooperate with study visits and 
procedures. 
11. Use of any of the restricted list of products known to strongly inhibit cytochrome P450 
(CYP) 3A4 drug metabolism (Appendix 15.4, Table 9) within 5 days prior to Day 1 dosing; or 
to strongly induce CYP3A4 metabolism (Appendix 15.4, Table 10) within 28 days prior to Day 
1 dosing; or to strongly inhibit P-glycoprotein (P-gp) transporter (Appendix 15.4, Table 11) 
within 5 days prior to Day 1 dosing; or digoxin within 5 days prior to Day 1 dosing. For patients 
who require chronic inhaled glucocorticoid therapy, Investigators should confer with the 
Medical Monitor for additional guidance. 
12. Serum bilirubin > ULN attributable to factors other than hemolysis and/or Gilbert's syndrome. 
13. Male patients with heart-rate corrected QT interval -Fridericia's method (QTcF) interval 
> 450 msec, or female patients with QTcF interval > 470 msec with the exception of patients 
with a left bundle branch block (LBBB). Medical Monitor approval needed in patients with a 
LBBB. 
14. Cardiac dysrhythmias judged as clinically significant by the Investigator or requiring therapy 
with drugs that are primarily substrates of CYP3A4. 
15. History of allergy to sulfonamides if characterized by acute hemolytic anemia, anaphylaxis, or 
rash of erythema multiforme type or Stevens-Johnson syndrome. 
16. Any other medical or psychological condition deemed by the Investigator to be likely to 
interfere with a patient's ability to understand and sign informed consent; cooperate with study 
visits, tests, and procedures; or otherwise safely and reliably participate in the study. 
Patients will not be permitted to enter the Extension Period if: 
17. The patient experienced AEs during the Core Period that are considered by the treating 
12 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Investigator or the Sponsor’s designated Medical Monitor or Responsible Medical Officer to 
pose a significant safety risk to the patient if treatment were to be extended. 
Investigational product, dosage, and mode of administration: 
AG-348 sulfate hydrate capsules will be provided as 5 mg, 25 mg, or 100 mg (free-base equivalent) of 
AG-348 sulfate hydrate without excipients in dark green opaque (5 mg), Swedish orange (25 mg), or 
white opaque (100 mg) gelatin capsules (size 2 capsules for all dose strengths). AG-348 will be 
administered PO BID. The number of capsules per dose will vary by assigned dose group. AG-348 will 
be administered with water and may be administered with or without food. 
Reference therapy, dosage and mode of administration:  
Not applicable. 
Duration of treatment:  
The duration of treatment for all patients in the Core Period will be up to 24 weeks. Patients who safely 
tolerate AG-348 and for whom the Investigator agrees with continuation of AG-348 treatment may be 
eligible to immediately roll over to the Extension Period for continued treatment (up to 2 years following 
completion of the Core Period). 
Criteria for evaluation: 
Safety: 
Monitoring of AEs in randomized patients, including determination of serious AEs (SAEs) and AEs 
leading to discontinuation; safety laboratory parameters (hematology, chemistry, urinalysis, 
coagulation); physical examination findings (including neurological examination); VS; 12-lead ECGs, 
and DXA scans. Adverse events will be graded using CTCAE, Version 4.03. Serum sex hormone levels 
(testosterone [total and free], estrone, and estradiol), bone turnover markers (serum osteocalcin-N-mid 
and serum C-terminal telopeptide [CTX]), 25-hydroxy vitamin D2 and D3, total cholesterol, high-
density lipoprotein-cholesterol (HDL-C), and triglycerides will be monitored for evidence of potential 
inhibition of aromatase by AG-348. Menstruating female patients will also keep a paper-based menstrual 
cycle diary throughout the study. 
Indicators of Clinical Activity: 
Monitoring of potential indicators of clinical activity will include evaluating changes in Hb, HCT, 
reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, ferritin, and transferrin saturation.  
Pharmacokinetics: 
Approximately the first 10 patients treated in the Core Period, contingent on clinical site feasibility, will 
undergo extensive PK sampling as detailed in Appendix 15.1, Table 7. The remainder of treated patients 
will undergo limited PK sampling as detailed in Appendix 15.1, Table 8. During the Core Period, serial 
blood sampling for determination of concentration-time profiles of AG-348 and its metabolite AGI-8702 
will be conducted following the first dose and the morning Day 15 dose, and additional trough levels of 
AG-348 and AGI-8702 will be obtained. During the Extension Period, predose PK samples will be 
drawn for the measurement of trough levels of AG-348 and AGI-8702 at each study visit (every 
3 months; see Appendix 15.1, Table 6). AG-348 and AGI-8702 will be analyzed using qualified assays 
13 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
to determine concentrations in plasma. Pharmacokinetic parameters on Day 1 and Day 15 will be 
computed using standard non-compartmental methods based on observed plasma AG-348 and AGI-8702 
concentrations. 
Pharmacodynamics: 
Pharmacodynamic assessments during the Core Period will include 2,3-DPG, ATP (secondary 
objectives), 
 
 
Approximately the first 10 patients treated during the Core Period will undergo extensive PD sampling 
as detailed in Appendix 15.1, Table 7. The remainder of treated patients will undergo limited PD 
sampling as detailed in Appendix 15.1, Table 8. During the Core Period, serial blood sampling for 
determination of levels of ATP and, 2,3-DPG will be conducted following the first dose and the morning 
Day 15 dose, and additional trough levels of ATP and 2,3-DPG will be obtained. During the Extension 
Period, predose PD samples will be drawn for the measurement of trough levels of 2,3-DPG, ATP, 
 
 at each study visit (every 3 months; see Appendix 15.1, Table 6). Adenosine triphosphate 
and 2,3 DPG will be analyzed using qualified assays to determine concentrations in whole blood. 
Pharmacodynamic parameters on Day 1 and Day 15 will be computed based on observed whole blood 
ATP and 2,3-DPG concentrations.  
Statistical methods: 
The primary objective during the Core Period of this Phase 2 study is to evaluate the safety and 
tolerability of up to 24 weeks of AG-348 in patients with PK deficiency. Therefore, analyses will be 
primarily descriptive in nature; no formal hypothesis testing will be conducted. All analyses will be 
conducted separately within each dose arm, or pooled when appropriate Analyses of safety and of 
indicators of clinical activity will be conducted for the Core Period, Extension Period, and overall, if 
applicable. For the Core Period, the data to be analyzed will include all collected data through 24 weeks 
of treatment for patients who directly enter the Extension period. For patients who do not enter the 
Extension Period, the analyses will include all collected data through the duration of treatment 
(24 weeks or less) plus 4 week follow up data. For patients who move directly from the Core to the 
Extension Period, the 4 week follow up data will be analyzed as part of the Extension Period. 
Summaries will be produced for disposition, baseline disease characteristics and demographic data 
including genotype, safety measurements, PK, PD parameters and indicators of clinical activity. Data 
14 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
from each AG-348 dose group will be analyzed separately, and pooled across all dose groups where 
appropriate. All data will also be listed by individual patient. Categorical variables will be summarized 
by frequency distributions (number and percentages) and continuous variables will be summarized by 
descriptive statistics (mean, standard deviation [SD], median, minimum, and maximum). 
Populations for analysis will include a Safety Analysis Set, a PK Analysis Set, and an Efficacy Analysis 
Set. The Safety Analysis set will include all patients who are enrolled and receive any dose of study 
treatment; this population will be the primary set for the analysis of safety data and the default analysis 
set for all data analyses unless otherwise stated. Patients will be classified according to treatment 
received, where treatment received is defined as the assigned treatment if it is received at least once, or 
as the first treatment received if assigned treatment is never received. The PK Analysis Set will include 
all patients in the Safety Analysis Set with sufficient plasma sample data to assess PK parameters. 
Results of the potential PD activity of AG-348 will also be based on the PK analysis set. The Efficacy 
Analysis Set will include all patients who are enrolled and achieve at least 50% compliance with their 
assigned dose intensity for at least 4 weeks of continued dosing. Additional efficacy analyses may be 
performed on subsets of patients with other degrees of compliance with their assigned dose intensity 
and/or duration of dosing. The Efficacy Analysis Set will be the primary set for the analysis of 
preliminary clinical activity data. Patients will be classified according to assigned treatment. 
 
 
 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) and the 
incidence of treatment-emergent AEs (TEAEs) (new or worsening from baseline) will be summarized by 
primary system organ class (SOC), preferred term, CTCAE Version 4.03 severity, outcome, action taken 
with study drug, and relationship to study drug by dose group. Separate summaries will be produced for 
all TEAEs, treatment-related AEs (those considered by the Investigator as at least possibly drug related), 
SAEs, discontinuations due to AEs, and AEs ≥ Grade 3 severity. Individual patient listings will be 
provided for any deaths, SAEs, AEs leading to interruption and/or reduction of study drug dose, and 
AEs leading to discontinuation of treatment. Analyses of AEs will be performed for those events that are 
considered treatment-emergent, where treatment-emergent is defined as any AE with onset beginning at 
the day of first administration of study drug, throughout the treatment period until 30 days after the last 
dose of study drug, or any event that was present at baseline but worsened in intensity or was 
subsequently considered drug-related by the Investigator through the end of the study. 
Descriptive statistics will be provided for clinical laboratory values (e.g., hematology, serum chemistry, 
coagulation studies, urinalysis) and VS data, presented as both actual values and changes from baseline 
relative to each on-study evaluation. Shift analyses will be conducted for selected laboratory parameters 
based on the baseline CTCAE grade to maximum CTCAE grade. Where applicable CTCAE terms do 
not exist, a grading system based on the upper and/or lower limits of normal will be used to classify the 
results. 
Electrocardiogram analyses will include individual patient listings and summaries of abnormal and 
clinically significant ECG results. Actual values and changes from baseline in the portion of the ECG 
wave from the beginning of the P wave to the beginning of the QRS complex (PR), QRS, heart rate-
15 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
corrected QT intervals (QTc) will be summarized by visit and dose arm.  
Data collected from the menstrual diaries such as the start and stop dates of the menses and the patient 
reported characteristics of the menses will be presented in a by-patient listing. Additional descriptions of 
the data may also be performed. 
Concomitant medications will be coded using the WHO Drug Dictionary. Concomitant medications 
prior to and after the start of the study drug will be listed by patient, and summarized by ATC term and 
dose arm. 
Descriptive statistics will be used to summarize PK parameters for the parent compound AG-348 and the 
metabolite AGI-8702 for each dose group, and where appropriate, for the entire population. 
Pharmacokinetic parameters will be summarized using the following descriptive statistics: n, mean, SD, 
coefficient of variation %, median, minimum, and maximum, geometric mean, and geometric coefficient 
of variation.  
Descriptive statistics will be used to summarize PD parameters for 2,3-DPG and ATP for each dose 
group, and where appropriate for the entire population. Pharmacodynamic parameters will be 
summarized using the following descriptive statistics: n, mean, SD, coefficient of variation %, median, 
minimum, and maximum, geometric mean, and geometric coefficient of variation %. 
Analyses of serum sex hormones will use appropriate graphic displays and statistical analyses to 
evaluate patient change from baseline for each parameter. These analyses will include summaries of 
actual values and change from baseline using appropriate descriptive statistics (mean, SD, median, min 
and max) as described above.  
Analyses evaluating indicators of potential clinical activity of AG-348 in patients with PK deficiency 
will include changes in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, 
ferritin, and transferrin saturation (serum iron/iron binding capacity). Characterization of Hb response 
(e.g., % of patients whose Hb increases by a certain amount), as well as time to Hb response, and 
duration of Hb response will be explored, among others. 
The study database will be locked and statistical analysis will be performed after all patients have 
completed the Core Period. The results of this analysis will be presented in a clinical study report (CSR). 
Additional data collected during the Extension Period after the CSR database lock will be analyzed for 
inclusion in a subsequent CSR addendum. 
Interim Review 
No formal statistical analysis will be conducted. Safety data will be reviewed on an ongoing basis by the 
DRT, who will meet to review safety, PK, PD, and preliminary clinical activity data at regular intervals 
(approximately every 6 weeks during the Core Period and approximately every 3 months during the 
Extension Period once all patients have completed the Core Period) throughout the duration of the study. 
The composition of the DRT, its meeting schedule, the data to be reviewed, and the decisions it is 
empowered to make have been described previously. 
Additional interim reviews of data may be conducted to support decision making concerning the current 
clinical study, the sponsor’s development programs in general, or in case of any safety concerns.  
 
16 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Sample Size 
Due to the rare disease setting, the minimal sample size may be determined by feasibility. Depending on 
possible early termination of 1 or both of the initial 2 arms or the addition of a 3rd dose arm, the study 
could enroll up to a maximum of 75 patients. 
17 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF 
FIGURES 
TABLE OF CONTENTS 
1. 
TITLE PAGE ................................................................................................................1 
2. 
SYNOPSIS ...................................................................................................................3 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES ...............18 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS.............................23 
5. 
INTRODUCTION ......................................................................................................27 
5.1. 
Pyruvate Kinase Deficiency .......................................................................................27 
5.2. 
AG-348 .......................................................................................................................28 
5.2.1. 
Summary of Nonclinical Data ....................................................................................28 
5.2.1.1. 
Pharmacology .............................................................................................................29 
5.2.1.2. 
Safety Pharmacology ..................................................................................................29 
5.2.1.3. 
Pharmacokinetics ........................................................................................................29 
5.2.1.4. 
Toxicology ..................................................................................................................30 
5.2.2. 
Summary of Clinical Data ..........................................................................................32 
5.2.2.1. 
Pharmacokinetics ........................................................................................................32 
5.2.2.2. 
Pharmacodynamics .....................................................................................................33 
5.2.2.3. 
Safety ..........................................................................................................................33 
5.3. 
Study Rationale ...........................................................................................................34 
5.3.1. 
Summary of Overall Safety Management Plan ..........................................................35 
6. 
TRIAL OBJECTIVES AND ENDPOINTS ...............................................................37 
6.1. 
Core Period .................................................................................................................37 
6.1.1. 
Primary Objective .......................................................................................................37 
6.1.2. 
Secondary Objectives .................................................................................................37 
 
37 
6.2. 
Extension Period .........................................................................................................37 
6.2.1. 
Primary Objective .......................................................................................................37 
6.2.2. 
Secondary Objectives .................................................................................................37 
 
38 
6.3. 
Study Measures and Endpoints ...................................................................................38 
18 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
6.3.1. 
Safety Measures and Endpoints ..................................................................................38 
6.3.2. 
Clinical Activity Measures and Endpoints .................................................................38 
6.3.3. 
Pharmacokinetic and Pharmacodynamic Measures and Endpoints ............................38 
 
39 
7. 
INVESTIGATIONAL PLAN .....................................................................................40 
7.1. 
Overall Study Design ..................................................................................................40 
7.2. 
Justification of the Study Design ................................................................................44 
7.3. 
Rationale for the Starting Dose, Dose Range, and Duration of Dosing .....................45 
7.4. 
Criteria for Study Termination ...................................................................................46 
8. 
STUDY POPULATION .............................................................................................48 
8.1. 
Number of Patients .....................................................................................................48 
8.2. 
Inclusion Criteria ........................................................................................................48 
8.3. 
Exclusion Criteria .......................................................................................................50 
8.4. 
Patient Identification and Registration .......................................................................52 
8.5. 
Patient Randomization ................................................................................................52 
8.6. 
Patient Withdrawal Criteria ........................................................................................53 
8.7. 
Replacement of Patients .............................................................................................53 
9. 
STUDY TREATMENT ..............................................................................................54 
9.1. 
Description of Study Drug ..........................................................................................54 
9.2. 
Study Drug Packaging and Labeling ..........................................................................54 
9.3. 
Study Drug Storage .....................................................................................................54 
9.4. 
Method of Assigning Patients to Treatment ...............................................................54 
9.5. 
Blinding ......................................................................................................................54 
9.6. 
Study Drug Preparation and Administration ..............................................................54 
9.7. 
Criteria for Dose Escalation, Dose Modification, or Discontinuation of Study 
Drug ............................................................................................................................55 
9.7.1. 
Dose Modification for Safety .....................................................................................56 
9.7.2. 
Dose Modification for Increase in Hemoglobin Level ...............................................56 
9.7.3. 
Stopping Criteria .........................................................................................................57 
9.8. 
Duration of Patient Participation ................................................................................57 
9.9. 
Treatment Compliance ................................................................................................57 
9.10. 
Study Drug Accountability .........................................................................................57 
9.11. 
Prior and Concomitant Medications and Treatments .................................................58 
19 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
9.11.1. 
Prior Medications and Procedures ..............................................................................58 
9.11.2. 
Prohibited Concomitant Therapy ................................................................................58 
9.11.3. 
Allowed Concomitant Therapy ...................................................................................60 
9.11.4. 
Potential for Phototoxicity ..........................................................................................60 
9.11.5. 
Management of Nausea, Vomiting, and Diarrhea ......................................................60 
9.11.6. 
Other Restrictions and Precautions .............................................................................61 
10. 
STUDY ASSESSMENTS ..........................................................................................62 
10.1. 
Schedule of Assessments ............................................................................................62 
10.2. 
Informed Consent and Confirmation of Eligibility ....................................................63 
10.3. 
Demographic Data, Medical and Medication History ................................................63 
10.4. 
PKR Enzymatic Assay and PKR Genotyping ............................................................64 
10.5. 
Safety Assessments .....................................................................................................64 
10.5.1. 
Physical Examination, Height, and Weight ................................................................64 
10.5.2. 
Vital Signs ..................................................................................................................64 
10.5.3. 
Electrocardiogram .......................................................................................................64 
10.5.4. 
DXA Scans .................................................................................................................64 
10.5.5. 
Safety Laboratory Assessments ..................................................................................65 
10.5.5.1. 
Hematology, Serum Chemistry, Coagulation Studies, and Urinalysis .......................65 
10.5.5.2. 
Screening Serology .....................................................................................................66 
10.5.6. 
Menstrual Cycle Diary ................................................................................................66 
10.5.7. 
Adverse Events ...........................................................................................................66 
10.6. 
Pharmacokinetic Assessments ....................................................................................67 
10.7. 
Pharmacodynamic Assessments .................................................................................67 
10.8. 
Ordering of Blood Sample Collection ........................................................................69 
10.9. 
Sample Processing, Storage, and Shipment ................................................................69 
11. 
ADVERSE EVENTS..................................................................................................70 
11.1. 
Definition of Adverse Events .....................................................................................70 
11.1.1. 
Adverse Event .............................................................................................................70 
11.1.2. 
Suspected Adverse Reaction .......................................................................................70 
11.1.3. 
Unexpected Adverse Event .........................................................................................70 
11.1.4. 
Serious Adverse Event ................................................................................................70 
11.1.4.1. 
Potential Severe Drug-Induced Liver Injury ..............................................................71 
11.2. 
Procedures for Reporting Adverse Events and Serious Adverse Events ....................71 
20 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
11.3. 
Pregnancy Reporting ..................................................................................................74 
12. 
STATISTICAL METHODS .......................................................................................75 
12.1. 
Sample Size Estimation ..............................................................................................75 
12.2. 
Populations for Analysis .............................................................................................75 
12.3. 
Procedures for Handling Missing, Unused, and Spurious Data .................................76 
12.4. 
Interim Analysis ..........................................................................................................76 
12.5. 
Statistical Methodology ..............................................................................................76 
12.5.1. 
General Methods .........................................................................................................76 
12.5.2. 
Disposition ..................................................................................................................77 
12.5.3. 
Exposure and Safety Analyses ....................................................................................77 
12.5.4. 
Pharmacokinetic Analyses ..........................................................................................78 
12.5.5. 
Pharmacodynamic Analyses .......................................................................................78 
12.5.6. 
Aromatase Hormone Analysis ....................................................................................78 
12.5.7. 
Clinical Activity ..........................................................................................................78 
 
79 
12.6. 
Procedures for Reporting Deviations to Original Statistical Analysis Plan ...............79 
13. 
ADMINSTRATIVE REQUIREMENTS ....................................................................80 
13.1. 
Good Clinical Practice ................................................................................................80 
13.2. 
Ethical Considerations ................................................................................................80 
13.3. 
Patient Information and Informed Consent ................................................................80 
13.4. 
Patient Confidentiality ................................................................................................80 
13.5. 
Protocol Compliance ..................................................................................................81 
13.6. 
Data Management .......................................................................................................81 
13.7. 
Source Document/Case Report Form Completion .....................................................82 
13.8. 
Direct Access to Source Data .....................................................................................82 
13.9. 
Record Retention ........................................................................................................82 
13.10. 
Liability and Insurance ...............................................................................................82 
13.11. 
Publication of Study Findings and Use of Information ..............................................83 
14. 
LIST OF REFERENCES ............................................................................................84 
15. 
APPENDICES ............................................................................................................86 
15.1. 
Schedules of Assessments ..........................................................................................87 
15.2. 
Eastern Cooperative Oncology Group Performance Status Scoring ..........................98 
15.3. 
National Cancer Institute Common Terminology Criteria for Adverse Events .........98 
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15.4. 
Potential Drug Interactions .........................................................................................99 
15.5. 
Declaration of Helsinki .............................................................................................102 
 
LIST OF TABLES 
Table 1: 
Dose Modification for Adverse Events Deemed at Least Possibly Related to 
AG-348 .......................................................................................................................56 
Table 2: 
Dose Reduction Table (by Dosing Arm) ....................................................................57 
Table 3: 
Summary of Assessments When Day 8 and/or Day 22 In-Clinic Visits Are 
Performed By Primary Care Physician (Core Period) ................................................63 
Table 4: 
Sample Size Estimation ..............................................................................................75 
Table 5: 
Schedule of Assessments: Core Period .......................................................................87 
Table 6: 
Schedule of Assessments: Extension Period ..............................................................93 
Table 7: 
Schedule of Assessments: Extensive PK/PD Sampling during the Core 
Period ..........................................................................................................................96 
Table 8: 
Schedule of Assessments: Limited PK/PD Sampling during the Core Period ...........97 
Table 9: 
Strong and Moderate CYP3A4 Inhibitors ..................................................................99 
Table 10: Strong CYP3A4 Inducers .........................................................................................100 
Table 11: Strong P-glycoprotein Inhibitors ..............................................................................100 
Table 12: Sensitive CYP3A4 Substrates...................................................................................101 
 
LIST OF FIGURES 
Figure 1:  Study Schema:  Core Period .........................................................................................7 
Figure 2: 
Red Cell Glycolysis ....................................................................................................27 
Figure 3: 
Study Schema:  Core Period .......................................................................................43 
Figure 4: 
PKR Enzymatic Reaction ...........................................................................................68 
 
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4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation or Specialist Term 
Explanation 
2,3-DPG 
2,3-diphosphoglycerate 
ACIP 
Advisory Committee on Immunization Practices 
ADP 
Adenosine diphosphate 
AE 
Adverse event 
ALC 
Absolute lymphocyte count 
ALP 
Alkaline phosphatase 
ALT 
Alanine aminotransferase 
ANC 
Absolute neutrophil count 
aPTT 
Activated partial thromboplastin time 
AST 
Aspartate aminotransferase 
ATP 
Adenosine triphosphate 
AUC 
Area under the curve 
AUC0-12hr 
Area under the plasma concentration versus time curve from 0 to 12 
hours 
AUC0 24hr 
Area under the plasma concentration versus time curve from 0 to 
24 hours 
AUC0-∞ 
Area under the plasma concentration versus time curve from 0 to 
infinity 
BCRP 
Breast cancer resistance protein 
BID 
Twice daily 
BP 
Blood pressure 
BUN 
Blood urea nitrogen 
CBC 
Complete blood count 
CDC 
Centers for Disease Control and Prevention 
Cmax 
Maximum plasma concentration 
CO2 
Carbon dioxide 
COHb 
Carboxyhemoglobin 
CSR 
Clinical study report 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTX 
Serum C-terminal telopeptide 
CV 
Cardiovascular 
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Abbreviation or Specialist Term 
Explanation 
DDI 
Drug-drug interaction 
CYP 
Cytochrome P450 
DILI 
Drug-induced liver injury 
DLT 
Dose-limiting toxicity 
DRT 
Data review team 
DXA 
Dual-energy x-ray absorptiometry 
ECG 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic case report form 
EDC 
Electronic Data Capture 
EPO 
Erythropoietin 
FDA 
Food and Drug Administration 
FSH 
Follicle stimulating hormone 
G6PD 
Glucose-6-phosphate-dehydrogenase 
GCP 
Good Clinical Practice 
GFR 
Glomerular filtration rate 
GLP 
Good Laboratory Practice 
Hb 
Hemoglobin 
HBsAg 
Hepatitis B surface antigen 
HCT 
Hematocrit 
HCV 
Hepatitis C virus 
HDL-C 
High-density lipoprotein-C 
HIV 
Human immunodeficiency virus 
Hp 
Haptoglobin 
IC50 
Concentration of drug that achieved half-maximal inhibition 
ICH 
International Conference on Harmonization 
IEC 
Independent Ethics Committee 
INR 
International normalized ratio 
IRB 
Institutional Review Board 
IUD 
Intrauterine device 
LBBB 
Left bundle branch block 
LDH 
Lactate dehydrogenase 
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Abbreviation or Specialist Term 
Explanation 
MAD 
Multiple ascending dose 
MCH 
Mean corpuscular hemoglobin 
MCHC 
Mean corpuscular hemoglobin concentration 
MCV 
Mean corpuscular volume 
MedDRA 
Medical Dictionary for Regulatory Activities 
mPKR 
PKR mutants 
MTD 
Maximum tolerated dose 
NCI 
National Cancer Institute 
NOEL 
No-observed-effect-level 
NOAEL 
No-observed-adverse-effect-level 
P-gp 
P-glycoprotein 
PCV13 
Pneumococcal Conjugate 
PD 
Pharmacodynamic 
PEP 
Phosphoenolpyruvate 
PK 
Pharmacokinetic 
PK deficiency 
Pyruvate kinase deficiency 
PKM2 
Pyruvate kinase isoform M2 
PKR 
Pyruvate kinase isoform R 
PO 
Oral(ly) 
PPSV23 
Pneumococcal polysaccharide 
PR 
The portion of the ECG wave from the beginning of the P wave to 
the beginning of the QRS complex 
q12h 
Every 12 hours 
q24h 
Every 24 hours 
QD 
Once-daily 
QTc 
Heart-rate corrected QT interval 
QTcB 
Corrected QT interval - Bazett correction formula 
QTcF 
Corrected QT interval - Fridericia’s method 
RBC 
Red blood cell (count) 
RDW 
Red cell distribution width 
SAD 
Single ascending dose 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
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Abbreviation or Specialist Term 
Explanation 
SD 
Standard deviation 
SOC 
System organ class 
t½ 
Apparent terminal half-life 
TIBC 
Total iron-binding capacity 
Tmax 
Time to maximum plasma concentration 
TEAE 
Treatment-emergent adverse event 
ULN 
Upper limit of normal 
VS 
Vital signs 
Vss 
Volume of distribution at steady-state 
Vz/F 
Mean apparent volume of distribution 
WBC 
White blood cell  
WMA 
World Medical Association 
WOCBP 
Women of childbearing potential 
WT 
Wild type 
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5. 
INTRODUCTION 
5.1. 
Pyruvate Kinase Deficiency 
Pyruvate kinase deficiency (PK deficiency) is a glycolytic enzymopathy that results in non-
spherocytic hemolytic anemia with a variable clinical presentation, ranging from mild or fully 
compensated forms to life-threatening neonatal anemia and life-long chronic hemolytic anemia 
associated with severe, debilitating co-morbidities. Pyruvate kinase deficiency is caused by 
mutations in the PKLR gene, which results in defective pyruvate kinase isoform R (PKR) in the 
red blood cell (RBC). As shown in the diagram below, PKR catalyzes the final and irreversible 
step in glycolysis, converting phosphoenolpyruvate (PEP) to pyruvate, and formation of 
adenosine triphosphate (ATP) from adenosine diphosphate (ADP). Mature RBCs rely almost 
exclusively on the process of glycolysis to generate the energy carrier molecule ATP. PKR is 
thus a key enzyme for maintaining energy homeostasis in erythrocytes, and it has been proposed 
that ATP levels are critical for optimally maintaining RBC membrane integrity (van Wijk and 
van Solinge 2005). 
Figure 2: 
Red Cell Glycolysis 
 
Abbreviations:  1,3 DPG = 1,3 diphosphoglycerate; 2,3 DPG = 2,3 diphosphoglycerate; 3-PG = 3-phosphoglycerate; 
ADP = adenosine diphosphate; ATP = adenosine triphosphate; FBP = fructose 1,6-bisphosphate; PEP = 
phosphoenolpyruvate; PKR = red blood cell-specific form of pyruvate kinase 
Note: Not all steps in glycolysis are shown. 
The majority of mutations in PKR that have been described have a deleterious effect on PKR 
catalytic activity, protein stability, and/or protein expression. Pyruvate kinase deficiency is 
associated with reduced RBC survival as well as impaired red cell maturation. Mature 
erythrocytes lack mitochondria relying predominantly on glycolysis to generate ATP. It is 
hypothesized that insufficient energy production promotes erythrocyte hemolysis due to impaired 
maintenance of cellular membrane homeostasis. 
Pyruvate kinase deficiency is an autosomal recessive disease, in which both homozygotes and 
compound heterozygotes develop hemolytic anemia. More than 200 different mutations in the 
PKLR gene have been identified in patients with PK deficiency to date, and the majority of these 
mutations are single nucleotide missense mutations (Zanella, et al. 2005). The prevalence of PK 
deficiency, based on the most common pyruvate kinase mutation in Southern Europe, is 
estimated at 51 cases per million in the Caucasian population (Beutler and Gelbart 2000). 
Some patients with PK deficiency have a clinically severe disease course that can present as a 
severe hemolytic anemia in early infancy, requiring immediate care which typically includes 
blood transfusion support (Christensen, et al. 2011; Ghidini, et al. 1991; Hennekam, et al. 1990). 
Infants with PK deficiency may have erythroblastosis fetalis with hepatosplenomegaly, 
extramedullary hematopoiesis, and severe indirect hyperbilirubinemia, which is associated with a 
Glucose
Pyruvate
PKR
`
ATP
FBP
1,3-DPG
2,3-DPG
3-PG
PEP
ADP
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high risk of kernicterus. Hyperbilirubinemia associated with the hemolytic state often requires 
supportive treatment with intensive phototherapy. For other infants, the hyperbilirubinemia 
caused by hemolysis can require exchange transfusion, as well as supportive transfusion to keep 
their hematocrit (HCT) levels >60%. 
Adults with PK deficiency are reported to have life-long hemolysis and the subsequent 
associated co-morbidities including anemia and transfusion dependence. Other co-morbidities 
include frequent miscarriages, aplastic crises, as well as symptoms associated with an acute on 
chronic hemolytic anemia (Rider, et al. 2011). Since unconjugated bilirubin is often chronically 
elevated, pigmented gallstones are common in children and adults. Severe and sometimes life-
threatening iron deposition occurs and is typically progressive. 
The current therapeutic options for patients with PK deficiency are supportive. Most require life-
long treatment, including blood transfusions at a frequency depending on the disease state. Long-
term surveillance for systemic iron overload, even in transfusion-independent patients, is 
standard as is the use of chelation therapy. Case reports of cure by allogeneic bone marrow 
transplant have been published (Tanphaichitr, et al. 2000) but have been infrequently performed. 
Splenectomy, while not curative, can reduce the need for supportive blood transfusions and 
decrease bilirubin levels in some patients. However, this intervention is associated with 
significant morbidity including infection risk, and possible increased risk of thrombosis. 
5.2. 
AG-348 
AG-348 is an orally available, allosteric activator of PKR. It is hypothesized that drug 
intervention with AG-348 restores glycolytic pathway activity and normalizes red cell 
metabolism in vivo. Biochemical experiments demonstrate that AG-348 is a potent, pan-activator 
of many PKR alleles associated with PK deficiency. Pyruvate kinsase deficiency red cells and 
their progenitors are characterized by changes in metabolism associated with defective 
glycolysis, including a build-up of PEP and 2,3-diphosphoglycerate (2,3-DPG), and lowered 
ATP levels. Treatment of PK deficiency patient red cells ex vivo with AG-348 results in 
increased ATP levels, and reductions in PEP and 2,3-DPG, consistent with pharmacological 
activation of PKR enzyme activity. In vitro studies against wild type (WT) PKR enzymes and ex 
vivo studies in blood from mice, monkeys and humans indicate that AG-348 activates WT PKR 
with a similar potency and efficacy as it does with the mutant versions. In vivo studies in mice 
confirm the potency of AG-348 in increasing WT PKR enzyme activity and modulating the 
levels of downstream markers such as ATP and 2,3-DPG. This therapeutic approach may be an 
effective way to correct the underlying pathology of PK deficiency and, importantly, provide 
clinical benefit to patients. 
5.2.1. 
Summary of Nonclinical Data 
A series of exploratory pharmacology studies were conducted to characterize the ability of 
AG-348 to activate WT PKR and anemia-associated PKR mutants (mPKR) in vitro, ex vivo, and 
in vivo. Additional details regarding nonclinical data can be found in the AG-348 Investigator’s 
Brochure 
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5.2.1.1. 
Pharmacology 
AG-348 is a potent, broad-spectrum activator of PKR with low nM potency against both WT and 
mutant enzymes. AG-348 is also able to effectively stabilize mPKR enzymes that are 
hypersensitive to thermal denaturation, suggesting that AG-348 may be able to increase PKR 
pathway activity by preventing destabilization and degradation of mPKR enzymes. Its major 
metabolite, AGI-8702, has low potency mixed activity against PKR and mutant isoforms and can 
increase the thermostability of some mPKR, albeit with significantly weaker potency than that of 
AG-348. 
The effect of AG-348 and AGI-8702 on PKR activity and a number of downstream pathway 
markers was evaluated in human RBCs. AG-348 dose-response curves in human whole blood 
showed potent activation of PKR, while AGI-8702 is a weak activator of the PKR enzyme. In 
whole blood from mice treated with AG-348, 13C-glucose labeling through the PKR reaction was 
increased by 80% demonstrating AG-348-mediated activation of the WT PKR enzyme. AG-348 
treatment of RBCs from healthy human donors potently increased levels of the PKR reaction 
product ATP consistent with activation of PKR.  
The effects of AG-348 on PKR activity and RBC metabolism also were assessed in blood 
samples from patients with PK deficiency. AG-348 activated PKR and induced metabolic 
changes (increased ATP levels and decreased 2,3-DPG levels) consistent with increased 
glycolytic pathway activity in RBCs from PK deficiency patients with different mutations in the 
PKR enzyme.  
Assessments of binding inhibition potential and enzymatic activity were evaluated in a panel of 
89 receptors, ion channels, and enzymes. Although AG-348 is a histamine H3 antagonist/inverse 
agonist and an aromatase inhibitor, no effects of histamine H3 modulation have been observed in 
safety pharmacology or toxicology studies. Effects consistent with aromatase inhibition were 
seen in toxicology studies (Section 5.2.1.4). 
5.2.1.2. 
Safety Pharmacology 
Safety pharmacology studies demonstrated that neither AG-348 nor AGI-8702 exhibited 
significant inhibition of the human ether-à-go-go related gene (hERG) current (concentration of 
drug that achieved half-maximal inhibition [IC50] 29.4 μM and > 10 μM, respectively). 
Additionally, in the monkey cardiovascular (CV) study, no CV effects were seen at any dose 
level. No effects were seen in AG-348 Good Laboratory Practice (GLP)-compliant Irwin and 
respiratory safety pharmacology studies in Sprague-Dawley rats. AG-348 administered via oral 
gavage had a dose-dependent emetic activity in the ferret at ≥ 30 mg/kg. 
5.2.1.3. 
Pharmacokinetics 
Absorption, distribution, metabolism, and excretion (ADME) studies of AG-348 were performed 
in Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys. Both oral (PO) and intravenous 
(IV) routes were evaluated at doses ranging from 1 to 50 mg/kg. A dose of 200 mg (50 mg/kg) 
was administered PO in the food-effect evaluation in monkeys. The PK of AG-348 in animal 
species is characterized by rapid oral absorption, medium to high total body plasma clearance 
(CLp), and high volume of distribution at steady-state (Vss) in rats, dogs, and monkeys. 
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AG-348 has a medium to long apparent terminal half-life (t1/2) in the rat, dog, and monkey. 
Medium to high oral bioavailability was observed for AG-348 in animals. A high-fat diet led to 
lower oral absorption of AG-348 in monkeys. 
AG-348 showed low brain penetration in rats (brain/plasma ratio of 5.7% to 9.1%) based on area 
under the plasma concentration versus time curve from 0 to 24 hours (AUC0 24hr) following 
single and repeated-dose administration, respectively. Metabolism appears to be the major 
elimination pathway for AG-348 with minimal biliary or urinary excretion.  
Both in vitro and in vivo, the predominant metabolite was the N-dealkylated product, AGI-8702. 
There were no metabolites unique to human liver microsomes and no direct Phase 2 metabolites. 
The plasma metabolite profile of AG-348 did not change with repeat or ascending dosing, 
suggesting that AG-348 is not susceptible to metabolic switching with increasing or prolonged 
dose administration. 
AG-348 is a weak direct inhibitor of cytochrome P450 (CYP) 2C8, CYP2C9, CYP2C19, 
CYP2D6, and CYP3A4/5 (testosterone 6β-hydroxylation). There was no direct inhibition of 
CYP1A2, CYP2B6, or CYP3A4/5 (midazolam 1′-hydroxylation) by AG-348. There was 
evidence of metabolism-dependent inhibition of CYP2C19 (largely reversible) and CYP3A4 
(largely irreversible) by AG-348. AG-348 is considered a weak time-dependent CYP3A4 
inhibitor. AGI-8702 is a weak direct inhibitor of CYP2C9 and CYP2C19. AG-348 has the 
potential to cause an induction-related drug-drug interaction (DDI) with sensitive CYP2B6 and 
CYP3A4 substrates. 
The routes of metabolism for AG-348 are via multiple CYPs with CYP3A4 contributing > 70% 
of the total. CYP1A2, CYP2C9, and CYP2C8 contribute approximately 6%, 10%, and 7% to the 
remaining metabolism of AG-348; other isoforms contribute < 4% each. 
AG-348 is a substrate for P-glycoprotein (P-gp), but not breast cancer resistance protein (BCRP). 
AG-348 is an inhibitor of P-gp (91% and 99% inhibition at 41 and 411 µM, respectively), but 
does not inhibit BCRP. AGI-8702 is not an inhibitor of P-gp or BCRP under the concentrations 
tested (5 and 100 μM). 
5.2.1.4. 
Toxicology 
In rats, the no-observed-effect-level/no-observed-adverse-effect-level (NOEL/NOAEL), 
determined as 2000 mg/kg, was associated with area under the plasma concentration versus time 
curve from 0 to 12 hours (AUC0-12hr) values 223- to 526-fold the projected human efficacious 
AUC0-12hr value. In dogs, clinical observations consistent with anaphylactoid reactions were seen, 
and the maximum tolerated dose (MTD) was 62.5 mg/kg, which was associated with an 
AUC0-12hr value 5.7-fold the projected efficacious AUC0-12hr value. The NOEL/NOAEL in dogs 
was 10 mg/kg, which was associated with an AUC0-12hr value 0.8-fold the projected efficacious 
AUC0-12hr value. In monkeys, the NOAEL was 1000 mg/kg, with only non-adverse emesis and 
body weight loss seen; this dose was associated with an AUC0-12hr value 70-fold the projected 
efficacious AUC0 12hr value. Based on the results of single-dose studies, the rat and the monkey 
were chosen as the most appropriate species for further evaluation in toxicology studies.  
Dose-limiting toxicity (DLT) in cynomolgus monkeys was defined in non-GLP 5-day and 
14-day repeat-dose studies as emesis, inappetence, and weight loss. These toxicities became dose 
limiting at AUC0-12hr values 27- to 34-fold the projected efficacious AUC0-12hr value, and 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
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precluded meaningful evaluation of other toxicities at this exposure level. Potential other effects 
at this exposure level were observed in a number hematology and serum chemistry parameters as 
well as in lymphoid organs. Additionally, minimal potential effects in kidneys (renal 
tubulointerstitial nephritis) and heart (myocardial degeneration), which could not be 
differentiated from spontaneous background lesions, were seen when monkeys were exposed to 
AG-348 AUC0-12hr values ≥ 27- to 34-fold the projected human efficacious AUC0-12hr value.  
In GLP-compliant 28-day monkey study, the dose of 150 mg/kg/day (75 mg/kg/dose twice daily 
[BID]) was the NOAEL. Effects were limited to increased liver weights without serum chemistry 
or microscopic correlate. At this dosage level, the Day 27 AUC0-12hr values were 8.9- and 
8.5-fold the projected efficacious AUC0-12hr value in males and females respectively. In the same 
study, the low dosage of 20 mg/kg/day (10 mg/kg/dose) resulted in AUC0-12hr values that 
approximated the efficacious AUC0-12hr value, and there were no test article-related effects seen. 
The next highest dose of 50 mg/kg/day (25 mg/kg/dose) was the NOEL and was associated with 
AUC0-12hr values 3.1- and 2.6-fold the projected efficacious AUC0-12hr value in males and females 
respectively.  
In the GLP-compliant 28-day rat study, the NOAEL in males was the highest dose tested, 
600 mg/kg/day (300 mg/kg/dose), and the NOAEL in females was the lowest dose tested, 
20 mg/kg/day (10 mg/kg/dose). At the 600 mg/kg/day dosage level in males, AG-348-related 
findings were limited to mild effects on hematology, serum chemistry, and urinalysis parameters, 
and microscopic findings in the adrenal gland (minimal to mild vacuolation of the adrenal zona 
glomerulosa and decreased thickness of the zona fasciculate), liver (minimal to mild 
hepatocellular hypertrophy), kidney (minimal tubular vacuolation), pancreas (minimal to 
moderate decreased zymogen granules), heart (minimal myocardial vacuolation), and prostate 
(minimal to mild decreased secretion). All findings were fully reversible over the 14-day 
recovery period with the exception of decreased serum glucose levels and decreased prostate 
secretion. In females, the highest dosage tested was 200 mg/kg/day (100 mg/kg/dose); adverse 
effects observed were similar to those observed in the 600 mg/kg/day males, with the exception 
that in females, fewer effects in hematology and serum chemistry parameters were seen, and also 
in females, adverse effects in the reproductive organs consistent with aromatase inhibition were 
observed.  
In the 13-week repeat-dose rat study, adverse effects in males were identified in the testes and 
consisted of seminiferous tubular degeneration, spermatid retention, and Leydig cell 
hypertrophy. These effects were defined as adverse at dose levels at which they are expected to 
impair fertility. These effects were either not present or present with incidence and severity 
similar to that of the vehicle group in lower dose levels. Adverse effects in females included 
uterine atrophy and increased folding of the luminal surface; these effects were defined as 
adverse at the dose level at which they are expected to impair fertility  
In the 13-week repeat-dose study in monkeys, no adverse effects were identified, and no new 
effects were identified when compared to the 4-week repeat-dose study. Similar to what occurred 
on the 4-week study, inappetence and emesis during the initial 1-2 weeks of dosing occurred, 
precluding evaluation of higher doses.  
AG-348 inhibits human aromatase activity with an IC50 of 2050 nM (based on human placental 
microsomes) and rat aromatase with an IC50 of 493 nM (based on rat ovarian microsomes). In the 
GLP-compliant 28-day rat study, histologic effects consistent with aromatase inhibition were 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
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seen in the female reproductive tract at the mid- and high-dosage levels (100 and 200 mg/kg/day) 
and included incomplete corpora lutea; ovarian follicular cysts; ovarian cystic, luteinized 
follicles; uterine atrophy; vaginal mucification; and altered cyclicity. Although these findings 
were minimal to mild and were fully reversible (over 14 days), they were considered adverse and 
the next lower dosage evaluated, 20 mg/kg/day (10 mg/kg/dose BID) was the NOAEL in 
females. The Day 27 AUC0-12hr value associated with this dosage level was 6.9-fold the projected 
human efficacious AUC0-12hr value. The potential for aromatase inhibition effects occurring in 
female rats at AUC0-12hr values > 6.9-fold and < 53-fold the projected efficacious AUC0-12hr value 
has been addressed in a 13-week rat study. In this study using doses between the NOAEL and 
LOAEL in the 28-day study, the NOAEL for histologic lesions of the uterus that may be 
associated with aromatase inhibition resulted in an AUC0-12hr that was 26-fold the projected 
efficacious value. Notably, due to the potency difference of AG-348 against rat versus human 
aromatase inhibition, there is potential for a 4-fold wider margin for aromatase inhibition in 
humans versus rats. AGI-8702 is not an aromatase inhibitor. 
5.2.2. 
Summary of Clinical Data 
To date, 72 healthy adult volunteers have been exposed to AG-348 in 2 clinical studies, a single 
ascending dose (SAD) study and a multiple ascending dose (MAD) study, with 31 of these 
subjects exposed to a single AG-348 dose under fasted conditions; 5 exposed to 2 doses of 
AG-348 under fasted and fed conditions; and 36 exposed to repeated doses of AG-348 for up to 
14 days1. The following discussion of clinical data refers only to healthy adult volunteer 
subjects, as this is the first clinical trial in which patients with PK deficiency will be treated with 
AG-348. 
5.2.2.1. 
Pharmacokinetics 
The pharmacokinetics (PK) of AG-348 showed low to moderate variability between subjects. 
After single doses of AG-348 from 30 to 2500 mg, AG-348 was rapidly absorbed as indicated by 
the short time to maximum plasma concentration (Tmax). There appeared to be some lengthening 
of the period of absorption at higher doses as indicated by higher Tmax and a less than 
proportional increase in maximum plasma concentration (Cmax). Dose-normalized area under the 
curve (AUC) generally remained constant over the dose range studied, suggesting that AG-348 
total exposure increased in a dose-proportional manner. The mean t1/2 ranged from 17.8 to 20.4 
hours when samples were collected through 72 hours and from 50.3 to 79.3 when samples were 
collected through 120 hours. However, this terminal elimination phase contributed little to 
overall exposure of AG-348, as indicated by the small difference between AUC0-12 and area 
under the plasma concentration versus time curve from 0 to infinity (AUC0-∞), suggesting a 
shorter effective half-life of approximately 3 to 6 hours. AG-348 was extensively distributed 
(mean apparent volume of distribution [Vz/F] range of 271 to 1148 L) and had a moderate rate of 
clearance (geometric mean clearance [CL/F] range of 10.3 to 13.9 L/hr). The fraction of AG-348 
excreted in urine ranged from 0.0145 to 0.0209 across the dose levels suggesting that renal 
excretion plays a minor role in the systemic elimination of AG-348. 
1 At the time of this document, results from Study AG348-C-002 in healthy volunteers have been unblinded, but the 
data have only partially been analysed and the Clinical Study Report is in preparation but not yet completed.  
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The preliminary repeat-dose PK of AG-348 at doses ranging from 15 mg every 12 hours (q12h) 
to 700 mg q12h also showed an increase in AG-348 exposure in a dose-proportional manner 
after the first dose. AG-348 exposure observed after repeated dosing for 14 days at doses of 
120 mg every 24 hours (q24h) to 700 mg q12h were lower than observed on Day 1 after the first 
dose. The magnitude of the effect was greater at higher doses; lower doses of 15 mg q12h and 
60 mg q12h did not show this effect. This observation may be related to the nonclinical finding 
that AG-348 is a potential inducer of human CYP3A4, an enzyme which biotransforms AG-348. 
It is possible that multiple doses of AG-348 leads to an increased rate of its own metabolism. 
Evaluation of the effect of food on the PK of a single 700 mg dose of AG-348 in 5 subjects who 
were administered the drug fasting and then, after an appropriate wash-out period, re-
administered the drug following ingestion of a standard US Food and Drug Administration 
(FDA) high fat meal, showed that food likely has a minimal effect on the PK of AG-348. 
5.2.2.2. 
Pharmacodynamics 
After a single dose of AG-348, a decrease in the concentration of 2,3-DPG was observed at 
3 hours post-dose, decreased in a dose-dependent manner to a minimum at 24-hour post-dose, 
and then returned to values similar to baseline by 72 to 120 hours post-dose. The mean decrease 
at 24 hours was approximately 300 µg/mL at the 700 through 2500 mg dose levels. Similar 
decreases were observed after the first dose of multiple doses and prior to doses in the MAD 
study. After the final dose of multiple doses, the concentration of 2,3-DPG returned to values 
similar to baseline between 72 and 120 hours post- dose. 
After a single dose of AG-348, a minimal increase in the concentration of ATP was observed at 
24 to 120 hours post-dose. In the MAD study, no increase was observed at 12 hours after the first 
dose; the concentration of ATP was increased on Day 8 to concentrations greater than in the 
single dose study, continued to trend upward on Day 11, and remained at a similar level through 
Day 14. The concentration of ATP remained elevated through 120 hours after the last dose on 
Day 14. The magnitude of the increase in ATP was similar across the dose range from 60 mg 
BID to 700 mg BID. 
5.2.2.3. 
Safety 
Overall, AG-348 has been well-tolerated among healthy volunteers at doses that produced strong 
pharmacodynamic (PD) effects on 2,3-DPG and ATP. 
After a single AG-348 dose, treatment-emergent adverse events (TEAEs) reported by > 1 subject 
at any time on study (either under fasted or fed conditions) included headache (22%), nausea 
(14%), and contact dermatitis and vomiting (each 6%). After repeated dosing of AG-348 for 
14 days, TEAEs that occurred in > 5% of all AG-348-treated subjects across all cohorts included 
headache (13.9%), nausea (13.9%), vomiting (8.3%), decreased appetite (8.3%), feeling hot 
(8.3%), restlessness (8.3%), and dizziness, fatigue, vessel puncture site bruise, hyperhidrosis, 
dermatitis allergic, and drug eruption (5.6% each). 
A dose-relationship was apparent with regard to the incidence of gastrointestinal events, 
primarily nausea and vomiting, with the incidence of such events increasing with increasing 
dose. Nausea and/or vomiting were observed only at doses ≥ 1400 mg in the single dose study 
and only at doses ≥ 700 BID in the MAD study. Nausea and vomiting were not observed at any 
dose ≤ 360 mg in either the single or multiple dose studies. 
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All but 1 TEAE reported to date has been mild or moderate (Grade 1 or 2) in intensity. The only 
Grade 3 TEAE was elevated liver transaminases (alanine aminotransferase [ALT]; aspartate 
aminotransferase [AST]) in a single subject in the MAD study treated with 700 mg AG-348 BID. 
The event was considered to be possibly related to AG-348, was declared a DLT, and led to 
study drug discontinuation, following which the elevated liver transaminases resolved. 
Altogether, 3 of 6 subjects treated with AG-348 at 700 mg BID discontinued study drug in the 
MAD study: the subject described above with Grade 3 elevated transaminases, and 2 others who 
withdrew themselves for nausea and vomiting. A fourth subject treated with AG-348 at 60 mg 
BID was discontinued from the study for a drug eruption. No AG-348-treated subject 
discontinued in the SAD study due to an adverse event (AE). 
No deaths or other serious AEs (SAEs) have been reported in any clinical study of AG-348. 
Furthermore, no DLTs were observed after a single administration of AG-348 at doses of 30 mg 
to 2500 mg or after repeat administration of AG-348 for 14 days at daily doses of 15 mg to 
700 mg BID except for the event of Grade 3 elevated liver function tests described above. 
Due to preclinical observations pertaining to the potential for inhibition of the aromatase enzyme 
(see Section 5.2.1.4), the AG348-C-002 multiple dose study included assessment of baseline and 
serial measures of free and total serum testosterone and serum estradiol and estrone. The 
unblinded serum hormone data are undergoing analysis, but at least some male subjects treated 
with all doses of AG-348 demonstrated modest increases in androgenic hormones and decreases 
in estrogens, compatible with a potential signal of aromatase inhibition, whereas the males who 
received placebo did not. These changes were reversible upon cessation of dosing. There were 
too few female subjects in the study who received AG-348 to permit any definitive conclusions. 
Additional analyses are ongoing. 
5.3. 
Study Rationale 
Study AG348-C-003 is the first study that will be conducted in patients with PK deficiency. This 
study is primarily intended to evaluate the safety and tolerability and potential indicators of 
clinical activity of AG-348 administered for up to 24 weeks. This study will also evaluate the PK 
profile of AG-348 and its metabolite AGI-8702, the PD responses in ATP and 2,3-DPG 
following administration of AG-348, and the clinical activity of AG-348 in PK deficiency 
patients. Two previous double-blind, placebo-controlled clinical trials of AG-348 conducted in 
healthy adult male and female volunteers (AG348-C-001, a SAD study; and AG348-C-002, a 
MAD study) have established an acceptable safety and tolerability profile for AG-348 for up to 
14 days of both once-daily (QD) and BID dosing at exposures that result in significant PD 
changes in whole blood levels of the glycolytic metabolites 2,3-DPG and ATP. Due to the 
potential for AG-348-mediated aromatase inhibition, dual-energy x-ray absorptiometry (DXA) 
scan (hip and spine) will be performed at Screening (if patient has not had prior DXA scan 
within 3 months of Day 1) to obtain T and Z scores. These data will serve as baseline measures 
of bone mineral density for all enrolling patients (see Section 7.1 for more details). 
The target population of this study consists of adult males and females with a diagnosis of PK 
deficiency, who are anemic but non-transfusion dependent. Non-transfusion dependent patients 
are preferred for this study in order to reduce any potential confounding effect of transfusion 
therapies on evaluation of potential indicators of clinical activity and PD responses. The safety, 
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tolerability, and PK/PD findings in this study will form the basis for subsequent clinical 
development of AG-348. 
The Extension Period is offered in order to allow for longer study of AG-348 in patients who 
experience a benefit and tolerate the drug. 
5.3.1. 
Summary of Overall Safety Management Plan 
Measures to minimize the risks to patients enrolled in the Core and Extension Periods have been 
taken with respect to the following study design elements: 
• The initial doses for Arms 1 and 2 have been selected on the basis of safety and 
tolerability observations already carefully made in two predecessor clinical trials in 
adult healthy male and female volunteers;  
• The specified inclusion/exclusion criteria have been carefully considered to avoid 
enrollment of subjects for whom exposure to the study drug might pose a hazard; 
• A designated Data Review Team (DRT) consisting of the overall study Coordinating 
Investigator, treating Investigators, Medical Monitor, Clinical Pharmacologist, 
Statistician, and Sponsor’s Responsible Medical Officer has been established to meet 
regularly at approximately 6-week intervals throughout the duration of the Core 
Period, and approximately every 3 months during the Extension Period once all 
patients have completed the Core Period to review the accumulating study data and 
will exercise options to suspend enrollment to one or both of the initial two study 
dose arms, discontinue enrollment to one or both of the initial two study dose arms, 
adjust the dose of patients in one or both of the initial two study arms, and/or 
implement one new study dose arm. If one new dosing arm is implemented in the 
Core Period, the dose selected will not exceed 360 mg BID, the highest dose that 
demonstrated acceptable safety and tolerance in the 14-day multiple BID dosing 
study in healthy volunteers. Group cohort stopping rules for terminating enrollment 
into an arm based on the severity (Common Terminology Criteria for Adverse Events 
[CTCAE]v4.03 grade) and frequency of AEs are defined;  
• Dose modification and stopping rules are defined for individual patients; 
• Guidance for permitted, prohibited, and cautionary concomitant medications is 
specified based on the estimated potential for DDIs from hepatic cytochrome enzyme 
interactions with AG-348. 
• Due to the potential for AG-348-mediated aromatase inhibition, bone mineral density 
will be monitored using DXA scans (hip and spine) at Baseline (if patient has not had 
prior DXA scan within 3 months of Day 1) and between Week 24 and Week 28. 
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In the event that any clear and unequivocal, previously unidentified/unexpected toxicities occur 
in pre-clinical toxicology studies, the Sponsor will notify the Investigators, Institutional Review 
Boards (IRBs)/Independent Ethics Committees (IECs), and regulatory authorities. The Sponsor 
will also report these to the DRT for review and discussion of appropriate actions, which may 
include, but are not necessarily limited to:  
• Amending the protocol to adjust the inclusion/exclusion criteria (e.g., to exclude 
patients with certain at-risk concurrent conditions); and/or  
• Amending the protocol to adjust safety monitoring procedures (e.g., to require 
additional monitoring of specified AEs, physical examinations, clinical laboratory 
testing, ECG monitoring, or other testing as appropriate); and/or  
• Adjusting the dose of an arm of the study as appropriate; and/or 
• Adjusting the dose modification and/or stopping rules (Section 9.7); and/or 
• Adjusting the patient withdrawal criteria (Section 8.6); or 
• Terminating the trial. 
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6. 
TRIAL OBJECTIVES AND ENDPOINTS 
6.1. 
Core Period 
6.1.1. 
Primary Objective 
The primary objective of the study is to: 
• Evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in 
patients with PK deficiency. 
6.1.2. 
Secondary Objectives 
The secondary objectives of the study are to: 
• Evaluate the PK of AG-348 and the metabolite AGI-8702. 
• Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348. 
• Evaluate indicators of clinical activity of AG-348 in patients with PK deficiency, 
including changes in hemoglobin (Hb), HCT, reticulocyte count, haptoglobin (Hp), 
carboxyhemoglobin (COHb), lactate dehydrogenase (LDH), total and indirect 
bilirubin, erythropoietin (EPO), ferritin, and transferrin saturation (serum iron/iron 
binding capacity). 
 
6.2. 
Extension Period 
6.2.1. 
Primary Objective 
The primary objective of the study is to: 
• Evaluate the safety and tolerability of up to 30 months of AG-348 administration in 
patients with PK deficiency. 
6.2.2. 
Secondary Objectives 
The secondary objectives of the study are to: 
• Evaluate the PK of AG-348 and the metabolite AGI-8702. 
• Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348. 
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• Evaluate indicators of clinical activity of AG-348 in patients with PK deficiency, 
including changes in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and 
indirect bilirubin, EPO, ferritin, and transferrin saturation (serum iron/iron binding 
capacity). 
6.3. 
Study Measures and Endpoints 
6.3.1. 
Safety Measures and Endpoints 
Safety will be evaluated by: 
• Monitoring of AEs, including determination of SAEs and AEs leading to 
discontinuation; safety laboratory parameters (hematology, chemistry, urinalysis, 
coagulation); physical examination findings; vital signs (VS); 12 lead 
electrocardiograms (ECGs); and DXA scans. Adverse events will be graded using 
CTCAE, Version 4.03. Serum sex hormone levels (testosterone [total and free], 
estrone, and estradiol), bone turnover markers (serum osteocalcin-N-mid and serum 
C-terminal telopeptide [CTX]), 25-hydroxy vitamin D2 and D3, total cholesterol, 
high-density lipoprotein-cholesterol (HDL-C), and triglycerides will be monitored for 
evidence of potential inhibition of aromatase by AG-348. Menstruating female 
patients will also keep a paper-based menstrual cycle diary throughout the Core and 
Extension Periods. 
6.3.2. 
Clinical Activity Measures and Endpoints 
• Monitoring of potential indicators of clinical activity will include evaluating changes 
in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, 
ferritin, and transferrin saturation. 
6.3.3. 
Pharmacokinetic and Pharmacodynamic Measures and Endpoints 
The PK and PD profile of AG-348 will be evaluated by: 
• Approximately the first 10 patients treated during the Core Period, contingent on 
clinical site feasibility, will undergo extensive PK sampling as detailed in 
Appendix 15.1, Table 7. The remainder of treated patients will undergo limited PK 
sampling as detailed in Appendix 15.1, Table 8. During the Core Period, serial blood 
sampling for determination of concentration-time profiles of AG-348 and its 
metabolite AGI-8702 will be conducted following the first dose and the morning 
Day 15 dose, and additional trough levels of AG-348 and AGI-8702 will be obtained. 
During the Extension Period, predose PK samples will be drawn for the measurement 
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of trough levels of AG-348 and AGI-8702 at each study visit (every 3 months; 
Appendix 15.1, Table 6). AG-348 and AGI-8702 will be analyzed using qualified 
assays to determine concentrations in plasma. Pharmacokinetic parameters on Day 1 
and Day 15 will be computed using standard non-compartmental methods based on 
observed plasma AG-348 and AGI-8702 concentrations. 
• Pharmacodynamic assessments during the Core Period will include 2,3-DPG, ATP 
(secondary objectives), 
 
 
 
Approximately the first 10 patients treated during the Core Period will undergo 
extensive PD sampling as detailed in Appendix 15.1, Table 7. The remainder of 
treated patients will undergo limited PD sampling as detailed in Appendix 15.1, 
Table 8. During the Core Period, serial blood sampling for determination of levels of 
ATP and, 2,3-DPG will be conducted following the first dose and the morning Day15 
dose, and additional trough levels of ATP and 2,3-DPG will be obtained. During the 
Extension Period, predose PD samples will be drawn for the measurement of trough 
levels of 2,3-DPG, ATP, 
 at each study visit (every 3 months; 
Appendix 15.1, Table 6). Adenosine triphosphate and 2,3-DPG will be analyzed 
using qualified assays to determine concentrations in whole blood. PD parameters on 
Day 1 and Day 15 will be computed based on observed whole blood ATP and 2,3-
DPG concentrations.  
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7. 
INVESTIGATIONAL PLAN 
7.1. 
Overall Study Design 
Study AG348-C-003 is a Phase 2, open-label, two-arm, multicenter, randomized, dose-ranging 
study in adult patients with PK deficiency; the study will be divided in to a Core Period and an 
Extension Period. During the Core Period, patients will receive multiple doses of AG-348 for up 
to 24 weeks; patients who are eligible can enter the Extension Period to receive AG-348 for up to 
2 years following the end of the Core Period. Patients with PK deficiency confirmed by RBC PK 
enzymatic assay performed at Screening will be eligible to participate in this study. At Week 25, 
patients who safely tolerate AG-348 and demonstrate clinical activity of AG-348 may be eligible 
to immediately roll over to the Extension Period for continued treatment. Patients who complete 
treatment at the end of the Core Period (24 weeks) will undergo follow-up assessment 4 weeks 
after the last dose of study drug. If a patient discontinues at any other time (including early 
discontinuation or discontinuation during the Core or Extension Period), follow-up assessments 
will be conducted 4 weeks after discontinuation. Patients with toxicity suspected to be related to 
study drug will continue follow-up until the AE resolves, is declared chronic by the Investigator, 
or the patient is lost to follow-up. 
For the Core Period, up to 25 patients will be initially randomized on an open-label, 1:1 basis to 
each of two BID doses of AG-348 (up to 50 patients; see Figure 3, Study Schema). The dose of 
Arm 1 is 300 mg of AG-348 administered orally q12h (BID). The dose of Arm 2 is 50 mg of 
AG-348 administered orally q12h (BID). Randomization will be stratified by PKR mutation in 
order to maintain balance as much as possible across the dose arms for the specific mutations 
expected to be most frequently enrolled. The PKR mutation stratification factor will consist of 
4 levels (R510Q, R486W, and R479H) and all other mutations (“other”). Mutation status is 
defined by the presence of at least one of the indicated mutations; patients with more than one 
stratified mutation will be assigned based on Sponsor’s discretion. 
The doses for each arm of the Core Period have been selected from the AG348-C-001 SAD 
study and AG348-C-002 MAD studies in healthy adult volunteers to represent the range of 
doses/exposures that were safely tolerated and resulted in maximal or near maximal PD effects 
on 2,3-DPG and ATP. 
Because PK deficiency is a rare disease with a limited eligible patient population and because the 
underlying pathophysiology and clinical phenotype of affected patients is heterogeneous due to 
the wide variety of mutations in PKR that cause the disease, it is deemed important to focus 
closely on dose findings in this first-in-patient study. Therefore, in addition to initiating this 
study with 2 different doses of AG-348 administered q12h, a DRT will be established to review 
study data on a frequent basis and adapt the study design, dose and schedule of AG-348 as 
indicated.  
The DRT will monitor safety on an on-going basis and meet at regular intervals of approximately 
every 6 weeks, or ad hoc as necessary, for as long as any patients are still in the Core Period to 
review AEs, VS, clinical laboratory (hematology, clinical chemistry, coagulation, and 
urinalysis), and ECGs. The DRT will also review available PK/PD data and indicators of clinical 
activity (e.g., changes from baseline in Hb). These DRT meetings will also include data review 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
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for all patients that may be under treatment in the Extension Period. If there are no patients still 
being treated in the Core Period, and the only patients on treatment are those in the Extension 
Period, then the frequency of the DRT meetings will reduce to approximately every 3 months in 
order to match the frequency of patient visits (and new data collection) in the Extension Period. 
The DRT will be comprised of the study Coordinating Investigator, treating Investigators, 
Medical Monitor, Clinical Pharmacologist, Statistician, and Sponsor’s Responsible Medical 
Officer. 
Beginning 6 weeks after the first patient is dosed in the Core Period or ad hoc as necessary, and 
proceeding according the schedule indicated above (approximately every 6 weeks during the 
Core Period, approximately every 3 months during the Extension Period once all patients have 
completed the Core Period), the DRT will review cumulative safety data, available PK/PD data, 
and clinical activity data. Based on the DRT’s recurring, the DRT may exercise one or more of 
the following options during the Core Period: 
• Continue treatment and enrollment in existing arms without change. 
• Add 1 new dose arm (Arm 3) to enroll up to 25 patients at a dose to be determined; 
the dose for Arm 3 may be lower or higher than Arm 1 and Arm 2 doses, but will not 
exceed 360 mg q12h; and the dose regimen may be less frequent than q12h. 
• Terminate or suspend enrollment to allow further review of clinical data in Arm 1 
and/or Arm 2 (and/or potential Arm 3). Enrollment in an arm could be terminated or 
suspended to allow further review, for example, for unacceptable safety/tolerability, 
poor PD response, or lack of signs of clinical activity. 
• Re-assign patient’s doses and schedule in a terminated arm to match the dose and 
schedule of another arm of the study. In this case, the patients in the terminated arm 
will remain in their original arm, i.e., they will not count towards the enrollment 
quota of the arm whose dose and schedule is being adopted. 
• Implement specific genotype restrictions to enrollment in one or more arms to ensure 
representation of patients with genotypes of greatest clinical relevance. 
The DRT may exercise one or more of the following options during the Extension Period: 
• Continue treatment without change; 
• Re-assign patients’ doses and schedule to an existing dose and schedule that has been 
determined to be safer, and/or have a better PD response, or produce signs of clinical 
activity; 
• Terminate or suspend treatment to allow further review of clinical data (eg, for 
unacceptable safety/tolerability, poor PD response, or lack of signs of clinical 
activity). 
The data that the DRT will review to make these decisions is expected to include, but are not 
necessarily limited to, the following:  
• Safety Observations: all AEs; VS, clinical laboratory (hematology, clinical chemistry, 
coagulation, and urinalysis), and ECGs;  
• PK and PD Observations: including changes in 2,3-DPG and ATP; 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
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• Indicators of Clinical Activity: including changes in Hb, HCT, reticulocyte count, Hp, 
COHb, LDH, EPO, total and indirect bilirubin, ferritin, and transferrin saturation.  
If a third dose arm is implemented, the dose of AG-348 selected will not exceed 360 mg BID, as 
this was the highest dose that demonstrated acceptable safety and tolerance in the 14-day 
multiple BID dosing study in healthy volunteers. The PK/PD sampling schedule in a potential 
third dosing arm will be determined by Sponsor’s discretion and may follow either the extensive 
or limited PK/PD sampling schedules as specified. 
The DRT will monitor the safety data in an ongoing manner as described and may make a 
decision to terminate enrollment in an arm if unacceptable AEs are observed, PD activity is 
limited, and/or markers of clinical activity indicate no effect. Unacceptable AEs are defined as 
≥ Grade 3 AEs (using National Cancer Institute (NCI) CTCAEv4.03) that are assessed as at least 
possibly related to AG-348, with the exception of Grade 3 headache, hot flash/flushing, nausea, 
vomiting, and/or diarrhea that is transient in duration (< 24 hours) or able to be medically 
managed to ≤ Grade 2 within 24 hours. The DRT may also exercise discretion to terminate 
enrollment in an arm if it observes a frequency of Grade 2 AEs that would make it difficult to 
support long-term dosing. 
Patients in the Extension Period will continue on the dose they were randomized to in the Core 
Period, unless the DRT had reason to establish a different dose/schedule during the course of the 
Core Period (the DRT will not propose a dose higher than 360 mg q12h).  
Due to the potential for AG-348-mediated aromatase inhibition, DXA scan (hip and spine) will 
be performed at Screening (if patient has not had prior DXA scan within 3 months of Day 1) to 
obtain T and Z scores. These data are intended to serve as a baseline measure of bone mineral 
density for all enrolling patients, and are deemed of particular importance for those who may 
enter the longer term Extension Period after completing 24 weeks of treatment (Core Period). All 
patients will have a second DXA scan in the interval between Weeks 24 and 28 for the Core 
Period. Patients in the Extension Period will have additional DXA scans at Months 18 and 30.. 
As the number of enrolling arms changes in the study (for example, from 2 to 3), the 
randomization scheme will adjust to enable balanced randomization into each actively accruing 
arm. Randomization and stratification will cease in the event that only a single arm is left 
enrolling. 
Depending on possible early termination of 1 or both of the initial 2 arms, or the addition of a 
third arm, the study could enroll up to a maximum of 75 patients. 
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Figure 3: 
Study Schema:  Core Period 
 
Abbreviations:  BID (q12h) = twice-daily (every 12 hours); DRT = data review team; PKR = pyruvate kinase red 
blood cell isoform; TBD = to be determined; w = weeks.  
The Investigator will monitor all patients for safety and tolerability. Modification of an 
individual patient’s dose of AG-348 will be based on AEs and observed changes in Hb levels as 
detailed in Section 9.7.1 and Section 9.7.2. 
Screening assessments will occur within 28 days prior to the first dose of study treatment. During 
the Core Treatment period, patients will attend visits at baseline (Day 1), weekly through Week 3 
(Days 8, 15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12), and 
monthly through Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate AG 348 through 
Week 24 (Core Period) may be eligible to immediately enter the Extension Period for continued 
treatment. Study visits for safety and clinical activity assessments will occur approximately 
every 3 months during the Extension Period for up to 2 years after the end of the Core Period. 
All patients will undergo a follow-up assessment 4 weeks after the last dose of AG-348, 
regardless of whether this was due to early discontinuation, the last dose in the Core Period for a 
patient who chooses not to continue in the Extension Period, or the last dose of the Extension 
Period. 
Safety assessments will include monitoring of AEs, including determination of SAEs and AEs 
leading to discontinuation; safety laboratory parameters (e.g., hematology, serum chemistry, 
coagulation studies, and urinalysis); physical examination findings; VS; 12 lead ECGs, and DXA 
scans. Additional safety assessments will include monitoring of sex hormone levels (testosterone 
[total and free], estrone, and estradiol), bone turnover markers (osteocalcin-N-mid and CTX), 
25-hydroxy vitamin D2 and D3 levels, total cholesterol, HDL-C, and triglycerides. Follow-up 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
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assessments will be conducted on Day 197 (Week 28) for patients who do not enter the 
Extension Period and will include physical examination, weight, performance status, VS, 12-lead 
ECGs, laboratory evaluations (hematology, Hp, EPO levels, serum chemistry, coagulation 
studies, urinalysis; lipids, hormonal testing), AEs, and transfusion record. These follow-up 
assessments will be performed approximately 4 weeks after discontinuation of AG-348 for 
patients who discontinue prior to completion of the Core Period and for those who discontinue in 
the Extension Period. Menstruating female patients will also be required to keep a paper-based 
menstrual cycle diary throughout the study.  
Pharmacokinetic assessments will include serial blood sampling for PK profiles of AG-348 and 
its metabolite AGI-8702. Pharmacodynamic evaluations will include serial blood sampling for 
determination of levels of ATP and 2,3 DPG. Extensive PK/PD sampling will be conducted on 
the first approximately 10 patients total treated in Arms 1 and 2 of the Core Period (see 
Appendix 15.1, Table 7) while limited PK/PD sampling will be conducted on the remainder of 
treated patients (see Appendix 15.1, Table 8). Limited trough sampling will be conducted every 
3 months during the Extension Period (Appendix 15.1, Table 6). 
 
 
 
 
7.2. 
Justification of the Study Design 
The primary and secondary objectives of this study are to evaluate the safety, tolerability, PK 
and PD, and indicators of clinical activity of AG-348 in patients with PK deficiency. The choice 
of dose and schedule of administration of AG -348 for Arms 1 and 2 was based on the highest 
safely tolerated dose (Arm 1: 300 mg BID) and the lowest dose with potentially relevant PD 
activity (Arm 2: 50 mg BID) from the forerunner AG348-C-002 MAD study in healthy 
volunteers. Decisions regarding continuing enrollment and treatment in these initial dose arms 
and/or implementation of an additional dose arm will be based on DRT review of safety, PK and 
PD data, and indicators of clinical activity collected from all patients treated in Arm 1 and 
Arm 2. This design was chosen to minimize risk to patients while allowing evaluation of safe 
and pharmacologically active dose levels of AG-348, and to allow the necessary flexibility to 
adjust dose and schedule should the safety, tolerability, PK, and/or PD be different in patients 
with PK deficiency compared with healthy volunteers.  
Additional safety measures intended to minimize risk to patients include monitoring of AEs by 
the DRT and specified provisions for individual patient dose modification as needed for safety 
and (potentially) large increases in Hb level (Section 9.7.1 and Section 9.7.2). Measures intended 
to maximize the opportunity for patients with demonstrated safety and tolerability to continue to 
derive benefit from any observed clinical activity of AG-348 include the option for continued 
treatment in the Extension Period.  
A comprehensive series of safety evaluations, including laboratory parameters, physical 
examinations, VS, 12-lead ECGs, and monitoring for SAEs and AEs, will be conducted to 
evaluate the safety profile of AG-348 and to aid in the determination of the recommended dose 
for continued development. 
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The study includes serial blood sampling across at least two different doses of the study drug to 
assess its PK and PD profiles.  
Consistent with the design of many Phase 2 studies, preliminary evaluation of the potential PD 
and clinical activity of AG-348 are secondary objectives of this study. The latter will include 
assessments of the PK/PD relationship between AG-348 and the biomarkers ATP and 2,3-DPG, 
 
7.3. 
Rationale for the Starting Dose, Dose Range, and Duration of Dosing 
Prior to execution of this study, Agios conducted two clinical studies of AG-348 in healthy 
volunteers, including a SAD study (AG48-C-001) and a MAD (14 day q12h) study 
(AG348-C-002). Available details of these studies are discussed in the current Investigator’s 
Brochure (IB). Between these two studies, 72 healthy human subjects have been dosed with 
AG-348. In vitro investigations, also reported in the IB, had previously demonstrated that 
AG-348 increased the activity of wild-type PKR approximately to the same extent as it did a 
series a recombinant mPKRs. Therefore it was deemed reasonable to study the safety, 
tolerability, PK, and PD of AG-348 in healthy subjects in a controlled Phase 1 setting as a more 
efficient means of obtaining information than in the actual rare disease population of patients 
with PK deficiency.  
The MAD study demonstrated that the exposures produced by AG-348 doses from 60 mg q12h 
to 360 mg q12h (including 120 mg q24h) resulted in maximal changes from baseline for the PD 
markers 2,3-DPG (reduction from baseline) and ATP (increase from baseline). The exposures 
resulting from doses less than 60 mg q12h were of lesser magnitude and the exposures resulting 
from doses greater than 360 mg q12h were of no greater magnitude than the aforementioned 
range. Therefore the starting doses for this first dose ranging study in patients with PK deficiency 
were selected to be 300 mg q12h (Arm 1) and 50 mg q12h (Arm 2). These doses were 
demonstrated to be safe and tolerable in the healthy volunteer studies. The availability of ATP is 
proposed as being critical for optimally maintaining RBC membrane integrity (see Section 5.1). 
The dose ranges from 50 mg q12h to 300 mg q12h may result in clinically effective modulation 
of PKR in PK deficiency patients if the mutated enzyme is responsive to AG-348 in a similar 
manner to the wild-type enzyme in healthy subjects. However, there are many different 
mutations in PKR that result in PK deficiency, and these mutations produce variable effects on 
the enzyme in terms of catalytic activity and thermal stability. It is not known if different 
mutations will respond clinically in a similar manner to the same exposure to AG-348. 
Therefore, it is prudent to study the range of safe and pharmacodynamically relevant doses as 
specified in this study, and to allow flexibility for the DRT to analyze the evolving study data to 
adapt the dose and schedule of administration of AG-348 to produce the optimal combination of 
safety, tolerability, and PD, and, potentially, clinical response. 
Justification for Duration of Dosing  
The initial treatment duration of 24 weeks (6 months) for the Core Period was chosen for this 
proof-of-concept trial for 2 principal reasons: 1) to begin establishing a safety database 
addressing the chronic administration of AG-348; and 2) to allow sufficient time for clinical 
response to treatment to appear.  
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It is anticipated that this treatment, if successful, may be taken for life, as PK deficiency is a 
genetically determined inborn error of metabolism. Therefore, it is important to begin to 
investigate the long-term safety of the treatment. As will be discussed below, the safety package 
supports the treatment duration of 6 months.  
Red blood cell turnover is typically 120 days (4 months), although it may be shorter in some 
populations of pyruvate kinase deficient RBCs (Mentzer, et al. 1971). Different PKR mutations 
produce a variety of physiologic consequences. In mutations where normal or nearly normal 
levels of PKR protein persist, but the protein’s catalytic function is impaired, an improvement in 
PKR functional activity might be seen relatively quickly upon exposure to AG-348. However, in 
cases where the PKR mutation results in an unstable mutant, PKR protein levels may be low and 
additional time may be required for stabilization of the newly synthesized mutant enzyme in 
developing bone marrow erythroblasts so that eventually more mature RBCs may be produced 
with more nearly normal levels of functional PKR protein.   
This study plans up to 24 weeks of dosing with AG-348 during the Core Period with the 
possibility of continued dosing beyond 24 weeks during the Extension Period in patients for 
whom AG-348 is safely tolerated and demonstrates clinical activity. The International 
Conference on Harmonisation (ICH) Guideline M3(R2) on Nonclinical Safety Studies for the 
Conduct of Human Clinical Trials and Marketing Authorisation for Pharmaceuticals, 
recommends that clinical trials of a duration between 2 weeks and 6 months should be supported 
by repeat-dose toxicity studies of at least equivalent duration, and clinical trials lasting beyond 
6 months should be supported by 6-month rodent and 9-month non-rodent studies. This guidance 
allows for clinical trials of longer duration to be initiated based on 3 months of nonclinical data, 
and for clinical dosing in serious or life-threatening indications to be extended based on complete 
chronic rodent data and in-life and necropsy data in the non-rodent (and complete histopathology 
in the non-rodent provided within an additional 3 months).  
For the current investigational product (AG-348), 13-week, repeat dose toxicology studies in the 
rat and monkey have been completed and are summarized in Section 5.2.1.4 of this protocol and 
in the current Investigator Brochure prepared to support initiation of this clinical study. 
Considering that PK deficiency is a serious condition with an unmet medical need, it is 
appropriate to initiate this clinical study with the available nonclinical data. Six-month rodent 
and nine-month monkey toxicology studies were initiated in January 2015, and the Sponsor will 
report the results of these studies in each applicable regulatory region as required before any 
patients will be treated for greater than 6 months (i.e., enter the Extension Period).  
7.4. 
Criteria for Study Termination 
This study may be prematurely terminated if, in the opinion of the Sponsor, there is sufficient 
reasonable cause. In the event of such action, written notification documenting the reason for 
study termination will be provided to each Investigator. 
Circumstances that may warrant termination include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to patients; 
• Insufficient adherence to protocol requirements; 
• Plans to modify, suspend, or discontinue the development of the study drug; 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
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• Other administrative reasons. 
Should the study be closed prematurely, all study materials must be returned to the Sponsor or 
the Sponsor’s designee. Patients who have participated in the Extension Period may be able to 
screen for eligibility in a study for a second generation allosteric PKR activator, if such a 
compound is available at that time, and after a suitable washout period has been observed. 
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8. 
STUDY POPULATION 
8.1. 
Number of Patients 
Up to approximately 75 patients may be enrolled in this study. 
8.2. 
Inclusion Criteria 
For entry into the Core Period, patients must meet all of the following criteria during the 
Screening or other specified period: 
1. Signed written informed consent obtained prior to performing any study procedure, 
including screening procedures.  
2. Male or female, aged 18 years and older. 
3. Known medical history of PK deficiency. 
4. All patients must have documented clinical laboratory confirmation of PK deficiency by 
RBC pyruvate kinase enzymatic assay performed at Screening by a designated central 
laboratory. Patients with prior documentation of PK deficiency by RBC enzymatic assay 
will have a reconfirmation of this result during Screening as a condition of enrollment. 
a. In the event that a patient’s screening pyruvate kinase enzymatic assay is negative 
(i.e., shows normal pyruvate kinase activity), the patient will be eligible for 
enrollment if the genotyping shows a mutant genotype that has been previously 
documented in the literature to be associated with PK deficiency. If the 
genotyping shows a previously undescribed mutation in the PKR gene, then the 
eligibility for enrollment will be determined on a case-by-case basis by the 
Coordinating Investigator and Medical Monitor in discussion with the 
Investigator. If no mutation is defined, then the patient will not be eligible. 
5. All patients must have genotypic characterization of the mutant PKR gene performed by 
the designated central laboratory at Screening. 
6. All patients must have genotypic characterization of the UGT1A1 gene performed by a 
designated central laboratory to document whether they may have underlying Gilbert's 
Disease. Patients with Gilbert’s Disease are eligible to enroll. 
7. Males must have Hb ≤ 12.0 g/dL; females must have Hb ≤ 11.0 g/dL. 
8. All patients must be considered transfusion independent as defined by: no greater than 
3 units of RBCs transfused in the 12-month period up to the first day of study dosing and 
no transfusions within 4 months of first day of study dosing. Patients who have received 
more transfusion support than described above will be evaluated for eligibility on a case-
by-case basis by the Medical Monitor. 
9. Eligible patients may still have their spleens in place, or may have undergone prior 
splenectomy. For splenectomized patients: 
a. Must have undergone their procedure at least 6 months prior to Screening. 
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b. Must be current in their vaccinations for Pneumococcal Conjugate (PCV13), 
Pneumococcal Polysaccharide (PPSV23), Quadrivalent Meningococcal vaccine, 
and Haemophilus influenzae type b  as recommended by Centers for Disease 
Control and Prevention's (CDC) Advisory Committee on Immunization Practices 
(ACIP) or immunization advisory groups in Canada and the European Union (for 
patients enrolled in Canada and the EU). 
[http://www.cdc.gov/vaccines/schedules/downloads/adult/adult-schedule.pdf] 
[Any missing vaccinations may be administered during the screening period. If the 
patient requires both PCV13 and PPSV23, PCV13 must be given before PPSV23, if 
possible. Administration of PPSV23 should follow PCV13 by at least 8 weeks; it is 
permissible to give PCV13 during Screening followed by PPSV23 following the 
initiation of AG-348 treatment.] 
10. Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2 (Appendix 15.2). 
11. Patients must be taking at least 1 mg of folic acid daily for at least 21 days prior to first 
dose and continued daily during study participation. 
12. Adequate organ function, defined as: 
a. Serum AST and ALT ≤ 1.5 × upper limit of normal (ULN) (unless the increased 
AST is assessed by the Investigator as due to hemolysis). 
b. Normal or elevated levels of serum bilirubin. In patients with serum bilirubin 
> ULN, the elevation must be attributed to hemolysis with or without Gilbert's 
syndrome and must not be choledocholithiasis, cholecystitis, biliary obstruction, 
or hepatocellular disease. 
c. Serum creatinine ≤ 1.25 × ULN. If serum creatinine > 1.25 × ULN, then 24-hour 
measured or calculated (Cockcroft-Gault) glomerular filtration rate 
(GFR) ≥ 60 mL/min. 
d. Absolute neutrophil count (ANC) > 1.0 × 109/L. 
e. Platelet count ≥ 100 × 109/L. 
f. Activated partial thromboplastin time (aPTT) and international normalized ratio 
(INR) ≤ 1.25 × ULN, unless the patient is receiving therapeutic anticoagulants. 
13. Women of childbearing potential (WOCBP) must agree to abstain from sexual 
intercourse or to use an acceptable/effective method of contraception (i.e., condom plus 
spermicide, condom plus oral contraceptive, condom plus intrauterine device [IUD], 
condom plus diaphragm with spermicide) from as soon as feasible during the Screening 
period until 30 days following the last dose of AG-348. Abstinence is an acceptable 
method only when this is in line with the normal life style of the patient, meaning that the 
patient plans to remain abstinent continuously throughout the duration of the study and 
for at least 30 days after the last dose of study drug. Periodic abstinence, e.g. calendar, 
sympathothermal and post-ovulation methods, and withdrawal are not acceptable 
methods of contraception. 
a. WOCBP include any female who has experienced menarche and who has not 
undergone successful surgical sterilization (hysterectomy, bilateral tubal ligation, 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
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and/or bilateral oophorectomy) or is not post-menopausal. Post-menopausal is 
defined as: 
i. Amenorrhea ≥ 12 consecutive months without another cause, and a 
documented serum follicle stimulating hormone (FSH) level 
> 35 mIU/mL; 
ii. Amenorrhea≥ 12 consecutive months in women ≥ 62 years old (FSH 
testing is not required). 
14. WOCBP must have a negative serum or urine pregnancy test within 72 hours before start 
of AG-348 dosing. 
15. Women must not be breastfeeding.  
16. Male patients, with the exception of those who have undergone vasectomy at least 
6 months prior to Screening, must agree to abstain from sexual intercourse or, if sexually 
active, to use a condom with spermicide as contraception (regardless of their female 
partner's childbearing potential or their partner's use of their own contraception) from 
Day 1 of dosing until 30 days following the last dose of AG-348. Abstinence is an 
acceptable method only when this is in line with the normal life style of the patient, 
meaning that the patient plans to remain abstinent continuously throughout the duration 
of the study and for at least 30 days after the last dose of study drug. Periodic abstinence, 
e.g. selective timing of intercourse based on partner's calendar, sympathothermal and 
post-ovulation methods, and withdrawal are not acceptable methods of contraception. 
For entry into the Extension Period, patients must meet the following criteria: 
17. Signed written informed consent obtained prior to performing any study procedure during 
the Extension Period. 
18. Patient must have completed 24 weeks of treatment during the Core Study and tolerated 
AG-348 (defined as having completed 24 weeks with or without permitted dose 
modifications). 
19. The patient’s treating Investigator agrees that there is a potential for clinical benefit to 
continued treatment and recommends participation in the Extension Period 
20. The Sponsor's designated Medical Monitor or Responsible Medical Officer approves the 
patient’s participation in the Extension Period  
21. As applicable, the patient must agree to continue to follow the same sexual 
abstinence/contraception rules as stated in Inclusion Criteria 13 and 16. 
8.3. 
Exclusion Criteria 
Patients who meet any of the following criteria at Screening or prior to dosing on Day 1 will not 
be enrolled in Core Period: 
1. Hemoglobin level > 12.0 g/dL if male; Hb > 11.0 g/dL if female. 
2. Additional diagnosis of any other congenital or acquired blood disorder, including 
glucose-6-phosphate-dehydrogenase (G6PD) deficiency, or any other hemolytic anemia 
process except for mild allo-immunization as a consequence of transfusion therapy. 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
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3. Iron overload (hemosiderosis or concurrent hemochromatosis) sufficiently severe to 
result in a clinical diagnosis by the Investigator of cardiac, hepatic, or pancreatic 
insufficiency. 
4. Prior bone marrow or stem cell transplant. 
5. Clinically symptomatic cholelithiasis or cholecystitis. (Prior cholecystectomy is not 
exclusionary. Patients with symptomatic cholelithiasis or cholecystitis may be rescreened 
once the disorder has been treated and clinical symptoms have resolved.) 
6. Currently enrolled in another therapeutic clinical trial involving on-going therapy with 
any investigational or marketed product or placebo. Concurrent participation in the 
Pyruvate Kinase Deficiency Natural History Study (NCT02053480) is permitted. 
7. Exposure to any investigational drug, device, or procedure within 28 days prior to 
Screening. 
8. Concurrent medical condition that could compromise participation in the study such as: 
a. Poorly controlled hypertension (defined as systolic blood pressure (BP) 
> 150 mm Hg or diastolic BP > 90 mm Hg) refractory to medical management. 
b. History of recent (within < 6 months from Screening date) congestive heart 
failure; myocardial infarction or unstable angina pectoris; or hemorrhagic, 
embolic, or thrombotic stroke; deep venous thrombosis; or pulmonary or arterial 
embolism. 
c. Currently active infection requiring the use of parenteral anti-microbial agents or 
that is ≥ Grade 3 (CTCAEv4.03) within 6 months of first dose. 
d. A pattern or frequency of post-splenectomy sepsis that in the assessment of the 
Investigator could reasonably be expected to interfere with the ability of the 
patient to complete the 24 week study participation. 
e. Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) 
antibody with signs of active Hepatitis B or C virus infection.  
f. Positive test for human immunodeficiency virus (HIV) 1 or 2 antibody. 
g. Diabetes mellitus judged to be in poor control by the Investigator or requiring 
> 3 anti-diabetic agents counting insulin; use of insulin per se is not exclusionary.  
h. History of any primary malignancy with the exception of: curatively treated non-
melanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; 
or other primary tumor treated with curative intent and no known active disease 
present and no treatment administered during the last 3 years. 
9. Undergone major surgery within 6 months of first dose.  
10. Current or recent history of psychiatric disorder that in the opinion of the Investigator or 
Medical Monitor could compromise the ability of the patient to cooperate with study 
visits and procedures. 
11. Use of any of the restricted list of products known to strongly inhibit CYP3A4 
metabolism (Appendix 15.4, Table 9) within 5 days prior to Day 1 dosing; or to strongly 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
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induce CYP3A4 metabolism (Appendix 15.4, Table 10) within 28 days prior to Day 1 
dosing; or to strongly inhibit P-gp transporter (Appendix 15.4, Table 11) within 5 days 
prior to Day 1 dosing; or digoxin within 5 days prior to Day 1 dosing. For patients who 
require chronic inhaled glucocorticoid therapy, Investigators should confer with the 
Medical Monitor for additional guidance. 
12. Serum bilirubin > ULN attributable to factors other than hemolysis and/or Gilbert's 
syndrome. 
13. Male patients with heart-rate corrected QT interval -Fridericia's method (QTcF) 
interval > 450 msec, or female patients with QTcF interval > 470 msec with the 
exception of patients with a left bundle branch block (LBBB). Medical Monitor approval 
needed in patients with a LBBB. 
14. Cardiac dysrhythmias judged as clinically significant by the Investigator or requiring 
therapy with drugs that are primarily substrates of CYP3A4. 
15. History of allergy to sulfonamides if characterized by acute hemolytic anemia, 
anaphylaxis, or rash of erythema multiforme type or Stevens-Johnson syndrome. 
16. Any other medical or psychological condition deemed by the Investigator to be likely to 
interfere with a patient’s ability to understand and sign informed consent; cooperate with 
study visits, tests, and procedures; or otherwise safely and reliably participate in the 
study. 
Patients will not be allowed to enter the Extension Period if: 
17. The patient experienced AEs during the Core Period that are considered by the treating 
Investigator or the Sponsor’s designated Medical Monitor or Responsible Medical 
Officer to pose a significant safety risk to the patient if treatment were to be extended. 
8.4. 
Patient Identification and Registration 
Patients who are candidates for enrollment into the study will be evaluated for eligibility by the 
Investigator to ensure that the inclusion and exclusion criteria (see Section 8.2 and Section 8.3, 
respectively) have been satisfied and that the patient is eligible for participation in this clinical 
study. The site will submit to the Sponsor an Eligibility form for each eligible patient and the 
Medical Monitor will confirm eligibility for all patients prior to receipt of the first dose of 
AG-348. 
8.5. 
Patient Randomization 
Patients who have been confirmed as eligible will be randomized in an equal ratio to a treatment 
arm (e.g., 1:1 or 1:1:1 depending on which arms are open). The site will provide a request for 
randomization form (including the patient’s confirmed genotype) to the study Medical Monitor. 
The randomization will be stratified by PKR mutation in order to maintain balance across the 
dose arms for the specific mutations expected to be most frequently enrolled. The PKR mutation 
stratification factor will consist of 4 levels (R510Q, R486W, and R479H) and all other mutations 
(“other”). Since this is an open label study, randomization will not be blinded. 
Please refer to the study manual for the randomization procedure. 
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8.6. 
Patient Withdrawal Criteria 
Patients have the right to withdraw from the study at any time for any reason. Patients will be 
withdrawn from study-related procedures and treatments under the following conditions: 
• Withdrawal of consent; 
• Experiences unacceptable toxicity; 
• Development of an intercurrent medical condition that precludes further participation 
in the trial; 
• Patient requires use of a prohibited concomitant medication (Section 9.11.2); 
• Investigator decision; 
• Protocol violation: non-adherence to protocol requirements; 
• Pregnancy; 
• Lost to follow-up. 
Should a patient decide to withdraw, all efforts will be made to complete and report the protocol-
defined study observations up to the time of the patient’s withdrawal as completely as possible 
and to determine the reason for withdrawal. 
In the event a patient is withdrawn from the study, the Medical Monitor must be informed. If 
there is a medical reason for withdrawal, the patient will remain under the supervision of the 
Investigator until satisfactory health is returned. 
When a patient withdraws from the study, the primary reason for discontinuation must be 
recorded in the appropriate section of the electronic case report form (eCRF) and all efforts will 
be made to complete and report final study observations as thoroughly as possible. 
All AEs should be followed until resolution or for a period of 30 days from the last dose of study 
drug, whichever is shorter. Patients with toxicity suspected to be related to study drug will 
continue follow-up until the AE resolves, is declared chronic by the Investigator, or the patient is 
lost to follow-up. 
8.7. 
Replacement of Patients 
Patients who drop out of the Core Period prior to completing the first 12 weeks of assigned 
dosing for reasons other than AEs may be replaced at the Sponsor’s discretion. 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
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9. 
STUDY TREATMENT 
9.1. 
Description of Study Drug 
AG-348 sulfate hydrate capsules will be provided as 5 mg, 25 mg, or 100 mg (free-base 
equivalent) of AG-348 sulfate hydrate without excipients in dark green opaque (5 mg), Swedish 
orange (25 mg), or white opaque (100 mg) gelatin capsules (size 2 capsules for all dose 
strengths). 
All study drugs are for investigational use only and are to be used only within the context of this 
study. All study drug products will be supplied by the Sponsor. Please see the Investigator’s 
Brochure for further details regarding study drug. 
9.2. 
Study Drug Packaging and Labeling 
AG-348 sulfate hydrate capsules are packaged in white, high-density polyethylene (HDPE) 
induction sealed bottles with a child-resistant screw cap. 
Packaging and labeling will be prepared to meet all regulatory requirements. 
9.3. 
Study Drug Storage 
AG-348 sulfate hydrate drug capsules must be stored at room temperature of 15 to 30°C 
(59 - 86°F). 
All study drug products must be stored in a secure, limited-access location and may be dispensed 
only by the Investigator or by a member of the staff specifically authorized by the Investigator. 
9.4. 
Method of Assigning Patients to Treatment 
Up to a maximum of 25 patients will be randomized to any one of the dosing arms in this study. 
Randomization will be balanced 1:1 or 1:1:1, depending on whether 2 or potentially 3 arms are 
open, and will be stratified by PKR mutation (see Section 8.5). The dose and schedule of 
AG-348 each patient receives will be dependent upon which dose arm is open for enrollment 
when the patient qualifies for and is randomized into the study. Patients in the Extension Period 
will continue on the dose they were randomized to in the Core Period, unless the DRT had 
reason to establish a different dose/schedule during the course of the Core Period (the DRT will 
not propose a dose higher than 360 mg q12h).  
9.5. 
Blinding 
This is an open-label study; no blinding methods will be used. 
9.6. 
Study Drug Preparation and Administration 
For the initial two treatment arms, (Arm 1 and Arm 2) in the Core Period, AG-348 will be 
administered orally BID (approximately q12h with a minimum of 10 hours between doses) over 
a 24 week treatment period. Starting with Day 1, dosing will be continuous; there will be no rest 
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periods. Patients who do not meet any of the treatment withdrawal criteria (see Section 8.5) may 
continue treatment for the entire 24-week treatment period. 
Patients will be dispensed the appropriate number of Sponsor-packaged, labeled bottles to allow 
for 28 days of dosing until the next scheduled visit.  
Patients will be given a dosing diary to be used for each 28-day dosing period. They should 
record relevant information regarding their study drug in the diary (e.g., confirmation that each 
daily dose was taken, reasons for missed doses). 
Treatment compliance will be assessed based on return of unused drug and the dosing diary (see 
Section 9.9). 
Patients should be instructed to take their daily dose at approximately the same times each day 
except for dosing on in-clinic visiting days.  
Patients who undergo extensive PK/PD sampling during the Core Period (see Appendix 15.1, 
Table 7) should be instructed from Week 3 on to bring the AM dose with them for in-clinic visits 
and to ingest the dose following PK/PD blood draws.  
Patients receiving limited PK/PD sampling during the Core Period (see Appendix 15.1, Table 8) 
should be instructed to bring the AM dose with them for all in-clinic visits and to take the AM 
dose following PK/PD blood draws.  
Patients receiving extensive PK/PD sampling on Day 1 and 15 will also have limited PK/PD on 
other visit days. As a general rule, regardless of extensive or limited schedule, patients will bring 
in the AM dose for all visits and take this dose following PK/PD blood draws.  
A minimum of 10 hours between the AM and PM dose will be required on those dosing days. 
Each dose should be taken with a glass of water and consumed over as short a time as possible. 
AG-348 may be taken with or without food. Patients should be instructed to swallow capsules 
whole and to not chew the capsules. For patients who have difficulty swallowing tablet(s), the 
Medical Monitor should be contacted to discuss administration. 
Patients will receive their first dose of AG-348 in the clinic on Day 1 and then may take the 
remaining doses on an outpatient basis (except for in-clinic visit days, as described above). 
9.7. 
Criteria for Dose Escalation, Dose Modification, or Discontinuation 
of Study Drug 
No intra-patient dose escalations will be permitted in this study unless the DRT decides to re-
assign patients’ doses and schedule in a terminated arm to match the dose and schedule of 
another arm of the study. In this case, the patients in the terminated arm will remain in their 
originally assigned arm; i.e., they will not count towards the enrollment quota of the arm whose 
dose and schedule is being adopted. All dosing modifications, as outlined below, will be 
implemented following discussions with the Medical Monitor. The same criteria for dose 
modifications or discontinuation of study drug apply in the Extension Period as in the Core 
Period. 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
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9.7.1. 
Dose Modification for Safety 
The Investigator will monitor all patients for safety and tolerability. Modification of the patient’s 
dose of AG-348 will be based on AEs and observed changes in Hb levels (see Section 9.7.2). 
Table 1: 
Dose Modification for Adverse Events Deemed at Least Possibly Related to 
AG-348 
Adverse Events(s) 
AG-348 Dose Adjustment 
Grade 1 
None required. 
Grade 2 
None required; Investigator and Medical Monitor judgment to manage as for 
Grade 3. 
Grade 3 
Suspend dosing;  
If event resolves to Grade 1 or baseline within approximately 14 days of 
suspension, resume dosing with 1 dose level reduction (see Table 2 below).  
If event does not resolve to Grade 1 or baseline within approximately 14 days of 
suspension, permanently discontinue dosing, unless the benefits outweigh the 
risks of resuming treatment and are approved by the Medical Monitor. 
Grade 4 
Permanently discontinue dosing, unless the benefits outweigh the risks of 
resuming treatment and are approved by the Medical Monitor. 
Dose modifications for Grade 2 and 3 AEs should be maintained long enough for the 
Investigator to be confident that the reduced dose is being well tolerated before considering a 
request of the Medical Monitor to permit subsequent re-escalation to the former dose level. 
Dosing for an individual patient will be discontinued permanently for Grade 3 and Grade 4 AEs 
that do not resolve to Grade 1 or baseline within approximately 14 days of suspension of dosing, 
unless the benefits outweigh the risks of resuming treatment and are approved by the Medical 
Monitor. No patients may be re-escalated to their former dose level after a dose modification 
without discussion with the Medical Monitor. If following the first dose reduction for a Grade 3 
AE the patient experiences a second occurrence of the same Grade 3 AE, then treatment with 
AG-348 must be immediately and permanently discontinued.  
It should be noted that if the initial dose of 300 mg BID selected for Arm 1 demonstrates an 
unacceptable safety profile resulting in multiple patients undergoing dose modifications, the 
DRT may exercise its option to re-assign these patients’ dose and schedule to match the dose and 
schedule of another study arm (for example, Arm 2 of the study, or to match the dose and 
schedule of a [potential] Arm 3, if implemented). 
9.7.2. 
Dose Modification for Increase in Hemoglobin Level 
It is presently unknown to what magnitude, how rapidly, or even whether, AG-348 will result in 
increased Hb levels in patients with PK deficiency across the variety of potential PKR mutations 
that may be encountered during this study. Moreover, it is unknown whether any potential 
increases in Hb experienced by patients in this study may be a safety risk. As a conservative 
measure, this study incorporates guidance for dose modification based on potential large 
increases in Hb levels that exceed the midpoint of the typical normal range by gender. The 
Investigator will monitor all patients for changes in Hb levels and should adjust the dose of 
AG-348 as outlined in Table 2 per the following guidelines and with Medical Monitor approval. 
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• Males: If Hb > 15 g/dL and confirmed with a second test, suspend dosing until Hb 
≤ 13.5 g/dL; then resume dosing with a 1 dose level reduction.  
• Females: If Hb > 13.5 g/dL and confirmed with a second test, suspend dosing until 
Hb ≤ 12.5 g/dL; then resume with a 1 dose level reduction.  
• The treating Investigator will discuss with the Medical Monitor questions relating to 
dose modifications on an as needed basis.  
Table 2: 
Dose Reduction Table (by Dosing Arm) 
Dose Group 
Starting Dose 
1st Dose Reduction 
2nd Dose Reduction 
Arm 1 
300 mg BID 
200 mg BID 
100 mg BID 
Arm 2 
50 mg BID 
25 mg BID 
TBD1 
Potential Arm 3 
TBD 
To approximately 
50-66% of initial dose 
To approximately 
25-33% of initial dose 
1 Dose to be determined by Medical Monitor. 
Hemoglobin levels above the ULN by gender should be reported as an AE, graded per the 
CTCAEv4.03, according to the guidance provided in Section 11.2. 
9.7.3. 
Stopping Criteria 
Dosing for an individual patient will be discontinued permanently for Grade 3 AEs that do not 
resolve to Grade 1 or baseline within approximately 14 days of suspension of dosing, and for 
Grade 4 AEs, unless the benefits outweigh the risks of resuming treatment and are approved by 
the Medical Monitor (Section 9.7.1). Other reasons for treatment termination are provided in 
Section 8.5. 
9.8. 
Duration of Patient Participation 
The duration of treatment for all patients will be up to 24 weeks in the Core Period. Patients who 
safely tolerate AG-348 and for whom the Investigator agrees with continuation of AG-348 
treatment may be eligible to immediately roll over to the Extension Period for continued 
treatment (up to 2 years following completion of the Core Period). 
9.9. 
Treatment Compliance 
During in-clinic visits, doses of AG-348 will be ingested by the patient under the supervision of 
clinical facility personnel. For at-home dosing, patients will be given a dosing diary to be used 
for the duration of the 24-week Core Treatment Period; the diary will also be used by patients 
who roll over to the Extension Period. Patients should record relevant information regarding their 
study drug in the diary (e.g., confirmation that each daily dose was taken, reasons for missed 
doses) and return the diary at each study visit. 
9.10. 
Study Drug Accountability 
Accountability for the study drug at the clinical facility is the responsibility of the Investigator. 
The Investigator will ensure that the study drug is used only in accordance with this protocol. 
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Where allowed, the Investigator may choose to assign drug accountability responsibilities to a 
pharmacist or other appropriate individual. 
The Investigator or delegate will maintain accurate drug accountability records indicating the 
drug’s delivery date to the site, inventory at the site, use by each patient, and return to Agios or 
its designee (or disposal of the drug, if approved by Agios). These records will adequately 
document that the patients were provided the doses as specified in the protocol and should 
reconcile all study drug received from Agios. Accountability records will include dates, 
quantities, batch/serial numbers, expiration dates (if applicable), and patient numbers. An 
unblinded monitor will review drug accountability at the site on a schedule agreed to by the 
Sponsor. 
Study drug must not be used for any purpose other than the present study.  
All unused and used study drug will be retained at the site until it is inventoried by the Study 
Monitor after database lock has occurred. All used, unused or expired study drug will be returned 
to Agios or its designee or, if authorized, disposed of at the study site per the site’s Standard 
Operating Procedures (SOPs) and documented. All material containing AG-348 will be treated 
and disposed of as hazardous waste in accordance with governing regulations. 
9.11. 
Prior and Concomitant Medications and Treatments 
9.11.1. 
Prior Medications and Procedures 
All medications administered and procedures conducted within 28 days prior to the first day of 
study drug administration are to be recorded on the source documentation and included in the 
eCRF. 
9.11.2. 
Prohibited Concomitant Therapy 
All concomitant medications and procedures administered from 28 days before administration of 
study drug through the last Follow-up Visit must be recorded in the appropriate section of the 
source documentation and eCRF along with dosage information, dates of administration, and 
reason for use.  
The following are prohibited at all times during participation in this study: 
• Investigational drugs must be discontinued 28 days prior to the first dose of study 
drug; 
• Products known to strongly inhibit CYP3A4 metabolism (listed in Appendix 15.4, 
Table 9) must be discontinued within 5 days prior to Day 1 dosing; 
• Products known to strongly induce CYP3A4 metabolism (listed in Appendix 15.4, 
Table 10) must be discontinued within 28 days prior to Day 1 dosing; 
• Products known to strongly inhibit P-gp transporter (listed in Appendix 15.4, 
Table 11) must be discontinued within 5 days prior to Day 1 dosing; 
• Digoxin must be discontinued within 5 days prior to Day 1 dosing; 
• Hematopoietic stimulating agents (EPOs, granulocyte colony stimulating factors, 
thrombopoietins, etc) must be discontinued no less than 28 days prior to the first dose 
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of study drug. [Folic acid 1 mg orally per day is required for all patients. B12 
injections are permitted for patients with a prior diagnosis of B12 deficiency 
syndromes, but must be repleted to stability of the Hb and mean corpuscular volume 
(MCV) prior to enrollment in the study]; 
• Anabolic steroids, including testosterone preparations, administered for anemia must 
be discontinued no less than 28 days prior to the first dose of study drug;  
• As the target population for this study consists of transfusion independent patients 
and transfusion of blood products could confound key endpoints of the study, blood 
transfusions of any type must be strictly avoided except in cases of compelling 
medical need. If medical circumstances permit, the Medical Monitor should be 
contacted for discussion before any transfusions are administered. 
Drugs that displace unconjugated bilirubin from albumin (including some common sulfa 
antibiotics [including sulfamethoxazole/trimethoprim], cephalosporins, salicylates, and 
aminophylline) should be used with caution with the understanding that patients with elevated 
levels of unconjugated bilirubin may potentially be at risk for kernicterus syndrome (Strauss, et 
al. 2006). 
Potential Drug Interactions 
Strong inhibitors of CYP3A4 (listed in Appendix 15.4, Table 9) are not permitted for use with 
AG-348. Based on modeling of AG-348 metabolism and tolerability data from the Phase 1 MAD 
study, moderate inhibitors of CYP3A4 do not appear to pose a risk to patients dosed with 
AG-348. 
In-vivo data from the Phase 1 MAD show AG-348 induces CYP3A4 and its own metabolism, 
presumably via CYP3A4. The use of strong CYP3A4 inducers in combination with AG-348 is 
expected to reduce AG-348’s efficacy. Therefore, administration of strong CYP3A4 inducers 
(listed in Appendix 15.4, Table 10) is not permitted with AG-348.  
Strong inhibitors of drug transport (listed in Appendix 15.4, Table 11) are not permitted for use 
with AG-348. 
Digoxin is not permitted for use with AG-348. 
Induction of CYP3A4 by AG-348 is expected to reduce the efficacy of certain sensitive co-
medications. Co-administration of AG-348 with the drugs in Appendix 15.4, Table 12 should be 
done with caution, as their efficacy may be reduced.  
Of note, women in the trial utilizing oral contraception must utilize barrier methods as per the 
Inclusion Criteria 14 (Section 8.2) while taking AG-348. 
Short-term (≤ 14 days at a time, and ≤ 28 days total during the 24 week treatment period) use of 
topical, inhaled, intra-nasal, and systemic glucocorticoids is permitted for acute medical 
indications. Every effort should be made to minimize total duration of glucocorticoid therapy and 
utilize alternative treatments. Patients must be off glucocorticoids for at least 28 days prior to 
Day 1 of AG-348 dosing as per Exclusion Criterion #11 (Section 8.3). For patients who require 
chronic inhaled glucocorticoid therapy, Investigators should confer with the Medical Monitor for 
additional guidance. 
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The expected patient co-medications deferoxamine, deferasirox, deferiprone, and oral penicillin 
are not expected to interact with AG-348. 
9.11.3. 
Allowed Concomitant Therapy 
Medications and treatments other than those specified above are permitted during the study. All 
intercurrent medical conditions will be treated at the discretion of the Investigator according to 
acceptable local standards of medical care. Subjects may receive analgesics, antiemetics, anti-
infectives (including penicillins), and antipyretics as medically indicated and consistent with the 
guidance in the previous two sections. Patients may continue iron chelation therapy with 
deferoxamine, deferasirox, or deferiprone. Patients must continue taking at least 1 mg of folic 
acid for the duration of the study. 
All concomitant medications, including transfusions of blood products, procedures performed 
during the study, including those used to treat AEs, will be recorded on the eCRF.  
9.11.4. 
Potential for Phototoxicity 
AG-348 may cause sensitivity to direct and indirect sunlight. Patients should be warned to avoid 
direct sun exposure. When exposure to sunlight is anticipated for longer than 15 minutes, the 
patient should be instructed to apply factor 30 or higher sunscreen to exposed areas and wear 
protective clothing and sunglasses. 
9.11.5. 
Management of Nausea, Vomiting, and Diarrhea 
As the primary objective of this study is to assess the safety and tolerability of up to 24 weeks of 
AG-348, routine use of prophylactic anti-emetic and antidiarrheal or other classes of medications 
is prohibited. However, if patients experience AEs relating to nausea, vomiting, or diarrhea these 
may be treated at the Investigator's clinical discretion with recommended medications as follows: 
• Nausea/vomiting: allowed agents include standard clinical dosing with palonosetron 
(Aloxi), dolasetron (Anzemet), or promethazine (Phenergen). Promethazine is a 
substrate for CYP2B6, and it is presently unknown if the potential for 2B6 induction 
after AG-348 dosing could be sufficient to reduce the therapeutic effect of 
promethazine. Not recommended are aprepitant (Emend; CYP3A4 inhibitor), 
ondansetron (Zofran; CYP3A4 inducer), chlorpromazine (Thorazine; CYP3A4 
inducer), prochlorperazine (Compazine; CYP3A4 substrate, QT prolongation has 
been reported), and granisetron (Kytril; CYP3A4 substrate, QT prolongation has been 
reported);  
• Diarrhea: recommended management includes standard clinical dosing with 
Kaopectate or other non-absorbable anti-diarrheals, diphenoxylate/atropine (Lomotil), 
or loperamide (Imodium). Loperamide is the least preferred choice because it is both 
a substrate and inhibitor for CYP3A4, a substrate for CYP2B6, and a substrate for 
P-gp. 
• For the use of any medications not specifically mentioned above the Investigator may 
confer with the Sponsor’s Medical Monitor. 
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9.11.6. 
Other Restrictions and Precautions 
Patients should be advised to refrain from altering their normal exercise routine for the first 
28 days of treatment with AG-348 as symptoms of anemia could theoretically temporarily 
worsen before the eventual potential beneficial effect of AG-348 on the Hb level becomes 
evident. The elevated blood levels of 2,3-DPG commonly seen in patients with PK deficiency 
may produce a right shift in the Hb-O2 dissociation curve. The effect of this is to enhance 
oxygen delivery at the tissue level, thus counteracting to some extent the physiologic 
consequences of the anemia. AG-348 was shown to produce rapid decreases (within the first 
12 hours following a single dose) in 2,3-DPG in two prior clinical trials with healthy adult male 
and female volunteers. In patients with PK deficiency who have elevated 2,3-DPG levels, it is 
theoretically possible that decreases in 2,3-DPG that precede correction of the anemia could 
result in a temporary decrease of oxygen delivery at the tissue level resulting in clinical 
symptoms (e.g., increased fatigue). 
As discussed in Section 5.2.1.1 of this protocol and in the Investigator Brochure, AG-348 has 
been identified as a histamine H3 receptor antagonist/inverse agonist. No effects of histamine H3 
modulation have been observed in safety pharmacology or toxicology studies. Nonetheless, 
patients should be monitored for potential AEs related to wakefulness and insomnia (Schwartz 
2011). 
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10. 
STUDY ASSESSMENTS 
10.1. 
Schedule of Assessments 
The Schedules of Assessments for this study are provided in Appendix 15.1.  
After obtaining written informed consent, patients will undergo screening evaluations. The 
Screening visit is to be conducted within approximately 28 days prior to first dose of study 
treatment.  
During the Core Treatment Period, patients will attend visits at baseline (Day 1), weekly through 
Week 3 (Days 8, 15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 
12) and monthly through Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate AG-348 
through Week 24 (Core Period) and for whom the Investigator agrees with continuation of 
AG-348 treatment may be eligible to immediately enter the Extension Period for continued 
treatment upon agreement of the treating Investigator and the Medical Monitor or Responsible 
Medical Officer. Study visits for safety and clinical activity assessments will occur 
approximately every 3 months during the Extension Period. All patients will undergo a follow-up 
assessment 4 weeks after the last dose of AG-348, regardless of whether this was due to early 
discontinuation, the last dose in the Core Period for a patient who chooses not to continue in the 
Extension Period, or the last dose of the Extension Period. 
Although not encouraged, as a convenience for patients who travel long distances to the study 
site, in-clinic visits on Day 8 and Day 22 may be performed by the patient’s primary care 
physician if necessary and must be approved by the Sponsor on a case by case basis. For details, 
please refer to Table 5 in Appendix 15.1 : Schedule of Assessments. For patients having their 
Day 8 and/or Day 22 visits performed by the primary care physician, the principal investigator 
will him or herself, or have a qualified research nurse or other designated site staff member, 
make telephone contact with the patient to inquire about any AEs. These must be recorded as if 
the patient appeared in the main study center. The Principal Investigator will exercise prudent 
clinical judgment in determining any clinical course of action to take based on any AEs 
discovered. These telephone contacts on Day 8 and Day 22 must be explained to the patient in 
advance and scheduled in advance to maximize the likelihood of successfully making contact. 
Having in-clinic visits on Days 8 and/or Day 22 performed by the primary care physician will 
necessitate re-scheduling certain assessments that the primary care physician’s office may not be 
reasonably expected to perform. Table 3 summarizes the details of the re-scheduling of these 
assessments as described in Table 5 in Appendix 15.1.  
During the Extension Period, all scheduled visits must be conducted by the Investigator and at 
the participating authorized investigative site; local primary care visits will not be allowed. 
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Table 3: 
Summary of Assessments When Day 8 and/or Day 22 In-Clinic Visits Are 
Performed By Primary Care Physician (Core Period) 
Day 8 Visit by Primary Care Physician 
Day 22 Visit by Primary Care Physician 
Primary 
Care Office 
Main 
Study 
Site 
Assessment 
to Move to 
Day 15 Main 
Study Site 
Assessments 
Not Required  
Primary Care 
Office 
Main 
Study 
Site 
Assessment 
to Move to 
Day 43 Main 
Study Site 
Assessments Not 
Required 
Hematology 
to central lab 
Phone 
contact 
with 
patient 
12-lead ECG 
VS; serum 
chemistry 
Hematology 
sample to 
central lab 
Phone 
contact 
with 
patient 
12-lead ECG 
VS; serum chemistry; 
coagulation, 
haptoglobin, EPO level, 
carboxyhemoglobin, 
PK/PD 
Abbreviations: ECG = electrocardiogram; EPO = erythropoietin; PK/PD = pharmacokinetics/pharmacodynamics; 
VS = vital signs 
Whenever more than one assessment is scheduled for the same nominal time, the assessments 
should be performed in the order of least invasive to most invasive assessment (e.g., VS first, 
ECG, blood draw). The timing of these assessments should allow the PK blood draw to occur at 
the exact nominal time. The order of procedures may be revised with prior discussion between 
Sponsor and Site. 
Minor adjustments to the timing, number of planned safety monitoring procedures (e.g., VS, 
ECG, blood draw), and PK/PD assessments may be made during the course of the study based on 
collected data to ensure appropriate safety monitoring and will not require a protocol 
amendment. These minor changes will require prior approval from the Sponsor’s Medical 
Monitor (or Responsible Medical Officer) as well as appropriate documentation in the study 
records. The addition of new safety monitoring procedures or other assessments will require a 
protocol amendment. 
10.2. 
Informed Consent and Confirmation of Eligibility 
A complete description of the study is to be presented to each potential patient and a signed and 
dated informed consent is to be obtained before any study specific procedures are performed. 
Separate informed consent forms will be used for the Core and Extension Periods. 
Patient’s eligibility will be confirmed at Screening and within 24 hours prior to study treatment 
dose. If a patient is determined to be ineligible, the patient will be excluded from participation. 
10.3. 
Demographic Data, Medical and Medication History 
Patient demographic data, including gender, date of birth, age, race, and ethnicity, will be 
obtained at Screening. Collection of demographic data will be modified by country regulatory 
requirements, as appropriate.  
Medication history, including all relevant prior medical history and current medical conditions, 
will be obtained at the Screening assessment and on Day -1; all concomitant medications 
administered and procedures conducted within 28 days prior to Day 1 should be reported in the 
source documentation and eCRF. Investigators will be asked to provide information on the 
patient’s history of any medical diagnoses (e.g., iron overload) and surgical procedures (e.g., 
splenectomy, cholecystectomy) pertaining to their diagnosis of PK deficiency and prior available 
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complete blood counts (CBCs) over the preceding 6 months and transfusion history over the 
preceding 12 months prior to the date of signing informed consent.  
10.4. 
PKR Enzymatic Assay and PKR Genotyping 
Assessments for PKR enzymatic activity and PKR genotyping will be performed at Screening 
only for confirmation of study eligibility. PKR enzymatic assays will be conducted at 
 
 PKR genotyping will be conducted at 
 
 
10.5. 
Safety Assessments 
10.5.1. 
Physical Examination, Height, and Weight 
A complete physical examination (including neurological examination; genital and rectal 
examinations will be performed at the discretion of the Investigator) will be obtained at 
Screening, Baseline, Week 12, and the Follow up Visit (Week 28). The neurological examination 
must include an assessment of general orientation and mental status including level of alertness 
(assess as normal or abnormal and specify any abnormality). Limited focused physical 
examinations will be performed at all other visits during the Core Period. Focused physical 
examinations, including neurological examination, will continue every 3 months during the 
Extension Period; a complete physical examination will be performed at Month 30. Any findings 
will be recorded on the eCRF. Height will be collected at Screening only. 
10.5.2. 
Vital Signs 
Vital signs, including systolic and diastolic BP, heart rate, respiratory rate, and temperature, will 
be obtained according to the according to the Schedules of Assessments (Appendix 15.1). Vital 
sign measurements will be repeated if clinically significant or machine/equipment errors occur. 
Out of range BP or heart rate measurements will be repeated at the Investigator’s discretion. Any 
confirmed, clinically significant vital sign measurements will be recorded as AEs. 
10.5.3. 
Electrocardiogram  
A 12-lead ECG will be obtained according to the Schedules of Assessments (Appendix 15.1). 
The ECGs will be measured using an ECG machine that calculates the heart rate and measures 
the portion of the ECG wave from the beginning of the P wave to the beginning of the QRS 
interval on an electrocardiogram (QRS) complex (PR), QRS, QT, QTcB (Bazett correction 
formula), and QTcF. Only QTcF (not QTcB) will be used for determination of eligibility. 
The 12-lead ECGs should be obtained following 5 minutes of recumbency. The Screening ECG 
will be performed at least 7 days prior to Day 1 dosing. ECGs will be repeated if clinically 
significant abnormalities are observed, if artifacts are present, or if machine/equipment errors 
occur. Any confirmed, clinically significant ECG findings will be recorded as AEs. 
10.5.4. 
DXA Scans 
DXA scans (hip and spine) will be performed at Screening to obtain T and Z scores and bone 
mineral density that will serve as a baseline measure for all enrolling patients. An additional 
DXA scan for the Core Period will be conducted in the interval between Week 24 and Week 28, 
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and scans will be conducted at Months 18 and 30 during the Extension Period as indicated in the 
Schedules of Assessments (Appendix 15.1). All redacted DXA scan reports must be held at the 
study site and will be collected by the Sponsor. 
10.5.5. 
Safety Laboratory Assessments 
10.5.5.1. Hematology, Serum Chemistry, Coagulation Studies, and Urinalysis 
Laboratory values obtained prior to Screening and RBC antibodies obtained at Screening will be 
performed at a local laboratory. On-study clinical laboratory evaluations are to be performed by 
a central laboratory. If Investigators believe that it is clinically indicated to obtain safety 
laboratory results from their own local laboratories on the day of the subject's visit, and before 
the results are returned from the designated central laboratory, they are free to exercise their 
discretion to do so. Investigators should be aware that since the Hb level is a secondary endpoint 
of the study, it behooves them to minimize blood volumes drawn, and samples for the central lab 
must still be collected, since it will serve as the official lab result for this study. 
Clinical laboratory evaluations are to be collected according to the Schedules of Assessments 
(Appendix 15.1). In addition, all clinically significant laboratory abnormalities noted on testing 
will be followed by repeat testing and further investigated according to the judgment of the 
Investigator. Please note that serum estradiol, free and total testosterone, and CBC will be 
collected in the AM at any 2 time points during Screening at least 2 days apart in addition to 
Baseline/Day 1 (total of 3 time points prior to Day 1 dosing).  
The following safety laboratory parameters are to be determined: 
Hematology: 
HCT, Hb, RBC count, absolute reticulocyte count, percent 
reticulocyte count, MCV, mean corpuscular Hb (MCH), mean 
corpuscular Hb concentration (MCHC), red cell distribution width 
(RDW), white blood cell (WBC) count with automated (or manual, 
as indicated) differential, ANC, and absolute lymphocyte count 
(ALC), and platelet count. In the event that the designated central 
laboratory for the study is unable to provide a valid result for any 
specific component of the defined CBC for a specific patient, the site 
may be asked to have the test performed at their local laboratory. 
The result of the local CBC will be entered into the study database 
along with the local normal reference range. G6PD and RBC 
antibody screen will be performed at Screening only 
Other 
EPO, Hp, COHb, 25-hydroxy vitamin D2 and D3. 
Serum Chemistry: 
alkaline phosphatase (ALP), sodium, potassium, chloride, calcium, 
magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, 
albumin, total protein, glucose, blood urea nitrogen (BUN), 
creatinine, uric acid, LDH, ALT, AST, total bilirubin, indirect 
bilirubin, (and estimated creatinine clearance or GFR for screening 
only, as appropriate). 
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Sex Hormones: 
testosterone (total and free), estrone, and estradiol. FSH will only be 
performed at Screening for female patients only for confirmation of 
post-menopausal status. 
Bone Turnover 
serum osteocalcin-N-mid and CTX. 
Lipids 
total cholesterol, HDL-C, triglycerides. 
Iron Panel 
iron, total iron-binding capacity (TIBC), transferrin saturation, 
ferritin. 
Coagulation Studies: 
fibrinogen, aPTT, INR. 
Urinalysis: 
color and appearance, pH, specific gravity, ketones, protein, glucose, 
bilirubin, nitrite, urobilinogen, pregnancy screen (dipstick), occult 
blood. Microscopic inspection of sediment will only be performed 
for cause or to investigate an abnormal dipstick finding per the 
Investigator’s discretion. 
 
10.5.5.2. Screening Serology 
A blood sample for serology, including HBsAg, HCV Ab screen, and HIV1 and HIV2 Ab, is to 
be collected from all patients at Screening.  
10.5.6. 
Menstrual Cycle Diary 
Menstruating female patients will be required to fill out a paper-based menstrual cycle diary each 
month in order to monitor any changes. Diaries will be dispensed and collected as indicated in 
the Schedules of Assessments (Appendix 15.1). Patients will record the start date, stop date, and 
any notable characteristics of each menstrual cycle. 
10.5.7. 
Adverse Events 
Each patient will be carefully monitored for the development of any AEs throughout the study 
from signing of the informed consent through all scheduled study follow-up visits, or withdrawal 
of consent, whichever occurs first. In addition, SAEs that are assessed as possibly or probably 
related to study treatment that occur > 30 days post-treatment also are to be reported. 
Adverse events will be evaluated by the Investigator and recorded as described in the Schedules 
of Assessments. On dosing visits, all AEs (elicited and spontaneously reported) will be 
continuously evaluated by the Investigator and recorded. At any non-dosing day visit, AEs will 
be evaluated by the Investigator and recorded. Any AEs already documented at a previous 
assessment and designated as ongoing will be reviewed at subsequent visits or assessment time 
points as necessary. If these AEs have resolved, this will be documented. 
All AEs will be graded using the CTCAE grading system (Appendix 15.3 ). 
Complete details on AE monitoring are provided in Section 11. 
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10.6. 
Pharmacokinetic Assessments 
The first approximately 10 patients treated in the Core Period, contingent on clinical site 
feasibility, will undergo extensive PK sampling as detailed in Appendix 15.1, Table 7. The 
remainder of treated patients will undergo limited PK sampling as detailed in Appendix 15.1, 
Table 8. The in-clinic visit on Day 22 may be performed by the patient’s primary care physician 
if necessary and must be approved by the Sponsor on a case by case basis. In this instance, PK 
sampling will not be required on Day 22. (Additional details regarding Day 8 and Day 22 visits 
performed by the patient’s primary care physician can be found in Table 5 in Appendix 15.1: 
Schedule of Assessments: Core Period.) During the Extension Period, predose PK samples will 
be drawn for the measurement of trough levels of AG-348 and AGI-8702 at each study visit 
(every 3 months; see Appendix 15.1, Table 6). 
The collection times for post-dose PK samples will start from the time that dosing is completed. 
(For example, a PK draw at 30 minutes will be collected 30 minutes after the last capsule has 
been ingested.) The completion time of each dose will be recorded. 
Procedures for sample collection and processing will be provided in a separate study manual. 
The actual time point of each sample collection will be recorded. 
Samples for PK and PD assessments may be retained for up to 2 years from collection.  
10.7. 
Pharmacodynamic Assessments 
The first approximately 10 patients treated in the Core Period, contingent on clinical site 
feasibility, will undergo extensive PD sampling for 2,3-DPG and ATP as detailed in 
Appendix 15.1, Table 7. The remainder of treated patients will undergo limited PD for 2,3-DPG 
and ATP sampling as detailed in Appendix 15.1, Table 8. During the Extension Period, predose 
PD samples will be drawn for the measurement of trough levels of 2,3-DPG, ATP, 
 
 at each study visit (every 3 months; see Appendix 15.1, Table 6). 
The collection times for post-dose PD samples will start from the time that dosing is completed. 
(For example, a PD draw at 30 minutes will be collected 30 minutes after the last capsule has 
been ingested.) The completion time of each dose will be recorded. 
Procedures for sample collection and processing will be provided in a separate study manual. 
The actual time point of each sample collection will be recorded. 
Pharmacodynamic assessments during the Core Period will include 2,3-DPG, ATP,
 
 
 The in-clinic visit on Day 22 may be performed by the patient’s primary 
care physician if necessary and must be approved by the Sponsor on a case by case basis. In this 
instance, PD sampling will not be required on Day 22. (Additional details regarding Day 8 and 
Day 22 visits performed by the patient’s primary care physician are stated in Table 5 in 
Appendix 15.1: Schedule of Assessments.) 
Figure 4 provides a brief schematic outlining the PKR reaction and how each of these PD 
assessments fits into a complete mechanistic understanding of the action of AG-348. 
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Figure 4: 
PKR Enzymatic Reaction 
 
The PKR enzyme catalyzes the PEP to pyruvate reaction, with concomitant formation of ATP. 
• Binding of AG-348 to the PKR tetramer can be assessed through an ex-vivo 
biochemical assay of cell lysates from AG-348 treated patients. Because WBCs 
contain a high level of pyruvate kinase from a non-PKR pyruvate kinase isoform, 
WBCs are first removed by filtration before the purified red cells are frozen.
 
 
It has been reported in the literature that there may be 
compensatory expression of PKM2 in the RBCs of some patients with PKR 
deficiency (Black, et al. 1979; Kahn, et al. 1975; Rijksen, et al. 1990).  Therefore, 
levels of PKM2 and appropriate reference proteins (e.g. actin, GAPDH) may be 
evaluated in these whole blood samples. 
• 
 
 
 
 
 
 
 
 
 
 
• AG-348 target engagement 
 has been 
shown in preclinical models and healthy volunteer clinical studies to result in 
accumulation of ATP and depletion of the upstream metabolite 2,3-DPG. Therefore, 
levels of these metabolites will be measured by mass spectrometry in frozen whole 
blood samples. 
The first approximately 10 patients treated during the Core Period, contingent on clinical site 
feasibility, will undergo extensive PD sampling as detailed in Appendix 15.1, Table 7. The 
remainder of treated patients will undergo limited PD sampling as detailed in Appendix 15.1, 
Table 8. The collection times for post-dose PD samples will start from the time that dosing is 
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completed. (For example, a PD draw at 30 minutes will be collected 30 minutes after the last 
capsule has been ingested.) The completion time of each dose will be recorded. 
Blood samples will be stored at the site and regularly transported at -80°C ±10 C to the 
bioanalytical laboratory for analysis. Procedures for sample collection and processing will be 
provided in a separate study manual. 
10.8. 
Ordering of Blood Sample Collection 
When more than 1 blood sample is collected at the same nominal time, the samples will be 
collected in the following order: 
1. Safety laboratory assessments 
2. PK  
3. PD (2,3 DPG, ATP) 
 
 
 
10.9. 
Sample Processing, Storage, and Shipment 
Instructions for the processing, storage and shipment of all study samples for central analysis 
will be provided in a separate study manual. 
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11. 
ADVERSE EVENTS 
Monitoring of AEs will be conducted throughout the study. Adverse events and SAEs will be 
recorded in the source documentation and eCRF from time of the signing informed consent 
through the Follow-up Visits for randomized patients. All AEs should be monitored until they 
are resolved or are clearly determined to be due to a patient’s stable or chronic condition or 
intercurrent illness(es). 
All SAEs will be followed through 30 days after the last dose of study treatment or until the SAE 
has resolved. Any SAEs that are assessed as possibly or probably related to study treatment that 
occur > 30 days post-treatment also are to be reported. 
11.1. 
Definition of Adverse Events 
11.1.1. 
Adverse Event 
An AE is any untoward medical occurrence associated with the use of a drug in humans, whether 
or not considered drug related. An AE (also referred to as an adverse experience) can be any 
unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a drug, without any judgment about causality. An AE can 
arise from any use of the drug (e.g., off-label use, use in combination with another drug) and 
from any route of administration, formulation, or dose, including an overdose. 
11.1.2. 
Suspected Adverse Reaction 
A suspected adverse reaction is any AE for which there is a reasonable possibility that the drug 
caused the AE. For the purposes of expedited safety reporting, ‘reasonable possibility’ means 
that there is evidence to suggest a causal relationship between the drug and the AE.  
11.1.3. 
Unexpected Adverse Event 
An unexpected AE is one for which the nature or severity of the event is not consistent with the 
applicable product information, e.g., the Investigator’s Brochure. 
11.1.4. 
Serious Adverse Event 
An AE or suspected adverse reaction is considered serious (SAE) if, in the view of either the 
Investigator or Sponsor, it results in any of the following outcomes: 
• Death; 
• Life-threatening. Life-threatening means that the patient was at immediate risk of 
death from the reaction as it occurred, i.e., it does not include a reaction which 
hypothetically might have caused death had it occurred in a more severe form; 
• In-patient hospitalization or prolongation of existing hospitalization. Hospitalization 
admissions and/or surgical operations scheduled to occur during the study period, but 
planned prior to study entry are not considered AEs if the illness or disease existed 
before the patient was enrolled in the study, provided that it did not deteriorate in an 
unexpected manner during the study (e.g., surgery performed earlier than planned); 
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• A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions; 
• Congenital anomaly/birth defect; 
• Important medical event. An important medical event is an event that may not result 
in death, be life-threatening, or require hospitalization but may be considered an SAE 
when, based upon appropriate medical judgment, it may jeopardize the patient or 
patient and 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 convulsions that do not result in in-patient hospitalization, 
or the development of drug dependency or drug abuse. 
11.1.4.1. Potential Severe Drug-Induced Liver Injury 
The document entitled FDA Guidance for Industry, Drug-Induced Liver Injury: Premarketing 
Clinical Evaluation (FDA 2009) provides guidance on how the measurement of various 
laboratory parameters may be used to assess a given drug’s potential to cause severe liver injury 
(i.e., irreversible liver failure that is fatal or requires liver transplantation). Such cases are 
suggested by the following three components: 
1. The drug causes hepatocellular injury, generally shown by a higher incidence of 3-fold or 
greater elevations above the ULN of ALT or AST than the (non-hepatotoxic) control 
drug or placebo; 
2. Among trial patients showing such aminotransferase elevations, often with 
aminotransferases much greater than 3×ULN, one or more also show elevation of serum 
total bilirubin to > 2×ULN, without initial findings of cholestasis (elevated serum ALP); 
3. No other reason can be found to explain the combination of increased aminotransferases 
and total bilirubin, such as viral hepatitis A, B, or C; pre-existing acute liver disease; or 
another drug capable of causing the observed injury.  
Clinical safety laboratory results compatible with the definition of drug-induced liver injury 
(DILI) stated above must be repeated for confirmation as soon as possible, and if confirmed, will 
be scored as an unacceptable AE and reported to FDA as a serious unexpected AE. 
11.2. 
Procedures for Reporting Adverse Events and Serious Adverse 
Events  
Each patient must be carefully monitored for the development of any AEs. This information 
should be obtained in the form of non-leading questions (e.g., “How are you feeling?”) and from 
signs and symptoms detected during each examination, observations of study personnel, and 
spontaneous reports from patients. 
All AEs (serious and non-serious) spontaneously reported by the patient and/or in response to an 
open question from study personnel or revealed by observation, physical examination, or other 
diagnostic procedures will be recorded in the source documentation and eCRF. Any clinically 
relevant deterioration in laboratory assessments or other clinical findings is considered an AE 
and must be recorded in the appropriate source documentation and eCRF. Treatment-emergent 
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abnormal clinical laboratory results should generally be reported as AEs if there are 
accompanying symptoms; if additional diagnostic evaluations or medical (including drug 
therapy) or surgical interventions are undertaken; if a change in study drug dosing or study drug 
discontinuation is required; or, if the laboratory result is considered clinically significant by the 
Investigator. Although it is an objective of this study to determine if treatment with AG-348 
results in increased Hb levels in patients with PK deficiency, overshoot of the Hb level above the 
ULN by gender is not recommended and should be reported as an AE, graded per the 
CTCAEv4.03. When possible, signs and symptoms indicating a common underlying pathology 
should be noted as one comprehensive event. 
All SAEs that occur during the course of the study must be promptly reported by the Investigator 
to 
 Global Safety and Pharmacovigilance (see below). Deaths and AEs assessed 
as life-threatening are to be reported immediately and SAEs that meet other criteria are to be 
reported within 24 hours from the point in time when the Investigator becomes aware of the 
SAE. All SAEs must be reported whether or not they are considered causally related to AG-348. 
Serious AE forms will be completed and the information collected will include subject number, a 
narrative description of the event, and an assessment by the Investigator as to the severity of the 
event and relatedness to study drug. Follow-up information on the SAE may be requested by the 
Sponsor or Medical Monitor. 
Contact Information: 
 Safety Management 
SAE Hotline (United States): 
 
SAE Fax (United States): 
 
SAE Hotline (France): 
 
SAE Fax (France): 
  
SAE Hotline (Netherlands): 
 
SAE Fax (Netherlands): 
 
SAE Hotline (Italy): 
 
SAE Fax (Italy): 
 
SAE Hotline (United Kingdom): 
 
SAE Fax (United Kingdom): 
 
E-mail: 
 
If there are serious, unexpected adverse drug reactions associated with the use of AG-348, the 
Sponsor will notify the appropriate regulatory agency(ies) and all participating Investigators on 
an expedited basis. The local IRB/IEC will be promptly notified based on local regulations 
where required by the IRB/IEC of all serious, unexpected adverse drug reactions involving risk 
to human subjects. 
All AEs, whether serious or not, will be described in the source documents in the database. All 
new events, as well as those that worsen in intensity or frequency relative to baseline, which 
occur after signing the informed consent through the final Follow-up Visit (Day 29 ± 3 days) 
must be recorded. Adverse events that are ongoing at the time of treatment discontinuation 
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should be followed up to 30 days after the last dose of study treatment. All SAEs will be 
followed up to 30 days after the last dose of study treatment, or until the SAE has resolved.  
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); 
• The date of onset of the event; 
• The date of resolution of the event; 
• Whether the event is serious or not; 
• Intensity of the event (see below for definitions); 
• Relationship of the event to study treatment (see below for definitions); 
• Action taken: none; change in the study drug administration (e.g., temporary 
interruption in dosing); drug treatment required; non-drug treatment required; 
hospitalization or prolongation of hospitalization required (complete SAE page); 
diagnostic procedure performed; patient discontinued from the study (complete End 
of Study visit); 
• Outcome: patient recovered without sequelae; patient recovered with sequelae; event 
ongoing; patient died (notify the Medical Monitor immediately, and complete the 
SAE form). 
Intensity of all AEs will be graded according to the NCI CTCAE Version 4.03 (Appendix 15.1). 
Relationship to study drug administration will be determined by the Investigator according to 
the following criteria: 
• Not Related:  Exposure to the study treatment did not occur, or the occurrence of the 
AE is not reasonably related in time, or the AE is considered unlikely to be related to 
the study treatment. 
• Possibly Related:  The study treatment and the AE were reasonably related in time, 
and the AE could be explained equally well by causes other than exposure to the 
study treatment. An attribution of possibly related means that there are facts in 
evidence to suggest a possible relationship. 
• Probably Related:  The study treatment and the AE were reasonably related in time, 
and the AE was more likely explained by exposure to the study treatment than by 
other causes, or the study treatment was the most likely cause of the AE. An 
attribution of probably related means that there are facts in evidence to suggest a 
probable relationship. 
For the purpose of safety analyses related to final database review, all AEs that are classified as 
possible or probable will be considered treatment-related AEs. 
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11.3. 
Pregnancy Reporting 
Pregnancy is neither an AE nor an SAE, unless a complication relating to the pregnancy occurs 
(e.g., spontaneous abortion, which may qualify as an SAE). However, any pregnancy in a 
participating female patient or a female partner of a participating male patient that occurs during 
this study or within 30 days following the last dose of AG-348 must be reported to the Sponsor 
or Medical Monitor within 30 days of being notified of the pregnancy. The Investigator must 
follow up and document the course and outcome of all pregnancies even if the patient was 
discontinued from the study or if the study has finished. The female patient or partner of a male 
patient should receive any necessary counseling regarding the risks of continuing the pregnancy 
and the possible effects on the fetus. Monitoring should continue until conclusion of the 
pregnancy. 
All outcomes of pregnancy must be reported by the Investigator to the Sponsor or Sponsor’s 
designee on a Pregnancy Outcome Report form within 30 days after he/she has gained 
knowledge of the delivery or elective abortion. 
Any SAE that occurs during pregnancy must be recorded on the SAE report form (e.g., maternal 
serious complications, spontaneous or therapeutic abortion, ectopic pregnancy, stillbirth, 
neonatal death, congenital anomaly, or birth defect) and reported within 24 hours in accordance 
with the procedure for reporting SAEs. 
All patients, male and female, must agree to use effective contraception during the entire study 
and for 30 days following the last dose of AG-348. Abstinence is an acceptable method only 
when this is in line with the normal life style of the patient, meaning that the patient plans to 
remain abstinent continuously throughout the duration of the study and for at least 30 days after 
the last dose of study drug. Periodic abstinence, e.g. calendar, sympathothermal and post-
ovulation methods, and withdrawal are not acceptable methods of contraception. 
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12. 
STATISTICAL METHODS 
The primary objective of this Phase 2 study is to evaluate the safety and tolerability of up to 
24 weeks of AG-348 in patients with PK deficiency. Therefore, analyses will be primarily 
descriptive in nature; no formal hypothesis testing will be conducted. All analyses will be 
conducted separately within each dose arm, or pooled where appropriate. Analyses of safety and 
of indicators of clinical activity will be conducted for the Core Period, Extension Period, and 
overall, if applicable. For the Core Period, the data to be analyzed will include all collected data 
through 24 weeks of treatment for patients who directly enter the Extension period. For patients 
who do not enter the Extension Period, the analyses will include all collected data through the 
duration of treatment (24 weeks or less) plus 4 week follow up data. For patients who move 
directly from the Core to the Extension Period, the 4 week follow up data will be analyzed as 
part of the Extension Period. 
12.1. 
Sample Size Estimation 
Due to the rare disease setting, the minimal sample size in each dose arm may be determined by 
feasibility. In order to evaluate the primary objective of safety and tolerability of AG-348 in 
Arm 1 and Arm 2, up to a maximum of 25 patients may be randomized onto each arm. The 
actual number of patients enrolled into Arms 1 and 2 will depend on the safety reviews and 
decisions made by the DRT. In addition, up to 25 additional patients may be enrolled to evaluate 
an additional dose arm (Arm 3; see Section 7.1). As for Arms 1 and 2, the actual enrollment in a 
potential Arm 3 will depend on the safety reviews and decisions made by the DRT. Therefore, up 
to approximately 75 total patients may be enrolled in this study across 2 to 3 dose arms. 
Table 4 provides the probability within a dose arm of detecting 1 or more AEs with varying 
sample size and the true underlying AE rates. 
Table 4: 
Sample Size Estimation 
Sample Size 
True Underlying AE Rate 
15% 
10% 
5% 
5 
56% 
41% 
23% 
10 
80% 
65% 
40% 
15 
91% 
79% 
54% 
25 
98% 
93% 
72% 
 
12.2. 
Populations for Analysis 
The following patient populations (i.e., analysis sets) will be evaluated and used for presentation 
of the data: 
• Safety Analysis Set: All patients who are enrolled and receive any dose of study 
treatment. The Safety Analysis Set will be the primary set for the analysis of safety 
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data. Patients will be classified according to treatment received, where treatment 
received is defined as the assigned treatment if it is received at least once, or as the 
first treatment received if assigned treatment is never received. Unless otherwise 
stated, the Safety Analysis Set will be the default analysis set for all data analyses. 
• Pharmacokinetic Analysis Set: All patients in the Safety Analysis Set with sufficient 
plasma sample data to assess PK parameters. Results of the potential PD activity of 
AG-348 will also be based on the PK analysis set.  
• Efficacy Analysis Set: All patients who are enrolled and achieve at least 50% 
compliance with their assigned dose intensity for at least 4 weeks of continuous 
dosing. Additional efficacy analyses may be performed on subsets of patients with 
other degrees of compliance with their assigned dose intensity and/or duration of 
dosing. The Efficacy Analysis Set will be the primary set for the analysis of 
preliminary clinical activity data. Patients will be classified according to assigned 
treatment. 
 
 
 If such analyses are performed, they will be 
described in a separate PK Statistical Analysis Plan (SAP) and may be reported separately in a 
stand-alone report. 
12.3. 
Procedures for Handling Missing, Unused, and Spurious Data 
No imputation will be performed for missing data elements. When summarizing AE data, partial 
dates will be imputed as described in the SAP. Additional rules addressing the handling of 
missing data will be detailed in the SAP. 
12.4. 
Interim Analysis 
No formal statistical interim analysis will be conducted. Safety data will be reviewed on an 
ongoing basis by the DRT, who will meet to review safety, PK, PD, and preliminary clinical 
activity data at regular intervals (approximately every 6 weeks during the Core Period and 
approximately every 3 months during the Extension Period once all patients have completed the 
Core Period) throughout the duration of the Core Period. The DRT’s decisions to suspend, 
terminate, or open a potential third dosing arm, or re-assign patients’ dosing in a terminated arm 
to match the dose and schedule of another arm of the study will be based on the totality of the 
data including, safety, PK, PD, and preliminary clinical activity (e.g., changes in Hb levels). 
Additional interim reviews of data may be conducted to support decision making concerning the 
current clinical study, the sponsor’s development programs in general, or in case of any safety 
concerns. 
12.5. 
Statistical Methodology 
12.5.1. 
General Methods 
This study will be primarily descriptive in nature; therefore, there will be no formal hypothesis 
testing. Summaries will be produced for disposition, baseline disease characteristics and 
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demographic data including genotype, safety measurements, PK, PD parameters and indicators 
of clinical activity. Data from each AG-348 dose group will be analyzed separately, and pooled 
across all dose groups where appropriate. All data will also be listed by individual patient. 
Categorical variables will be summarized by frequency distributions (number and percentages) 
and continuous variables will be summarized by descriptive statistics (mean, standard deviation 
[SD], median, minimum, and maximum). 
The study database will be locked and statistical analysis will be performed after all patients 
have completed the Core Period. The results of this analysis will be presented in a clinical study 
report (CSR). Additional data collected during the Extension Period after the CSR database lock 
will be analyzed for inclusion in a subsequent CSR addendum. 
12.5.2. 
Disposition 
A summary of the disposition of patients will be presented, including the number enrolled, the 
number treated, and the reasons for study discontinuation. Entry criteria and protocol deviations 
will be listed. 
12.5.3. 
Exposure and Safety Analyses 
Patients will receive multiple PO doses of AG-348 over a 24-week treatment period. The actual 
dose and duration in days of AG-348, as well as the dose intensity (computed as the ratio of 
actual dose received and actual duration) and the relative dose intensity (computed as the ratio of 
dose intensity and planned dose received/planned duration) will be listed and summarized using 
descriptive statistics by dose arm. 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) 
and the incidence of TEAEs (new or worsening from baseline) will be summarized by primary 
system organ class (SOC), preferred term, CTCAE Version 4.03 severity, outcome, action taken 
with study drug, and relationship to the study drug by dose group. Separate summaries will be 
produced for all TEAEs, treatment-related AEs (those considered by the Investigator as at least 
possibly drug related), SAEs, discontinuations due to AEs, and AEs ≥ Grade 3 severity. 
Individual patient listings will be provided for deaths, SAEs, AEs leading to interruption and/or 
reduction of study drug dose, and AEs leading to discontinuation of treatment. 
Analyses of AEs will be performed for those events that are considered treatment-emergent, 
where treatment-emergent is defined as any AE with onset beginning at the day of first 
administration of study drug, throughout the treatment period until 30 days after the last dose of 
study drug, or any event that was present at baseline but worsened in intensity or was 
subsequently considered drug-related by the Investigator through the end of the study. 
Descriptive statistics will be provided for clinical laboratory values (e.g., hematology, serum 
chemistry, coagulation studies, urinalysis) and VS data, presented as both actual values and 
changes from baseline relative to each on-study evaluation. Shift analyses will be conducted for 
selected laboratory parameters based on the baseline CTCAE grade to maximum CTCAE grade. 
Where applicable CTCAE terms do not exist, a grading system based on the upper and/or lower 
limits of normal will be used to classify the results. 
No formal hypothesis-testing analysis of AE incidence rates will be performed. 
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Electrocardiogram analyses will include individual patient listings and summaries of abnormal 
and clinically significant ECG results. Actual values and changes from baseline in PR, QRS, and 
heart-rate corrected QT interval (QTc) intervals will be summarized by visit and dose arm. Full 
details of the QTc analysis including correction methods used will be described in the SAP. 
Data collected from the menstrual diaries such as the start and stop dates of the menses and the 
patient reported characteristics of the menses will be presented in a by-patient listing. Additional 
descriptions of the data may also be performed. 
Concomitant medications will be coded using the WHO Drug Dictionary. Concomitant 
medications prior to and after the start of the study drug will be listed by patient, and 
summarized by ATC term and dose arm. 
12.5.4. 
Pharmacokinetic Analyses 
Descriptive statistics will be used to summarize PK parameters for the parent compound AG-348 
and the metabolite AGI-8702 for each dose group, and where appropriate, for the entire 
population. Pharmacokinetic parameters will be summarized using the following descriptive 
statistics: n, mean, SD, coefficient of variation %, median, minimum and maximum, geometric 
mean, and geometric coefficient of variation. PK analyses will be described in a separate PK 
SAP. 
12.5.5. 
Pharmacodynamic Analyses 
Descriptive statistics will be used to summarize PD parameters for 2,3-DPG and ATP for each 
dose group, and where appropriate for the entire population. Pharmacodynamic parameters will 
be summarized using the following descriptive statistics: n, mean, SD, coefficient of variation %, 
median, minimum, and maximum, geometric mean, and geometric coefficient of variation %. PD 
analyses will be described in a separate PD SAP. 
12.5.6. 
Aromatase Hormone Analysis  
The analyses of serum sex hormones will use appropriate graphic displays and statistical 
analyses to evaluate patient change from baseline for each parameter. These analyses will 
include summaries of actual values and change from baseline using appropriate descriptive 
statistics (mean, SD, median, min and max) as described above.  
 
These analyses will present information by each dose arm, and analyses of a pooled 
AG-348 cohort. Additional details regarding these analyses will be provided in the SAP. 
12.5.7. 
Clinical Activity 
Details on analyses to evaluate indicators of potential clinical activity of AG-348 in patients with 
PK deficiency will be described in the SAP. These will include changes in Hb, HCT, reticulocyte 
count, Hp, COHb, LDH, total and indirect bilirubin, EPO, ferritin, and transferrin saturation 
(serum iron/iron binding capacity). Characterization of Hb response which will include, but is 
not limited to percent of patients with increase in Hb, time to Hb response, and duration of Hb 
response will be explored. 
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12.6. 
Procedures for Reporting Deviations to Original Statistical Analysis 
Plan 
All deviations from the original SAP will be provided in the final CSR. 
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13. 
ADMINSTRATIVE REQUIREMENTS 
13.1. 
Good Clinical Practice 
The study will be conducted in accordance with the ICH for Good Clinical Practice (GCP) and 
the appropriate regulatory requirement(s). The Investigator will be thoroughly familiar with the 
appropriate use of the study drug as described in the protocol and Investigator’s Brochure. 
Essential clinical documents will be maintained to demonstrate the validity of the study and the 
integrity of the data collected. Master files should be established at the beginning of the study, 
maintained for the duration of the study, and retained according to the appropriate regulations. 
13.2. 
Ethical Considerations 
The study will be conducted in accordance with ethical principles founded in the Declaration of 
Helsinki (Appendix 15.5). 
The Investigator must obtain IRB approval for the investigation and must submit written 
documentation of the approval to the Sponsor before he or she can enroll any patient into the 
study. The IRB will review all appropriate study documentation in order to safeguard the rights, 
safety, and well-being of the patients. The study will only be conducted at sites where IRB 
approval has been obtained. The protocol, Investigator’s Brochure, informed consent, 
advertisements (if applicable), written information given to the patients, signed Dose Escalation 
Interim Safety Reports, annual progress reports, and any revisions to these documents will be 
provided to the IRB. The IRB is to be notified of any amendment to the protocol in accordance 
with local requirements. Progress reports and notifications of serious unexpected adverse drug 
reactions are to be provided to the IRB according to local regulations and guidelines. 
13.3. 
Patient Information and Informed Consent 
The Investigator or trained designee will ensure that the patient is given full and adequate oral 
and written information about the nature, purpose, possible risk, and benefit of the study. Patients 
must also be notified that they are free to discontinue from the study at any time. The patient 
should be given the opportunity to ask questions and allowed time to consider the information 
provided. 
After the study has been fully explained, written informed consent will be obtained from the 
patient prior to study participation.  
The patient’s signed and dated informed consent must be obtained before conducting any study-
related procedures. The Investigator must maintain the original, signed consent form. A copy of 
the signed form must be given to the patient. 
The method of obtaining and documenting the informed consent and the contents of the consent 
will comply with ICH-GCP and all applicable regulatory requirement(s). 
13.4. 
Patient Confidentiality 
In order to maintain patient privacy, all source documents, study drug accountability records, 
study reports and communications will identify the patient by the assigned patient number. The 
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Investigator will grant monitor(s) and auditor(s) from the Sponsor or its designee and regulatory 
authority(ies) access to the patient’s original medical records for verification of data gathered on 
the source documents and to audit the data collection process. The patient’s confidentiality will 
be maintained and will not be made publicly available to the extent permitted by the applicable 
laws and regulations. 
13.5. 
Protocol Compliance 
The Investigator will conduct the study in compliance with the protocol. Modifications to the 
protocol should not be made without agreement of both the Investigator and the Sponsor. 
Changes to the protocol will require written IRB approval/favorable opinion prior to 
implementation, except when the modification is needed to eliminate an immediate hazard(s) to 
patients. The IRB may provide, if applicable, where regulatory authority(ies) permit, expedited 
review and approval/favorable opinion for minor change(s) in ongoing studies that have the 
approval/favorable opinion of the IRB. The Sponsor or designee will submit all protocol 
modifications to the regulatory authority(ies) in accordance with the governing regulations.  
When immediate deviation from the protocol is required to eliminate an immediate hazard(s) to 
patients, the Investigator will contact the Sponsor’s Medical Monitor (or Responsible Medical 
Officer), if circumstances permit, to discuss the planned course of action. Any departures from 
the protocol must be fully documented in the source documents/database. 
13.6. 
Data Management 
A paper or eCRF will be completed for each patient or an electronic data capture (EDC) system 
will be used. The EDC system (Medidata Rave®) is a software tool designed to ensure quality 
assurance and facilitate data capture during clinical trials. Through a system regulated workflow 
that includes barcode scanning and interfaces to medical equipment to avoid manual data entry, 
study operations performance is controlled and captured in real time. The system is fully Code of 
Federal Regulations (CFR) 21 Part 11 compliant. Source documentation supporting the data 
should indicate participation in the study and should document the dates and details of study 
procedures, AEs, and patient status. The Investigator, or trained designee should complete and 
the Investigator should verify the source documents as the information is collected. Any 
outstanding entries must be completed immediately after the final examination. An explanation 
should be given for all missing data. The Investigator will ensure the accuracy, completeness, 
and timeliness of the data reported to the Sponsor. Data collection processes and procedures will 
be reviewed and validated to ensure completeness, accuracy, reliability, and consistency. A 
complete audit trail will be maintained of all data changes. The Investigator or designee will 
cooperate with the Sponsor’s representative(s) for the periodic review of study documents to 
ensure the accuracy and completeness of the data capture system at each scheduled monitoring 
visit. Electronic consistency checks and manual review will be used to identify any errors or 
inconsistencies in the data. This information will be provided to the respective study sites by 
means of electronic or manual queries. The Investigator or designee will prepare and maintain 
adequate and accurate study documents (medical records, ECGs, AE, and concomitant 
medication reporting, raw data collection forms, etc.) designed to record all observations and 
other pertinent data for each patient receiving study treatment. The Investigator will allow 
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Sponsor representatives, contract designees, authorized regulatory authority inspectors, and the 
IRB to have direct access to all documents pertaining to the study. 
13.7. 
Source Document/Case Report Form Completion 
Source documents will be completed for each study patient. It is the Investigator’s responsibility 
to ensure the accuracy, completeness, and timeliness of the data reported in the patient’s source 
document. The source document should indicate the patient’s participation in the study and 
should document the dates and details of study procedures, AEs and patient status. 
The Investigator, or designated representative, should complete the source document as soon as 
possible after information is collected, preferably on the same day that a study patient is seen for 
an examination, treatment, or any other study procedure. Any outstanding entries must be 
completed immediately after the final examination. An explanation should be given for all 
missing data. 
The Investigator will sign and date each required assessment for all study patients. The 
Investigator will retain all completed source documents. 
13.8. 
Direct Access to Source Data 
The study will be monitored by the Sponsor or its designee. Monitoring will be done by personal 
visits from a representative of the Sponsor (site monitor) and will include on-site review of the 
source documents for completeness and clarity, cross-checking with source documents, and 
clarification of administrative matters will be performed. The review of medical records will be 
performed in a manner to ensure that patient confidentiality is maintained. 
The site monitor will ensure that the investigation is conducted according to protocol design and 
regulatory requirements by frequent communications (letter, telephone, e-mail, and fax). 
All unused study drug and other study materials are to be returned to the Sponsor or designee 
after the study has been completed and the database has been locked. 
Regulatory authorities, the IRB, and/or the Sponsor’s clinical quality assurance group or 
designee may request access to all source documents, database, and any other applicable study 
documentation for an on-site audit or inspection. Direct access to these documents must be 
guaranteed by the Investigator, who must provide support at all times for these activities. 
13.9. 
Record Retention 
The Investigator will maintain all study records according to ICH-GCP and applicable regulatory 
requirement(s). Records will be retained for at least 2 years after the last marketing application 
approval or 2 years after formal discontinuation of the clinical development of the investigational 
product or according to applicable regulatory requirement(s). If the Investigator withdraws from 
the responsibility of keeping the study records, custody must be transferred to a person willing to 
accept the responsibility. The Sponsor must be notified in writing if a custodial change occurs. 
13.10. 
Liability and Insurance 
The Sponsor has subscribed to an insurance policy covering, in its terms and provisions, its legal 
liability for injuries caused to participating persons and arising out of this research performed 
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strictly in accordance with the scientific protocol as well as with applicable law and professional 
standards.  
13.11. 
Publication of Study Findings and Use of Information 
All information regarding AG-348 supplied by the Sponsor or designee to the Investigator is 
privileged and confidential information. The Investigator agrees to use this information to 
accomplish the study and will not use it for other purposes without consent from the Sponsor. It 
is understood that there is an obligation to provide the Sponsor with complete data obtained 
during the study. The information obtained from the clinical study will be used towards the 
development of AG-348 and may be disclosed to regulatory authority(ies), other Investigators, 
corporate partners, or consultants as required. 
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14. 
LIST OF REFERENCES 
 
Beutler E, and Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene 
frequency in the general white population. Blood. 2000;95(11):3585-8. 
Black JA, Rittenberg MB, Bigley RH, and Koler RD. Hemolytic anemia due to pyruvate kinase 
deficiency: characterization of the enzymatic activity from eight patients. Am J Hum Genet. 
1979;31(3):300-10. 
Christensen RD, Yaish HM, Johnson CB, Bianchi P, and Zanella A. Six children with pyruvate 
kinase deficiency from one small town: molecular characterization of the PK-LR gene. J Pediatr. 
2011;159(4):695-7. 
FDA. Guidance for Industry: Drug-Induced Liver Injury: Premarketing Clinical Evaluation. 
2009. 
Ghidini A, Sirtori M, Romero R, Yarkoni S, Solomon L, and Hobbins JC. Hepatosplenomegaly 
as the only prenatal finding in a fetus with pyruvate kinase deficiency anemia. Am J Perinatol. 
1991;8(1):44-6. 
Hennekam RC, Beemer FA, Cats BP, Jansen G, and Staal GE. Hydrops fetalis associated with 
red cell pyruvate kinase deficiency. Genet Couns. 1990;1(1):75-9. 
Kahn A, Marie J, Galand C, and Boivin P. Molecular mechanism of erythrocyte pyruvate kinase 
deficiency. Humangenetik. 1975;29(4):271-80. 
Mentzer WC, Jr., Baehner RL, Schmidt-Schonbein H, Robinson SH, and Nathan DG. Selective 
reticulocyte destruction in erythrocyte pyruvate kinase deficiency. J Clin Invest. 1971;50(3):688-
99. 
Rider NL, Strauss KA, Brown K, Finkenstedt A, Puffenberger EG, Hendrickson CL, et al. 
Erythrocyte pyruvate kinase deficiency in an old-order Amish cohort: longitudinal risk and 
disease management. Am J Hematol. 2011;86(10):827-34. 
Rijksen G, Veerman AJ, Schipper-Kester GP, and Staal GE. Diagnosis of pyruvate kinase 
deficiency in a transfusion-dependent patient with severe hemolytic anemia. Am J Hematol. 
1990;35(3):187-93. 
Schwartz JC. The histamine H3 receptor: from discovery to clinical trials with pitolisant. Br J 
Pharmacol. 2011;163(4):713-21. 
Strauss KA, Robinson DL, Vreman HJ, Puffenberger EG, Hart G, and Morton DH. Management 
of hyperbilirubinemia and prevention of kernicterus in 20 patients with Crigler-Najjar disease. 
Eur J Pediatr. 2006;165(5):306-19. 
Tanphaichitr VS, Suvatte V, Issaragrisil S, Mahasandana C, Veerakul G, Chongkolwatana V, et 
al. Successful bone marrow transplantation in a child with red blood cell pyruvate kinase 
deficiency. Bone Marrow Transplant. 2000;26(6):689-90. 
van Wijk R, and van Solinge WW. The energy-less red blood cell is lost: erythrocyte enzyme 
abnormalities of glycolysis. Blood. 2005;106(13):4034-42. 
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Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
Zanella A, Fermo E, Bianchi P, and Valentini G. Red cell pyruvate kinase deficiency: molecular 
and clinical aspects. Br J Haematol. 2005;130(1):11-25. 
 
85 
 


Protocol AG348-C-003, Amendment 3, Version 4.0 
10 November 2015 
 
15. 
APPENDICES 
 
86 
 


Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
10 November 2015 
 
15.1. 
Schedules of Assessments 
Table 5: 
Schedule of Assessments: Core Period 
Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up1 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
-26 to -12 
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Written Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
PK enzyme assay 
(confirmation of PK 
deficiency) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
PKR Genotype 
(for randomization) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
UGT1A1 Genotype 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medical/Surgical 
History (General and 
PK deficiency-specific)
4 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medication History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Transfusion History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Confirmation of 
Vaccinations 
(Splenectomized 
Patients) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Examination5/ 
Height5 and Weight 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
X 
X 
Performance Status 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
X 
X 
87 
 


Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
10 November 2015 
 
Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up1 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
-26 to -12 
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Vital signs  
(BP, HR, RR, T)6  
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG7 
X 
 
X 
X 
 
X 
 
 
 
 
 
X 
X 
DXA Scan8 
X 
 
 
 
 
 
 
 
 
 
 
X9 
 
Laboratory Evaluations
10 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBsAg, HCV Ab, 
HIV1 and 2 Ab 
X 
 
 
 
 
 
 
 
 
 
 
 
 
RBC antibody 
Screen  
X 
 
 
 
 
 
 
 
 
 
 
 
 
Hematology (CBC)11 
X 
X12 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin13 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
EPO levels14 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
G6PD screen 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Serum Chemistry15 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Iron Panel16 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Carboxyhemoglobin 
(COHb) 
 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
 
Coagulation Studies
17 
X 
 
X 
 
X 
 
 
 
X 
 
 
X 
X 
Urinalysis18 
X 
 
X 
 
X 
 
 
 
X 
 
 
X 
X 
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Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
10 November 2015 
 
Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up1 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
-26 to -12 
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Serum or Urine 
Pregnancy19 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
Lipids20 
 
 
X 
 
 
 
X 
 
X 
 
 
X 
X 
Hormonal Testing21 
X 
X22 
X 
 
 
 
 
 
X 
 
 
X 
X 
Serum 
osteocalcin-N-mid 
and CTX23 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
25-hydroxy Vitamin 
D2 and D3 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Randomization24 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Study Drug 
Administration 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Dispense Study Drug25 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
PK blood sampling26 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
PD Assessments26 
 
 
 
 
 
 
 
 
 
 
 
 
 
2,3-DPG/ATP 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
Dispense/Collect 
Menstrual Cycle Diary
28 
 
 
X 
 
 
 
X 
 
X 
X 
X 
X 
X 
Adverse Events29 
 
 
Continuous 
X 
89 
 


Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
10 November 2015 
 
Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up1 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
-26 to -12 
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Transfusion Record30 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
Medications/Procedures 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Rollover to Extension 
Period31 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Abbreviations:  Ab = antibody; ATP = adenosine triphosphate; BP = blood pressure; CBC= complete blood count; COHb = carboxyhemoglobin; 
CTX = C-terminal telopeptide; D = day; DPG = diphosphoglycerate; DXA = Dual-energy x-ray absorptiometry; ECG = electrocardiogram; 
EPO = erythropoietin; FSH = follicle stimulating hormone; G6PD = glucose-6-phosphate-dehydrogenase; HBsAg = hepatitis B surface antigen; 
HCV Ab = hepatitis C virus antibody; HDL-C = high-density lipoprotein-cholesterol; HIV = human immunodeficiency virus; HR = heart rate; PD = 
pharmacodynamic; PK = pharmacokinetic; PK deficiency = pyruvate kinase deficiency; PKR = pyruvate kinase isoform R; RR = resting rate; W = week. 
Whenever more than one assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive 
assessment (e.g., VS, ECG, blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time. The order of 
procedures may be revised with prior discussion between Sponsor and site. 
 
1 The Week 28 Follow-up Visit will only be conducted for patients who do not enter the Extension Period. 
2 To be performed at least 2 days after the first Screening Visit. 
3 In-clinic visits on Day 8 and Day 22 may be performed by the patient’s primary care physician if necessary and must be approved by the Sponsor on a case by 
case basis; in these instances PK/PD sampling would not be required and dispensing of study medication would not be performed. For the Day 8 visit 
performed by the patient’s primary care physician, the primary care medical office will collect a blood sample for hematology using the blood sample 
collection and shipping supplies from the kit prepared by the designated central laboratory. The kit will be sent to the primary care physician’s office. No 
other testing or procedures will be asked of the primary care physician on Day 8 [VS and serum chemistry will not be required]. The 12-lead ECG scheduled 
for Day 8 will instead be performed at the main study center on Day 15. For the Day 22 visit performed by the patient’s primary care physician, the primary 
care medical office will collect a blood sample for hematology using the blood sample collection and shipping supplies from the kit prepared by the 
designated central laboratory. The kit will be sent to the primary care physician’s office. No other testing or procedures will be asked of the primary care 
physician on Day 22. [VS, serum chemistry, coagulation, haptoglobin, EPO level, carboxyhemoglobin, and PK/PD samples will not be required.] The 12-lead 
electrocardiogram scheduled for Day 22 will instead be performed at the main study center on Day 43 (Week 6). For patients having their Day 8 and/or 
Day 22 visits performed by the primary care physician, the Principal Investigator will him or herself, or have a qualified research nurse or other designated 
site staff member, make telephone contact with the patient to inquire about any adverse events. These must be recorded as if the patient appeared in the main 
study center. The Principal Investigator will exercise prudent clinical judgment in determining any clinical course of action to take based on any adverse 
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Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
10 November 2015 
 
events discovered. These telephone contacts on Day 8 and Day 22 must be explained to the patient in advance and scheduled in advance to maximize the 
likelihood of successfully making contact. 
4 Medical history, including all relevant prior medical history, current medical conditions, and hematology profile (CBCs) over prior 6 months, will be obtained 
at the Screening assessment. 
5 A complete physical examination (including neurological examination; genital and rectal examinations will be performed at the discretion of the Investigator) 
will be obtained at Screening, Baseline, Week 12, and the Follow up Visit (Week 28), or Week 24 for patients rolling over to the Extension Period. The 
neurological examination must include an assessment of general orientation and mental status including level of alertness (assess as normal or abnormal and 
specify any abnormality). Limited focused physical examinations will be performed at all other specified visits. Height to be collected at Screening only. 
6 Vital signs will include systolic and diastolic blood pressure, heart rate, respiratory rate, and temperature.  
7 12-lead ECGs are to be conducted after 5 minutes of recumbency. Screening ECG will be performed at least 7 days prior to Day 1 dosing. 
8 If a DXA scan of the hip and spine with T and Z scores has been performed within 3 months preceding the first day of dosing, this may be used to meet this 
requirement.  
9 Week 24 DXA scan may be performed anytime between Weeks 24 and 28 and must be performed at the same imaging center on the same DXA machine as 
the original Screening DXA scan. 
10 Laboratory evaluations (hematology, serum chemistry, coagulation studies, and urinalysis) are to be collected in the morning. These should be collected 
following an overnight fast on Baseline Day 1 Week 6 (Day 43), Week 12 (Day 85), Week 24 (Day 169), and Follow-up Week 28 (Day 197), when the lipid 
samples are also included. 
11 Three Screening/Baseline samples will be collected for complete blood count (CBC). Samples will be collected in the AM on 3 different days; the samples 
collected on Baseline/Day 1 may comprise one of these; samples may be taken at the same time as Screening/Baseline hormone assessments (any 2 time 
points during Screening at least 2 days apart). CBC will include HCT, Hb, red blood cell (RBC) count, absolute reticulocyte count, percent reticulocyte count, 
mean corpuscular volume (MCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW), white 
blood cell (WBC) count with automated (or manual, as indicated) differential, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and 
platelet count. In the event that the designated central laboratory for the study is unable to provide a valid result for any specific component of the defined 
CBC for a specific patient, the site may be asked to have the test performed at their local laboratory. The result of the local CBC will be entered into the study 
database along with the local normal reference range. 
12 The second Screening hematology (CBC) should be drawn in the morning (does not have to be fasting), and may be drawn at the same time the patient returns 
for the second estradiol and free and total testosterone sample. 
13 Haptoglobin will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28. 
14 Erythropoietin (EPO) levels will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 
28. 
15 Alkaline phosphatase, sodium, potassium, chloride, calcium, magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, albumin, total protein, glucose, 
blood urea nitrogen (BUN), creatinine, uric acid, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total 
bilirubin and indirect bilirubin, (and estimated creatinine clearance or glomerular filtration rate for screening only, as appropriate). 
16 Iron, total iron-binding capacity (TIBC), transferrin saturation, and ferritin will be performed prior to dosing on Day 1, at the end of Week 12 and at the end 
of Week 24. 
17 Fibrinogen, activated partial thromboplastin time (aPTT), and international normalized ratio (INR) will be performed at Screening, prior to dosing on Day 1, 
at the end of Week 2, the end of Week 12, the end of Week 24, and the end of Week 28. 
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Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
10 November 2015 
 
18 Color, appearance, pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite, urobilinogen, and occult blood. Microscopic inspection of sediment 
should only be performed for cause or to investigate an abnormal dipstick finding per the Investigator’s discretion. Urinalysis will be performed at Screening, 
prior to dosing on Day 1, at the end of Week 2, the end of Week 12, the end of Week 24, and the end of Week 28. 
19 Must be repeated at any point throughout the study period if pregnancy is clinically suspected. 
20 Total cholesterol, HDL-C, and triglyceride samples will be collected in the morning following an overnight fast. 
21 Three Screening/Baseline samples will be collected for estradiol and testosterone (total and free). Samples will be collected in the AM on 3 different days; the 
samples collected on Baseline/Day 1 may comprise one of these; the Screening samples may be collected at any 2 time points during Screening at least 2 days 
apart. Serum estrone, estradiol, and free and total testosterone will then follow the schedule indicated on Day 1 and Weeks 12, 24, and 28. FSH will only be 
performed at Screening for female patients only for confirmation of post-menopausal status. 
22 The second Screening hormone testing will consist of estradiol and testosterone (free and total) only. Samples should be drawn in the AM (does not need to 
be fasting), and may be drawn at the same time the patient returns for the second CBC sample. 
23 Serum osteocalcin-N-mid and CTX will be drawn in the AM each time, approximately between 8-10 AM, and after an overnight fast of 10-12 hours. 
24 Randomization will be performed following PKR genotyping and prior to and as close as feasible to dosing on Day 1. 
25 Study drug will be dispensed on a 28-day schedule, or on an alternate schedule (< 28 days) as needed to accommodate patient visit schedule and dose 
modifications. 
26 For the first 10 patients treated, extensive PK/PD sampling will be conducted on Days 1 and 15 (see Appendix 15.1, Table 7 for details), followed by limited 
PK/PD sampling from Week 3 to Week 24 (see Appendix 15.1, Table 8 for details). Limited PK/PD sampling will be conducted on the remainder of patients 
treated (see Appendix 15.1, Table 8). See Section 10.6, Section 10.7, and Section 10.9 for details on blood sampling for PK and PD assessments, respectively, 
and guidelines on sample processing and storage. 
 
 
28 Menstruating female patients will record their menstrual cycles (start, stop, characteristics) monthly. Paper-based menstrual cycle diaries will be dispensed at 
study visits approximately every month. The previous month’s diary will be collected at these visits as well. 
29 All randomized patients will be evaluated for AEs from the time they sign informed consent until they complete all scheduled study follow-up visits or 
withdraw consent, whichever occurs first.  
30 All transfusions must be recorded in the eCRF 
31 Patient must have completed 24 weeks of treatment and tolerated AG-348 (may have had dose modifications). Investigator and Medical Monitor or 
Responsible Medical Officer must agree with the patient continuing on treatment and patient must sign a separate ICF for the Extension Period. 
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Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
10 November 2015 
 
Table 6: 
Schedule of Assessments: Extension Period 
Visit 
Month 9 
Month 12 
Month 15 
Month 18 
Month 21 
Month 24 
Month 27 
Month 30 
Follow-up 
Approximate Study Day 
259 
365 
455 
545 
635 
724 
814 
904 
934 
Visit Window 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
Physical Examination/Weight1 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Performance Status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs  
(BP, HR, RR, T)2 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG3 
 
X 
 
X 
 
X 
 
X 
X 
DXA Scan 
 
 
 
X 
 
 
 
X 
 
Laboratory Evaluations4 
 
 
 
 
 
 
 
 
 
Hematology (CBC)5 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin 
 
X 
 
X 
 
X 
 
X 
X 
EPO levels6 
 
X 
 
X 
 
X 
 
X 
X 
Serum Chemistry7 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Iron Panel8 
 
X 
 
X 
 
X 
 
X 
 
Carboxyhemoglobin (COHb) 
 
X 
 
X 
 
X 
 
X 
 
Coagulation Studies9 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urinalysis10 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum or Urine Pregnancy11 
 
 
 
 
 
 
 
 
 
Lipids12 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Hormonal Testing13 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum osteocalcin-N-mid and 
CTX14 
 
 
 
X 
 
 
 
X 
 
Study Drug Administration 
X 
X 
X 
X 
X 
X 
X 
X 
 
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Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
10 November 2015 
 
Visit 
Month 9 
Month 12 
Month 15 
Month 18 
Month 21 
Month 24 
Month 27 
Month 30 
Follow-up 
Approximate Study Day 
259 
365 
455 
545 
635 
724 
814 
904 
934 
Visit Window 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
Dispense Study Drug15 
X 
X 
X 
X 
X 
X 
X 
 
 
PK blood sampling16 
X 
X 
X 
X 
X 
X 
X 
X 
 
PD Assessments17 
(2,3-DPG/ATP, 
) 
X 
X 
X 
X 
X 
X 
X 
X 
 
Dispense/Collect Menstrual 
Cycle Diary18 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Events19 
Continuous 
X 
Transfusion Record20 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
Medications/Procedures 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; BP = blood pressure; CBC= complete blood count; COHb = carboxyhemoglobin; CTX = C-terminal telopeptide; 
DPG = diphosphoglycerate; DXA = Dual-energy x-ray absorptiometry; ECG = electrocardiogram; EPO = erythropoietin; HDL-C = high-density lipoprotein-
cholesterol; HIV = human immunodeficiency virus; HR = heart rate; PD = pharmacodynamic; PK = pharmacokinetic; PK deficiency = pyruvate kinase 
deficiency; PKR = pyruvate kinase isoform R; RR = resting rate; W = week. 
Whenever more than one assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive 
assessment (e.g., VS, ECG, blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time. The order of 
procedures may be revised with prior discussion between Sponsor and site. 
 
1 A focused physical examination (including neurological examination; genital and rectal examinations will be performed at the discretion of the Investigator) 
will be obtained every 3 months; a complete physical examination will be performed at the Month 30 visit. The neurological examination must include an 
assessment of general orientation and mental status including level of alertness (assess as normal or abnormal and specify any abnormality). 
2 Vital signs will include systolic and diastolic blood pressure, heart rate, respiratory rate, and temperature 
3 12-lead ECGs are to be conducted after 5 minutes of recumbency.  
4 Laboratory evaluations (hematology, serum chemistry, coagulation studies, and urinalysis) are to be collected in the morning. These should be collected 
following an overnight fast. 
5 CBC will include HCT, Hb, red blood cell (RBC) count, absolute reticulocyte count, percent reticulocyte count, mean corpuscular volume (MCV), mean 
corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW), white blood cell (WBC) count with automated (or 
manual, as indicated) differential, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and platelet count. In the event that the designated 
central laboratory for the study is unable to provide a valid result for any specific component of the defined CBC for a specific patient, the site may be asked 
to have the test performed at their local laboratory. The result of the local CBC will be entered into the study database along with the local normal reference 
range. 
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Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
10 November 2015 
 
6 Erythropoietin (EPO) levels will be performed prior to dosing at Month 12, Month 18, Month 24, and Month 30. 
7 Alkaline phosphatase, sodium, potassium, chloride, calcium, magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, albumin, total protein, glucose, 
blood urea nitrogen (BUN), creatinine, uric acid, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total 
bilirubin and indirect bilirubin, (and estimated creatinine clearance or glomerular filtration rate, as appropriate). 
8 Iron, total iron-binding capacity (TIBC), transferrin saturation, and ferritin will be performed prior to dosing at Month 12, Month 18, Month 24, and Month 
30. 
9 Fibrinogen, activated partial thromboplastin time (aPTT), and international normalized ratio (INR) will be performed at each study visit. 
10 Color, appearance, pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite, urobilinogen, and occult blood. Microscopic inspection of sediment 
should only be performed for cause or to investigate an abnormal dipstick finding per the Investigator’s discretion. Urinalysis will be performed prior to 
dosing at each study visit. 
11 Must be obtained at any point throughout the study period if pregnancy is clinically suspected. Effective contraception must be continued throughout the 
Extension Period in all patients and any pregnancies must be reported (see Section 11.3). 
12 Total cholesterol, HDL-C, and triglyceride samples will be collected in the morning following an overnight fast. 
13 Serum estrone, estradiol, and free and total testosterone.  
14 Serum osteocalcin-N-mid and CTX will be drawn in the AM each time, approximately between 8-10 AM, and after an overnight fast of 10-12 hours. 
15 Study drug will be dispensed on a 3-month schedule, or on an alternate schedule (< 3 months) as needed to accommodate patient visit schedule and dose 
modifications. 
16 Predose; PK sampling will only include AG-348 and AGI-8702 concentrations. 
17 Predose. 
18 Menstruating female patients will record their menstrual cycles (start, stop, characteristics) monthly. Paper-based menstrual cycle diaries will be dispensed 
and collected at each study visit. 
19 All randomized patients will be evaluated for AEs from the time they sign informed consent until they complete all scheduled study follow-up visits or 
withdraw consent, whichever occurs first.  
20 All transfusions must be recorded in the eCRF. 
95 
 


Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
10 November 2015 
 
Table 7: 
Schedule of Assessments: Extensive PK/PD Sampling during the Core Period 
Sample Timing/Interval 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Visit 
Baseline / D1 
W2 / D15 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
Study Day 
1/15 
22 
43 
64 
85 
113 
141 
169 
Visit Window 
± 2 D (D15) 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Timing 
Pre-
dose1 
30 
min2 
1 hr2 
2 hr2 
4 hr3 
8 hr3 
12 
hr3 
Pre-
dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
PK blood sample 
X 
X 
X 
X 
X 
X 
X4 
X 
X 
X 
X 
X 
X 
X 
2,3 DPG/ATP 
X 
X 
X 
X 
X 
X 
X4 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; D = day; DPG = diphosphoglycerate; PD = pharmacodynamic; PK = pharmacokinetic; 
 
 W = week. 
 The acceptable time window will be within 60 minutes prior to study treatment dose administration for the pre-dose PK/PD sample. 
2 The acceptable time window will be within ± 5 minutes of the scheduled collection time for the 30 minute, 1 and 2 hour PK/PD samples. 
3 The acceptable time window will be within ± 30 minutes of the scheduled collection time for the 4, 8, and 12 hour PK/PD samples. 
4 To be collected on Day 1 only. 
 
  
6 If the 12 hour time point cannot be collected at site on Day 1, an 8 hour time point may be collected instead. 
 
 
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Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
10 November 2015 
 
Table 8: 
Schedule of Assessments: Limited PK/PD Sampling during the Core Period 
Sample Timing/Interval 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Visit 
Baseline / 
D1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
Study Day 
1 
15 
22 
43 
64 
85 
113 
141 
169 
Visit Window 
- 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Timing 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
PK blood sample 
X 
X 
X 
X 
X 
X 
X 
X 
X 
2,3 DPG/ATP 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; D = day; DPG = diphosphoglycerate; PD = pharmacodynamic; PK = pharmacokinetic; 
 
 W = week. 
 
1 The predose blood sample for plasma PK/PD analysis should be collected within 60 minutes prior to study treatment dose administration. 
 
 
 
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Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
10 November 2015 
 
15.2. 
Eastern Cooperative Oncology Group Performance Status Scoring 
 
Grade 
Symptomatology 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a 
light or sedentary nature, e.g., light house work, office work 
2 
Ambulatory and capable of all self-care but unable to carry out any work activities. Up and 
about more than 50% of waking hours 
3 
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours 
4 
Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair 
5 
Dead 
Source: Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, et al. Toxicity and Response 
Criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol 1982;5:649-655. 
 
15.3. 
National Cancer Institute Common Terminology Criteria for 
Adverse Events 
 
The NCI CTCAE, Version 4.03, can be accessed using the following link:   
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_8.5x11.pdf 
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15.4. 
Potential Drug Interactions 
Strong inhibitors of CYP3A4 (listed in Table 9) are not permitted for use with AG-348. Based on 
modeling of AG-348 metabolism and tolerability data from the Phase 1 MAD study, it is thought 
that moderate inhibitors of CYP3A4 do not pose a risk to patients dosed with AG-348. 
In-vivo data from the Phase 1 MAD show AG-348 induces CYP3A4 and induces its own 
metabolism, presumably via CYP3A4. The use of strong CYP3A4 inducers in combination with 
AG-348 is expected to reduce AG-348’s efficacy. Therefore, administration of strong CYP3A4 
inducers (listed in Table 10) with AG-348 is not permitted. Strong inhibitors of drug transport 
(listed in Table 11) are not permitted for use with AG-348. 
Digoxin is not permitted for use with AG-348. 
Induction of CYP3A4 by AG-348 is expected to reduce the efficacy of certain sensitive co-
medications. Co-administration of AG-348 with the drugs in Table 12 should be done with 
caution, as their efficacy may be reduced.  
Of note, in accordance with Inclusion Criteria 14, women in the trial utilizing oral contraception 
must utilize barrier methods while taking AG-348. 
The expected patient co-medications deferoxamine, deferasirox, deferiprone, and oral penicillin 
are not expected to interact with AG-348. 
Table 9: 
Strong and Moderate CYP3A4 Inhibitors 
Strong CYP3A4 Inhibitors: Contraindicated 
Moderate CYP3A4 Inhibitors: No Action 
Indinavir 
Aprepitant 
Nelfinavir 
Erythromycin1 
Ritonavir 
Fluconazole 
Clarithromycin 
Verapamil1 
Itraconazole 
Diltiazem1 
Ketoconazole 
 
Nefazodone 
 
Saquinavir 
 
Suboxone 
 
Telithromycin 
 
Grapefruit juice2 
 
Strong Inhibitor; > 5 fold increase in AUC 
Moderate Inhibitor; > 2 fold, < 5 fold increase in AUC 
1 Erythromycin, verapamil and diltiazem are contraindicated because they are strong P-gp inhibitors (see Table 11) 
2 Although classified as a moderate CYP3A4 inhibitor, grapefruit and grapefruit juice are prohibited 
 
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Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
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Table 10: 
Strong CYP3A4 Inducers 
Strong CYP3A4 Inducers:  Contraindicated 
Efavirenz 
Phenytoin 
Nevirapine 
Pioglitazone 
Carbamazepine 
Rifabutin 
Glucocorticoids1 
Rifampin 
Modafinil 
St. John's Wort 
Oxcarbazepine 
Troglitazone 
Phenobarbital 
 
1 Short-term (≤ 14 days at a time, and ≤ 28 days total during the 24 week treatment period) use of topical, inhaled, 
intra-nasal, and systemic glucocorticoids is permitted for acute medical indications. Every effort should be made to 
minimize total duration of glucocorticoid therapy and utilize alternative treatments. Patients must be off 
glucocorticoids for at least 28 days prior to Day 1 of AG-348 dosing as per exclusion criterion #11 (Section 8.3). 
For patients who require chronic inhaled glucocorticoid therapy, Investigators should confer with the Medical 
Monitor for additional guidance. 
 
Table 11: 
Strong P-glycoprotein Inhibitors 
Strong P-gp Inhibitors:  Contraindicated 
Amiodarone 
Felodipine 
Azithromycin 
Itraconazole 
Captopril 
Ketoconazole 
Carvedilol 
Lopinavir 
Clarithromycin 
Ritonavir 
Conivaptan 
Quercetin 
Cyclosporine 
Quinidine 
Diltiazem 
Ranolazine 
Dronedarone 
Ticagrelor 
Erythromycin 
Verapamil 
 
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Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
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Table 12: 
Sensitive CYP3A4 Substrates 
Sensitive CYP3A4 Substrates:  Substitute or Use with Caution 
 
Antihistamines: 
Miscellaneous: 
 
 
 
Chlorpheniramine 
Alfentanil 
Finasteride 
Salmeterol 
 
 
Aprepitant 
Gleevec 
Sildenafil 
Benzodiazepines: 
Calcium Channel 
Blockers: 
Aripiprazole 
Haloperidol 
Sirolimus 
Alprazolam 
Amlodipine 
Boceprevir 
Irinotecan 
Sorafenib 
Diazepam→3OH 
Lercanidipine 
Buspirone 
LAAM 
Sunitinib 
Midazolam 
Nifedipine 
Cafergot 
Lidocaine 
Tamoxifen 
Triazolam 
Nisoldipine 
Caffeine→TMU 
Methadone 
Taxol 
Nitrendipine 
Cilostazol 
Nateglinide 
Telaprevir 
Immune Modulators: 
 
Cocaine 
Nevirapine 
Terfenadine 
Tacrolimus (FK506) 
HMG CoA 
Reductase 
Inhibitors: 
Codeine-N-
demethylation 
Ondansetron 
Torisel 
 
Atorvastatin 
Dapsone 
Pimozide 
Trazodone 
Steroid 6beta-OH: 
Cerivastatin  
Dextromethorphan 
Propranolol 
Vemurafenib 
Estradiol 
Lovastatin 
Docetaxel 
Quetiapine 
Vincristine 
Hydrocortisone (and 
other glucocorticoids) 
Simvastatin 
Domperidone 
Quinine 
Zaleplon 
Progesterone 
 
Eplerenone 
Risperidone 
Ziprasidone 
Testosterone 
 
Fentanyl 
Romidepsin 
Zolpidem 
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Protocol AG348-C-003, Amendment 3, Protocol Version 4.0 
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15.5. 
Declaration of Helsinki 
WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI: 
Recommendations Guiding Medical Doctors in Biomedical Research Involving Human Subjects 
Adopted by the 18th World Medical Association (WMA) General Assembly, Helsinki, Finland, 
June 1964 and amended by the 29th WMA General Assembly, Tokyo, Japan, October 1975, 35th 
WMA General Assembly, Venice, Italy, October 1983, and the 41st WMA General Assembly, 
Hong Kong, September 1989, the 48th WMA General Assembly, Somerset West, Republic of 
South Africa, October 1996; 52nd WMA General Assembly, Edinburgh, Scotland, October 2000; 
53rd WMA General Assembly, Washington 2002 (Note of Clarification on paragraph 29 added); 
55th WMA General Assembly, Tokyo 2004 (Note of Clarification on Paragraph 30 added); and 
59th WMA General Assembly, Seoul, October 2008. 
A. INTRODUCTION  
1. The World Medical Association (WMA) has developed the Declaration of Helsinki as a 
statement of ethical principles for medical research involving human subjects, including 
research on identifiable human material and data.  
The Declaration is intended to be read as a whole and each of its constituent paragraphs 
should not be applied without consideration of all other relevant paragraphs.  
2. Although the Declaration is addressed primarily to physicians, the WMA encourages 
other participants in medical research involving human subjects to adopt these principles.  
3. It is the duty of the physician to promote and safeguard the health of subjects, including 
those who are involved in medical research. The physician's knowledge and conscience 
are dedicated to the fulfilment of this duty.  
4. The Declaration of Geneva of the WMA binds the physician with the words, "The health 
of my subject will be my first consideration," and the International Code of Medical 
Ethics declares that, "A physician shall act in the subject's best interest when providing 
medical care."  
5. Medical progress is based on research that ultimately must include studies involving 
human subjects. Populations that are underrepresented in medical research should be 
provided appropriate access to participation in research.  
6. In medical research involving human subjects, the well-being of the individual research 
subject must take precedence over all other interests.  
7. The primary purpose of medical research involving human subjects is to understand the 
causes, development and effects of diseases and improve preventive, diagnostic and 
therapeutic interventions (methods, procedures and treatments). Even the best current 
interventions must be evaluated continually through research for their safety, 
effectiveness, efficiency, accessibility and quality.  
8. In medical practice and in medical research, most interventions involve risks and 
burdens.  
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9. Medical research is subject to ethical standards that promote respect for all human 
subjects and protect their health and rights. Some research populations are particularly 
vulnerable and need special protection. These include those who cannot give or refuse 
consent for themselves and those who may be vulnerable to coercion or undue influence.  
10. Physicians should consider the ethical, legal and regulatory norms and standards for 
research involving human subjects in their own countries as well as applicable 
international norms and standards. No national or international ethical, legal or regulatory 
requirement should reduce or eliminate any of the protections for research subjects set 
forth in this Declaration.  
B. BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH  
11. It is the duty of physicians who participate in medical research to protect the life, health, 
dignity, integrity, right to self-determination, privacy, and confidentiality of personal 
information of research subjects.  
12. Medical research involving human subjects must conform to generally accepted scientific 
principles, be based on a thorough knowledge of the scientific literature, other relevant 
sources of information, and adequate laboratory and, as appropriate, animal 
experimentation. The welfare of animals used for research must be respected.  
13. Appropriate caution must be exercised in the conduct of medical research that may harm 
the environment.  
14. The design and performance of each research study involving human subjects must be 
clearly described in a research protocol. The protocol should contain a statement of the 
ethical considerations involved and should indicate how the principles in this Declaration 
have been addressed. The protocol should include information regarding funding, 
Sponsors, institutional affiliations, other potential conflicts of interest, incentives for 
subjects and provisions for treating and/or compensating subjects who are harmed as a 
consequence of participation in the research study. The protocol should describe 
arrangements for post-study access by study subjects to interventions identified as 
beneficial in the study or access to other appropriate care or benefits.  
15. The research protocol must be submitted for consideration, comment, guidance and 
approval to a research ethics committee before the study begins. This committee must be 
independent of the researcher, the Sponsor and any other undue influence. It must take 
into consideration the laws and regulations of the country or countries in which the 
research is to be performed as well as applicable international norms and standards but 
these must not be allowed to reduce or eliminate any of the protections for research 
subjects set forth in this Declaration. The committee must have the right to monitor 
ongoing studies. The researcher must provide monitoring information to the committee, 
especially information about any SAEs. No change to the protocol may be made without 
consideration and approval by the committee.  
16. Medical research involving human subjects must be conducted only by individuals with 
the appropriate scientific training and qualifications. Research on subjects or healthy 
volunteers requires the supervision of a competent and appropriately qualified physician 
or other health care professional. The responsibility for the protection of research subjects 
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must always rest with the physician or other health care professional and never the 
research subjects, even though they have given consent.  
17. Medical research involving a disadvantaged or vulnerable population or community is 
only justified if the research is responsive to the health needs and priorities of this 
population or community and if there is a reasonable likelihood that this population or 
community stands to benefit from the results of the research.  
18. Every medical research study involving human subjects must be preceded by careful 
assessment of predictable risks and burdens to the individuals and communities involved 
in the research in comparison with foreseeable benefits to them and to other individuals 
or communities affected by the condition under investigation.  
19. Every clinical trial must be registered in a publicly accessible database before recruitment 
of the first subject.  
20. Physicians may not participate in a research study involving human subjects unless they 
are confident that the risks involved have been adequately assessed and can be 
satisfactorily managed. Physicians must immediately stop a study when the risks are 
found to outweigh the potential benefits or when there is conclusive proof of positive and 
beneficial results.  
21. Medical research involving human subjects may only be conducted if the importance of 
the objective outweighs the inherent risks and burdens to the research subjects.  
22. Participation by competent individuals as subjects in medical research must be voluntary. 
Although it may be appropriate to consult family members or community leaders, no 
competent individual may be enrolled in a research study unless he or she freely agrees.  
23. Every precaution must be taken to protect the privacy of research subjects and the 
confidentiality of their personal information and to minimize the impact of the study on 
their physical, mental and social integrity.  
24. In medical research involving competent human subjects, each potential subject must be 
adequately informed of the aims, methods, sources of funding, any possible conflicts of 
interest, institutional affiliations of the researcher, the anticipated benefits and potential 
risks of the study and the discomfort it may entail, and any other relevant aspects of the 
study. The potential subject must be informed of the right to refuse to participate in the 
study or to withdraw consent to participate at any time without reprisal. Special attention 
should be given to the specific information needs of individual potential subjects as well 
as to the methods used to deliver the information. After ensuring that the potential subject 
has understood the information, the physician or another appropriately qualified 
individual must then seek the potential subject's freely-given informed consent, 
preferably in writing. If the consent cannot be expressed in writing, the non-written 
consent must be formally documented and witnessed.  
25. For medical research using identifiable human material or data, physicians must normally 
seek consent for the collection, analysis, storage and/or reuse. There may be situations 
where consent would be impossible or impractical to obtain for such research or would 
pose a threat to the validity of the research. In such situations the research may be done 
only after consideration and approval of a research ethics committee.  
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26. When seeking informed consent for participation in a research study the physician should 
be particularly cautious if the potential subject is in a dependent relationship with the 
physician or may consent under duress. In such situations the informed consent should be 
sought by an appropriately qualified individual who is completely independent of this 
relationship.  
27. For a potential research subject who is incompetent, the physician must seek informed 
consent from the legally authorized representative. These individuals must not be 
included in a research study that has no likelihood of benefit for them unless it is intended 
to promote the health of the population represented by the potential subject, the research 
cannot instead be performed with competent persons, and the research entails only 
minimal risk and minimal burden.  
28. When a potential research subject who is deemed incompetent is able to give assent to 
decisions about participation in research, the physician must seek that assent in addition 
to the consent of the legally authorized representative. The potential subject's dissent 
should be respected.  
29. Research involving subjects who are physically or mentally incapable of giving consent, 
for example, unconscious subjects, may be done only if the physical or mental condition 
that prevents giving informed consent is a necessary characteristic of the research 
population. In such circumstances the physician should seek informed consent from the 
legally authorized representative. If no such representative is available and if the research 
cannot be delayed, the study may proceed without informed consent provided that the 
specific reasons for involving subjects with a condition that renders them unable to give 
informed consent have been stated in the research protocol and the study has been 
approved by a research ethics committee. Consent to remain in the research should be 
obtained as soon as possible from the subject or a legally authorized representative.  
30. Authors, editors and publishers all have ethical obligations with regard to the publication 
of the results of research. Authors have a duty to make publicly available the results of 
their research on human subjects and are accountable for the completeness and accuracy 
of their reports. They should adhere to accepted guidelines for ethical reporting. Negative 
and inconclusive as well as positive results should be published or otherwise made 
publicly available. Sources of funding, institutional affiliations and conflicts of interest 
should be declared in the publication. Reports of research not in accordance with the 
principles of this Declaration should not be accepted for publication.  
C. ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH COMBINED WITH 
MEDICAL CARE  
31. The physician may combine medical research with medical care only to the extent that 
the research is justified by its potential preventive, diagnostic or therapeutic value and if 
the physician has good reason to believe that participation in the research study will not 
adversely affect the health of the subjects who serve as research subjects.  
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32. The benefits, risks, burdens and effectiveness of a new intervention must be tested 
against those of the best current proven intervention, except in the following 
circumstances:  
• The use of placebo, or no treatment, is acceptable in studies where no current proven 
intervention exists; or  
• Where for compelling and scientifically sound methodological reasons the use of 
placebo is necessary to determine the efficacy or safety of an intervention and the 
subjects who receive placebo or no treatment will not be subject to any risk of serious 
or irreversible harm. Extreme care must be taken to avoid abuse of this option.  
33. At the conclusion of the study, subjects entered into the study are entitled to be informed 
about the outcome of the study and to share any benefits that result from it, for example, 
access to interventions identified as beneficial in the study or to other appropriate care or 
benefits.  
34. The physician must fully inform the subject which aspects of the care are related to the 
research. The refusal of a subject to participate in a study or the subject's decision to 
withdraw from the study must never interfere with the subject-physician relationship.  
35. In the treatment of a subject, where proven interventions do not exist or have been 
ineffective, the physician, after seeking expert advice, with informed consent from the 
subject or a legally authorized representative, may use an unproven intervention if in the 
physician's judgement it offers hope of saving life, re-establishing health or alleviating 
suffering. Where possible, this intervention should be made the object of research, 
designed to evaluate its safety and efficacy. In all cases, new information should be 
recorded and, where appropriate, made publicly available. 
106 
 


 
1. 
TITLE PAGE 
Clinical Study Protocol AG348-C-003 
EudraCT No. 2015-000484-13 
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, 
Pharmacokinetic and Pharmacodynamic Study of AG-348 in Adult Patients 
with Pyruvate Kinase Deficiency 
Study Sponsor: 
Agios Pharmaceuticals, Inc. 
88 Sidney Street 
Cambridge, MA 02139-4169 
Phone:  617-649-8600 
Fax:  617-649-8618 
Responsible Medical Officer: 
, MD, PhD 
 
 
Agios Pharmaceuticals, Inc. 
Phone: 
 
Email:  
 
Study Medical Monitor 
, MD 
 
On behalf of Agios Pharmaceuticals, Inc. 
Mobile Phone: 
 
Office Phone: 
 
Email: 
  
Document Version (Date): 
Revised 
Version 1.0 (05 January 2015) 
Amendment 1, Protocol Version 2.0 (02 February 2015) Final 
Amendment 2, Protocol Version 3.0 (05 Aug 2015) Final 
This study will be conducted according to the protocol and in compliance with Good Clinical 
Practice, the ethical principles stated in the Declaration of Helsinki, and other applicable 
regulatory requirements. 
CONFIDENTIALITY NOTE: 
The information contained in this document is confidential and proprietary to Agios Pharmaceuticals, Inc. Any 
distribution, copying, or disclosure is strictly prohibited unless such disclosure is required by federal regulations or 
state law. Persons to whom the information is disclosed must know that it is confidential and that it may not be 
further disclosed by them. 
1 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
INVESTIGATOR’S AGREEMENT 
 
I understand that all documentation provided to me by Agios 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). No changes will be made to the study protocol without the 
prior written approval of Agios and the IRB, except where necessary to eliminate an immediate 
hazard to the patient. 
I have read, understood, and agree to conduct this study as outlined in the protocol and in 
accordance with the guidelines and all applicable government regulations. 
 
 
Investigator Name (printed) 
 
Investigator Signature 
 
Date 
 
 
 
 
Investigational site or name of institution and location (printed) 
2 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
2. 
SYNOPSIS 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Title of Study:  
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, Pharmacokinetic and 
Pharmacodynamic Study of AG-348 in Adult Patients with Pyruvate Kinase Deficiency 
Study Center(s): 
This study will be conducted at multiple study centers. 
Phase of Development: 2 
Objectives: 
Primary: 
• 
Evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in patients with 
pyruvate kinase deficiency (PK deficiency). 
Secondary: 
• 
Evaluate the pharmacokinetics (PK) of AG-348 and the metabolite AGI-8702. 
• 
Evaluate the pharmacodynamic (PD) response of adenosine triphosphate (ATP) and 
2,3-diphosphoglycerate (2,3-DPG) after administration of AG-348. 
• 
Evaluate indicators of clinical activity of AG-348 in patients with PK deficiency, including changes 
in hemoglobin (Hb), hematocrit (HCT), reticulocyte count, haptoglobin (Hp), carboxyhemoglobin 
(COHb), lactate dehydrogenase (LDH), total and indirect bilirubin, erythropoietin (EPO), ferritin, 
and transferrin saturation (serum iron/iron binding capacity). 
Methodology: 
Study AG348-C-003 is a Phase 2, open label, two arm, multicenter, randomized, dose-ranging study 
during which adult patients with PK deficiency will receive multiple doses of AG-348 for up to 
24 weeks. Patients with PK deficiency confirmed by red blood cell PK enzymatic assay performed at 
Screening will be eligible to participate in this study. At Week 25, patients who safely tolerate AG-348 
and demonstrate clinical activity of AG-348 may be eligible to immediately roll over to a safety 
extension for continued treatment. Patients who finish treatment at the end of 24 weeks or sooner will 
undergo follow-up assessment 4 weeks after the last dose of study drug. Patients with toxicity suspected 
to be related to study drug will continue follow-up until the adverse event (AE) resolves, is declared 
chronic by the Investigator, or the patient is lost to follow-up.  
3 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Initially, up to 25 patients will be randomized on an open-label 1:1 basis to each of two twice-daily 
(BID) doses of AG-348 (up to 50 patients total; see Figure 1, Study Schema). The dose of Arm 1 is 
300 mg of AG-348 administered orally every 12 hours (q12h, BID). The dose of Arm 2 is 50 mg of 
AG-348 administered orally q12h (BID). Randomization will be stratified by PKR mutation in order to 
maintain balance as much as possible across the dose arms for the specific mutations expected to be 
most frequently enrolled. The PKR mutation stratification factor will consist of 4 levels (R510Q, 
R486W, and R479H) and all other mutations (“other”). Mutation status is defined by the presence of at 
least one of the indicated mutations; patients with more than one stratified mutation will be assigned 
based on Sponsor’s discretion.  
The doses for each arm have been selected from the forerunner AG348-C-001 single ascending dose 
(SAD) and AG348-C-002 multiple ascending dose (MAD) studies in healthy adult volunteers to 
represent the range of doses/exposures that were safely tolerated and resulted in maximal or near-
maximal PD effects on 2,3-DPG and ATP. 
Because PK deficiency is a rare disease with a limited eligible patient population and because the 
underlying pathophysiology and clinical phenotype of affected patients is heterogenous due to the wide 
variety of mutations in PKR that cause the disease, it is important to focus closely on dose findings in 
this first-in-patient study. Therefore, in addition to initiating this study with 2 different doses of AG-348 
administered q12h, a Data Review Team (DRT) will be established to review study data on a regular 
basis and adapt the study design, dose and schedule of AG-348. 
The DRT will monitor safety on an on-going basis and meet at regular intervals (approximately every 
6 weeks), or ad hoc, as necessary, to review AEs, vital signs (VS), clinical laboratory (hematology, 
clinical chemistry, coagulation, and urinalysis), and electrocardiograms (ECGs) on enrolled patients. 
The DRT will also review available PK/PD data and indicators of clinical activity (e.g., changes from 
baseline in Hb). The DRT will be comprised of the study Coordinating Investigator, treating 
Investigators, Medical Monitor, Clinical Pharmacologist, Statistician, and Sponsor’s Responsible 
Medical Officer.  
Approximately every 6 weeks beginning six weeks after the first patient is dosed or ad hoc as necessary, 
the DRT will review cumulative safety data, available PK/PD data, and clinical activity data. Based on 
the DRT’s recurring 6 week reviews, the DRT may exercise one or more of the following options: 
• 
Continue treatment and enrollment in existing arms without change. 
• 
Add 1 new dose arm (Arm 3) to enroll up to 25 patients at a dose to be determined; the dose for 
Arm 3 may be lower or higher than Arm 1 and Arm 2 doses, but will not exceed 360 mg q12h; 
and the dose regimen may be less frequent than q12h. 
• 
Terminate or suspend enrollment to allow further review of clinical data in Arm 1 and/or Arm 2 
(and/or potential Arm 3). Enrollment in an arm could be terminated or suspended to allow 
further review, for example, for unacceptable safety/tolerability, poor PD response, or lack of 
signs of clinical activity. 
• 
Re-assign patients’ doses and schedule in a terminated arm to match the dose and schedule of 
another arm of the study. In this case, the patients in the terminated arm will remain in their 
original arm, i.e., they will not count towards the enrollment quota of the arm whose dose and 
4 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
schedule is being adopted. 
• 
Implement specific genotype restrictions to enrollment in one or more arms to ensure 
representation of patients with genotypes of greatest clinical relevance. 
The DRT will perform these evaluations on a recurring 6-week basis. The data that the DRT will review 
to make these decisions is expected to include, but are not necessarily limited to, the following:  
• 
Safety Observations: all AEs; VS, clinical laboratory (hematology, clinical chemistry, 
coagulation, and urinalysis), and ECG;  
• 
PK and PD Observations: including changes in 2,3-DPG and ATP; 
• 
Indicators of Clinical Activity: including changes in Hb, HCT, reticulocyte count, Hp, COHb, 
LDH, EPO, total and indirect bilirubin, ferritin, and transferrin saturation.  
If a third dose arm is implemented, the dose of AG-348 selected will not exceed 360 mg BID, as this 
was the highest dose that demonstrated acceptable safety and tolerance in the 14-day multiple BID 
dosing study in healthy volunteers. The PK/PD sampling schedule in a potential third dosing arm will be 
determined by Sponsor’s discretion and may follow either the extensive or limited PK/PD sampling 
schedules as specified. 
The DRT will monitor the data in an ongoing manner as described and may make a decision to terminate 
enrollment in an arm if unacceptable AEs are observed, PD activity is limited, and/or markers of clinical 
activity indicate no effect. Unacceptable AEs are defined as ≥ Grade 3 AEs (using National Cancer 
Institute Common Terminology Criteria for Adverse Events [NCI CTCAEv4.03]) that are assessed as at 
least possibly related to AG-348, with the exception of Grade 3 headache, hot flash/flushing, nausea, 
vomiting, and/or diarrhea that is transient in duration (< 24 hours) or able to be medically managed to 
≤ Grade 2 within 24 hours. The DRT may also exercise discretion to terminate enrollment in an arm if it 
observes a frequency of Grade 2 AEs that would make it difficult to support long-term dosing. 
Due to the potential for AG-348-mediated aromatase inhibition, dual-energy x-ray absorptiometry 
(DXA) scan (hip and spine) will be performed at Screening (if patient has not had prior DXA scan 
within 3 months of Day 1) to obtain T and Z scores. These data are intended to serve as a baseline 
measure of bone mineral density for all enrolling patients, and are deemed of particular importance for 
those who may enter the longer term safety extension after completing the current study. All patients 
will have a second DXA scan in the interval between Weeks 24 and 28. The safety extension will 
address additional follow-up DXA scanning. 
As the number of enrolling arms changes in the study (for example, from 2 to 3), the randomization 
scheme will adjust to enable balanced randomization into each actively accruing arm. Randomization 
and stratification will cease in the event that only a single arm is left enrolling. 
Depending on possible early termination of 1 or both of the initial 2 arms, or the addition of a third arm, 
the study could enroll up to a maximum of 75 patients. 
5 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Figure 1:  Study Schema 
 
Abbreviations:  BID (q12h) = twice-daily (every 12 hours); DRT = data review team; PKR = pyruvate kinase red blood cell 
isoform; TBD= to be determined; w = week(s). 
 
Visit Schedule 
Screening assessments will occur within 28 days prior to the first dose of study treatment. During the 
Treatment period, patients will attend visits at baseline (Day 1), weekly through Week 3 (Days 8, 15, 
and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12), and monthly through 
Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate AG-348 and demonstrate evidence of 
clinical activity of AG-348 through Week 24 may be eligible to immediately enter a safety extension for 
continued treatment. For patients who finish treatment at the end of 24 weeks or sooner, or who elect not 
to enter the safety extension, study discharge will occur 4 weeks (Week 28 or earlier) following the last 
dose of study treatment at the final follow-up assessment. 
Dose Modifications for Safety and/or Increase in Hb Level 
The Investigator will monitor all patients for safety and tolerability. Modification of an individual 
patient’s dose of AG-348 will be based on AEs and/or observed changes in Hb level as described in 
Section 9.7.1 and Section 9.7.2. 
6 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Number of patients (planned): Up to approximately 75 patients. 
Diagnosis and main criteria for inclusion: 
Inclusion criteria: 
For entry into the study, patients must meet all of the following criteria during the Screening or other 
specified period: 
1. Signed written informed consent obtained prior to performing any study procedure, including 
screening procedures.  
2. Male or female, aged 18 years and older. 
3. Known medical history of PK deficiency.  
4. All patients must have documented clinical laboratory confirmation of PK deficiency by red 
blood cell pyruvate kinase enzymatic assay performed at Screening by a designated central 
laboratory. Patients with prior documentation of PK deficiency by red blood cell (RBC) 
enzymatic assay will have a reconfirmation of this result during Screening as a condition of 
enrollment. 
a. In the event that a patient’s screening pyruvate kinase enzymatic assay is negative (i.e., 
shows normal pyruvate kinase activity), the patient will be eligible for enrollment if the 
genotyping shows a mutant genotype that has been previously documented in the 
literature to be associated with pyruvate kinase deficiency. If the genotyping shows a 
previously undescribed mutation in the PKR gene, then the eligibility for enrollment 
will be determined on a case-by-case basis by the Coordinating Investigator and 
Medical Monitor in discussion with the Investigator. If no mutation is defined, then the 
patient will not be eligible. 
5. All patients must have genotypic characterization of the mutant PKR gene performed by a 
designated central laboratory at Screening, unless genotype is available from the patient’s 
participation in the Pyruvate Kinase Deficiency Natural History Study (NCT02053480). 
6. All patients must have genotypic characterization of the UGT1A1 gene performed by a 
designated central laboratory to document whether they may have underlying Gilbert's Disease. 
Patients with Gilbert’s Disease are eligible to enroll. 
7. Males must have Hb ≤ 12.0 g/dL; females must have Hb ≤ 11.0 g/dL.  
8. All patients must be considered transfusion independent as defined by: no greater than 3 units of 
RBCs transfused in the 12-month period up to the first day of study dosing and no transfusions 
within 4 months of first day of study dosing. Patients who have received more transfusion 
support than described above will evaluated for eligibility on a case-by-case basis by the 
7 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Medical Monitor. 
9. Eligible patients may still have their spleens in place, or may have undergone prior splenectomy. 
For splenectomized patients: 
a. Must have undergone their procedure at least 6 months prior to Screening. 
b. Must be current in their vaccinations for Pneumococcal Conjugate (PCV13), 
Pneumococcal Polysaccharide (PPSV23), Quadrivalent Meningococcal vaccine, and 
Haemophilus influenzae type b (Hib) as recommended by Centers for Disease Control 
and Prevention's (CDC) Advisory Committee on Immunization Practices (ACIP) or 
immunization advisory groups in Canada and the European Union (for patients enrolled 
in Canada and the EU). [http://www.cdc.gov/vaccines/schedules/downloads/adult/adult-
schedule.pdf] [Any missing vaccinations may be administered during the screening 
period.] 
10. Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2. (Appendix 15.2) 
11. Patients must be taking at least 1 mg of folic acid daily for at least 21 days prior to first dose and 
continue daily during study participation. 
12. Adequate organ function, defined as: 
a. Serum aspartate transaminase (AST) and alanine aminotransferase (ALT) ≤ 1.5 × upper 
limit of normal (ULN) (unless the increased AST is assessed by the Investigator as due 
to hemolysis). 
b. Normal or elevated levels of serum bilirubin. In patients with serum bilirubin > ULN, 
the elevation must be attributed to hemolysis with or without Gilbert's syndrome and 
must not be choledocholithiasis, cholecystitis, biliary obstruction, or hepatocellular 
disease. 
c. Serum creatinine ≤ 1.25 × ULN. If serum creatinine > 1.25 × ULN, then 24-hour 
measured or calculated (Cockcroft-Gault) glomerular filtration rate (GFR) ≥ 60 mL/min. 
d. Absolute neutrophil count (ANC) > 1.0 × 109/L. 
e. Platelet count ≥ 100 × 109/L. 
f. Activated partial thromboplastin time (aPTT) and international normalized ratio (INR) 
≤ 1.25 × ULN, unless the patient is receiving therapeutic anticoagulants. 
13. Women of childbearing potential (WOCBP) must agree to abstain from sexual intercourse or to 
use an acceptable/effective method of contraception (i.e., condom plus spermicide, condom plus 
oral contraceptive, condom plus intrauterine device [IUD], condom plus diaphragm with 
spermicide) from as soon as feasible during the Screening period until 30 days following the last 
dose of AG-348. Abstinence is an acceptable method only when this is in line with the normal 
life style of the patient, meaning that the patient plans to remain abstinent continuously 
throughout the duration of the study and for at least 30 days after the last dose of study drug. 
Periodic abstinence, e.g., calendar, sympathothermal and post-ovulation methods, and 
8 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
withdrawal are not acceptable methods of contraception. 
a. WOCBP include any female who has experienced menarche and who has not undergone 
successful surgical sterilization (hysterectomy, bilateral tubal ligation, and/or bilateral 
oophorectomy) or is not post-menopausal. Post-menopausal is defined as: 
i. 
Amenorrhea ≥ 12 consecutive months without another cause, and a documented 
serum follicle stimulating hormone (FSH) level > 35 mIU/mL; 
ii. 
Amenorrhea ≥ 12 consecutive months in women ≥ 62 years old (FSH testing is 
not required). 
14. WOCBP must have a negative serum or urine pregnancy test within 72 hours before start of 
AG-348 dosing. 
15. Women must not be breastfeeding.  
16. Male patients, with the exception of those who have undergone vasectomy at least 6 months 
prior to Screening, must agree to abstain from sexual intercourse or, if sexually active, to use a 
condom with spermicide as contraception (regardless of their female partner's childbearing 
potential or their partner's use of their own contraception) from Day 1 of dosing until 30 days 
following the last dose of AG-348. Abstinence is an acceptable method only when this is in line 
with the normal life style of the patient, meaning that the patient plans to remain abstinent 
continuously throughout the duration of the study and for at least 30 days after the last dose of 
study drug. Periodic abstinence, e.g., selective timing of intercourse based on partner’s calendar, 
sympathothermal and post-ovulation methods, and withdrawal are not acceptable methods of 
contraception. 
Exclusion criteria: 
Patients who meet any of the following criteria at Screening or prior to dosing on Day 1 will not be 
enrolled in the study: 
1. Hemoglobin level > 12.0 g/dL if male; Hb > 11.0 g/dL if female. 
2. Additional diagnosis of any other congenital or acquired blood disorder, including 
glucose-6-phosphate-dehydrogenase (G6PD) deficiency, or any other hemolytic anemia 
process except for mild allo-immunization as a consequence of transfusion therapy. 
3. Iron overload (hemosiderosis or concurrent hemochromatosis) sufficiently severe to result in a 
clinical diagnosis by the Investigator of cardiac, hepatic, or pancreatic insufficiency. 
4. Prior bone marrow or stem cell transplant. 
5. Clinically symptomatic cholelithiasis or cholecystitis. (Prior cholecystectomy is not 
exclusionary. Patients with symptomatic cholelithiasis or cholecystitis may be rescreened once 
9 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
the disorder has been treated and clinical symptoms have resolved.) 
6. Currently enrolled in another therapeutic clinical trial involving on-going therapy with any 
investigational or marketed product or placebo. Concurrent participation in the Pyruvate Kinase 
Deficiency Natural History Study (NCT02053480) is permitted. 
7. Exposure to any investigational drug, device, or procedure within 28 days prior to Screening. 
8. Concurrent medical condition that could compromise participation in the study such as: 
a. Poorly controlled hypertension (defined as systolic blood pressure (BP) > 150 mm Hg 
or diastolic BP > 90 mm Hg) refractory to medical management. 
b. History of recent (within < 6 months from Screening date) congestive heart failure; 
myocardial infarction or unstable angina pectoris; or hemorrhagic, embolic, or 
thrombotic stroke; deep venous thrombosis; or pulmonary or arterial embolism.  
c. Currently active infection requiring the use of parenteral anti-microbial agents or that is 
greater than Grade 3 (CTCAEv4.03) within 6 months of first dose. 
d. A pattern or frequency of post-splenectomy sepsis that in the assessment of the 
Investigator could reasonably be expected to interfere with the ability of the patient to 
complete the 24 week study participation. 
e. Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) 
antibody with signs of active Hepatitis B or C virus infection.  
f. Positive test for human immunodeficiency virus (HIV) 1 or 2 antibody. 
g. Diabetes mellitus judged to be in poor control by the Investigator or requiring > 3 anti-
diabetic agents counting insulin (all insulins are considered one agent); use of insulin 
per se is not exclusionary.  
h. History of any primary malignancy with the exception of: curatively treated non-
melanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; or 
other primary tumor treated with curative intent and no known active disease present 
and no treatment administered during the last 3 years. 
9. Undergone major surgery within 6 months of first dose.  
10. Current or recent history of psychiatric disorder that in the opinion of the Investigator or 
Medical Monitor could compromise the ability of the patient to cooperate with study visits and 
procedures. 
11. Use of any of the restricted list of products known to strongly inhibit CYP3A4 drug metabolism 
(Appendix 15.4, Table 8) within 5 days prior to Day 1 dosing; or to strongly induce CYP3A4 
10 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
metabolism (Appendix 15.4, Table 9) within 28 days prior to Day 1 dosing; or to strongly inhibit 
P-glycoprotein (P-gp) transporter (Appendix 15.4, Table 10) within 5 days prior to Day 1 
dosing; or digoxin within 5 days prior to Day 1 dosing. For patients who require chronic inhaled 
glucocorticoid therapy, Investigators should confer with the Medical Monitor for additional 
guidance. 
12. Serum bilirubin > ULN attributable to factors other than hemolysis and/or Gilbert's syndrome. 
13. Male patients with heart-rate corrected QT (Fridericia's correction factor) QTcF interval 
> 450 msec, or female patients with QTcF interval > 470 msec with the exception of patients 
with a left bundle branch block (LBBB). Medical Monitor approval needed in patients with a 
LBBB. 
14. Cardiac dysrhythmias judged as clinically significant by the Investigator or requiring therapy 
with drugs that are primarily substrates of CYP3A4. 
15. History of allergy to sulfonamides if characterized by acute hemolytic anemia, anaphylaxis, or 
rash of erythema multiforme type or Stevens-Johnson syndrome. 
16. Any other medical or psychological condition deemed by the Investigator to be likely to 
interfere with a patient's ability to understand and sign informed consent; cooperate with study 
visits, tests, and procedures; or otherwise safely and reliably participate in the study. 
Investigational product, dosage, and mode of administration: 
AG-348 sulfate hydrate capsules will be provided as 5 mg, 25 mg, or 100 mg (free-base equivalent) of 
AG-348 sulfate hydrate without excipients in dark green opaque (5 mg), Swedish orange (25 mg), or 
white opaque (100 mg) gelatin capsules (size 2 capsules for all dose strengths). AG-348 will be 
administered orally BID. The number of capsules per dose will vary by assigned dose group. Patients 
will receive multiple oral (PO) doses of AG-348 over a 24-week treatment period. AG-348 will be 
administered with water and may be administered with or without food. 
Reference therapy, dosage and mode of administration:  
Not applicable. 
Duration of treatment:  
The duration of treatment for all patients on this study will be up to 24 weeks. Patients who safely 
tolerate and demonstrate one or more indicators of clinical activity of AG-348 may be eligible to 
immediately roll over to a safety extension for continued treatment. 
Criteria for evaluation: 
Safety: 
Monitoring of AEs in randomized patients, including determination of serious adverse events (SAEs) 
and AEs leading to discontinuation; safety laboratory parameters (hematology, chemistry, urinalysis, 
coagulation); physical examination findings (including neurological examination); VS; 12-lead ECGs, 
11 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
and DXA scans. Adverse events will be graded using CTCAE, Version 4.03. Serum sex hormone levels 
(testosterone [total and free], estrone, and estradiol), bone turnover markers (serum osteocalcin-N-mid 
and serum C-terminal telopeptide [CTX]), 25-hydroxy vitamin D2 and D3, total cholesterol, high-
density lipoprotein-cholesterol (HDL-C), and triglycerides will be monitored for evidence of potential 
inhibition of aromatase by AG-348. Menstruating female patients will also keep a paper-based menstrual 
cycle diary throughout the study. 
Indicators of Clinical Activity: 
Monitoring of potential indicators of clinical activity will include evaluating changes in Hb, HCT, 
reticulocyte count, COHb, LDH, total and indirect bilirubin, EPO, ferritin, and transferrin saturation.  
Pharmacokinetics: 
Approximately the first 10 patients treated, contingent on clinical site feasibility, will undergo extensive 
PK sampling as detailed in Appendix 15.1, Table 6. The remainder of treated patients will undergo 
limited PK sampling as detailed in Appendix 15.1, Table 7. Serial blood sampling for determination of 
concentration-time profiles of AG-348 and its metabolite AGI-8702 will be conducted following the first 
dose and the morning Day 15 dose, and additional trough levels of AG-348 and AGI-8702 will be 
obtained. AG-348 and AGI-8702 will be analyzed using qualified assays to determine concentrations in 
plasma. Pharmacokinetic parameters on Day 1 and Day 15 will be computed using standard non-
compartmental methods based on observed plasma AG-348 and AGI-8702 concentrations. 
Pharmacodynamics: 
Pharmacodynamic assessments will include 2,3-DPG, ATP (secondary objectives), 
 
. Approximately 
the first 10 patients treated will undergo extensive PD sampling as detailed in Appendix 15.1, Table 6. 
The remainder of treated patients will undergo limited PD sampling as detailed in Appendix 15.1, 
Table 7. Serial blood sampling for determination of levels of ATP and, 2,3-DPG will be conducted 
following the first dose and the morning Day 15 dose, and additional trough levels of ATP and 2,3-DPG 
will be obtained. Adenosine triphosphate and 2,3 DPG will be analyzed using qualified assays to 
determine concentrations in whole blood. Pharmacodynamic parameters on Day 1 and Day 15 will be 
computed based on observed whole blood ATP and 2,3-DPG concentrations.  
Statistical methods: 
The primary objective of this Phase 2 study is to evaluate the safety and tolerability of up to 24 weeks of 
12 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
AG-348 in patients with PK deficiency. Therefore, analyses will be primarily descriptive in nature; no 
formal hypothesis testing will be conducted. All analyses will be conducted separately within each dose 
arm, or pooled when appropriate. 
Summaries will be produced for disposition, baseline disease characteristics and demographic data 
including genotype, safety measurements, PK, PD parameters and indicators of clinical activity. Data 
from each AG-348 dose group will be analyzed separately, and pooled across all dose groups where 
appropriate. All data will also be listed by individual patient. Categorical variables will be summarized 
by frequency distributions (number and percentages) and continuous variables will be summarized by 
descriptive statistics (mean, standard deviation [SD], median, minimum, and maximum). 
Populations for analysis will include a Safety Analysis Set, a PK Analysis Set, and an Efficacy Analysis 
Set. The Safety Analysis set will include all patients who are enrolled and receive any dose of study 
treatment; this population will be the primary set for the analysis of safety data and the default analysis 
set for all data analyses unless otherwise stated. Patients will be classified according to treatment 
received, where treatment received is defined as the assigned treatment if it is received at least once, or 
as the first treatment received if assigned treatment is never received. The PK Analysis Set will include 
all patients in the Safety Analysis Set with sufficient plasma sample data to assess PK parameters. 
Results of the potential PD activity of AG-348 will also be based on the PK analysis set. The Efficacy 
Analysis Set will include all patients who are enrolled and achieve at least 50% compliance with their 
assigned dose intensity for at least 4 weeks of continued dosing. Additional efficacy analyses may be 
performed on subsets of patients with other degrees of compliance with their assigned dose intensity 
and/or duration of dosing. The Efficacy Analysis Set will be the primary set for the analysis of 
preliminary clinical activity data. Patients will be classified according to assigned treatment. 
 
 
 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) and the 
incidence of treatment-emergent AEs (TEAEs) (new or worsening from baseline) will be summarized by 
primary system organ class (SOC), preferred term (PT), CTCAE Version 4.03 severity, outcome, action 
taken with study drug, and relationship to study drug by dose group. Separate summaries will be 
produced for all TEAEs, treatment-related AEs (those considered by the Investigator as at least possibly 
drug related), SAEs, discontinuations due to AEs, and AEs ≥ Grade 3 severity. Individual patient listings 
will be provided for any deaths, SAEs, AEs leading to interruption and/or reduction of study drug dose, 
and AEs leading to discontinuation of treatment. Analyses of AEs will be performed for those events 
that are considered treatment-emergent, where treatment-emergent is defined as any AE with onset 
beginning at the day of first administration of study drug, throughout the treatment period until 30 days 
after the last dose of study drug, or any event that was present at baseline but worsened in intensity or 
was subsequently considered drug-related by the Investigator through the end of the study. 
Descriptive statistics will be provided for clinical laboratory values (e.g., hematology, serum chemistry, 
coagulation studies, urinalysis) and VS data, presented as both actual values and changes from baseline 
relative to each on-study evaluation. Shift analyses will be conducted for selected laboratory parameters 
based on the baseline CTCAE grade to maximum CTCAE grade. Where applicable CTCAE terms do 
13 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
not exist, a grading system based on the upper and/or lower limits of normal will be used to classify the 
results. 
Electrocardiogram analyses will include individual patient listings and summaries of abnormal and 
clinically significant ECG results. Actual values and changes from baseline in PR, QRS, QTc intervals 
will be summarized by visit and dose arm.  
Data collected from the menstrual diaries such as the start and stop dates of the menses and the patient 
reported characteristics of the menses will be presented in a by-patient listing. Additional descriptions of 
the data may also be performed. 
Concomitant medications will be coded using the WHO Drug Dictionary. Concomitant medications 
prior to and after the start of the study drug will be listed by patient, and summarized by ATC term and 
dose arm. 
Descriptive statistics will be used to summarize PK parameters for the parent compound AG-348 and the 
metabolite AGI-8702 for each dose group, and where appropriate, for the entire population. 
Pharmacokinetic parameters will be summarized using the following descriptive statistics: n, mean, 
standard deviation (SD), coefficient of variation %, median, minimum, and maximum, geometric mean, 
and geometric coefficient of variation.  
Descriptive statistics will be used to summarize PD parameters for 2,3-DPG and ATP for each dose 
group, and where appropriate for the entire population. Pharmacodynamic parameters will be 
summarized using the following descriptive statistics: n, mean, SD, coefficient of variation %, median, 
minimum, and maximum, geometric mean, and geometric coefficient of variation %. 
Analyses of serum sex hormones will use appropriate graphic displays and statistical analyses to 
evaluate patient change from baseline for each parameter. These analyses will include summaries of 
actual values and change from baseline using appropriate descriptive statistics (mean, SD, median, min 
and max) as described above.  
Analyses evaluating indicators of potential clinical activity of AG-348 in patients with PK deficiency 
will include changes in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, 
ferritin, and transferrin saturation (serum iron/iron binding capacity). Characterization of Hb response 
(e.g., % of patients whose Hb increases by a certain amount), as well as time to Hb response, and 
duration of Hb response will be explored, among others. 
Interim Review 
No formal statistical analysis will be conducted. Safety data will be reviewed on an ongoing basis by the 
DRT, who will meet to review safety, PK, PD, and preliminary clinical activity data at regular intervals 
(approximately every 6 weeks) throughout the duration of the study. The composition of the DRT, its 
meeting schedule, the data to be reviewed, and the decisions it is empowered to make have been 
described previously. 
Additional interim reviews of data may be conducted to support decision making concerning the current 
clinical study, the sponsor’s development programs in general, or in case of any safety concerns.  
Sample Size 
Due to the rare disease setting, the minimal sample size may be determined by feasibility. Depending on 
14 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
possible early termination of 1 or both of the initial 2 arms or the addition of a 3rd dose arm, the study 
could enroll up to a maximum of 75 patients. 
15 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF 
FIGURES 
TABLE OF CONTENTS 
1. 
TITLE PAGE ................................................................................................................1 
2. 
SYNOPSIS ...................................................................................................................3 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES ...............16 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS.............................21 
5. 
INTRODUCTION ......................................................................................................25 
5.1. 
Pyruvate Kinase Deficiency .......................................................................................25 
5.2. 
AG-348 .......................................................................................................................26 
5.2.1. 
Summary of Nonclinical Data ....................................................................................26 
5.2.1.1. 
Pharmacology .............................................................................................................26 
5.2.1.2. 
Safety Pharmacology ..................................................................................................27 
5.2.1.3. 
Pharmacokinetics ........................................................................................................27 
5.2.1.4. 
Toxicology ..................................................................................................................28 
5.2.2. 
Summary of Clinical Data ..........................................................................................30 
5.2.2.1. 
Pharmacokinetics ........................................................................................................30 
5.2.2.2. 
Pharmacodynamics .....................................................................................................31 
5.2.2.3. 
Safety ..........................................................................................................................31 
5.3. 
Study Rationale ...........................................................................................................32 
5.3.1. 
Summary of Overall Safety Management Plan ..........................................................33 
6. 
TRIAL OBJECTIVES AND ENDPOINTS ...............................................................35 
6.1. 
Primary Objective .......................................................................................................35 
6.2. 
Secondary Objectives .................................................................................................35 
 
35 
6.4. 
Study Measures and Endpoints ...................................................................................35 
6.4.1. 
Safety Measures and Endpoints ..................................................................................35 
6.4.2. 
Clinical Activity Measures and Endpoints .................................................................36 
6.4.3. 
Pharmacokinetic and Pharmacodynamic Measures and Endpoints ............................36 
 
36 
7. 
INVESTIGATIONAL PLAN .....................................................................................37 
16 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
7.1. 
Overall Study Design ..................................................................................................37 
7.2. 
Justification of the Study Design ................................................................................40 
7.3. 
Rationale for the Starting Dose, Dose Range, and Duration of Dosing .....................41 
7.4. 
Criteria for Study Termination ...................................................................................42 
8. 
STUDY POPULATION .............................................................................................44 
8.1. 
Number of Patients .....................................................................................................44 
8.2. 
Inclusion Criteria ........................................................................................................44 
8.3. 
Exclusion Criteria .......................................................................................................46 
8.4. 
Patient Identification and Registration .......................................................................48 
8.5. 
Patient Randomization ................................................................................................48 
8.6. 
Patient Withdrawal Criteria ........................................................................................48 
8.7. 
Replacement of Patients .............................................................................................49 
9. 
STUDY TREATMENT ..............................................................................................50 
9.1. 
Description of Study Drug ..........................................................................................50 
9.2. 
Study Drug Packaging and Labeling ..........................................................................50 
9.3. 
Study Drug Storage .....................................................................................................50 
9.4. 
Method of Assigning Patients to Treatment ...............................................................50 
9.5. 
Blinding ......................................................................................................................50 
9.6. 
Study Drug Preparation and Administration ..............................................................50 
9.7. 
Criteria for Dose Escalation, Dose Modification, or Discontinuation of Study 
Drug ............................................................................................................................51 
9.7.1. 
Dose Modification for Safety .....................................................................................51 
9.7.2. 
Dose Modification for Increase in Hemoglobin Level ...............................................52 
9.7.3. 
Stopping Criteria .........................................................................................................53 
9.8. 
Duration of Patient Participation ................................................................................53 
9.9. 
Treatment Compliance ................................................................................................53 
9.10. 
Study Drug Accountability .........................................................................................53 
9.11. 
Prior and Concomitant Medications and Treatments .................................................54 
9.11.1. 
Prior Medications and Procedures ..............................................................................54 
9.11.2. 
Prohibited Concomitant Therapy ................................................................................54 
9.11.3. 
Allowed Concomitant Therapy ...................................................................................56 
9.11.4. 
Potential for Phototoxicity ..........................................................................................56 
9.11.5. 
Management of Nausea, Vomiting, and Diarrhea ......................................................56 
17 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
9.11.6. 
Other Restrictions and Precautions .............................................................................56 
10. 
STUDY ASSESSMENTS ..........................................................................................58 
10.1. 
Schedule of Assessments ............................................................................................58 
10.2. 
Informed Consent and Confirmation of Eligibility ....................................................59 
10.3. 
Demographic Data, Medical and Medication History ................................................59 
10.4. 
PKR Enzymatic Assay and PKR Genotyping ............................................................60 
10.5. 
Safety Assessments .....................................................................................................60 
10.5.1. 
Physical Examination, Height, and Weight ................................................................60 
10.5.2. 
Vital Signs ..................................................................................................................60 
10.5.3. 
Electrocardiogram .......................................................................................................60 
10.5.4. 
DXA Scans .................................................................................................................60 
10.5.5. 
Safety Laboratory Assessments ..................................................................................61 
10.5.5.1. 
Hematology, Serum Chemistry, Coagulation Studies, and Urinalysis .......................61 
10.5.5.2. 
Screening Serology .....................................................................................................62 
10.5.6. 
Menstrual Cycle Diary ................................................................................................62 
10.5.7. 
Adverse Events ...........................................................................................................62 
10.6. 
Pharmacokinetic Assessments ....................................................................................63 
10.6.1. 
Blood Sample Collection and Pharmacokinetic Measurements During Dose 
Escalation ....................................................................................................................63 
10.7. 
Pharmacodynamic Assessments .................................................................................63 
10.8. 
Ordering of Blood Sample Collection ........................................................................65 
10.9. 
Sample Processing, Storage, and Shipment ................................................................65 
11. 
ADVERSE EVENTS..................................................................................................66 
11.1. 
Definition of Adverse Events .....................................................................................66 
11.1.1. 
Adverse Event (AE) ....................................................................................................66 
11.1.2. 
Suspected Adverse Reaction .......................................................................................66 
11.1.3. 
Unexpected Adverse Event .........................................................................................66 
11.1.4. 
Serious Adverse Event ................................................................................................66 
11.1.4.1. 
Potential Severe Drug-Induced Liver Injury ..............................................................67 
11.2. 
Procedures for Reporting Adverse Events and Serious Adverse Events ....................67 
11.3. 
Pregnancy Reporting ..................................................................................................70 
12. 
STATISTICAL METHODS .......................................................................................71 
12.1. 
Sample Size Estimation ..............................................................................................71 
18 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
12.2. 
Populations for Analysis .............................................................................................71 
12.3. 
Procedures for Handling Missing, Unused, and Spurious Data .................................72 
12.4. 
Interim Analysis ..........................................................................................................72 
12.5. 
Statistical Methodology ..............................................................................................72 
12.5.1. 
General Methods .........................................................................................................72 
12.5.2. 
Disposition ..................................................................................................................73 
12.5.3. 
Exposure and Safety Analyses ....................................................................................73 
12.5.4. 
Pharmacokinetic Analyses ..........................................................................................74 
12.5.5. 
Pharmacodynamic Analyses .......................................................................................74 
12.5.6. 
Aromatase Hormone Analysis ....................................................................................74 
12.5.7. 
Clinical Activity ..........................................................................................................74 
 
74 
12.6. 
Procedures for Reporting Deviations to Original Statistical Analysis Plan ...............74 
13. 
ADMINSTRATIVE REQUIREMENTS ....................................................................75 
13.1. 
Good Clinical Practice ................................................................................................75 
13.2. 
Ethical Considerations ................................................................................................75 
13.3. 
Patient Information and Informed Consent ................................................................75 
13.4. 
Patient Confidentiality ................................................................................................76 
13.5. 
Protocol Compliance ..................................................................................................76 
13.6. 
Data Management .......................................................................................................76 
13.7. 
Source Document/Case Report Form Completion .....................................................77 
13.8. 
Direct Access to Source Data .....................................................................................77 
13.9. 
Record Retention ........................................................................................................77 
13.10. 
Liability and Insurance ...............................................................................................78 
13.11. 
Publication of Study Findings and Use of Information ..............................................78 
14. 
LIST OF REFERENCES ............................................................................................79 
15. 
APPENDICES ............................................................................................................80 
15.1. 
Schedules of Assessments ..........................................................................................81 
15.2. 
Eastern Cooperative Oncology Group Performance Status Scoring ..........................89 
15.3. 
National Cancer Institute Common Terminology Criteria for Adverse Events .........89 
15.4. 
Potential Drug Interactions .........................................................................................90 
15.5. 
Declaration of Helsinki ...............................................................................................93 
 
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LIST OF TABLES 
Table 1: 
Dose Modification for Adverse Events Deemed at Least Possibly Related to 
AG-348 .......................................................................................................................52 
Table 2: 
Dose Reduction Table (by Dosing Arm) ....................................................................53 
Table 3: 
Summary of Assessments When Day 8 and/or Day 22 In-Clinic Visits Are 
Performed By Primary Care Physician .......................................................................59 
Table 4: 
Sample Size Estimation ..............................................................................................71 
Table 5: 
Schedule of Assessments: ...........................................................................................81 
Table 6: 
Schedule of Assessments: Extensive PK/PD Sampling .............................................87 
Table 7: 
Schedule of Assessments: Limited PK/PD Sampling ................................................88 
Table 8: 
Strong and Moderate CYP3A4 Inhibitors ..................................................................90 
Table 9: 
Strong CYP3A4 Inducers ...........................................................................................91 
Table 10: Strong P-glycoprotein Inhibitors ................................................................................91 
Table 11:  Sensitive CYP3A4 Substrates.....................................................................................92 
 
LIST OF FIGURES 
Figure 1:  Study Schema ...............................................................................................................6 
Figure 2: 
Red Cell Glycolysis ....................................................................................................25 
Figure 3: 
Study Schema .............................................................................................................39 
Figure 4: 
PKR Enzymatic Reaction ...........................................................................................64 
 
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4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation or Specialist Term 
Explanation 
2,3-DPG 
2,3-diphosphoglycerate 
ACIP 
Advisory Committee on Immunization Practices 
ADME 
Absorption, distribution, metabolism, excretion 
ADP 
Adenosine diphosphate 
AE 
Adverse event 
ALC 
Absolute lymphocyte count 
ALP 
Alkaline phosphatase 
ALT 
Alanine aminotransferase 
ANC 
Absolute neutrophil count 
aPTT 
Activated partial thromboplastin time 
AST 
Aspartate aminotransferase 
ATP 
Adenosine triphosphate 
AUC 
Area under the curve 
AUC0-12hr 
Area under the plasma concentration versus time curve from 0 to 12 
hours 
AUC0 24hr 
Area under the plasma concentration versus time curve from 0 to 
24 hours 
AUC0-∞ 
Area under the plasma concentration versus time curve from 0 to 
infinity 
BCRP 
Breast cancer resistance protein 
BID 
Twice-daily 
BMI 
Body mass index 
BP 
Blood pressure 
BUN 
Blood urea nitrogen 
CDC 
Centers for Disease Control and Prevention 
CFR 
Code of Federal Regulations 
CLP 
Total body plasma clearance 
Cmax 
Maximum plasma concentration 
CO2 
Carbon dioxide 
COHb 
Carboxyhemoglobin 
CTCAE 
Common Terminology Criteria for Adverse Events 
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Abbreviation or Specialist Term 
Explanation 
CTX 
Serum C-terminal telopeptide 
CV 
Cardiovascular 
DDI 
Drug-drug interaction 
CYP 
Cytochrome P450 
DILI 
Drug-induced liver injury 
DLT 
Dose-limiting toxicity 
DRT 
Data review team 
DXA 
Dual-energy x-ray absorptiometry 
ECG 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic case report form 
EDC 
Electronic Data Capture 
EPO 
Erythropoietin 
F 
Oral bioavailability 
FDA 
Food and Drug Administration 
FSH 
Follicle stimulating hormone 
G6PD 
Glucose-6-phosphate-dehydrogenase 
GCP 
Good Clinical Practice 
GFR 
Glomerular filtration rate 
GLP 
Good Laboratory Practice 
Hb 
Hemoglobin 
HBsAg 
Hepatitis B surface antigen 
HCT 
Hematocrit 
HCV 
Hepatitis C virus 
HDL-C 
High-density lipoprotein-C 
HDPE 
High density polyethylene 
hERG 
Human ether à-go-go related gene 
Hib 
Haemophilus influenzae type b 
HIV 
Human immunodeficiency virus 
Hp 
Haptoglobin 
IC50 
Concentration of drug that achieved half-maximal inhibition 
ICH 
International Conference on Harmonization 
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Abbreviation or Specialist Term 
Explanation 
INR 
International normalized ratio 
IRB 
Institutional Review Board 
IUD 
Intrauterine device 
IV 
Intravenous 
LDH 
Lactate dehydrogenase 
MAD 
Multiple ascending dose 
MCH 
Mean corpuscular hemoglobin 
MCHC 
Mean corpuscular hemoglobin concentration 
MCV 
Mean corpuscular volume 
MedDRA 
Medical Dictionary for Regulatory Activities 
mPKR 
PKR mutants 
MTD 
Maximum tolerated dose 
NCI 
National Cancer Institute 
NOEL 
No-observed-effect-level 
NOAEL 
No-observed-adverse-effect-level 
P-gp 
P-glycoprotein 
PCV13 
Pneumococcal Conjugate 
PD 
Pharmacodynamic 
PEP 
Phosphoenolpyruvate 
PK 
Pharmacokinetic 
PK deficiency 
Pyruvate kinase deficiency 
PKR 
Pyruvate kinase isoform R 
PO 
Oral 
PPSV23 
Pneumococcal polysaccharide 
PR 
The portion of the ECG wave from the beginning of the P wave to 
the beginning of the QRS complex 
PT 
Preferred term 
q12h 
Every 12 hours 
q24h 
Every 24 hours 
QD 
Once-daily 
QRS 
QRS interval on an electrocardiogram 
QTc 
Heart-rate corrected QT interval 
QTcB 
Corrected QT interval - Bazett correction formula 
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Abbreviation or Specialist Term 
Explanation 
QTcF 
Corrected QT interval - Fridericia’s method 
RBC 
Red blood cell (count) 
RDW 
Red cell distribution width 
SAD 
Single ascending dose 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SD 
Standard deviation 
SOC 
System organ class 
SOP(s) 
Standard Operating Procedure(s) 
t½ 
Apparent terminal half-life 
TIBC 
Total iron-binding capacity 
Tmax 
Time to maximum concentration 
TEAE 
Treatment-emergent adverse event 
ULN 
Upper limit of normal 
VS 
Vital signs 
Vss 
Volume of distribution at steady-state 
Vz/F 
Mean apparent volume of distribution 
WBC 
White blood cell  
WMA 
World Medical Association 
WOCBP 
Women of childbearing potential 
WT 
Wild type 
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5. 
INTRODUCTION 
5.1. 
Pyruvate Kinase Deficiency 
Pyruvate kinase deficiency (PK deficiency) is a glycolytic enzymopathy that results in non-
spherocytic hemolytic anemia with a variable clinical presentation, ranging from mild or fully 
compensated forms to life-threatening neonatal anemia and life-long chronic hemolytic anemia 
associated with severe, debilitating co-morbidities. PK deficiency is caused by mutations in the 
PKLR gene, which results in defective pyruvate kinase isoform R (PKR) in the red blood cell 
(RBC). As shown in the diagram below, PKR catalyzes the final and irreversible step in 
glycolysis, converting phosphoenolpyruvate (PEP) to pyruvate, and formation of adenosine 
triphosphate (ATP) from adenosine diphosphate (ADP). Mature RBCs rely almost exclusively on 
the process of glycolysis to generate the energy carrier molecule ATP. PKR is thus a key enzyme 
for maintaining energy homeostasis in erythrocytes, and it has been proposed that ATP levels are 
critical for optimally maintaining RBC membrane integrity (van Wijk and van Solinge 2005). 
Figure 2: 
Red Cell Glycolysis 
 
Abbreviations:  1,3 DPG = 1,3 diphosphoglycerate; 2,3 DPG = 2,3 diphosphoglycerate; 3-PG = 3-phosphoglycerate; 
ADP = adenosine diphosphate; ATP = adenosine triphosphate; FBP = fructose 1,6-bisphosphate; PEP = 
phosphoenolpyruvate; PKR = red blood cell-specific form of pyruvate kinase 
Note: Not all steps in glycolysis are shown. 
The majority of mutations in PKR that have been described have a deleterious effect on PKR 
catalytic activity, protein stability, and/or protein expression. PK deficiency is associated with 
reduced RBC survival as well as impaired red cell maturation. Mature erythrocytes lack 
mitochondria relying predominantly on glycolysis to generate ATP. It is hypothesized that 
insufficient energy production promotes erythrocyte hemolysis due to impaired maintenance of 
cellular membrane homeostasis. 
PK deficiency is an autosomal recessive disease, in which both homozygotes and compound 
heterozygotes develop hemolytic anemia. More than 200 different mutations in the PKLR gene 
have been identified in patients with PK deficiency to date, and the majority of these mutations 
are single nucleotide missense mutations (Zanella, et al. 2005). The prevalence of PK deficiency, 
based on the most common pyruvate kinase mutation in Southern Europe, is estimated at 51 
cases per million in the Caucasian population (Beutler and Gelbart 2000). 
Some patients with PK deficiency have a clinically severe disease course that can present as a 
severe hemolytic anemia in early infancy, requiring immediate care which typically includes 
blood transfusion support (Christensen, et al. 2011; Ghidini, et al. 1991; Hennekam, et al. 1990). 
Infants with PK deficiency may have erythroblastosis fetalis with hepatosplenomegaly, 
extramedullary hematopoiesis, and severe indirect hyperbilirubinemia, which is associated with a 
high risk of kernicterus. Hyperbilirubinemia associated with the hemolytic state often requires 
Glucose
Pyruvate
PKR
`
ATP
FBP
1,3-DPG
2,3-DPG
3-PG
PEP
ADP
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supportive treatment with intensive phototherapy. For other infants, the hyperbilirubinemia 
caused by hemolysis can require exchange transfusion, as well as supportive transfusion to keep 
their hematocrit (HCT) levels >60%. 
Adults with PK deficiency are reported to have life-long hemolysis and the subsequent 
associated co-morbidities including anemia and transfusion dependence. Other co-morbidities 
include frequent miscarriages, aplastic crises, as well as symptoms associated with an acute on 
chronic hemolytic anemia (Rider, et al. 2011). Since unconjugated bilirubin is often chronically 
elevated, pigmented gallstones are common in children and adults. Severe and sometimes life-
threatening iron deposition occurs and is typically progressive. 
The current therapeutic options for patients with PK deficiency are supportive. Most require life-
long treatment, including blood transfusions at a frequency depending on the disease state. Long-
term surveillance for systemic iron overload, even in transfusion-independent patients, is 
standard as is the use of chelation therapy. Case reports of cure by allogeneic bone marrow 
transplant have been published (Tanphaichitr, et al. 2000) but have been infrequently performed. 
Splenectomy, while not curative, can reduce the need for supportive blood transfusions and 
decrease bilirubin levels in some patients. However, this intervention is associated with 
significant morbidity including infection risk, and possible increased risk of thrombosis. 
5.2. 
AG-348 
AG-348 is an orally available, allosteric activator of PKR. It is hypothesized that drug 
intervention with AG-348 restores glycolytic pathway activity and normalizes red cell 
metabolism in vivo. Biochemical experiments demonstrate that AG-348 is a potent, pan-activator 
of many PKR alleles associated with PK deficiency. PK deficiency red cells and their 
progenitors are characterized by changes in metabolism associated with defective glycolysis, 
including a build-up of PEP and 2,3-diphosphoglycerate (2,3-DPG), and lowered ATP levels. 
Treatment of PK deficiency patient red cells ex vivo with AG-348 results in increased ATP 
levels, and reductions in PEP and 2,3-DPG, consistent with pharmacological activation of PKR 
enzyme activity. In vitro studies against wild type (WT) PKR enzymes and ex vivo studies in 
blood from mice, monkeys and humans indicate that AG-348 activates WT PKR with a similar 
potency and efficacy as it does with the mutant versions. In vivo studies in mice confirm the 
potency of AG-348 in increasing WT PKR enzyme activity and modulating the levels of 
downstream markers such as ATP and 2,3-DPG. This therapeutic approach may be an effective 
way to correct the underlying pathology of PK deficiency and, importantly, provide clinical 
benefit to patients. 
5.2.1. 
Summary of Nonclinical Data 
A series of exploratory pharmacology studies were conducted to characterize the ability of 
AG-348 to activate WT PKR and anemia-associated PKR mutants (mPKR) in vitro, ex vivo, and 
in vivo. Additional details regarding nonclinical data can be found in the AG-348 Investigator’s 
Brochure 
5.2.1.1. 
Pharmacology 
AG-348 is a potent, broad-spectrum activator of PKR with low nM potency against both WT and 
mutant enzymes. AG-348 is also able to effectively stabilize mPKR enzymes that are 
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hypersensitive to thermal denaturation, suggesting that AG-348 may be able to increase PKR 
pathway activity by preventing destabilization and degradation of mPKR enzymes. Its major 
metabolite, AGI-8702, has low potency mixed activity against PKR and mutant isoforms and can 
increase the thermostability of some mPKR, albeit with significantly weaker potency than that of 
AG-348. 
The effect of AG-348 and AGI-8702 on PKR activity and a number of downstream pathway 
markers was evaluated in human RBCs. AG-348 dose-response curves in human whole blood 
showed potent activation of PKR, while AGI-8702 is a weak activator of the PKR enzyme. In 
whole blood from mice treated with AG-348, 13C-glucose labeling through the PKR reaction was 
increased by 80% demonstrating AG-348-mediated activation of the WT PKR enzyme. AG-348 
treatment of RBCs from healthy human donors potently increased levels of the PKR reaction 
product ATP consistent with activation of PKR.  
The effects of AG-348 on PKR activity and RBC metabolism also were assessed in blood 
samples from patients with PK deficiency. AG-348 activated PKR and induced metabolic 
changes (increased ATP levels and decreased 2,3-DPG levels) consistent with increased 
glycolytic pathway activity in RBCs from PK deficiency patients with different mutations in the 
PKR enzyme.  
Assessments of binding inhibition potential and enzymatic activity were evaluated in a panel of 
89 receptors, ion channels, and enzymes. Although AG-348 is a histamine H3 antagonist/inverse 
agonist and an aromatase inhibitor, no effects of histamine H3 modulation have been observed in 
safety pharmacology or toxicology studies. Effects consistent with aromatase inhibition were 
seen in toxicology studies (Section 5.2.1.4). 
5.2.1.2. 
Safety Pharmacology 
Safety pharmacology studies demonstrated that neither AG-348 nor AGI-8702 exhibited 
significant inhibition of the human ether-à-go-go related gene (hERG) current (concentration of 
drug that achieved half-maximal inhibition [IC50] 29.4 μM and > 10 μM, respectively). 
Additionally, in the monkey cardiovascular (CV) study, no CV effects were seen at any dose 
level. No effects were seen in AG-348 Good Laboratory Practice (GLP)-compliant Irwin and 
respiratory safety pharmacology studies in Sprague-Dawley rats. AG-348 administered via oral 
gavage had a dose-dependent emetic activity in the ferret at ≥ 30 mg/kg. 
5.2.1.3. 
Pharmacokinetics 
Absorption, distribution, metabolism, and excretion (ADME) studies of AG-348 were performed 
in Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys. Both oral (PO) and intravenous 
(IV) routes were evaluated at doses ranging from 1 to 50 mg/kg. A dose of 200 mg (50 mg/kg) 
was administered PO in the food-effect evaluation in monkeys. The PK of AG-348 in animal 
species is characterized by rapid oral absorption, medium to high total body plasma clearance 
(CLp), and high volume of distribution at steady-state (Vss) in rats, dogs, and monkeys. 
AG-348 has a medium to long apparent terminal half-life (t1/2) in the rat, dog, and monkey. 
Medium to high oral bioavailability (F) was observed for AG-348 in animals. A high-fat diet led 
to lower oral absorption of AG-348 in monkeys. 
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AG-348 showed low brain penetration in rats (brain/plasma ratio of 5.7% to 9.1%) based on area 
under the plasma concentration versus time curve from 0 to 24 hours (AUC0 24hr) following 
single and repeated-dose administration, respectively. Metabolism appears to be the major 
elimination pathway for AG-348 with minimal biliary or urinary excretion.  
Both in vitro and in vivo, the predominant metabolite was the N-dealkylated product, AGI-8702. 
There were no metabolites unique to human liver microsomes and no direct Phase 2 metabolites. 
The plasma metabolite profile of AG-348 did not change with repeat or ascending dosing, 
suggesting that AG-348 is not susceptible to metabolic switching with increasing or prolonged 
dose administration. 
AG-348 is a weak direct inhibitor of cytochrome P450 (CYP) 2C8, CYP2C9, CYP2C19, 
CYP2D6, and CYP3A4/5 (testosterone 6β-hydroxylation). There was no direct inhibition of 
CYP1A2, CYP2B6, or CYP3A4/5 (midazolam 1′-hydroxylation) by AG-348. There was 
evidence of metabolism-dependent inhibition of CYP2C19 (largely reversible) and CYP3A4 
(largely irreversible) by AG-348. AG-348 is considered a weak time-dependent CYP3A4 
inhibitor. AGI-8702 is a weak direct inhibitor of CYP2C9 and CYP2C19. AG-348 has the 
potential to cause an induction-related drug-drug interaction (DDI) with sensitive CYP2B6 and 
CYP3A4 substrates. 
The routes of metabolism for AG-348 are via multiple CYPs with CYP3A4 contributing > 70% 
of the total. CYP1A2, CYP2C9, and CYP2C8 contribute approximately 6%, 10%, and 7% to the 
remaining metabolism of AG-348; other isoforms contribute < 4% each. 
AG-348 is a substrate for P-glycoprotein (P-gp), but not breast cancer resistance protein (BCRP). 
AG-348 is an inhibitor of P-gp (91% and 99% inhibition at 41 and 411 µM, respectively), but 
does not inhibit BCRP. AGI-8702 is not an inhibitor of P-gp or BCRP under the concentrations 
tested (5 and 100 μM). 
5.2.1.4. 
Toxicology 
In rats, the no-observed-effect level/no-observed-adverse-effect level (NOEL/NOAEL), 
determined as 2000 mg/kg, was associated with area under the plasma concentration versus time 
curve from 0 to 12 hours (AUC0-12hr) values 223- to 526-fold the projected human efficacious 
AUC0-12hr value. In dogs, clinical observations consistent with anaphylactoid reactions were seen, 
and the maximum tolerated dose (MTD) was 62.5 mg/kg, which was associated with an 
AUC0-12hr value 5.7-fold the projected efficacious AUC0-12hr value. The NOEL/NOAEL in dogs 
was 10 mg/kg, which was associated with an AUC0-12hr value 0.8-fold the projected efficacious 
AUC0-12hr value. In monkeys, the NOAEL was 1000 mg/kg, with only non-adverse emesis and 
body weight loss seen; this dose was associated with an AUC0-12hr value 70-fold the projected 
efficacious AUC0 12hr value. Based on the results of single-dose studies, the rat and the monkey 
were chosen as the most appropriate species for further evaluation in toxicology studies.  
Dose-limiting toxicity (DLT) in cynomolgus monkeys was defined in non-GLP 5-day and 
14-day repeat-dose studies as emesis, inappetence, and weight loss. These toxicities became dose 
limiting at AUC0-12hr values 27- to 34-fold the projected efficacious AUC0-12hr value, and 
precluded meaningful evaluation of other toxicities at this exposure level. Potential other effects 
at this exposure level were observed in a number hematology and serum chemistry parameters as 
well as in lymphoid organs. Additionally, minimal potential effects in kidneys (renal 
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tubulointerstitial nephritis) and heart (myocardial degeneration), which could not be 
differentiated from spontaneous background lesions, were seen when monkeys were exposed to 
AG-348 AUC0-12hr values ≥ 27- to 34-fold the projected human efficacious AUC0-12hr value.  
In GLP-compliant 28-day monkey study, the dose of 150 mg/kg/day (75 mg/kg/dose twice daily 
[BID]) was the NOAEL. Effects were limited to increased liver weights without serum chemistry 
or microscopic correlate. At this dosage level, the Day 27 AUC0-12hr values were 8.9- and 
8.5-fold the projected efficacious AUC0-12hr value in males and females respectively. In the same 
study, the low dosage of 20 mg/kg/day (10 mg/kg/dose) resulted in AUC0-12hr values that 
approximated the efficacious AUC0-12hr value, and there were no test article-related effects seen. 
The next highest dose of 50 mg/kg/day (25 mg/kg/dose) was the NOEL and was associated with 
AUC0-12hr values 3.1- and 2.6-fold the projected efficacious AUC0-12hr value in males and females 
respectively.  
In the GLP-compliant 28-day rat study, the NOAEL in males was the highest dose tested, 
600 mg/kg/day (300 mg/kg/dose), and the NOAEL in females was the lowest dose tested, 
20 mg/kg/day (10 mg/kg/dose). At the 600 mg/kg/day dosage level in males, AG-348-related 
findings were limited to mild effects on hematology, serum chemistry, and urinalysis parameters, 
and microscopic findings in the adrenal gland (minimal to mild vacuolation of the adrenal zona 
glomerulosa and decreased thickness of the zona fasciculate), liver (minimal to mild 
hepatocellular hypertrophy), kidney (minimal tubular vacuolation), pancreas (minimal to 
moderate decreased zymogen granules), heart (minimal myocardial vacuolation), and prostate 
(minimal to mild decreased secretion). All findings were fully reversible over the 14-day 
recovery period with the exception of decreased serum glucose levels and decreased prostate 
secretion. In females, the highest dosage tested was 200 mg/kg/day (100 mg/kg/dose); adverse 
effects observed were similar to those observed in the 600 mg/kg/day males, with the exception 
that in females, fewer effects in hematology and serum chemistry parameters were seen, and also 
in females, adverse effects in the reproductive organs consistent with aromatase inhibition were 
observed.  
In the 13-week repeat-dose rat study, adverse effects in males were identified in the testes and 
consisted of seminiferous tubular degeneration, spermatid retention, and Leydig cell 
hypertrophy. These effects were defined as adverse at dose levels at which they are expected to 
impair fertility. These effects were either not present or present with incidence and severity 
similar to that of the vehicle group in lower dose levels. Adverse effects in females included 
uterine atrophy and increased folding of the luminal surface; these effects were defined as 
adverse at the dose level at which they are expected to impair fertility  
In the 13-week repeat-dose study in monkeys, no adverse effects were identified, and no new 
effects were identified when compared to the 4-week repeat-dose study. Similar to what occurred 
on the 4-week study, inappetence and emesis during the initial 1-2 weeks of dosing occurred, 
precluding evaluation of higher doses.  
AG-348 inhibits human aromatase activity with an IC50 of 2050 nM (based on human placental 
microsomes) and rat aromatase with an IC50 of 493 nM (based on rat ovarian microsomes). In the 
GLP-compliant 28-day rat study, histologic effects consistent with aromatase inhibition were 
seen in the female reproductive tract at the mid- and high-dosage levels (100 and 200 mg/kg/day) 
and included incomplete corpora lutea; ovarian follicular cysts; ovarian cystic, luteinized 
follicles; uterine atrophy; vaginal mucification; and altered cyclicity. Although these findings 
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were minimal to mild and were fully reversible (over 14 days), they were considered adverse and 
the next lower dosage evaluated, 20 mg/kg/day (10 mg/kg/dose BID) was the NOAEL in 
females. The Day 27 AUC0-12hr value associated with this dosage level was 6.9-fold the projected 
human efficacious AUC0-12hr value. The potential for aromatase inhibition effects occurring in 
female rats at AUC0-12hr values > 6.9-fold and < 53-fold the projected efficacious AUC0-12hr value 
has been addressed in a 13-week rat study. In this study using doses between the NOAEL and 
LOAEL in the 28-day study, the NOAEL for histologic lesions of the uterus that may be 
associated with aromatase inhibition resulted in an AUC0-12hr that was 26-fold the projected 
efficacious value. Notably, due to the potency difference of AG-348 against rat versus human 
aromatase inhibition, there is potential for a 4-fold wider margin for aromatase inhibition in 
humans versus rats. AGI-8702 is not an aromatase inhibitor. 
5.2.2. 
Summary of Clinical Data 
To date, 72 healthy adult volunteers have been exposed to AG-348 in 2 clinical studies, a single 
ascending dose (SAD) study and a multiple ascending dose (MAD) study, with 31 of these 
subjects exposed to a single AG-348 dose under fasted conditions; 5 exposed to 2 doses of 
AG-348 under fasted and fed conditions; and 36 exposed to repeated doses of AG-348 for up to 
14 days1. The following discussion of clinical data refers only to healthy adult volunteer 
subjects, as this is the first clinical trial in which patients with PK deficiency will be treated with 
AG-348. 
5.2.2.1. 
Pharmacokinetics 
The PK of AG-348 showed low to moderate variability between subjects. After single doses of 
AG-348 from 30 to 2500 mg, AG-348 was rapidly absorbed as indicated by the short time to 
maximum plasma concentration (Tmax). There appeared to be some lengthening of the period of 
absorption at higher doses as indicated by higher Tmax and a less than proportional increase in 
maximum concentration (Cmax). Dose-normalized area under the curve (AUC) generally 
remained constant over the dose range studied, suggesting that AG-348 total exposure increased 
in a dose-proportional manner. The mean t1/2 ranged from 17.8 to 20.4 hours when samples were 
collected through 72 hours and from 50.3 to 79.3 when samples were collected through 
120 hours. However, this terminal elimination phase contributed little to overall exposure of 
AG-348, as indicated by the small difference between AUC0-12 and area under the plasma 
concentration versus time curve from 0 to infinity (AUC0-∞), suggesting a shorter effective half-
life of approximately 3 to 6 hours. AG-348 was extensively distributed (mean apparent volume 
of distribution [Vz/F] range of 271 to 1148 L) and had a moderate rate of clearance (geometric 
mean clearance [CL/F] range of 10.3 to 13.9 L/hr). The fraction of AG-348 excreted in urine 
ranged from 0.0145 to 0.0209 across the dose levels suggesting that renal excretion plays a 
minor role in the systemic elimination of AG-348. 
The preliminary repeat-dose PK of AG-348 at doses ranging from 15 mg every 12 hours (q12h) 
to 700 mg q12h also showed an increase in AG-348 exposure in a dose-proportional manner 
after the first dose. AG-348 exposure observed after repeated dosing for 14 days at doses of 
1 At the time of this document, results from Study AG348-C-002 in healthy volunteers have been unblinded, but the 
data have only partially been analysed and the Clinical Study Report is in preparation but not yet completed.  
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120 mg every 24 hours (q24h) to 700 mg q12h were lower than observed on Day 1 after the first 
dose. The magnitude of the effect was greater at higher doses; lower doses of 15 mg q12h and 
60 mg q12h did not show this effect. This observation may be related to the nonclinical finding 
that AG-348 is a potential inducer of human CYP3A4, an enzyme which biotransforms AG-348. 
It is possible that multiple doses of AG-348 leads to an increased rate of its own metabolism. 
Evaluation of the effect of food on the PK of a single 700 mg dose of AG-348 in 5 subjects who 
were administered the drug fasting and then, after an appropriate wash-out period, re-
administered the drug following ingestion of a standard US Food and Drug Administration 
(FDA) high fat meal, showed that food likely has a minimal effect on the PK of AG-348. 
5.2.2.2. 
Pharmacodynamics 
After a single dose of AG-348, a decrease in the concentration of 2,3-DPG was observed at 
3 hours post-dose, decreased in a dose-dependent manner to a minimum at 24-hour post-dose, 
and then returned to values similar to baseline by 72 to 120 hours post-dose. The mean decrease 
at 24 hours was approximately 300 µg/mL at the 700 through 2500 mg dose levels. Similar 
decreases were observed after the first dose of multiple doses and prior to doses in the MAD 
study. After the final dose of multiple doses, the concentration of 2,3-DPG returned to values 
similar to baseline between 72 and 120 hours post- dose. 
After a single dose of AG-348, a minimal increase in the concentration of ATP was observed at 
24 to 120 hours post-dose. In the MAD study, no increase was observed at 12 hours after the first 
dose; the concentration of ATP was increased on Day 8 to concentrations greater than in the 
single dose study, continued to trend upward on Day 11, and remained at a similar level through 
Day 14. The concentration of ATP remained elevated through 120 hours after the last dose on 
Day 14. The magnitude of the increase in ATP was similar across the dose range from 60 mg 
BID to 700 mg BID. 
5.2.2.3. 
Safety 
Overall, AG-348 has been well-tolerated among healthy volunteers at doses that produced strong 
pharmacodynamic effects on 2,3-DPG and ATP. 
After a single AG-348 dose, treatment-emergent adverse events (TEAEs) reported by > 1 subject 
at any time on study (either under fasted or fed conditions) included headache (22%), nausea 
(14%), and contact dermatitis and vomiting (each 6%). After repeated dosing of AG-348 for 
14 days, TEAEs that occurred in > 5% of all AG-348-treated subjects across all cohorts included 
headache (13.9%), nausea (13.9%), vomiting (8.3%), decreased appetite (8.3%), feeling hot 
(8.3%), restlessness (8.3%), and dizziness, fatigue, vessel puncture site bruise, hyperhidrosis, 
dermatitis allergic, and drug eruption (5.6% each). 
A dose-relationship was apparent with regard to the incidence of gastrointestinal events, 
primarily nausea and vomiting, with the incidence of such events increasing with increasing 
dose. Nausea and/or vomiting were observed only at doses ≥ 1400 mg in the single dose study 
and only at doses ≥ 700 BID in the MAD study. Nausea and vomiting were not observed at any 
dose ≤ 360 mg in either the single or multiple dose studies. 
All but 1 TEAE reported to date has been mild or moderate (Grade 1 or 2) in intensity. The only 
Grade 3 TEAE was elevated liver transaminases (alanine aminotransferase [ALT]; aspartate 
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aminotransferase [AST]) in a single subject in the MAD study treated with 700 mg AG-348 BID. 
The event was considered to be possibly related to AG-348, was declared a DLT, and led to 
study drug discontinuation, following which the elevated liver transaminases resolved. 
Altogether, 3 of 6 subjects treated with AG-348 at 700 mg BID discontinued study drug in the 
MAD study: the subject described above with Grade 3 elevated transaminases, and 2 others who 
withdrew themselves for nausea and vomiting. A fourth subject treated with AG-348 at 60 mg 
BID was discontinued from the study for a drug eruption. No AG-348-treated subject 
discontinued in the SAD study due to an adverse event (AE). 
No deaths or other serious adverse events (SAEs) have been reported in any clinical study of 
AG-348. Furthermore, no DLTs were observed after a single administration of AG-348 at doses 
of 30 mg to 2500 mg or after repeat administration of AG-348 for 14 days at daily doses of 
15 mg to 700 mg BID except for the event of Grade 3 elevated liver function tests described 
above. 
Due to preclinical observations pertaining to the potential for inhibition of the aromatase enzyme 
(see Section 5.2.1.4), the AG348-C-002 multiple dose study included assessment of baseline and 
serial measures of free and total serum testosterone and serum estradiol and estrone. The 
unblinded serum hormone data are undergoing analysis, but at least some male subjects treated 
with all doses of AG-348 demonstrated modest increases in androgenic hormones and decreases 
in estrogens, compatible with a potential signal of aromatase inhibition, whereas the males who 
received placebo did not. These changes were reversible upon cessation of dosing. There were 
too few female subjects in the study who received AG-348 to permit any definitive conclusions. 
Additional analyses are ongoing. 
5.3. 
Study Rationale 
Study AG348-C-003 is the first study that will be conducted in patients with PK deficiency. This 
study is primarily intended to evaluate the safety and tolerability and potential indicators of 
clinical activity of AG-348 administered for up to 24 weeks. This study will also evaluate the PK 
profile of AG-348 and its metabolite AGI-8702, the PD responses in ATP and 2,3-DPG 
following administration of AG-348, and the clinical activity of AG-348 in PK deficiency 
patients. Two previous double-blind, placebo-controlled clinical trials of AG-348 conducted in 
healthy adult male and female volunteers (AG348-C-001, a SAD study; and AG348-C-002, a 
MAD study) have established an acceptable safety and tolerability profile for AG-348 for up to 
14 days of both once-daily (QD) and BID dosing at exposures that result in significant 
pharmacodynamic (PD) changes in whole blood levels of the glycolytic metabolites 2,3-DPG 
and ATP. Due to the potential for AG-348-mediated aromatase inhibition, dual-energy x-ray 
absorptiometry (DXA) scan (hip and spine) will be performed at Screening (if patient has not 
had prior DXA scan within 3 months of Day 1) to obtain T and Z scores. These data will serve as 
baseline measures of bone mineral density for all enrolling patients (see Section 7.1 for more 
details). 
The target population of this study consists of adult males and females with a diagnosis of PK 
deficiency, who are anemic but non-transfusion dependent. Non-transfusion dependent patients 
are preferred for this study in order to reduce any potential confounding effect of transfusion 
therapies on evaluation of potential indicators of clinical activity and PD responses. The safety, 
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tolerability, and PK/PD findings in this study will form the basis for subsequent clinical 
development of AG-348. 
5.3.1. 
Summary of Overall Safety Management Plan 
Measures to minimize the risks to patients enrolled in this study have been taken with respect to 
the following study design elements: 
• The initial doses for Arms 1 and 2 have been selected on the basis of safety and 
tolerability observations already carefully made in two predecessor clinical trials in 
adult healthy male and female volunteers;  
• The specified inclusion/exclusion criteria have been carefully considered to avoid 
enrollment of subjects for whom exposure to the study drug might pose a hazard; 
• A designated Data Review Team (DRT) consisting of the overall study Coordinating 
Investigator, treating Investigators, Medical Monitor, Clinical Pharmacologist, 
Statistician, and Sponsor’s Responsible Medical Officer has been established to meet 
regularly at approximately 6-week intervals throughout the duration of the study to 
review the accumulating study data and will exercise options to suspend enrollment to 
one or both of the initial two study dose arms, discontinue enrollment to one or both 
of the initial two study dose arms, adjust the dose of patients in one or both of the 
initial two study arms, and/or implement one new study dose arm. If one new dosing 
arm is implemented, the dose selected will not exceed 360 mg BID, the highest dose 
that demonstrated acceptable safety and tolerance in the 14-day multiple BID dosing 
study in healthy volunteers. Group cohort stopping rules for terminating enrollment 
into an arm based on the severity (CTCAEv4.03 grade) and frequency of AEs are 
defined;  
• Dose modification and stopping rules are defined for individual patients; 
• Guidance for permitted, prohibited, and cautionary concomitant medications is 
specified based on the estimated potential for drug-drug interactions from hepatic 
cytochrome enzyme interactions with AG-348. 
• Due to the potential for AG-348-mediated aromatase inhibition, bone mineral density 
will be monitored using DXA scans (hip and spine) at Baseline (if patient has not had 
prior DXA scan within 3 months of Day 1) and between Week 24 and Week 28. 
In the event that any clear and unequivocal, previously unidentified/unexpected toxicities occur 
in pre-clinical toxicology studies, the Sponsor will notify the Investigators, IRBs/ECs, and 
regulatory authorities. The Sponsor will also report these to the DRT for review and discussion 
of appropriate actions, which may include, but are not necessarily limited to:  
• Amending the protocol to adjust the inclusion/exclusion criteria (e.g., to exclude 
patients with certain at-risk concurrent conditions); and/or  
• Amending the protocol to adjust safety monitoring procedures (e.g., to require 
additional monitoring of specified adverse events, physical examinations, clinical 
laboratory testing, ECG monitoring, or other testing as appropriate); and/or  
• Adjusting the dose of an arm of the study as appropriate; and/or 
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• Adjusting the dose modification and/or stopping rules (Section 9.7); and/or 
• Adjusting the patient withdrawal criteria (Section 8.6); or 
• Terminating the trial.    
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6. 
TRIAL OBJECTIVES AND ENDPOINTS 
6.1. 
Primary Objective 
The primary objective of the study is to: 
• Evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in 
patients with PK deficiency. 
6.2. 
Secondary Objectives 
The secondary objectives of the study are to: 
• Evaluate the pharmacokinetics (PK) of AG-348 and the metabolite AGI-8702. 
• Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348. 
• Evaluate indicators of clinical activity of AG-348 in patients with PK deficiency, 
including changes in hemoglobin (Hb), HCT, reticulocyte count, haptoglobin (Hp), 
carboxyhemoglobin (COHb), lactate dehydrogenase (LDH), total and indirect 
bilirubin, erythropoietin (EPO), ferritin, and transferrin saturation (serum iron/iron 
binding capacity). 
 
6.4. 
Study Measures and Endpoints 
6.4.1. 
Safety Measures and Endpoints 
Safety will be evaluated by: 
• Monitoring of AEs, including determination of SAEs and AEs leading to 
discontinuation; safety laboratory parameters (hematology, chemistry, urinalysis, 
coagulation); physical examination findings; vital signs (VS); 12 lead 
electrocardiograms (ECGs); and DXA scans. Adverse events will be graded using 
Common Toxicity Criteria for Adverse Events (CTCAE), Version 4.03. Serum sex 
hormone levels (testosterone [total and free], estrone, and estradiol), bone turnover 
markers (serum osteocalcin-N-mid and serum C-terminal telopeptide [CTX]), 25-
hydroxy vitamin D2 and D3, total cholesterol, high-density lipoprotein-cholesterol 
(HDL-C), and triglycerides will be monitored for evidence of potential inhibition of 
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aromatase by AG-348. Menstruating female patients will also keep a paper-based 
menstrual cycle diary throughout the study. 
6.4.2. 
Clinical Activity Measures and Endpoints 
• Monitoring of potential indicators of clinical activity will include evaluating changes 
in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, 
ferritin, and transferrin saturation. 
6.4.3. 
Pharmacokinetic and Pharmacodynamic Measures and Endpoints 
The PK and PD profile of AG-348 will be evaluated by: 
• Approximately the first 10 patients treated, contingent on clinical site feasibility, will 
undergo extensive PK sampling as detailed in Appendix 15.1, Table 6. The remainder 
of treated patients will undergo limited PK sampling as detailed in Appendix 15.1, 
Table 7. Serial blood sampling for determination of concentration-time profiles of 
AG-348 and its metabolite AGI-8702 will be conducted following the first dose and 
the morning Day 15 dose, and additional trough levels of AG-348 and AGI-8702 will 
be obtained. AG-348 and AGI-8702 will be analyzed using qualified assays to 
determine concentrations in plasma. Pharmacokinetic parameters on Day 1 and 
Day 15 will be computed using standard non-compartmental methods based on 
observed plasma AG-348 and AGI-8702 concentrations. 
• Pharmacodynamic assessments will include 2,3-DPG, ATP (secondary objectives), 
 
 
Approximately the first 10 patients 
treated will undergo extensive PD sampling as detailed in Appendix 15.1, Table 6. 
The remainder of treated patients will undergo limited PD sampling as detailed in 
Appendix 15.1, Table 7. Serial blood sampling for determination of levels of ATP 
and, 2,3-DPG will be conducted following the first dose and the morning Day15 dose, 
and additional trough levels of ATP and 2,3-DPG will be obtained. ATP and 2,3 DPG 
will be analyzed using qualified assays to determine concentrations in whole blood. 
PD parameters on Day 1 and Day 15 will be computed based on observed whole 
blood ATP and 2,3-DPG concentrations.  
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7. 
INVESTIGATIONAL PLAN 
7.1. 
Overall Study Design 
Study AG348-C-003 is a Phase 2, open-label, two-arm, multicenter, randomized, dose-ranging 
study during which adult patients with PK deficiency will receive multiple doses of AG-348 for 
up to 24 weeks. Patients with PK deficiency confirmed by red blood cell PK enzymatic assay 
performed at Screening will be eligible to participate in this study. At Week 25, patients who 
safely tolerate AG-348 and demonstrate clinical activity of AG-348 may be eligible to 
immediately roll over to a safety extension for continued treatment. Patients who finish treatment 
at the end of 24 weeks or sooner will undergo follow-up assessment 4 weeks after the last dose 
of study drug. Patients with toxicity suspected to be related to study drug will continue follow-up 
until the AE resolves, is declared chronic by the Investigator, or the patient is lost to follow-up. 
Initially, up to 25 patients will be randomized on an open-label, 1:1 basis to each of two BID 
doses of AG-348 (up to 50 patients; see Figure 3, Study Schema). The dose of Arm 1 is 300 mg 
of AG-348 administered orally q12h (BID). The dose of Arm 2 is 50 mg of AG-348 
administered orally q12h (BID). Randomization will be stratified by PKR mutation in order to 
maintain balance as much as possible across the dose arms for the specific mutations expected to 
be most frequently enrolled. The PKR mutation stratification factor will consist of 4 levels 
(R510Q, R486W, and R479H) and all other mutations (“other”). Mutation status is defined by 
the presence of at least one of the indicated mutations; patients with more than one stratified 
mutation will be assigned based on Sponsor’s discretion. 
The doses for each arm have been selected from the forerunner AG348-C-001 SAD study and 
AG348-C-002 MAD studies in healthy adult volunteers to represent the range of doses/exposures 
that were safely tolerated and resulted in maximal or near maximal PD effects on 2,3-DPG and 
ATP. 
Because PK deficiency is a rare disease with a limited eligible patient population and because the 
underlying pathophysiology and clinical phenotype of affected patients is heterogeneous due to 
the wide variety of mutations in PKR that cause the disease, it is deemed important to focus 
closely on dose findings in this first-in-patient study. Therefore, in addition to initiating this 
study with 2 different doses of AG-348 administered q12h, a DRT will be established to review 
study data on a frequent basis and adapt the study design, dose and schedule of AG-348 as 
indicated.  
The DRT will monitor safety on an on-going basis and meet at regular intervals (approximately 
every 6 weeks), or ad hoc as necessary, to review AEs, VS, clinical laboratory (hematology, 
clinical chemistry, coagulation, and urinalysis), and ECGs on enrolled patients. The DRT will 
also review available PK/PD data and indicators of clinical activity (e.g., changes from baseline 
in Hb). The DRT will be comprised of the study Coordinating Investigator, treating 
Investigators, Medical Monitor, Clinical Pharmacologist, Statistician, and Sponsor’s Responsible 
Medical Officer. 
Approximately every 6 weeks, beginning 6 weeks after the first patient is doses or ad hoc as 
necessary, the DRT will review cumulative safety data, available PK/PD data, and clinical 
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activity data. Based on the DRT’s recurring 6-week reviews, the DRT may exercise one or more 
of the following options: 
• Continue treatment and enrollment in existing arms without change. 
• Add 1 new dose arm (Arm 3) to enroll up to 25 patients at a dose to be determined; 
the dose for Arm 3 may be lower or higher than Arm 1 and Arm 2 doses, but will not 
exceed 360 mg q12h; and the dose regimen may be less frequent than q12h. 
• Terminate or suspend enrollment to allow further review of clinical data in Arm 1 
and/or Arm 2 (and/or potential Arm 3). Enrollment in an arm could be terminated or 
suspended to allow further review, for example, for unacceptable safety/tolerability, 
poor PD response, or lack of signs of clinical activity. 
• Re-assign patient’s doses and schedule in a terminated arm to match the dose and 
schedule of another arm of the study. In this case, the patients in the terminated arm 
will remain in their original arm, i.e., they will not count towards the enrollment 
quota of the arm whose dose and schedule is being adopted. 
• Implement specific genotype restrictions to enrollment in one or more arms to ensure 
representation of patients with genotypes of greatest clinical relevance. 
The DRT will perform these evaluations on a recurring 6-week basis. The data that the DRT will 
review to make these decisions is expected to include, but are not necessarily limited to, the 
following:  
• Safety Observations: all AEs; VS, clinical laboratory (hematology, clinical chemistry, 
coagulation, and urinalysis), and ECGs;  
• PK and PD Observations: including changes in 2,3-DPG and ATP; 
• Indicators of Clinical Activity: including changes in Hb, HCT, reticulocyte count, Hp, 
COHb, LDH, EPO, total and indirect bilirubin, ferritin, and transferrin saturation.  
If a third dose arm is implemented, the dose of AG-348 selected will not exceed 360 mg BID, as 
this was the highest dose that demonstrated acceptable safety and tolerance in the 14-day 
multiple BID dosing study in healthy volunteers. The PK/PD sampling schedule in a potential 
third dosing arm will be determined by Sponsor’s discretion and may follow either the extensive 
or limited PK/PD sampling schedules as specified. 
The DRT will monitor the safety data in an ongoing manner as described and may make a 
decision to terminate enrollment in an arm if unacceptable AEs are observed, PD activity is 
limited, and/or markers of clinical activity indicate no effect. Unacceptable AEs are defined as 
≥ Grade 3 AEs (using National Cancer Institute Common Terminology Criteria for Adverse 
Events [NCI CTCAEv4.03]) that are assessed as at least possibly related to AG-348, with the 
exception of Grade 3 headache, hot flash/flushing, nausea, vomiting, and/or diarrhea that is 
transient in duration (< 24 hours) or able to be medically managed to ≤ Grade 2 within 24 hours. 
The DRT may also exercise discretion to terminate enrollment in an arm if it observes a 
frequency of Grade 2 AEs that would make it difficult to support long-term dosing. 
Due to the potential for AG-348-mediated aromatase inhibition, DXA scan (hip and spine) will 
be performed at Screening (if patient has not had prior DXA scan within 3 months of Day 1) to 
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obtain T and Z scores. These data are intended to serve as a baseline measure of bone mineral 
density for all enrolling patients, and are deemed of particular importance for those who may 
enter the longer term safety extension after completing 24 weeks of treatment. All patients will 
have a second DXA scan in the interval between Weeks 24 and 28. The safety extension will 
address additional follow-up DXA scanning. 
As the number of enrolling arms changes in the study (for example, from 2 to 3), the 
randomization scheme will adjust to enable balanced randomization into each actively accruing 
arm. Randomization and stratification will cease in the event that only a single arm is left 
enrolling. 
Depending on possible early termination of 1 or both of the initial 2 arms, or the addition of a 
third arm, the study could enroll up to a maximum of 75 patients. 
Figure 3: 
Study Schema 
 
Abbreviations:  BID (q12h) = twice-daily (every 12 hours); DRT = data review team; PKR = pyruvate kinase red 
blood cell isoform; TBD = to be determined; w = weeks.  
The Investigator will monitor all patients for safety and tolerability. Modification of an 
individual patient’s dose of AG-348 will be based on AEs and observed changes in Hb levels as 
detailed in Section 9.7.1 and Section 9.7.2. 
Screening assessments will occur within 28 days prior to the first dose of study treatment. During 
the Treatment period, patients will attend visits at baseline (Day 1), weekly through Week 3 
(Days 8, 15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12), and 
monthly through Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate AG 348 and 
demonstrate evidence of clinical activity of AG-348 through Week 24 may be eligible to 
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immediately enter a safety extension for continued treatment. For patients who finish treatment 
at the end of 24 weeks or sooner, or who elect not to enter the safety extension, study discharge 
will occur 4 weeks (Week 28 or earlier) following the last dose of study treatment at the final 
follow-up assessment. 
Safety assessments will include monitoring of AEs, including determination of SAEs and AEs 
leading to discontinuation; safety laboratory parameters (e.g., hematology, serum chemistry, 
coagulation studies, and urinalysis); physical examination findings; VS; 12 lead ECGs, and DXA 
scans. Additional safety assessments will include monitoring of sex hormone levels (testosterone 
[total and free], estrone, and estradiol), bone turnover markers (osteocalcin-N-mid and CTX), 25-
hydroxy vitamin D2 and D3 levels, total cholesterol, high-density lipoprotein-cholesterol (HDL-
C), and triglycerides. Follow-up assessments will be conducted on Day 197 (Week 28) and will 
include physical examination, weight, performance status, VS, 12-lead ECGs, laboratory 
evaluations (hematology, Hp, EPO levels, serum chemistry, coagulation studies, urinalysis; 
lipids, hormonal testing), AEs, and transfusion record. Menstruating female patients will also be 
required to keep a paper-based menstrual cycle diary throughout the study. 
Pharmacokinetic assessments will include serial blood sampling for PK profiles of AG-348 and 
its metabolite AGI-8702. Pharmacodynamic evaluations will include serial blood sampling for 
determination of levels of ATP and 2,3 DPG. Extensive PK/PD sampling will be conducted on 
the first approximately 10 patients total treated in Arms 1 and 2 (see Appendix 15.1, Table 6) 
while more limited PK/PD sampling will be conducted on additional patients treated if 
enrollment in Arm 1 or Arm 2 is expanded or if an alternate dosing arm is added (see 
Appendix 15.1, Table 7).  
 
 
 
 
7.2. 
Justification of the Study Design 
The primary and secondary objectives of this study are to evaluate the safety, tolerability, PK 
and PD, and indicators of clinical activity of AG-348 in patients with PK deficiency. The choice 
of dose and schedule of administration of AG -348 for Arms 1 and 2 was based on the highest 
safely tolerated dose (Arm 1: 300 mg BID) and the lowest dose with potentially relevant PD 
activity (Arm 2: 50 mg BID) from the forerunner AG348-C-002 MAD study in healthy 
volunteers. Decisions regarding continuing enrollment and treatment in these initial dose arms 
and/or implementation of an additional dose arm will be based on DRT review of safety, PK and 
PD data, and indicators of clinical activity collected from all patients treated in Arm 1 and 
Arm 2. This design was chosen to minimize risk to patients while allowing evaluation of safe 
and pharmacologically active dose levels of AG-348, and to allow the necessary flexibility to 
adjust dose and schedule should the safety, tolerability, PK, and/or PD be different in patients 
with PK deficiency compared with healthy volunteers.  
Additional safety measures intended to minimize risk to patients include monitoring of AEs by 
the DRT and specified provisions for individual patient dose modification as needed for safety 
and (potentially) large increases in Hb level (Section 9.7.1 and Section 9.7.2). Measures intended 
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Protocol AG348-C-003, Amendment 2, Version 3.0 
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to maximize the opportunity for patients with demonstrated safety and tolerability to continue to 
derive benefit from any observed clinical activity of AG-348 include the option for continued 
treatment on a safety extension.  
A comprehensive series of safety evaluations, including laboratory parameters, physical 
examinations, VS, 12-lead ECGs, and monitoring for SAEs and AEs, will be conducted to 
evaluate the safety profile of AG-348 and to aid in the determination of the recommended dose 
for continued development. 
The study includes serial blood sampling across at least two different doses of the study drug to 
assess its PK and PD profiles.  
Consistent with the design of many Phase 2 studies, preliminary evaluation of the potential PD 
and clinical activity of AG-348 are secondary objectives of this study. The latter will include 
assessments of the PK/PD relationship between AG-348 and the biomarkers ATP and 2,3-DPG, 
 
7.3. 
Rationale for the Starting Dose, Dose Range, and Duration of Dosing 
Prior to execution of this study, Agios conducted two clinical studies of AG-348 in healthy 
volunteers, including a SAD study (AG48-C-001) and a MAD (14 day q12h) study 
(AG348-C-002). Available details of these studies are discussed in the current Investigator’s 
Brochure (IB). Between these two studies, 72 healthy human subjects have been dosed with 
AG-348. In vitro investigations, also reported in the IB, had previously demonstrated that 
AG-348 increased the activity of wild-type PKR approximately to the same extent as it did a 
series a recombinant mPKRs. Therefore it was deemed reasonable to study the safety, 
tolerability, PK, and PD of AG-348 in healthy subjects in a controlled Phase 1 setting as a more 
efficient means of obtaining information than in the actual rare disease population of patients 
with PK deficiency.  
The MAD study demonstrated that the exposures produced by AG-348 doses from 60 mg q12h 
to 360 mg q12h (including 120 mg q24h) resulted in maximal changes from baseline for the PD 
markers 2,3-DPG (reduction from baseline) and ATP (increase from baseline). The exposures 
resulting from doses less than 60 mg q12h were of lesser magnitude and the exposures resulting 
from doses greater than 360 mg q12h were of no greater magnitude than the aforementioned 
range. Therefore the starting doses for this first dose ranging study in patients with PK deficiency 
were selected to be 300 mg q12h (Arm 1) and 50 mg q12h (Arm 2). These doses were 
demonstrated to be safe and tolerable in the healthy volunteer studies. The availability of ATP is 
proposed as being critical for optimally maintaining RBC membrane integrity (see Section 5.1). 
The dose ranges from 50 mg q12h to 300 mg q12h may result in clinically effective modulation 
of PKR in PK deficiency patients if the mutated enzyme is responsive to AG-348 in a similar 
manner to the wild-type enzyme in healthy subjects. However, there are many different 
mutations in PKR that result in PK deficiency, and these mutations produce variable effects on 
the enzyme in terms of catalytic activity and thermal stability. It is not known if different 
mutations will respond clinically in a similar manner to the same exposure to AG-348. 
Therefore, it is prudent to study the range of safe and pharmacodynamically relevant doses as 
specified in this study, and to allow flexibility for the DRT to analyze the evolving study data to 
adapt the dose and schedule of administration of AG-348 to produce the optimal combination of 
safety, tolerability, and PD, and, potentially, clinical response. 
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Justification for Duration of Dosing  
The treatment duration of 24 weeks (6 months) was chosen for this proof-of-concept trial for 
2 principal reasons: 1) to begin establishing a safety database addressing the chronic 
administration of AG-348; and 2) to allow sufficient time for clinical response to treatment to 
appear.  
It is anticipated that this treatment, if successful, may be taken for life, as PK deficiency is a 
genetically determined inborn error of metabolism. Therefore, it is important to begin to 
investigate the long-term safety of the treatment. As will be discussed below, the safety package 
supports the treatment duration of 6 months.  
Red blood cell turnover is typically 120 days (4 months), although it may be shorter in some 
populations of pyruvate kinase deficient RBCs (Mentzer, et al. 1971). Different PKR mutations 
produce a variety of physiologic consequences. In mutations where normal or nearly normal 
levels of PKR protein persist, but the protein’s catalytic function is impaired, an improvement in 
PKR functional activity might be seen relatively quickly upon exposure to AG-348. However, in 
cases where the PKR mutation results in an unstable mutant, PKR protein levels may be low and 
additional time may be required for stabilization of the newly synthesized mutant enzyme in 
developing bone marrow erythroblasts so that eventually more mature RBCs may be produced 
with more nearly normal levels of functional PKR protein.   
This study plans up to 24 weeks of dosing with AG-348 with the possibility of continued dosing 
beyond 24 weeks on a safety extension in patients for whom AG-348 is safely tolerated and 
demonstrates clinical activity. The ICH Guideline M3(R2) on Nonclinical Safety Studies for the 
Conduct of Human Clinical Trials and Marketing Authorisation for Pharmaceuticals, 
recommends that clinical trials of a duration between 2 weeks and 6 months should be supported 
by repeat-dose toxicity studies of at least equivalent duration, and clinical trials lasting beyond 
6 months should be supported by 6-month rodent and 9-month non-rodent studies. This guidance 
allows for clinical trials of longer duration to be initiated based on 3 months of nonclinical data, 
and for clinical dosing in serious or life-threatening indications to be extended based on complete 
chronic rodent data and in-life and necropsy data in the non-rodent (and complete histopathology 
in the non-rodent provided within an additional 3 months).  
For the current investigational product (AG-348), 13-week, repeat dose toxicology studies in the 
rat and monkey have been completed and are summarized in Section 5.2.1.4 of this protocol and 
in the current Investigator Brochure prepared to support initiation of this clinical study. 
Considering that PK deficiency is a serious condition with an unmet medical need, it is 
appropriate to initiate this clinical study with the available nonclinical data. Six-month rodent 
and nine-month monkey toxicology studies were initiated in January 2015, and the Sponsor will 
report the results of these studies in each applicable regulatory region as required.  
7.4. 
Criteria for Study Termination 
This study may be prematurely terminated if, in the opinion of the Sponsor, there is sufficient 
reasonable cause. In the event of such action, written notification documenting the reason for 
study termination will be provided to each Investigator. 
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Circumstances that may warrant termination include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to patients; 
• Insufficient adherence to protocol requirements; 
• Plans to modify, suspend, or discontinue the development of the study drug; 
• Other administrative reasons. 
Should the study be closed prematurely, all study materials must be returned to the Sponsor or 
the Sponsor’s designee. 
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8. 
STUDY POPULATION 
8.1. 
Number of Patients 
Up to approximately 75 patients may be enrolled in this study. 
8.2. 
Inclusion Criteria 
For entry into the study, patients must meet all of the following criteria during the Screening or 
other specified period: 
1. Signed written informed consent obtained prior to performing any study procedure, 
including screening procedures.  
2. Male or female, aged 18 years and older. 
3. Known medical history of PK deficiency. 
4. All patients must have documented clinical laboratory confirmation of PK deficiency by 
RBC pyruvate kinase enzymatic assay performed at Screening by a designated central 
laboratory. Patients with prior documentation of PK deficiency by RBC enzymatic assay 
will have a reconfirmation of this result during Screening as a condition of enrollment. 
a. In the event that a patient’s screening pyruvate kinase enzymatic assay is negative 
(i.e., shows normal pyruvate kinase activity), the patient will be eligible for 
enrollment if the genotyping shows a mutant genotype that has been previously 
documented in the literature to be associated with pyruvate kinase deficiency. If 
the genotyping shows a previously undescribed mutation in the PKR gene, then 
the eligibility for enrollment will be determined on a case-by-case basis by the 
Coordinating Investigator and Medical Monitor in discussion with the 
Investigator. If no mutation is defined, then the patient will not be eligible. 
5. All patients must have genotypic characterization of the mutant PKR gene performed by 
a designated central laboratory at Screening, unless genotype is available from the 
patient's participation in the Pyruvate Kinase Deficiency Natural History Study 
(NCT02053480). 
6. All patients must have genotypic characterization of the UGT1A1 gene performed by a 
designated central laboratory to document whether they may have underlying Gilbert's 
Disease. Patients with Gilbert’s Disease are eligible to enroll. 
7. Males must have Hb ≤ 12.0 g/dL; females must have Hb ≤ 11.0 g/dL. 
8. All patients must be considered transfusion independent as defined by: no greater than 
3 units of RBCs transfused in the 12-month period up to the first day of study dosing and 
no transfusions within 4 months of first day of study dosing. Patients who have received 
more transfusion support than described above will evaluated for eligibility on a case-by-
case basis by the Medical Monitor. 
9. Eligible patients may still have their spleens in place, or may have undergone prior 
splenectomy. For splenectomized patients: 
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a. Must have undergone their procedure at least 6 months prior to Screening. 
b. Must be current in their vaccinations for Pneumococcal Conjugate (PCV13), 
Pneumococcal Polysaccharide (PPSV23), Quadrivalent Meningococcal vaccine, 
and Haemophilus influenzae type b (Hib) as recommended by Centers for Disease 
Control and Prevention's (CDC) Advisory Committee on Immunization Practices 
(ACIP) or immunization advisory groups in Canada and the European Union (for 
patients enrolled in Canada and the EU). 
[http://www.cdc.gov/vaccines/schedules/downloads/adult/adult-schedule.pdf] 
[Any missing vaccinations may be administered during the screening period.] 
10. Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2 (Appendix 15.2). 
11. Patients must be taking at least 1 mg of folic acid daily for at least 21 days prior to first 
dose and continued daily during study participation. 
12. Adequate organ function, defined as: 
a. Serum AST and ALT ≤ 1.5 × upper limit of normal (ULN) (unless the increased 
AST is assessed by the Investigator as due to hemolysis). 
b. Normal or elevated levels of serum bilirubin. In patients with serum bilirubin 
> ULN, the elevation must be attributed to hemolysis with or without Gilbert's 
syndrome and must not be choledocholithiasis, cholecystitis, biliary obstruction, 
or hepatocellular disease. 
c. Serum creatinine ≤ 1.25 × ULN. If serum creatinine > 1.25 × ULN, then 24-hour 
measured or calculated (Cockcroft-Gault) glomerular filtration rate 
(GFR) ≥ 60 mL/min. 
d. Absolute neutrophil count (ANC) > 1.0 × 109/L. 
e. Platelet count ≥ 100 × 109/L. 
f. Activated partial thromboplastin time (aPTT) and international normalized ratio 
(INR) ≤ 1.25 × ULN, unless the patient is receiving therapeutic anticoagulants. 
13. Women of childbearing potential (WOCBP) must agree to abstain from sexual 
intercourse or to use an acceptable/effective method of contraception (i.e., condom plus 
spermicide, condom plus oral contraceptive, condom plus intrauterine device [IUD], 
condom plus diaphragm with spermicide) from as soon as feasible during the Screening 
period until 30 days following the last dose of AG-348. Abstinence is an acceptable 
method only when this is in line with the normal life style of the patient, meaning that the 
patient plans to remain abstinent continuously throughout the duration of the study and 
for at least 30 days after the last dose of study drug. Periodic abstinence, e.g. calendar, 
sympathothermal and post-ovulation methods, and withdrawal are not acceptable 
methods of contraception. 
a. WOCBP include any female who has experienced menarche and who has not 
undergone successful surgical sterilization (hysterectomy, bilateral tubal ligation, 
and/or bilateral oophorectomy) or is not post-menopausal. Post-menopausal is 
defined as: 
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i. Amenorrhea ≥ 12 consecutive months without another cause, and a 
documented serum follicle stimulating hormone (FSH) level 
> 35 mIU/mL; 
ii. Amenorrhea≥ 12 consecutive months in women ≥ 62 years old (FSH 
testing is not required). 
14. WOCBP must have a negative serum or urine pregnancy test within 72 hours before start 
of AG-348 dosing. 
15. Women must not be breastfeeding.  
16. Male patients, with the exception of those who have undergone vasectomy at least 
6 months prior to Screening, must agree to abstain from sexual intercourse or, if sexually 
active, to use a condom with spermicide as contraception (regardless of their female 
partner's childbearing potential or their partner's use of their own contraception) from 
Day 1 of dosing until 30 days following the last dose of AG-348. Abstinence is an 
acceptable method only when this is in line with the normal life style of the patient, 
meaning that the patient plans to remain abstinent continuously throughout the duration 
of the study and for at least 30 days after the last dose of study drug. Periodic abstinence, 
e.g. selective timing of intercourse based on partner's calendar, sympathothermal and 
post-ovulation methods, and withdrawal are not acceptable methods of contraception. 
8.3. 
Exclusion Criteria 
Patients who meet any of the following criteria at Screening or prior to dosing on Day 1 will not 
be enrolled in the study: 
1. Hemoglobin level > 12.0 g/dL if male; Hb > 11.0 g/dL if female. 
2. Additional diagnosis of any other congenital or acquired blood disorder, including 
glucose-6-phosphate-dehydrogenase (G6PD) deficiency, or any other hemolytic anemia 
process except for mild allo-immunization as a consequence of transfusion therapy. 
3. Iron overload (hemosiderosis or concurrent hemochromatosis) sufficiently severe to 
result in a clinical diagnosis by the Investigator of cardiac, hepatic, or pancreatic 
insufficiency. 
4. Prior bone marrow or stem cell transplant. 
5. Clinically symptomatic cholelithiasis or cholecystitis. (Prior cholecystectomy is not 
exclusionary. Patients with symptomatic cholelithiasis or cholecystitis may be rescreened 
once the disorder has been treated and clinical symptoms have resolved.) 
6. Currently enrolled in another therapeutic clinical trial involving on-going therapy with 
any investigational or marketed product or placebo. Concurrent participation in the 
Pyruvate Kinase Deficiency Natural History Study (NCT02053480) is permitted. 
7. Exposure to any investigational drug, device, or procedure within 28 days prior to 
Screening. 
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8. Concurrent medical condition that could compromise participation in the study such as: 
a. Poorly controlled hypertension (defined as systolic blood pressure (BP) 
> 150 mm Hg or diastolic BP > 90 mm Hg) refractory to medical management. 
b. History of recent (within < 6 months from Screening date) congestive heart 
failure; myocardial infarction or unstable angina pectoris; or hemorrhagic, 
embolic, or thrombotic stroke; deep venous thrombosis; or pulmonary or arterial 
embolism. 
c. Currently active infection requiring the use of parenteral anti-microbial agents or 
that is ≥ Grade 3 (CTCAEv4.03) within 6 months of first dose. 
d. A pattern or frequency of post-splenectomy sepsis that in the assessment of the 
Investigator could reasonably be expected to interfere with the ability of the 
patient to complete the 24 week study participation. 
e. Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) 
antibody with signs of active Hepatitis B or C virus infection.  
f. Positive test for human immunodeficiency virus (HIV) 1 or 2 antibody. 
g. Diabetes mellitus judged to be in poor control by the Investigator or requiring 
> 3 anti-diabetic agents counting insulin; use of insulin per se is not exclusionary.  
h. History of any primary malignancy with the exception of: curatively treated non-
melanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; 
or other primary tumor treated with curative intent and no known active disease 
present and no treatment administered during the last 3 years. 
9. Undergone major surgery within 6 months of first dose.  
10. Current or recent history of psychiatric disorder that in the opinion of the Investigator or 
Medical Monitor could compromise the ability of the patient to cooperate with study 
visits and procedures. 
11. Use of any of the restricted list of products known to strongly inhibit CYP3A4 
metabolism (Appendix 15.4, Table 8) within 5 days prior to Day 1 dosing; or to strongly 
induce CYP3A4 metabolism (Appendix 15.4, Table 9) within 28 days prior to Day 1 
dosing; or to strongly inhibit P-gp transporter (Appendix 15.4, Table 10) within 5 days 
prior to Day 1 dosing; or digoxin within 5 days prior to Day 1 dosing. For patients who 
require chronic inhaled glucocorticoid therapy, Investigators should confer with the 
Medical Monitor for additional guidance. 
12. Serum bilirubin > ULN attributable to factors other than hemolysis and/or Gilbert's 
syndrome. 
13. Male patients with heart-rate corrected QT (Fridericia's correction factor) QTcF 
interval > 450 msec, or female patients with QTcF interval > 470 msec with the 
exception of patients with a left bundle branch block (LBBB). Medical Monitor approval 
needed in patients with a LBBB. 
14. Cardiac dysrhythmias judged as clinically significant by the Investigator or requiring 
therapy with drugs that are primarily substrates of CYP3A4. 
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15. History of allergy to sulfonamides if characterized by acute hemolytic anemia, 
anaphylaxis, or rash of erythema multiforme type or Stevens-Johnson syndrome. 
16. Any other medical or psychological condition deemed by the Investigator to be likely to 
interfere with a patient’s ability to understand and sign informed consent; cooperate with 
study visits, tests, and procedures; or otherwise safely and reliably participate in the 
study. 
8.4. 
Patient Identification and Registration 
Patients who are candidates for enrollment into the study will be evaluated for eligibility by the 
Investigator to ensure that the inclusion and exclusion criteria (see Section 8.2 and Section 8.3, 
respectively) have been satisfied and that the patient is eligible for participation in this clinical 
study. The site will submit to the Sponsor an Eligibility form for each eligible patient and the 
Medical Monitor will confirm eligibility for all patients prior to receipt of the first dose of 
AG-348. 
8.5. 
Patient Randomization 
Patients who have been confirmed as eligible will be randomized in an equal ratio to a treatment 
arm (e.g., 1:1 or 1:1:1 depending on which arms are open). The site will provide a request for 
randomization form (including the patient’s confirmed genotype) to the study Medical Monitor. 
The randomization will be stratified by PKR mutation in order to maintain balance across the 
dose arms for the specific mutations expected to be most frequently enrolled. The PKR mutation 
stratification factor will consist of 4 levels (R510Q, R486W, and R479H) and all other mutations 
(“other”). Since this is an open label study, randomization will not be blinded. 
Please refer to the study manual for the randomization procedure. 
8.6. 
Patient Withdrawal Criteria 
Patients have the right to withdraw from the study at any time for any reason. Patients will be 
withdrawn from study-related procedures and treatments under the following conditions: 
• Withdrawal of consent; 
• Experiences unacceptable toxicity; 
• Development of an intercurrent medical condition that precludes further participation 
in the trial; 
• Patient requires use of a prohibited concomitant medication (Section 9.11.2); 
• Investigator decision; 
• Protocol violation: non-adherence to protocol requirements; 
• Pregnancy; 
• Lost to follow-up. 
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Should a patient decide to withdraw, all efforts will be made to complete and report the protocol-
defined study observations up to the time of the patient’s withdrawal as completely as possible 
and to determine the reason for withdrawal. 
In the event a patient is withdrawn from the study, the Medical Monitor must be informed. If 
there is a medical reason for withdrawal, the patient will remain under the supervision of the 
Investigator until satisfactory health is returned. 
When a patient withdraws from the study, the primary reason for discontinuation must be 
recorded in the appropriate section of the electronic case report form (eCRF) and all efforts will 
be made to complete and report final study observations as thoroughly as possible. 
All AEs should be followed until resolution or for a period of 30 days from the last dose of study 
drug, whichever is shorter. Patients with toxicity suspected to be related to study drug will 
continue follow-up until the AE resolves, is declared chronic by the Investigator, or the patient is 
lost to follow-up. 
8.7. 
Replacement of Patients 
Patients who drop out of the study prior to completing the first 12 weeks of assigned dosing for 
reasons other than AEs may be replaced at the Sponsor’s discretion. 
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9. 
STUDY TREATMENT 
9.1. 
Description of Study Drug 
AG-348 sulfate hydrate capsules will be provided as 5 mg, 25 mg, or 100 mg (free-base 
equivalent) of AG-348 sulfate hydrate without excipients in dark green opaque (5 mg), Swedish 
orange (25 mg), or white opaque (100 mg) gelatin capsules (size 2 capsules for all dose 
strengths). 
All study drugs are for investigational use only and are to be used only within the context of this 
study. All study drug products will be supplied by the Sponsor. Please see the Investigator’s 
Brochure for further details regarding study drug. 
9.2. 
Study Drug Packaging and Labeling 
AG-348 sulfate hydrate capsules are packaged in white, high-density polyethylene (HDPE) 
induction sealed bottles with a child-resistant screw cap. 
Packaging and labeling will be prepared to meet all regulatory requirements. 
9.3. 
Study Drug Storage 
AG-348 sulfate hydrate drug capsules must be stored at room temperature of 15 to 30°C 
(59 - 86°F). 
All study drug products must be stored in a secure, limited-access location and may be dispensed 
only by the Investigator or by a member of the staff specifically authorized by the Investigator. 
9.4. 
Method of Assigning Patients to Treatment 
Up to a maximum of 25 patients will be randomized to any one of the dosing arms in this study. 
Randomization will be balanced 1:1 or 1:1:1, depending on whether 2 or potentially 3 arms are 
open, and will be stratified by PKR mutation (see Section 8.5). The dose and schedule of 
AG-348 each patient receives will be dependent upon which dose arm is open for enrollment 
when the patient qualifies for and is randomized into the study.  
9.5. 
Blinding 
This is an open-label study; no blinding methods will be used. 
9.6. 
Study Drug Preparation and Administration 
For the initial two treatment arms, (Arm 1 and Arm 2), AG-348 will be administered orally BID 
(approximately every 12 hours with a minimum of 10 hours between doses) over a 24 week 
treatment period. Starting with Day 1, dosing will be continuous; there will be no rest periods. 
Patients who do not meet any of the treatment withdrawal criteria (see Section 8.5) may continue 
treatment for the entire 24-week treatment period. 
Patients will be dispensed the appropriate number of Sponsor-packaged, labeled bottles to allow 
for 28 days of dosing until the next scheduled visit.  
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Patients will be given a dosing diary to be used for each 28-day dosing period. They should 
record relevant information regarding their study drug in the diary (e.g., confirmation that each 
daily dose was taken, reasons for missed doses). 
Treatment compliance will be assessed based on return of unused drug and the dosing diary (see 
Section 9.9). 
Patients should be instructed to take their daily dose at approximately the same times each day 
except for dosing on in-clinic visiting days.  
Patients who undergo extensive PK/PD sampling (see Appendix 15.1, Table 6) should be 
instructed from Week 3 on to bring the AM dose with them for in-clinic visits and to ingest the 
dose following PK/PD blood draws.  
Patients receiving limited PK/PD sampling (see Appendix 15.1, Table 7) should be instructed to 
bring the AM dose with them for all in-clinic visits and to take the AM dose following PK/PD 
blood draws.  
Patients receiving extensive PK/PD sampling on Day 1 and 15 will also have limited PKPD on 
other visit days. As a general rule, regardless of extensive or limited schedule, patients will bring 
in the AM dose for all visits and take this dose following PK/PD blood draws.  
A minimum of 10 hours between the AM and PM dose will be required on those dosing days. 
Each dose should be taken with a glass of water and consumed over as short a time as possible. 
AG-348 may be taken with or without food. Patients should be instructed to swallow capsules 
whole and to not chew the capsules. For patients who have difficulty swallowing tablet(s), the 
Medical Monitor should be contacted to discuss administration. 
Patients will receive their first dose of AG -348 in the clinic on Day 1 and then may take the 
remaining doses on an outpatient basis. 
9.7. 
Criteria for Dose Escalation, Dose Modification, or Discontinuation 
of Study Drug 
No intra-patient dose escalations will be permitted in this study unless the DRT decides to re-
assign patients’ doses and schedule in a terminated arm to match the dose and schedule of 
another arm of the study. In this case, the patients in the terminated arm will remain in their 
originally assigned arm; i.e., they will not count towards the enrollment quota of the arm whose 
dose and schedule is being adopted. All dosing modifications, as outlined below, will be 
implemented following discussions with the Medical Monitor. 
9.7.1. 
Dose Modification for Safety 
The Investigator will monitor all patients for safety and tolerability. Modification of the patient’s 
dose of AG-348 will be based on AEs and observed changes in Hb levels (see Section 9.7.2). 
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Table 1: 
Dose Modification for Adverse Events Deemed at Least Possibly Related to 
AG-348 
Adverse Events(s) 
AG-348 Dose Adjustment 
Grade 1 
None required. 
Grade 2 
None required; Investigator and Medical Monitor judgment to manage as for 
Grade 3. 
Grade 3 
Suspend dosing;  
If event resolves to Grade 1 or baseline within approximately 14 days of 
suspension, resume dosing with 1 dose level reduction (see Table 2 below).  
If event does not resolve to Grade 1 or baseline within approximately 14 days of 
suspension, permanently discontinue dosing, unless the benefits outweigh the 
risks of resuming treatment and are approved by the Medical Monitor. 
Grade 4 
Permanently discontinue dosing, unless the benefits outweigh the risks of 
resuming treatment and are approved by the Medical Monitor. 
Dose modifications for Grade 2 and 3 AEs should be maintained long enough for the 
Investigator to be confident that the reduced dose is being well tolerated before considering a 
request of the Medical Monitor to permit subsequent re-escalation to the former dose level. 
Dosing for an individual patient will be discontinued permanently for Grade 3 and Grade 4 AEs 
that do not resolve to Grade 1 or baseline within approximately 14 days of suspension of dosing, 
unless the benefits outweigh the risks of resuming treatment and are approved by the Medical 
Monitor. No patients may be re-escalated to their former dose level after a dose modification 
without discussion with the Medical Monitor. If following the first dose reduction for a Grade 3 
AE the patient experiences a second occurrence of the same Grade 3 AE, then treatment with 
AG-348 must be immediately and permanently discontinued.  
It should be noted that if the initial dose of 300 mg BID selected for Arm 1 demonstrates an 
unacceptable safety profile resulting in multiple subjects undergoing dose modifications, the 
DRT may exercise its option to re-assign these patients’ dose and schedule to match the dose and 
schedule of another study arm (for example, Arm 2 of the study, or to match the dose and 
schedule of a [potential] Arm 3, if implemented). 
9.7.2. 
Dose Modification for Increase in Hemoglobin Level 
It is presently unknown to what magnitude, how rapidly, or even whether, AG-348 will result in 
increased Hb levels in patients with PK deficiency across the variety of potential PKR mutations 
that may be encountered during this study. Moreover, it is unknown whether any potential 
increases in Hb experienced by patients in this study may be a safety risk. As a conservative 
measure, this study incorporates guidance for dose modification based on potential large 
increases in Hb levels that exceed the midpoint of the typical normal range by gender. The 
Investigator will monitor all patients for changes in Hb levels and should adjust the dose of 
AG-348 as outlined in Table 2 per the following guidelines and with Medical Monitor approval. 
• Males: If Hb > 15 g/dL and confirmed with a second test, suspend dosing until Hgb 
≤ 13.5 g/dL; then resume dosing with a 1 dose level reduction.  
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Protocol AG348-C-003, Amendment 2, Version 3.0 
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• Females: If Hb > 13.5 g/dL and confirmed with a second test, suspend dosing until 
Hb ≤ 12.5 g/dL; then resume with a 1 dose level reduction.  
• The treating Investigator will discuss with the Medical Monitor questions relating to 
dose modifications on an as needed basis.  
Table 2: 
Dose Reduction Table (by Dosing Arm) 
Dose Group 
Starting Dose 
1st Dose Reduction 
2nd Dose Reduction 
Arm 1 
300 mg BID 
200 mg BID 
100 mg BID 
Arm 2 
50 mg BID 
25 mg BID 
TBD1 
Potential Arm 3 
TBD 
To approximately 
50-66% of initial dose 
To approximately 
25-33% of initial dose 
1 Dose to be determined by Medical Monitor. 
Hemoglobin levels above the ULN by gender should be reported as an AE, graded per the 
CTCAEv4.03, according to the guidance provided in Section 11.2. 
9.7.3. 
Stopping Criteria 
Dosing for an individual patient will be discontinued permanently for Grade 3 AEs that do not 
resolve to Grade 1 or baseline within approximately 14 days of suspension of dosing, and for 
Grade 4 AEs, unless the benefits outweigh the risks of resuming treatment and are approved by 
the Medical Monitor (Section 9.7.1). Other reasons for treatment termination are provided in 
Section 8.5. 
9.8. 
Duration of Patient Participation 
The duration of treatment for all patients on this study will be up to 24 weeks. Patients who 
safely tolerate and demonstrate one or more indicators of clinical activity of AG-348 may be 
eligible to immediately roll over to a safety extension for continued treatment. 
9.9. 
Treatment Compliance 
During in-clinic visits, doses of AG-348 will be ingested by the patient under the supervision of 
clinical facility personnel. For at-home dosing, patients will be given a dosing diary to be used 
for the duration of the 24-week treatment period. Patients should record relevant information 
regarding their study drug in the diary (e.g., confirmation that each daily dose was taken, reasons 
for missed doses) and return the diary at each study visit. 
9.10. 
Study Drug Accountability 
Accountability for the study drug at the clinical facility is the responsibility of the Investigator. 
The Investigator will ensure that the study drug is used only in accordance with this protocol. 
Where allowed, the Investigator may choose to assign drug accountability responsibilities to a 
pharmacist or other appropriate individual. 
The Investigator or delegate will maintain accurate drug accountability records indicating the 
drug’s delivery date to the site, inventory at the site, use by each patient, and return to Agios or 
its designee (or disposal of the drug, if approved by Agios). These records will adequately 
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document that the patients were provided the doses as specified in the protocol and should 
reconcile all study drug received from Agios. Accountability records will include dates, 
quantities, batch/serial numbers, expiration dates (if applicable), and patient numbers. An 
unblinded monitor will review drug accountability at the site on a schedule agreed to by the 
Sponsor. 
Study drug must not be used for any purpose other than the present study.  
All unused and used study drug will be retained at the site until it is inventoried by the Study 
Monitor after database lock has occurred. All used, unused or expired study drug will be returned 
to Agios or its designee or, if authorized, disposed of at the study site per the site’s Standard 
Operating Procedures (SOPs) and documented. All material containing AG-348 will be treated 
and disposed of as hazardous waste in accordance with governing regulations. 
9.11. 
Prior and Concomitant Medications and Treatments 
9.11.1. 
Prior Medications and Procedures 
All medications administered and procedures conducted within 28 days prior to the first day of 
study drug administration are to be recorded on the source documentation and included in the 
eCRF. 
9.11.2. 
Prohibited Concomitant Therapy 
All concomitant medications and procedures administered from 28 days before administration of 
study drug through the last Follow-up Visit (Week 28/Day 197) must be recorded in the 
appropriate section of the source documentation and eCRF along with dosage information, dates 
of administration, and reason for use.  
The following are prohibited at all times during participation in this study: 
• Investigational drugs must be discontinued 28 days prior to the first dose of study 
drug; 
• Products known to strongly inhibit CYP3A4 metabolism (listed in Appendix 15.4, 
Table 8) must be discontinued within 5 days prior to Day 1 dosing; 
• Products known to strongly induce CYP3A4 metabolism (listed in Appendix 15.4, 
Table 9) must be discontinued within 28 days prior to Day 1 dosing; 
• Products known to strongly inhibit P-gp transporter (listed in Appendix 15.4, 
Table 10) must be discontinued within 5 days prior to Day 1 dosing; 
• Digoxin must be discontinued within 5 days prior to Day 1 dosing; 
• Hematopoietic stimulating agents (erythropoietins, granulocyte colony stimulating 
factors, thrombopoietins, etc) must be discontinued no less than 28 days prior to the 
first dose of study drug. [Folic acid 1 mg orally per day is required for all patients. 
B12 injections are permitted for patients with a prior diagnosis of B12 deficiency 
syndromes, but must be repleted to stability of the Hb and mean corpuscular volume  
(MCV) prior to enrollment in the study]; 
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• Anabolic steroids, including testosterone preparations, administered for anemia must 
be discontinued no less than 28 days prior to the first dose of study drug;  
• As the target population for this study consists of transfusion independent patients 
and transfusion of blood products could confound key endpoints of the study, blood 
transfusions of any type must be strictly avoided except in cases of compelling 
medical need. If medical circumstances permit, the Medical Monitor should be 
contacted for discussion before any transfusions are administered. 
Drugs that displace unconjugated bilirubin from albumin (including some common sulfa 
antibiotics [including sulfamethoxazole/trimethoprim], cephalosporins, salicylates, and 
aminophylline) should be used with caution with the understanding that patients with elevated 
levels of unconjugated bilirubin may potentially be at risk for kernicterus syndrome (Strauss, et 
al. 2006). 
Potential Drug Interactions 
Strong inhibitors of CYP3A4 (listed in Appendix 15.4, Table 8) are not permitted for use with 
AG-348. Based on modeling of AG-348 metabolism and tolerability data from the Phase 1 MAD 
study, moderate inhibitors of CYP3A4 do not appear to pose a risk to patients dosed with 
AG-348. 
In-vivo data from the Phase 1 MAD show AG-348 induces CYP3A4 and its own metabolism, 
presumably via CYP3A4. The use of strong CYP3A4 inducers in combination with AG-348 is 
expected to reduce AG-348’s efficacy. Therefore, administration of strong CYP3A4 inducers 
(listed in Appendix 15.4, Table 9) is not permitted with AG-348.  
Strong inhibitors of drug transport (listed in Appendix 15.4, Table 10) are not permitted for use 
with AG-348. 
Digoxin is not permitted for use with AG-348. 
Induction of CYP3A4 by AG-348 is expected to reduce the efficacy of certain sensitive co-
medications. Co-administration of AG-348 with the drugs in Appendix 15.4, Table 11 should be 
done with caution, as their efficacy may be reduced.  
Of note, women in the trial utilizing oral contraception must utilize barrier methods as per the 
Inclusion Criteria 14 (Section 8.2) while taking AG-348. 
Short-term (≤ 14 days at a time, and ≤ 28 days total during the 24 week treatment period) use of 
topical, inhaled, intra-nasal, and systemic glucocorticoids is permitted for acute medical 
indications. Every effort should be made to minimize total duration of glucocorticoid therapy and 
utilize alternative treatments. Patients must be off glucocorticoids for at least 28 days prior to 
Day 1 of AG-348 dosing as per Exclusion Criterion #11 (Section 8.3). For patients who require 
chronic inhaled glucocorticoid therapy, Investigators should confer with the Medical Monitor for 
additional guidance. 
The expected patient co-medications deferoxamine, deferasirox, deferiprone, and oral penicillin 
are not expected to interact with AG-348. 
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9.11.3. 
Allowed Concomitant Therapy 
Medications and treatments other than those specified above are permitted during the study. All 
intercurrent medical conditions will be treated at the discretion of the Investigator according to 
acceptable local standards of medical care. Subjects may receive analgesics, antiemetics, anti-
infectives (including penicillins), and antipyretics as medically indicated and consistent with the 
guidance in the previous two sections. Patients may continue iron chelation therapy with 
deferoxamine and deferasirox. Patients must continue taking 1 mg of folic acid for the duration 
of the study. 
All concomitant medications, including transfusions of blood products, procedures performed 
during the study, including those used to treat AEs, will be recorded on the eCRF.  
9.11.4. 
Potential for Phototoxicity 
AG-348 may cause sensitivity to direct and indirect sunlight. Patients should be warned to avoid 
direct sun exposure. When exposure to sunlight is anticipated for longer than 15 minutes, the 
patient should be instructed to apply factor 30 or higher sunscreen to exposed areas and wear 
protective clothing and sunglasses. 
9.11.5. 
Management of Nausea, Vomiting, and Diarrhea 
As the primary objective of this study is to assess the safety and tolerability of up to 24 weeks of 
AG-348, routine use of prophylactic anti-emetic and antidiarrheal or other classes of medications 
is prohibited. However, if patients experience AEs relating to nausea, vomiting, or diarrhea these 
may be treated at the Investigator's clinical discretion with recommended medications as follows: 
• Nausea/vomiting: allowed agents include standard clinical dosing with palonosetron 
(Aloxi), dolasetron (Anzemet), or promethazine (Phenergen). Promethazine is a 
substrate for CYP2B6, and it is presently unknown if the potential for 2B6 induction 
after AG-348 dosing could be sufficient to reduce the therapeutic effect of 
promethazine. Not recommended are aprepitant (Emend; CYP3A4 inhibitor), 
ondansetron (Zofran; CYP3A4 inducer), chlorpromazine (Thorazine; CYP3A4 
inducer), prochlorperazine (Compazine; CYP3A4 substrate, QT prolongation has 
been reported), and granisetron (Kytril; CYP3A4 substrate, QT prolongation has been 
reported);  
• Diarrhea: recommended management includes standard clinical dosing with 
Kaopectate or other non-absorbable anti-diarrheals, diphenoxylate/atropine (Lomotil), 
or loperamide (Imodium). Loperamide is the least preferred choice because it is both 
a substrate and inhibitor for CYP3A4, a substrate for CYP2B6, and a substrate for 
P-gp. 
• For the use of any medications not specifically mentioned above the Investigator may 
confer with the Sponsor’s Medical Monitor. 
9.11.6. 
Other Restrictions and Precautions 
Patients should be advised to refrain from altering their normal exercise routine for the first 
28 days of treatment with AG-348 as symptoms of anemia could theoretically temporarily 
worsen before the eventual potential beneficial effect of AG-348 on the Hb level becomes 
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evident. The elevated blood levels of 2,3-DPG commonly seen in patients with PK deficiency 
may produce a right shift in the Hb-O2 dissociation curve. The effect of this is to enhance 
oxygen delivery at the tissue level, thus counteracting to some extent the physiologic 
consequences of the anemia. AG-348 was shown to produce rapid decreases (within the first 
12 hours following a single dose) in 2,3-DPG in two prior clinical trials with healthy adult male 
and female volunteers. In patients with PK deficiency who have elevated 2,3-DPG levels, it is 
theoretically possible that decreases in 2,3-DPG that precede correction of the anemia could 
result in a temporary decrease of oxygen delivery at the tissue level resulting in clinical 
symptoms (e.g., increased fatigue). 
As discussed in Section 5.2.1.1 of this protocol and in the Investigator Brochure, AG-348 has 
been identified as a histamine H3 receptor antagonist/inverse agonist. No effects of histamine H3 
modulation have been observed in safety pharmacology or toxicology studies. Nonetheless, 
patients should be monitored for potential adverse events related to wakefulness and insomnia 
(Schwartz 2011). 
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10. 
STUDY ASSESSMENTS 
10.1. 
Schedule of Assessments 
The Schedules of Assessments for this study are provided in Appendix 15.1.  
After obtaining written informed consent, patients will undergo screening evaluations. The 
Screening visit is to be conducted within approximately 28 days prior to first dose of study 
treatment.  
During the Treatment period, patients will attend visits at baseline (Day 1), weekly through 
Week 3 (Days 8, 15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 
12) and monthly through Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate and 
demonstrate one or more indicators of clinical activity of AG-348 through Week 24 may be 
eligible to immediately enter a safety extension for continued treatment upon agreement of the 
treating Investigator and the Medical Monitor. For patients who finish treatment, Study 
Discharge will occur 4 weeks (Week 28 or earlier) following the last dose of study treatment at 
the final follow-up assessment. 
Although not encouraged, as a convenience for patients who travel long distances to the study 
site, in-clinic visits on Day 8 and Day 22 may be performed by the patient’s primary care 
physician if necessary and must be approved by the Sponsor on a case by case basis. For details, 
please refer to Table 5 in Appendix 15.1 : Schedule of Assessments.  For patients having their 
Day 8 and/or Day 22 visits performed by the primary care physician, the principal investigator 
will him or herself, or have a qualified research nurse or other designated site staff member, 
make telephone contact with the patient to inquire about any adverse events. These must be 
recorded as if the patient appeared in the main study center. The Principal Investigator will 
exercise prudent clinical judgment in determining any clinical course of action to take based on 
any AEs discovered. These telephone contacts on Day 8 and Day 22 must be explained to the 
patient in advance and scheduled in advance to maximize the likelihood of successfully making 
contact. Having in-clinic visits on Days 8 and/or Day 22 performed by the primary care 
physician will necessitate re-scheduling certain assessments that the primary care physician’s 
office may not be reasonably expected to perform. Table 3 summarizes the details of the 
re-scheduling of these assessments as described in Table 5 in Appendix 15.1.  
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Table 3: 
Summary of Assessments When Day 8 and/or Day 22 In-Clinic Visits Are 
Performed By Primary Care Physician 
Day 8 Visit by Primary Care Physician 
Day 22 Visit by Primary Care Physician 
Primary 
Care Office 
Main 
Study 
Site 
Assessment 
to Move to 
Day 15 Main 
Study Site 
Assessments 
Not Required  
Primary Care 
Office 
Main 
Study 
Site 
Assessment 
to Move to 
Day 43 Main 
Study Site 
Assessments Not 
Required 
Hematology 
to central lab 
Phone 
contact 
with 
patient 
12-lead ECG 
VS; serum 
chemistry 
Hematology 
sample to 
central lab 
Phone 
contact 
with 
patient 
12-lead ECG 
VS; serum chemistry; 
coagulation, 
haptoglobin, EPO level, 
carboxyhemoglobin, 
PK/PD 
Abbreviations: ECG = electrocardiogram; EPO = erythropoietin; PK/PD = pharmacokinetics/pharmacodynamics; 
VS = vital signs 
Whenever more than one assessment is scheduled for the same nominal time, the assessments 
should be performed in the order of least invasive to most invasive assessment (e.g., VS first, 
ECG, blood draw). The timing of these assessments should allow the PK blood draw to occur at 
the exact nominal time. The order of procedures may be revised with prior discussion between 
Sponsor and Site. 
Minor adjustments to the timing, number of planned safety monitoring procedures (e.g., VS, 
ECG, blood draw), and PK/PD assessments may be made during the course of the study based on 
collected data to ensure appropriate safety monitoring and will not require a protocol 
amendment. These minor changes will require prior approval from the Sponsor’s Medical 
Monitor (or Responsible Medical Officer) as well as appropriate documentation in the study 
records. The addition of new safety monitoring procedures or other assessments will require a 
protocol amendment. 
10.2. 
Informed Consent and Confirmation of Eligibility 
A complete description of the study is to be presented to each potential patient and a signed and 
dated informed consent is to be obtained before any study specific procedures are performed.  
Patient’s eligibility will be confirmed at Screening and within 24 hours prior to study treatment 
dose. If a patient is determined to be ineligible, the patient will be excluded from participation. 
10.3. 
Demographic Data, Medical and Medication History 
Patient demographic data, including gender, date of birth, age, race, and ethnicity, will be 
obtained at Screening. Collection of demographic data will be modified by country regulatory 
requirements, as appropriate.  
Medication history, including all relevant prior medical history and current medical conditions, 
will be obtained at the Screening assessment and on Day -1; all concomitant medications 
administered and procedures conducted within 28 days prior to Day 1 should be reported in the 
source documentation and eCRF. Investigators will be asked to provide information on the 
patient’s history of any medical diagnoses (e.g., iron overload) and surgical procedures (e.g., 
splenectomy, cholecystectomy) pertaining to their diagnosis of PK deficiency and prior available 
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complete blood counts (CBCs) over the preceding 6 months and transfusion history over the 
preceding 12 months prior to the date of signing informed consent.  
10.4. 
PKR Enzymatic Assay and PKR Genotyping 
Assessments for PKR enzymatic activity and PKR genotyping will be performed at Screening 
only for confirmation of study eligibility. PKR enzymatic assays will be conducted at 
 
 
. PKR genotyping will be conducted at 
 
 
10.5. 
Safety Assessments 
10.5.1. 
Physical Examination, Height, and Weight 
A complete physical examination (including neurological examination; genital and rectal 
examinations will be performed at the discretion of the Investigator) will be obtained at 
Screening, Baseline, Week 12, and the Follow up Visit (Week 28). The neurological examination 
must include an assessment of general orientation and mental status including level of alertness 
(assess as normal or abnormal and specify any abnormality). For patients rolling over to the 
safety extension, the last physical examination will occur at Week 24. Limited focused physical 
examinations will be performed at all other visits. Any findings will be recorded on the eCRF. 
Height will be collected at Screening only. 
10.5.2. 
Vital Signs 
Vital signs, including systolic and diastolic BP, heart rate, respiratory rate, and temperature, will 
be obtained according to the according to the Schedule of Assessments (Appendix 15.1). Vital 
sign measurements will be repeated if clinically significant or machine/equipment errors occur. 
Out of range BP or heart rate measurements will be repeated at the Investigator’s discretion. Any 
confirmed, clinically significant vital sign measurements will be recorded as AEs. 
10.5.3. 
Electrocardiogram  
A 12-lead ECG will be obtained according to the Schedule of Assessments (Appendix 15.1). The 
ECGs will be measured using an ECG machine that calculates the heart rate and measures PR, 
QRS, QT, QTcB (Bazett correction formula), and QTcF (Frederica’s correction). Only QTcF 
(not QTcB) will be used for determination of eligibility. 
The 12-lead ECGs should be obtained following 5 minutes of recumbency. The Screening ECG 
will be performed at least 7 days prior to Day 1 dosing. ECGs will be repeated if clinically 
significant abnormalities are observed, if artifacts are present, or if machine/equipment errors 
occur. Any confirmed, clinically significant ECG findings will be recorded as AEs. 
10.5.4. 
DXA Scans 
DXA scans (hip and spine) will be performed at Screening to obtain T and Z scores that will 
serve as a baseline measure of bone mineral density for all enrolling patients. A second DXA 
scan will be conducted in the interval between Week 24 and Week 28 as indicated in the 
Schedule of Assessments (Appendix 15.1). All redacted DXA scan reports must be held at the 
study site and will be collected by the Sponsor. 
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10.5.5. 
Safety Laboratory Assessments 
10.5.5.1. Hematology, Serum Chemistry, Coagulation Studies, and Urinalysis 
Laboratory values obtained prior to Screening and RBC antibodies obtained at Screening will be 
performed at a local laboratory. On-study clinical laboratory evaluations are to be performed by 
a central laboratory. If Investigators believe that it is clinically indicated to obtain safety 
laboratory results from their own local laboratories on the day of the subject's visit, and before 
the results are returned from the designated central laboratory, they are free to exercise their 
discretion to do so. Investigators should be aware that since the Hb level is a secondary endpoint 
of the study, it behooves them to minimize blood volumes drawn, and samples for the central lab 
must still be collected, since it will serve as the official lab result for this study. 
Clinical laboratory evaluations are to be collected according to the Schedule of Assessments 
(Appendix 15.1). In addition, all clinically significant laboratory abnormalities noted on testing 
will be followed by repeat testing and further investigated according to the judgment of the 
Investigator. Please note that serum estradiol, free and total testosterone, and complete blood 
count (CBC) will be collected in the AM at any 2 time points during Screening at least 2 days 
apart in addition to Baseline/Day 1 (total of 3 time points prior to Day 1 dosing).  
The following safety laboratory parameters are to be determined: 
Hematology: 
HCT, Hb, RBC count, absolute reticulocyte count, percent 
reticulocyte count, MCV, mean corpuscular Hb (MCH), mean 
corpuscular Hb concentration (MCHC), red cell distribution width 
(RDW), white blood cell (WBC) count with automated (or manual, 
as indicated) differential, ANC, and absolute lymphocyte count 
(ALC), and platelet count. In the event that the designated central 
laboratory for the study is unable to provide a valid result for any 
specific component of the defined CBC for a specific patient, the site 
may be asked to have the test performed at their local laboratory. 
The result of the local CBC will be entered into the study database 
along with the local normal reference range. G6PD and RBC 
antibody screen will be performed at Screening only 
Other 
EPO, Hp, COHb, 25-hydroxy vitamin D2 and D3. 
Serum Chemistry: 
alkaline phosphatase (ALP), sodium, potassium, chloride, calcium, 
magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, 
albumin, total protein, glucose, blood urea nitrogen (BUN), 
creatinine, uric acid, LDH, ALT, AST, total bilirubin, indirect 
bilirubin, (and estimated creatinine clearance or glomerular filtration 
rate for screening only, as appropriate). 
Sex Hormones: 
testosterone (total and free), estrone, and estradiol. FSH will only be 
performed at Screening for female patients only for confirmation of 
post-menopausal status. 
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Bone Turnover 
serum osteocalcin-N-mid and CTX. 
Lipids 
total cholesterol, HDL-C, triglycerides. 
Iron Panel 
iron (Fe), total iron-binding capacity (TIBC), transferrin saturation, 
ferritin. 
Coagulation Studies: 
fibrinogen, aPTT, INR. 
Urinalysis: 
color and appearance, pH, specific gravity, ketones, protein, glucose, 
bilirubin, nitrite, urobilinogen, pregnancy screen (dipstick), occult 
blood. Microscopic inspection of sediment will only be performed 
for cause or to investigate an abnormal dipstick finding per the 
Investigator’s discretion. 
 
10.5.5.2. Screening Serology 
A blood sample for serology, including HBsAg, HCV Ab screen, and HIV1 and HIV2 Ab, is to 
be collected from all patients at Screening.  
10.5.6. 
Menstrual Cycle Diary 
Menstruating female patients will be required to fill out a paper-based menstrual cycle diary each 
month in order to monitor any changes. Diaries will be dispensed and collected as indicated in 
the Schedule of Assessments (Appendix 15.1). Patients will record the start date, stop date, and 
any notable characteristics of each menstrual cycle. 
10.5.7. 
Adverse Events 
Each patient will be carefully monitored for the development of any AEs throughout the study 
from signing of the informed consent through all scheduled study follow-up visits, or withdrawal 
of consent, whichever occurs first. In addition, SAEs that are assessed as possibly or probably 
related to study treatment that occur > 30 days post-treatment also are to be reported. 
AEs will be evaluated by the Investigator and recorded as described in the Schedule of 
Assessments. On dosing visits, all AEs (elicited and spontaneously reported) will be 
continuously evaluated by the Investigator and recorded. At any non-dosing day visit, AEs will 
be evaluated by the Investigator and recorded. Any AEs already documented at a previous 
assessment and designated as ongoing will be reviewed at subsequent visits or assessment time 
points as necessary. If these AEs have resolved, this will be documented. 
All AEs will be graded using the CTCAE grading system (Appendix 15.2). 
Complete details on AE monitoring are provided in Section 11. 
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10.6. 
Pharmacokinetic Assessments 
10.6.1. 
Blood Sample Collection and Pharmacokinetic Measurements During Dose 
Escalation 
The first approximately 10 patients treated, contingent on clinical site feasibility, will undergo 
extensive PK sampling as detailed in Appendix 15.1, Table 6. The remainder of treated patients 
will undergo limited PK sampling as detailed in Appendix 15.1, Table 7. The in-clinic visit on 
Day 22 may be performed by the patient’s primary care physician if necessary and must be 
approved by the Sponsor on a case by case basis. In this instance, PK sampling will not be 
required on Day 22. (Additional details regarding Day 8 and Day 22 visits performed by the 
patient’s primary care physician can be found in Table 5 in Appendix 15.1: Schedule of 
Assessments.) 
The collection times for post-dose PK samples will start from the time that dosing is completed. 
(For example, a PK draw at 30 minutes will be collected 30 minutes after the last capsule has 
been ingested.) The completion time of each dose will be recorded. 
Procedures for sample collection and processing will be provided in a separate study manual. 
The actual time point of each sample collection will be recorded. 
Samples for PK and PD assessments may be retained for up to 2 years from collection.  
10.7. 
Pharmacodynamic Assessments 
The first approximately 10 patients treated, contingent on clinical site feasibility, will undergo 
extensive PD sampling for 2,3-DPG and ATP as detailed in Appendix 15.1, Table 6. The 
remainder of treated patients will undergo limited PD for 2,3-DPG and ATP sampling as detailed 
in Appendix 15.4, Table 7. 
The collection times for post-dose PD samples will start from the time that dosing is completed. 
(For example, a PD draw at 30 minutes will be collected 30 minutes after the last capsule has 
been ingested.) The completion time of each dose will be recorded. 
Procedures for sample collection and processing will be provided in a separate study manual. 
The actual time point of each sample collection will be recorded. 
Pharmacodynamic assessments will include 2,3-DPG, ATP,
 
 
The in-clinic visit on Day 22 may be performed 
by the patient’s primary care physician if necessary and must be approved by the Sponsor on a 
case by case basis. In this instance, PD sampling will not be required on Day 22. (Additional 
details regarding Day 8 and Day 22 visits performed by the patient’s primary care physician are 
stated in Table 5 in Appendix 15.1: Schedule of Assessments.) 
Figure 4 provides a brief schematic outlining the PKR reaction and how each of these PD 
assessments fits into a complete mechanistic understanding of the action of AG-348. 
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Figure 4: 
PKR Enzymatic Reaction 
 
The PKR enzyme catalyzes the PEP to pyruvate reaction, with concomitant formation of ATP. 
• Binding of AG-348 to the PKR tetramer can be assessed through an ex-vivo 
biochemical assay of cell lysates from AG-348 treated patients. Because WBCs 
contain a high level of pyruvate kinase from a non-PKR pyruvate kinase isoform, 
WBCs are first removed by filtration before the purified red cells are frozen. 
 
 
 
• 
 
 
 
 
 
 
 
 
 
• AG-348 target engagement 
 has been 
shown in preclinical models and healthy volunteer clinical studies to result in 
accumulation of ATP and depletion of the upstream metabolite 2,3-DPG. Therefore, 
levels of these metabolites will be measured by mass spectrometry in frozen whole 
blood samples. 
The first approximately 10 patients treated, contingent on clinical site feasibility, will undergo 
extensive PD sampling as detailed in Appendix 15.1, Table 6. The remainder of treated patients 
will undergo limited PD sampling as detailed in Appendix 15.1, Table 7. The collection times for 
post-dose PD samples will start from the time that dosing is completed. (For example, a PD draw 
at 30 minutes will be collected 30 minutes after the last capsule has been ingested.) The 
completion time of each dose will be recorded. 
Blood samples will be stored at the site and regularly transported at -80°C ±10 C to the 
bioanalytical laboratory for analysis. Procedures for sample collection and processing will be 
provided in a separate study manual. 
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10.8. 
Ordering of Blood Sample Collection 
When more than 1 blood sample is collected at the same nominal time, the samples will be 
collected in the following order: 
1. Safety laboratory assessments 
2. PK  
3. PD (2,3 DPG, ATP) 
 
 
 
10.9. 
Sample Processing, Storage, and Shipment 
Instructions for the processing, storage and shipment of all study samples for central analysis 
will be provided in a separate study manual. 
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11. 
ADVERSE EVENTS 
Monitoring of AEs will be conducted throughout the study. Adverse events and SAEs will be 
recorded in the source documentation and eCRF from time of the signing informed consent 
through the Follow-up Visits for randomized patients. All AEs should be monitored until they 
are resolved or are clearly determined to be due to a patient’s stable or chronic condition or 
intercurrent illness(es). 
All SAEs will be followed through 30 days after the last dose of study treatment or until the SAE 
has resolved. Any SAEs that are assessed as possibly or probably related to study treatment that 
occur > 30 days post-treatment also are to be reported. 
11.1. 
Definition of Adverse Events 
11.1.1. 
Adverse Event (AE) 
An AE is any untoward medical occurrence associated with the use of a drug in humans, whether 
or not considered drug related. An AE (also referred to as an adverse experience) can be any 
unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a drug, without any judgment about causality. An AE can 
arise from any use of the drug (e.g., off-label use, use in combination with another drug) and 
from any route of administration, formulation, or dose, including an overdose. 
11.1.2. 
Suspected Adverse Reaction 
A suspected adverse reaction is any AE for which there is a reasonable possibility that the drug 
caused the AE. For the purposes of expedited safety reporting, ‘reasonable possibility’ means 
that there is evidence to suggest a causal relationship between the drug and the AE.  
11.1.3. 
Unexpected Adverse Event 
An unexpected AE is one for which the nature or severity of the event is not consistent with the 
applicable product information, e.g., the Investigator’s Brochure. 
11.1.4. 
Serious Adverse Event 
An AE or suspected adverse reaction is considered serious (SAE) if, in the view of either the 
Investigator or Sponsor, it results in any of the following outcomes: 
• Death; 
• Life-threatening. Life-threatening means that the patient was at immediate risk of 
death from the reaction as it occurred, i.e., it does not include a reaction which 
hypothetically might have caused death had it occurred in a more severe form; 
• In-patient hospitalization or prolongation of existing hospitalization. Hospitalization 
admissions and/or surgical operations scheduled to occur during the study period, but 
planned prior to study entry are not considered AEs if the illness or disease existed 
before the patient was enrolled in the study, provided that it did not deteriorate in an 
unexpected manner during the study (e.g., surgery performed earlier than planned); 
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• A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions; 
• Congenital anomaly/birth defect; 
• Important medical event. An important medical event is an event that may not result 
in death, be life-threatening, or require hospitalization but may be considered an SAE 
when, based upon appropriate medical judgment, it may jeopardize the patient or 
patient and 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 convulsions that do not result in in-patient hospitalization, 
or the development of drug dependency or drug abuse. 
11.1.4.1. Potential Severe Drug-Induced Liver Injury 
The document entitled FDA Guidance for Industry, Drug-Induced Liver Injury: Premarketing 
Clinical Evaluation (FDA 2009) provides guidance on how the measurement of various 
laboratory parameters may be used to assess a given drug’s potential to cause severe liver injury 
(i.e., irreversible liver failure that is fatal or requires liver transplantation). Such cases are 
suggested by the following three components: 
1. The drug causes hepatocellular injury, generally shown by a higher incidence of 3-fold or 
greater elevations above the ULN of ALT or AST than the (non-hepatotoxic) control 
drug or placebo; 
2. Among trial patients showing such aminotransferase elevations, often with 
aminotransferases much greater than 3×ULN, one or more also show elevation of serum 
total bilirubin to > 2×ULN, without initial findings of cholestasis (elevated serum 
elevated serum ALP); 
3. No other reason can be found to explain the combination of increased aminotransferases 
and total bilirubin, such as viral hepatitis A, B, or C; pre-existing acute liver disease; or 
another drug capable of causing the observed injury.  
Clinical safety laboratory results compatible with the definition of drug-induced liver injury 
(DILI) stated above must be repeated for confirmation as soon as possible, and if confirmed, will 
be scored as an unacceptable AE and reported to FDA as a serious unexpected AE. 
11.2. 
Procedures for Reporting Adverse Events and Serious Adverse 
Events  
Each patient must be carefully monitored for the development of any AEs. This information 
should be obtained in the form of non-leading questions (e.g., “How are you feeling?”) and from 
signs and symptoms detected during each examination, observations of study personnel, and 
spontaneous reports from patients. 
All AEs (serious and non-serious) spontaneously reported by the patient and/or in response to an 
open question from study personnel or revealed by observation, physical examination, or other 
diagnostic procedures will be recorded in the source documentation and eCRF. Any clinically 
relevant deterioration in laboratory assessments or other clinical findings is considered an AE 
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and must be recorded in the appropriate source documentation and eCRF. Treatment-emergent 
abnormal clinical laboratory results should generally be reported as AEs if there are 
accompanying symptoms; if additional diagnostic evaluations or medical (including drug 
therapy) or surgical interventions are undertaken; if a change in study drug dosing or study drug 
discontinuation is required; or, if the laboratory result is considered clinically significant by the 
Investigator. Although it is an objective of this study to determine if treatment with AG-348 
results in increased Hb levels in patients with PK deficiency, overshoot of the Hb level above the 
ULN by gender is not recommended and should be reported as an AE, graded per the 
CTCAEv4.03. When possible, signs and symptoms indicating a common underlying pathology 
should be noted as one comprehensive event. 
All SAEs that occur during the course of the study must be promptly reported by the Investigator 
to 
 Global Safety and Pharmacovigilance (see below). Deaths and AEs assessed 
as life-threatening are to be reported immediately and SAEs that meet other criteria are to be 
reported within 24 hours from the point in time when the Investigator becomes aware of the 
SAE. All SAEs must be reported whether or not they are considered causally related to AG-348. 
Serious adverse event forms will be completed and the information collected will include subject 
number, a narrative description of the event, and an assessment by the Investigator as to the 
severity of the event and relatedness to study drug. Follow-up information on the SAE may be 
requested by the Sponsor or Medical Monitor. 
Contact Information: 
 Safety Management 
SAE Hotline (United States): 
 
SAE Fax (United States): 
 
SAE Hotline (France): 
 
SAE Fax (France): 
  
SAE Hotline (Netherlands): 
 
SAE Fax (Netherlands): 
 
SAE Hotline (Italy): 
 
SAE Fax (Italy): 
 
SAE Hotline (United Kingdom): 
 
SAE Fax (United Kingdom): 
 
E-mail: 
 
If there are serious, unexpected adverse drug reactions associated with the use of AG-348, the 
Sponsor will notify the appropriate regulatory agency(ies) and all participating Investigators on 
an expedited basis. The local 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. 
All AEs, whether serious or not, will be described in the source documents in the database. All 
new events, as well as those that worsen in intensity or frequency relative to baseline, which 
occur after signing the informed consent through the final Follow-up Visit (Day 29 ± 3 days) 
must be recorded. Adverse events that are ongoing at the time of treatment discontinuation 
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should be followed up to 30 days after the last dose of study treatment. All SAEs will be 
followed up to 30 days after the last dose of study treatment, or until the SAE has resolved.  
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); 
• The date of onset of the event; 
• The date of resolution of the event; 
• Whether the event is serious or not; 
• Intensity of the event (see below for definitions); 
• Relationship of the event to study treatment (see below for definitions); 
• Action taken: none; change in the study drug administration (e.g., temporary 
interruption in dosing); drug treatment required; non-drug treatment required; 
hospitalization or prolongation of hospitalization required (complete SAE page); 
diagnostic procedure performed; patient discontinued from the study (complete End 
of Study visit); 
• Outcome: patient recovered without sequelae; patient recovered with sequelae; event 
ongoing; patient died (notify the Medical Monitor immediately, and complete the 
SAE form). 
Intensity of all AEs will be graded according to the NCI CTCAE Version 4.03 (Appendix 15.1). 
Relationship to study drug administration will be determined by the Investigator according to 
the following criteria: 
• Not Related:  Exposure to the study treatment did not occur, or the occurrence of the 
AE is not reasonably related in time, or the AE is considered unlikely to be related to 
the study treatment. 
• Possibly Related:  The study treatment and the AE were reasonably related in time, 
and the AE could be explained equally well by causes other than exposure to the 
study treatment. An attribution of possibly related means that there are facts in 
evidence to suggest a possible relationship. 
• Probably Related:  The study treatment and the AE were reasonably related in time, 
and the AE was more likely explained by exposure to the study treatment than by 
other causes, or the study treatment was the most likely cause of the AE. An 
attribution of probably related means that there are facts in evidence to suggest a 
probable relationship. 
For the purpose of safety analyses related to final database review, all AEs that are classified as 
possible or probable will be considered treatment-related AEs. 
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11.3. 
Pregnancy Reporting 
Pregnancy is neither an AE nor an SAE, unless a complication relating to the pregnancy occurs 
(e.g., spontaneous abortion, which may qualify as an SAE). However, any pregnancy in a 
participating female patient or a female partner of a participating male patient that occurs during 
this study or within 30 days following the last dose of AG-348 must be reported to the Sponsor 
or Medical Monitor within 30 days of being notified of the pregnancy. The Investigator must 
follow up and document the course and outcome of all pregnancies even if the patient was 
discontinued from the study or if the study has finished. The female patient or partner of a male 
patient should receive any necessary counseling regarding the risks of continuing the pregnancy 
and the possible effects on the fetus. Monitoring should continue until conclusion of the 
pregnancy. 
All outcomes of pregnancy must be reported by the Investigator to the Sponsor or Sponsor’s 
designee on a Pregnancy Outcome Report form within 30 days after he/she has gained 
knowledge of the delivery or elective abortion. 
Any SAE that occurs during pregnancy must be recorded on the SAE report form (e.g., maternal 
serious complications, spontaneous or therapeutic abortion, ectopic pregnancy, stillbirth, 
neonatal death, congenital anomaly, or birth defect) and reported within 24 hours in accordance 
with the procedure for reporting SAEs. 
All patients, male and female, must agree to use effective contraception during the entire study 
and for 30 days following the last dose of AG-348. Abstinence is an acceptable method only 
when this is in line with the normal life style of the patient, meaning that the patient plans to 
remain abstinent continuously throughout the duration of the study and for at least 30 days after 
the last dose of study drug. Periodic abstinence, e.g. calendar, sympathothermal and post-
ovulation methods, and withdrawal are not acceptable methods of contraception. 
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Protocol AG348-C-003, Amendment 2, Version 3.0 
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12. 
STATISTICAL METHODS 
The primary objective of this Phase 2 study is to evaluate the safety and tolerability of up to 
24 weeks of AG-348 in patients with PK deficiency. Therefore, analyses will be primarily 
descriptive in nature; no formal hypothesis testing will be conducted. All analyses will be 
conducted separately within each dose arm, or pooled where appropriate. 
12.1. 
Sample Size Estimation 
Due to the rare disease setting, the minimal sample size in each dose arm may be determined by 
feasibility. In order to evaluate the primary objective of safety and tolerability of AG-348 in 
Arm 1 and Arm 2, up to a maximum of 25 patients may be randomized onto each arm. The 
actual number of patients enrolled into Arms 1 and 2 will depend on the safety reviews and 
decisions made by the DRT. In addition, up to 25 additional patients may be enrolled to evaluate 
an additional dose arm (Arm 3; see Section 7.1). As for Arms 1 and 2, the actual enrollment in a 
potential Arm 3 will depend on the safety reviews and decisions made by the DRT. Therefore, up 
to approximately 75 total patients may be enrolled in this study across 2 to 3 dose arms. 
Table 4 provides the probability within a dose arm of detecting 1 or more AEs with varying 
sample size and the true underlying AE rates. 
Table 4: 
Sample Size Estimation 
Sample Size 
True Underlying AE Rate 
15% 
10% 
5% 
5 
56% 
41% 
23% 
10 
80% 
65% 
40% 
15 
91% 
79% 
54% 
25 
98% 
93% 
72% 
 
12.2. 
Populations for Analysis 
The following patient populations (i.e., analysis sets) will be evaluated and used for presentation 
of the data: 
• Safety Analysis Set: All patients who are enrolled and receive any dose of study 
treatment. The Safety Analysis Set will be the primary set for the analysis of safety 
data. Patients will be classified according to treatment received, where treatment 
received is defined as the assigned treatment if it is received at least once, or as the 
first treatment received if assigned treatment is never received. Unless otherwise 
stated, the Safety Analysis Set will be the default analysis set for all data analyses. 
• Pharmacokinetic (PK) Analysis Set: All patients in the Safety Analysis Set with 
sufficient plasma sample data to assess PK parameters. Results of the potential PD 
activity of AG-348 will also be based on the PK analysis set.  
71 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
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• Efficacy Analysis Set: All patients who are enrolled and achieve at least 50% 
compliance with their assigned dose intensity for at least 4 weeks of continuous 
dosing. Additional efficacy analyses may be performed on subsets of patients with 
other degrees of compliance with their assigned dose intensity and/or duration of 
dosing. The Efficacy Analysis Set will be the primary set for the analysis of 
preliminary clinical activity data. Patients will be classified according to assigned 
treatment. 
 
 If such analyses are performed, they will be 
described in a separate PK Statistical Analysis Plan (SAP) and may be reported separately in a 
stand-alone report. 
12.3. 
Procedures for Handling Missing, Unused, and Spurious Data 
No imputation will be performed for missing data elements. When summarizing AE data, partial 
dates will be imputed as described in the SAP. Additional rules addressing the handling of 
missing data will be detailed in the SAP. 
12.4. 
Interim Analysis 
No formal statistical interim analysis will be conducted. Safety data will be reviewed on an 
ongoing basis by the DRT, who will meet to review safety, PK, PD, and preliminary clinical 
activity data at regular intervals (approximately every 6 weeks) throughout the duration of the 
study. The DRT’s decisions to suspend, terminate, or open a potential third dosing arm, or re-
assign patients’ dosing in a terminated arm to match the dose and schedule of another arm of the 
study will be based on the totality of the data including, safety, PK, PD, and preliminary clinical 
activity (e.g., changes in Hb levels). 
Additional interim reviews of data may be conducted to support decision making concerning the 
current clinical study, the sponsor’s development programs in general, or in case of any safety 
concerns. 
12.5. 
Statistical Methodology 
12.5.1. 
General Methods 
This study will be primarily descriptive in nature; therefore, there will be no formal hypothesis 
testing. Summaries will be produced for disposition, baseline disease characteristics and 
demographic data including genotype, safety measurements, PK, PD parameters and indicators 
of clinical activity. Data from each AG-348 dose group will be analyzed separately, and pooled 
across all dose groups where appropriate. All data will also be listed by individual patient. 
Categorical variables will be summarized by frequency distributions (number and percentages) 
and continuous variables will be summarized by descriptive statistics (mean, standard deviation 
[SD], median, minimum, and maximum). 
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Protocol AG348-C-003, Amendment 2, Version 3.0 
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12.5.2. 
Disposition 
A summary of the disposition of patients will be presented, including the number enrolled, the 
number treated, and the reasons for study discontinuation. Entry criteria and protocol deviations 
will be listed. 
12.5.3. 
Exposure and Safety Analyses 
Patients will receive multiple PO doses of AG-348 over a 24-week treatment period. The actual 
dose and duration in days of AG-348, as well as the dose intensity (computed as the ratio of 
actual dose received and actual duration) and the relative dose intensity (computed as the ratio of 
dose intensity and planned dose received/planned duration) will be listed and summarized using 
descriptive statistics by dose arm. 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) 
and the incidence of TEAEs (new or worsening from baseline) will be summarized by primary 
system organ class (SOC), preferred term (PT), CTCAE Version 4.03 severity, outcome, action 
taken with study drug, and relationship to the study drug by dose group. Separate summaries will 
be produced for all TEAEs, treatment-related AEs (those considered by the Investigator as at 
least possibly drug related), SAEs, discontinuations due to AEs, and AEs ≥ Grade 3 severity. 
Individual patient listings will be provided for deaths, SAEs, AEs leading to interruption and/or 
reduction of study drug dose, and AEs leading to discontinuation of treatment. 
Analyses of AEs will be performed for those events that are considered treatment-emergent, 
where treatment-emergent is defined as any AE with onset beginning at the day of first 
administration of study drug, throughout the treatment period until 30 days after the last dose of 
study drug, or any event that was present at baseline but worsened in intensity or was 
subsequently considered drug-related by the Investigator through the end of the study. 
Descriptive statistics will be provided for clinical laboratory values (e.g., hematology, serum 
chemistry, coagulation studies, urinalysis) and VS data, presented as both actual values and 
changes from baseline relative to each on-study evaluation. Shift analyses will be conducted for 
selected laboratory parameters based on the baseline CTCAE grade to maximum CTCAE grade. 
Where applicable CTCAE terms do not exist, a grading system based on the upper and/or lower 
limits of normal will be used to classify the results. 
No formal hypothesis-testing analysis of AE incidence rates will be performed. 
Electrocardiogram analyses will include individual patient listings and summaries of abnormal 
and clinically significant ECG results. Actual values and changes from baseline in PR, QRS, and 
heart-rate corrected QT interval (QTc) intervals will be summarized by visit and dose arm. Full 
details of the QTc analysis including correction methods used will be described in the SAP. 
Data collected from the menstrual diaries such as the start and stop dates of the menses and the 
patient reported characteristics of the menses will be presented in a by-patient listing. Additional 
descriptions of the data may also be performed. 
Concomitant medications will be coded using the WHO Drug Dictionary. Concomitant 
medications prior to and after the start of the study drug will be listed by patient, and 
summarized by ATC term and dose arm. 
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Protocol AG348-C-003, Amendment 2, Version 3.0 
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12.5.4. 
Pharmacokinetic Analyses 
Descriptive statistics will be used to summarize PK parameters for the parent compound AG-348 
and the metabolite AGI-8702 for each dose group, and where appropriate, for the entire 
population. Pharmacokinetic parameters will be summarized using the following descriptive 
statistics: n, mean, SD, coefficient of variation %, median, minimum and maximum, geometric 
mean, and geometric coefficient of variation. PK analyses will be described in a separate PK 
SAP. 
12.5.5. 
Pharmacodynamic Analyses 
Descriptive statistics will be used to summarize PD parameters for 2,3-DPG and ATP for each 
dose group, and where appropriate for the entire population. Pharmacodynamic parameters will 
be summarized using the following descriptive statistics: n, mean, SD, coefficient of variation %, 
median, minimum, and maximum, geometric mean, and geometric coefficient of variation %. PD 
analyses will be described in a separate PD SAP. 
12.5.6. 
Aromatase Hormone Analysis  
The analyses of serum sex hormones will use appropriate graphic displays and statistical 
analyses to evaluate patient change from baseline for each parameter. These analyses will 
include summaries of actual values and change from baseline using appropriate descriptive 
statistics (mean, SD, median, min and max) as described above.  
 
These analyses will present information by each dose arm, and analyses of a pooled 
AG-348 cohort. Additional details regarding these analyses will be provided in the SAP. 
12.5.7. 
Clinical Activity 
Details on analyses to evaluate indicators of potential clinical activity of AG-348 in patients with 
PK deficiency will be described in the SAP. These will include changes in Hb, HCT, reticulocyte 
count, Hp, COHb, LDH, total and indirect bilirubin, EPO, ferritin, and transferrin saturation 
(serum iron/iron binding capacity). Characterization of Hb response which will include, but is 
not limited to percent of patients with increase in Hb, time to Hb response, and duration of Hb 
response will be explored. 
12.6. 
Procedures for Reporting Deviations to Original Statistical Analysis 
Plan 
All deviations from the original SAP will be provided in the final clinical study report. 
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Protocol AG348-C-003, Amendment 2, Version 3.0 
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13. 
ADMINSTRATIVE REQUIREMENTS 
13.1. 
Good Clinical Practice 
The study will be conducted in accordance with the International Conference on Harmonization 
(ICH) for Good Clinical Practice (GCP) and the appropriate regulatory requirement(s). The 
Investigator will be thoroughly familiar with the appropriate use of the study drug as described in 
the protocol and Investigator’s Brochure. Essential clinical documents will be maintained to 
demonstrate the validity of the study and the integrity of the data collected. Master files should 
be established at the beginning of the study, maintained for the duration of the study, and 
retained according to the appropriate regulations. 
13.2. 
Ethical Considerations 
The study will be conducted in accordance with ethical principles founded in the Declaration of 
Helsinki (Appendix 15.5). 
The Investigator must obtain IRB approval for the investigation and must submit written 
documentation of the approval to the Sponsor before he or she can enroll any patient into the 
study. The IRB will review all appropriate study documentation in order to safeguard the rights, 
safety, and well-being of the patients. The study will only be conducted at sites where IRB 
approval has been obtained. The protocol, Investigator’s Brochure, informed consent, 
advertisements (if applicable), written information given to the patients, signed Dose Escalation 
Interim Safety Reports, annual progress reports, and any revisions to these documents will be 
provided to the IRB. The IRB is to be notified of any amendment to the protocol in accordance 
with local requirements. Progress reports and notifications of serious unexpected adverse drug 
reactions are to be provided to the IRB according to local regulations and guidelines. 
13.3. 
Patient Information and Informed Consent 
The Investigator or trained designee will ensure that the patient is given full and adequate oral 
and written information about the nature, purpose, possible risk, and benefit of the study. Patients 
must also be notified that they are free to discontinue from the study at any time. The patient 
should be given the opportunity to ask questions and allowed time to consider the information 
provided. 
After the study has been fully explained, written informed consent will be obtained from the 
patient prior to study participation.  
The patient’s signed and dated informed consent must be obtained before conducting any study-
related procedures. The Investigator must maintain the original, signed consent form. A copy of 
the signed form must be given to the patient. 
The method of obtaining and documenting the informed consent and the contents of the consent 
will comply with ICH-GCP and all applicable regulatory requirement(s). 
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Protocol AG348-C-003, Amendment 2, Version 3.0 
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13.4. 
Patient Confidentiality 
In order to maintain patient privacy, all source documents, study drug accountability records, 
study reports and communications will identify the patient by the assigned patient number. The 
Investigator will grant monitor(s) and auditor(s) from the Sponsor or its designee and regulatory 
authority(ies) access to the patient’s original medical records for verification of data gathered on 
the source documents and to audit the data collection process. The patient’s confidentiality will 
be maintained and will not be made publicly available to the extent permitted by the applicable 
laws and regulations. 
13.5. 
Protocol Compliance 
The Investigator will conduct the study in compliance with the protocol. Modifications to the 
protocol should not be made without agreement of both the Investigator and the Sponsor. 
Changes to the protocol will require written IRB approval/favorable opinion prior to 
implementation, except when the modification is needed to eliminate an immediate hazard(s) to 
patients. The IRB may provide, if applicable, where regulatory authority(ies) permit, expedited 
review and approval/favorable opinion for minor change(s) in ongoing studies that have the 
approval/favorable opinion of the IRB. The Sponsor or designee will submit all protocol 
modifications to the regulatory authority(ies) in accordance with the governing regulations.  
When immediate deviation from the protocol is required to eliminate an immediate hazard(s) to 
patients, the Investigator will contact the Sponsor’s Medical Monitor (or Responsible Medical 
Officer), if circumstances permit, to discuss the planned course of action. Any departures from 
the protocol must be fully documented in the source documents/database. 
13.6. 
Data Management 
A paper or eCRF will be completed for each patient or an electronic data capture system will be 
used. The electronic data capture system (EDC) (Medidata Rave®) is a software tool designed to 
ensure quality assurance and facilitate data capture during clinical trials. Through a system 
regulated workflow that includes barcode scanning and interfaces to medical equipment to avoid 
manual data entry, study operations performance is controlled and captured in real time. The 
system is fully Code of Federal Regulations (CFR) 21 Part 11 compliant. Source documentation 
supporting the data should indicate participation in the study and should document the dates and 
details of study procedures, AEs, and patient status. The Investigator, or trained designee should 
complete and the Investigator should verify the source documents as the information is collected. 
Any outstanding entries must be completed immediately after the final examination. An 
explanation should be given for all missing data. The Investigator will ensure the accuracy, 
completeness, and timeliness of the data reported to the Sponsor. Data collection processes and 
procedures will be reviewed and validated to ensure completeness, accuracy, reliability, and 
consistency. A complete audit trail will be maintained of all data changes. The Investigator or 
designee will cooperate with the Sponsor’s representative(s) for the periodic review of study 
documents to ensure the accuracy and completeness of the data capture system at each scheduled 
monitoring visit. Electronic consistency checks and manual review will be used to identify any 
errors or inconsistencies in the data. This information will be provided to the respective study 
sites by means of electronic or manual queries. The Investigator or designee will prepare and 
maintain adequate and accurate study documents (medical records, ECGs, AE, and concomitant 
76 
 


Protocol AG348-C-003, Amendment 2, Version 3.0 
05 August 2015 
 
medication reporting, raw data collection forms, etc.) designed to record all observations and 
other pertinent data for each patient receiving study treatment. The Investigator will allow 
Sponsor representatives, contract designees, authorized regulatory authority inspectors, and the 
IRB to have direct access to all documents pertaining to the study. 
13.7. 
Source Document/Case Report Form Completion 
Source documents will be completed for each study patient. It is the Investigator’s responsibility 
to ensure the accuracy, completeness, and timeliness of the data reported in the patient’s source 
document. The source document should indicate the patient’s participation in the study and 
should document the dates and details of study procedures, AEs and patient status. 
The Investigator, or designated representative, should complete the source document as soon as 
possible after information is collected, preferably on the same day that a study patient is seen for 
an examination, treatment, or any other study procedure. Any outstanding entries must be 
completed immediately after the final examination. An explanation should be given for all 
missing data. 
The Investigator will sign and date each required assessment for all study patients. The 
Investigator will retain all completed source documents. 
13.8. 
Direct Access to Source Data 
The study will be monitored by the Sponsor or its designee. Monitoring will be done by personal 
visits from a representative of the Sponsor (site monitor) and will include on-site review of the 
source documents for completeness and clarity, cross-checking with source documents, and 
clarification of administrative matters will be performed. The review of medical records will be 
performed in a manner to ensure that patient confidentiality is maintained. 
The site monitor will ensure that the investigation is conducted according to protocol design and 
regulatory requirements by frequent communications (letter, telephone, e-mail, and fax). 
All unused study drug and other study materials are to be returned to the Sponsor or designee 
after the study has been completed and the database has been locked. 
Regulatory authorities, the IRB, and/or the Sponsor’s clinical quality assurance group or 
designee may request access to all source documents, database, and any other applicable study 
documentation for an on-site audit or inspection. Direct access to these documents must be 
guaranteed by the Investigator, who must provide support at all times for these activities. 
13.9. 
Record Retention 
The Investigator will maintain all study records according to ICH-GCP and applicable regulatory 
requirement(s). Records will be retained for at least 2 years after the last marketing application 
approval or 2 years after formal discontinuation of the clinical development of the investigational 
product or according to applicable regulatory requirement(s). If the Investigator withdraws from 
the responsibility of keeping the study records, custody must be transferred to a person willing to 
accept the responsibility. The Sponsor must be notified in writing if a custodial change occurs. 
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Protocol AG348-C-003, Amendment 2, Version 3.0 
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13.10. 
Liability and Insurance 
The Sponsor has subscribed to an insurance policy covering, in its terms and provisions, its legal 
liability for injuries caused to participating persons and arising out of this research performed 
strictly in accordance with the scientific protocol as well as with applicable law and professional 
standards.  
13.11. 
Publication of Study Findings and Use of Information 
All information regarding AG-348 supplied by the Sponsor or designee to the Investigator is 
privileged and confidential information. The Investigator agrees to use this information to 
accomplish the study and will not use it for other purposes without consent from the Sponsor. It 
is understood that there is an obligation to provide the Sponsor with complete data obtained 
during the study. The information obtained from the clinical study will be used towards the 
development of AG-348 and may be disclosed to regulatory authority(ies), other Investigators, 
corporate partners, or consultants as required. 
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Protocol AG348-C-003, Amendment 2, Version 3.0 
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14. 
LIST OF REFERENCES 
 
Beutler E, and Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene 
frequency in the general white population. Blood. 2000;95(11):3585-8. 
Christensen RD, Yaish HM, Johnson CB, Bianchi P, and Zanella A. Six children with pyruvate 
kinase deficiency from one small town: molecular characterization of the PK-LR gene. J Pediatr. 
2011;159(4):695-7. 
FDA. Guidance for Industry: Drug-Induced Liver Injury: Premarketing Clinical Evaluation. 
2009. 
Ghidini A, Sirtori M, Romero R, Yarkoni S, Solomon L, and Hobbins JC. Hepatosplenomegaly 
as the only prenatal finding in a fetus with pyruvate kinase deficiency anemia. Am J Perinatol. 
1991;8(1):44-6. 
Hennekam RC, Beemer FA, Cats BP, Jansen G, and Staal GE. Hydrops fetalis associated with 
red cell pyruvate kinase deficiency. Genet Couns. 1990;1(1):75-9. 
Mentzer WC, Jr., Baehner RL, Schmidt-Schonbein H, Robinson SH, and Nathan DG. Selective 
reticulocyte destruction in erythrocyte pyruvate kinase deficiency. J Clin Invest. 1971;50(3):688-
99. 
Rider NL, Strauss KA, Brown K, Finkenstedt A, Puffenberger EG, Hendrickson CL, et al. 
Erythrocyte pyruvate kinase deficiency in an old-order Amish cohort: longitudinal risk and 
disease management. Am J Hematol. 2011;86(10):827-34. 
Schwartz JC. The histamine H3 receptor: from discovery to clinical trials with pitolisant. Br J 
Pharmacol. 2011;163(4):713-21. 
Strauss KA, Robinson DL, Vreman HJ, Puffenberger EG, Hart G, and Morton DH. Management 
of hyperbilirubinemia and prevention of kernicterus in 20 patients with Crigler-Najjar disease. 
Eur J Pediatr. 2006;165(5):306-19. 
Tanphaichitr VS, Suvatte V, Issaragrisil S, Mahasandana C, Veerakul G, Chongkolwatana V, et 
al. Successful bone marrow transplantation in a child with red blood cell pyruvate kinase 
deficiency. Bone Marrow Transplant. 2000;26(6):689-90. 
van Wijk R, and van Solinge WW. The energy-less red blood cell is lost: erythrocyte enzyme 
abnormalities of glycolysis. Blood. 2005;106(13):4034-42. 
Zanella A, Fermo E, Bianchi P, and Valentini G. Red cell pyruvate kinase deficiency: molecular 
and clinical aspects. Br J Haematol. 2005;130(1):11-25. 
 
 
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Protocol AG348-C-003, Amendment 2, Version 3.0 
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15. 
APPENDICES 
 
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Protocol AG348-C-003, Amendment 2, Protocol Version 3.0 
05 August 2015 
 
15.1. 
Schedules of Assessments 
Table 5: 
Schedule of Assessments:  
Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
-26 to -11 
1 
82 
15 
222 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Written Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
PK enzyme assay 
(confirmation of PK 
deficiency) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
PKR Genotype 
(for randomization) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
UGT1A1 Genotype 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medical/Surgical 
History (General and 
PK deficiency-specific)
3 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medication History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Transfusion History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Confirmation of 
Vaccinations 
(Splenectomized 
Patients) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Examination4/ 
Height4 and Weight 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
X 
X 
Performance Status 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
X 
X 
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Protocol AG348-C-003, Amendment 2, Protocol Version 3.0 
05 August 2015 
 
Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
-26 to -11 
1 
82 
15 
222 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Vital signs  
(BP, HR, RR, T)5  
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG6 
X 
 
X 
X 
 
X 
 
 
 
 
 
X 
X 
DXA Scan7 
X 
 
 
 
 
 
 
 
 
 
 
X8 
 
Laboratory Evaluations
9 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBsAg, HCV Ab, 
HIV1 and 2 Ab 
X 
 
 
 
 
 
 
 
 
 
 
 
 
RBC antibody 
Screen  
X 
 
 
 
 
 
 
 
 
 
 
 
 
Hematology (CBC)10 
X 
X11 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin12 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
EPO levels13 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
G6PD screen 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Serum Chemistry14 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Iron Panel15 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Carboxyhemoglobin 
(COHb) 
 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
 
Coagulation Studies
16 
X 
 
X 
 
X 
 
 
 
X 
 
 
X 
X 
Urinalysis17 
X 
 
X 
 
X 
 
 
 
X 
 
 
X 
X 
Serum or Urine 
Pregnancy18 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
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Protocol AG348-C-003, Amendment 2, Protocol Version 3.0 
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Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
-26 to -11 
1 
82 
15 
222 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Lipids19 
 
 
X 
 
 
 
X 
 
X 
 
 
X 
X 
Hormonal Testing20 
X 
X21 
X 
 
 
 
 
 
X 
 
 
X 
X 
Serum 
osteocalcin-N-mid 
and CTX22 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
25-hydroxy Vitamin 
D2 and D3 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Randomization23 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Study Drug 
Administration 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Dispense Study Drug24 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
PK blood sampling25 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
PD Assessments25 
 
 
 
 
 
 
 
 
 
 
 
 
 
2,3-DPG/ATP 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
Dispense/Collect 
Menstrual Cycle Diary
27 
 
 
X 
 
 
 
X 
 
X 
X 
X 
X 
X 
Adverse Events28 
 
 
Continuous 
X 
Transfusion Record 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
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Protocol AG348-C-003, Amendment 2, Protocol Version 3.0 
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Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
-26 to -11 
1 
82 
15 
222 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Concomitant 
Medications/Procedures 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Rollover to safety 
extension  
 
 
 
 
 
 
 
 
 
 
 
X 
 
Abbreviations:  Ab = antibody; ATP = adenosine triphosphate; BP = blood pressure; CBC= complete blood count; COHb = carboxyhemoglobin; 
CTX = C-terminal telopeptide; D = day; DPG = diphosphoglycerate; DXA = Dual-energy x-ray absorptiometry; ECG = electrocardiogram; 
EPO = erythropoietin; FSH = follicle stimulating hormone; G6PD = glucose-6-phosphate-dehydrogenase; HBsAg = hepatitis B surface antigen; 
HCV Ab = hepatitis C virus antibody; HDL-C = high-density lipoprotein-C; HIV = human immunodeficiency virus; HR = heart rate; PD = pharmacodynamic; 
PK = pharmacokinetic; PK deficiency = pyruvate kinase deficiency; PKR = pyruvate kinase isoform R; RR = resting rate; W = week. 
Whenever more than one assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive 
assessment (e.g., VS, ECG, blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time. The order of 
procedures may be revised with prior discussion between Sponsor and site. 
 
1 To be performed at least 2 days after the first Screening Visit. 
2 In-clinic visits on Day 8 and Day 22 may be performed by the patient’s primary care physician if necessary and must be approved by the Sponsor on a case by 
case basis; in these instances PK/PD sampling would not be required and dispensing of study medication would not be performed. For the Day 8 visit 
performed by the patient’s primary care physician, the primary care medical office will collect a blood sample for hematology using the blood sample 
collection and shipping supplies from the kit prepared by the designated central laboratory.  The kit will be sent to the primary care physician’s office. No 
other testing or procedures will be asked of the primary care physician on Day 8 [VS and serum chemistry will not be required]. The 12-lead ECG scheduled 
for Day 8 will instead be performed at the main study center on Day 15. For the Day 22 visit performed by the patient’s primary care physician, the primary 
care medical office will collect a blood sample for hematology using the blood sample collection and shipping supplies from the kit prepared by the 
designated central laboratory. The kit will be sent to the primary care physician’s office. No other testing or procedures will be asked of the primary care 
physician on Day 22. [VS, serum chemistry, coagulation, haptoglobin, EPO level, carboxyhemoglobin, and PK/PD samples will not be required.] The 12-lead 
electrocardiogram scheduled for Day 22 will instead be performed at the main study center on Day 43 (Week 6). For patients having their Day 8 and/or 
Day 22 visits performed by the primary care physician, the Principal Investigator will him or herself, or have a qualified research nurse or other designated 
site staff member, make telephone contact with the patient to inquire about any adverse events. These must be recorded as if the patient appeared in the main 
study center. The Principal Investigator will exercise prudent clinical judgment in determining any clinical course of action to take based on any adverse 
events discovered. These telephone contacts on Day 8 and Day 22 must be explained to the patient in advance and scheduled in advance to maximize the 
likelihood of successfully making contact. 
3 Medical history, including all relevant prior medical history, current medical conditions, and hematology profile (CBCs) over prior 6 months, will be obtained 
at the Screening assessment. 
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4 A complete physical examination (including neurological examination; genital and rectal examinations will be performed at the discretion of the Investigator) 
will be obtained at Screening, Baseline, Week 12, and the Follow up Visit (Week 28), or Week 24 for patients rolling over to the safety extension. The 
neurological examination must include an assessment of general orientation and mental status including level of alertness (assess as normal or abnormal and 
specify any abnormality). Limited focused physical examinations will be performed at all other specified visits. Height to be collected at Screening only. 
5 Vital signs will include systolic and diastolic blood pressure, heart rate, respiratory rate, and temperature.  
6 12-lead ECGs are to be conducted after 5 minutes of recumbency. Screening ECG will be performed at least 7 days prior to Day 1 dosing. 
7 If a DXA scan of the hip and spine with T and Z scores has been performed within 3 months preceding the first day of dosing, this may be used to meet this 
requirement.  
8 Week 24 DXA scan may be performed anytime between Weeks 24 and 28 and must be performed at the same imaging center on the same DXA machine as 
the original Screening DXA scan. 
9 Laboratory evaluations (hematology, serum chemistry, coagulation studies, and urinalysis) are to be collected in the morning. These should be collected 
following an overnight fast on Baseline Day 1 Week 6 (Day 43), Week 12 (Day 85), Week 24 (Day 169), and Follow-up Week 28 (Day 197), when the lipid 
samples are also included. 
10 Three Screening/Baseline samples will be collected for complete blood count (CBC). Samples will be collected in the AM on 3 different days; the samples 
collected on Baseline/Day 1 may comprise one of these; samples may be taken at the same time as Screening/Baseline hormone assessments (any 2 time 
points during Screening at least 2 days apart). CBC will include HCT, Hb, red blood cell (RBC) count, absolute reticulocyte count, percent reticulocyte count, 
mean corpuscular volume (MCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW), white 
blood cell (WBC) count with automated (or manual, as indicated) differential, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and 
platelet count. In the event that the designated central laboratory for the study is unable to provide a valid result for any specific component of the defined 
CBC for a specific patient, the site may be asked to have the test performed at their local laboratory. The result of the local CBC will be entered into the study 
database along with the local normal reference range. 
11 The second Screening hematology (CBC) should be drawn in the morning (does not have to be fasting), and may be drawn at the same time the patient returns 
for the second estradiol and free and total testosterone sample. 
12 Haptoglobin will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28. 
13 Erythropoietin (EPO) levels will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 
28. 
14 Alkaline phosphatase, sodium, potassium, chloride, calcium, magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, albumin, total protein, glucose, 
blood urea nitrogen (BUN), creatinine, uric acid, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total 
bilirubin and indirect bilirubin, (and estimated creatinine clearance or glomerular filtration rate for screening only, as appropriate). 
15 Iron (Fe), total iron-binding capacity (TIBC), transferrin saturation, and ferritin will be performed prior to dosing on Day 1, at the end of Week 12 and at the 
end of Week 24. 
16  Fibrinogen, activated partial thromboplastin time (aPTT), and international normalized ratio (INR) will be performed at Screening, prior to dosing on Day 1, 
at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28. 
17 Color, appearance, pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite, urobilinogen, and occult blood. Microscopic inspection of sediment 
should only be performed for cause or to investigate an abnormal dipstick finding per the Investigator’s discretion. Urinalysis will be performed at Screening, 
prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28. 
18 Must be repeated at any point throughout the study period if pregnancy is clinically suspected. 
19 Total cholesterol, HDL-C, and triglyceride samples will be collected in the morning following an overnight fast. 
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Protocol AG348-C-003, Amendment 2, Protocol Version 3.0 
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20 Three Screening/Baseline samples will be collected for estradiol and testosterone (total and free). Samples will be collected in the AM on 3 different days; the 
samples collected on Baseline/Day 1 may comprise one of these; the Screening samples may be collected at any 2 time points during Screening at least 2 days 
apart. Serum estrone, estradiol, and free and total testosterone will then follow the schedule indicated on Day 1 and Weeks 12, 24, and 28. FSH will only be 
performed at Screening for female patients only for confirmation of post-menopausal status. 
21 The second Screening hormone testing will consist of estradiol and testosterone (free and total) only. Samples should be drawn in the AM (does not need to 
be fasting), and may be drawn at the same time the patient returns for the second CBC sample. 
22 Serum osteocalcin-N-mid and CTX will be drawn in the AM each time, approximately between 8-10 AM, and after an overnight fast of 10-12 hours. 
23 Randomization will be performed following PKR genotyping and prior to and as close as feasible to dosing on Day 1. 
24 Study drug will be dispensed on a 28-day schedule, or on an alternate schedule (< 28 days) as needed to accommodate patient visit schedule and dose 
modifications. 
25 For the first 10 patients treated, extensive PK/PD sampling will be conducted on Days 1 and 15 (see Appendix 15.1, Table 6 for details), followed by limited 
PK/PD sampling from Week 3 to Week 24 (see Appendix 15.1, Table 7 for details). Limited PK/PD sampling will be conducted on the remainder of patients 
treated (see Appendix 15.1 , Table 7). See Section 10.6.1, Section 10.7, and Section 10.9 for details on blood sampling for PK and PD assessments, 
respectively, and guidelines on sample processing and storage. 
 
 
27 Menstruating female patients will record their menstrual cycles (start, stop, characteristics) monthly. Paper-based menstrual cycle diaries will be dispensed at 
study visits approximately every month. The previous month’s diary will be collected at these visits as well. 
28 All randomized patients will be evaluated for AEs from the time they sign informed consent until they complete all scheduled study follow-up visits or 
withdraw consent, whichever occurs first.  
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Table 6: 
Schedule of Assessments: Extensive PK/PD Sampling 
Sample Timing/Interval 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Visit 
Baseline / D1 
W2 / D15 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
Study Day 
1/15 
22 
43 
64 
85 
113 
141 
169 
Visit Window 
± 2 D (D15) 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Timing 
Pre-
dose1 
30 
min2 
1 hr2 
2 hr2 
4 hr3 
8 hr3 
12 
hr3 
Pre-
dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
PK blood sample 
X 
X 
X 
X 
X 
X 
X4 
X 
X 
X 
X 
X 
X 
X 
2,3 DPG/ATP 
X 
X 
X 
X 
X 
X 
X4 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; D = day; DPG = diphosphoglycerate; PD = pharmacodynamic; PK = pharmacokinetic; PKR = pyruvate kinase 
isoform R; W = week. 
1 The acceptable time window will be within 60 minutes prior to study treatment dose administration for the pre-dose PK/PD sample. 
2 The acceptable time window will be within ± 5 minutes of the scheduled collection time for the 30 minute, 1 and 2 hour PK/PD samples. 
3 The acceptable time window will be within ± 30 minutes of the scheduled collection time for the 4, 8, and 12 hour PK/PD samples. 
4 To be collected on Day 1 only. 
 
  
6 If the 12 hour time point cannot be collected at site on Day 1, an 8 hour time point may be collected instead. 
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Table 7: 
Schedule of Assessments: Limited PK/PD Sampling 
Sample Timing/Interval 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Visit 
Baseline / 
D1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
Study Day 
1 
15 
22 
43 
64 
85 
113 
141 
169 
Visit Window 
- 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Timing 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
PK blood sample 
X 
X 
X 
X 
X 
X 
X 
X 
X 
2,3 DPG/ATP 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; D = day; DPG = diphosphoglycerate; PD = pharmacodynamic; PK = pharmacokinetic; PKR = pyruvate kinase 
isoform R; W = week. 
 
1 The predose blood sample for plasma PK/PD analysis should be collected within 60 minutes prior to study treatment dose administration. 
 
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15.2. 
Eastern Cooperative Oncology Group Performance Status Scoring 
 
Grade 
Symptomatology 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a 
light or sedentary nature, e.g., light house work, office work 
2 
Ambulatory and capable of all self-care but unable to carry out any work activities. Up and 
about more than 50% of waking hours 
3 
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours 
4 
Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair 
5 
Dead 
Source: Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, et al. Toxicity and Response 
Criteria of The Eastern Cooperative Oncology Group. Am J Clin Oncol 1982;5:649-655. 
 
15.3. 
National Cancer Institute Common Terminology Criteria for 
Adverse Events 
 
The NCI CTCAE, Version 4.03, can be accessed using the following link:   
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_8.5x11.pdf 
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Protocol AG348-C-003, Amendment 2, Protocol Version 3.0 
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15.4. 
Potential Drug Interactions 
Strong inhibitors of CYP3A4 (listed in Table 8) are not permitted for use with AG-348. Based on 
modeling of AG-348 metabolism and tolerability data from the Phase 1 MAD study, it is thought 
that moderate inhibitors of CYP3A4 do not pose a risk to patients dosed with AG-348. 
In-vivo data from the Phase 1 MAD show AG-348 induces CYP3A4 and induces its own 
metabolism, presumably via CYP3A4. The use of strong CYP3A4 inducers in combination with 
AG-348 is expected to reduce AG-348’s efficacy. Therefore, administration of strong CYP3A4 
inducers (listed in Table 9) with AG-348 is not permitted. Strong inhibitors of drug transport 
(listed in Table 10) are not permitted for use with AG-348. 
Digoxin is not permitted for use with AG-348. 
Induction of CYP3A4 by AG-348 is expected to reduce the efficacy of certain sensitive co-
medications. Co-administration of AG-348 with the drugs in Table 11 should be done with 
caution, as their efficacy may be reduced.  
Of note, in accordance with Inclusion Criteria 14, women in the trial utilizing oral contraception 
must utilize barrier methods while taking AG-348. 
The expected patient co-medications deferoxamine, deferasirox, deferiprone, and oral penicillin 
are not expected to interact with AG-348. 
Table 8: 
Strong and Moderate CYP3A4 Inhibitors 
Strong CYP3A4 Inhibitors: Contraindicated 
Moderate CYP3A4 Inhibitors: No Action 
Indinavir 
Aprepitant 
Nelfinavir 
Erythromycin 
Ritonavir 
Fluconazole 
Clarithromycin 
Verapamil1 
Itraconazole 
Diltiazem1 
Ketoconazole 
 
Nefazodone 
 
Saquinavir 
 
Suboxone 
 
Telithromycin 
 
Grapefruit juice2 
 
Strong Inhibitor; > 5 fold increase in AUC 
Moderate Inhibitor; > 2 fold, < 5 fold increase in AUC 
1 Verapamil and diltiazem are contraindicated because they are strong P-gp inhibitors (see Table 10) 
2 Although classified as a moderate CYP3A4 inhibitor, grapefruit and grapefruit juice are prohibited 
 
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Table 9: 
Strong CYP3A4 Inducers 
Strong CYP3A4 Inducers:  Contraindicated 
Efavirenz 
Phenytoin 
Nevirapine 
Pioglitazone 
Carbamazepine 
Rifabutin 
Glucocorticoids1 
Rifampin 
Modafinil 
St. John's Wort 
Oxcarbazepine 
Troglitazone 
Phenobarbital 
 
1 Short-term (≤ 14 days at a time, and ≤ 28 days total during the 24 week treatment period) use of topical, inhaled, 
intra-nasal, and systemic glucocorticoids is permitted for acute medical indications. Every effort should be made to 
minimize total duration of glucocorticoid therapy and utilize alternative treatments. Patients must be off 
glucocorticoids for at least 28 days prior to Day 1 of AG-348 dosing as per exclusion criterion #11 (Section 8.3). 
For patients who require chronic inhaled glucocorticoid therapy, Investigators should confer with the Medical 
Monitor for additional guidance. 
 
Table 10: 
Strong P-glycoprotein Inhibitors 
Strong P-gp Inhibitors:  Contraindicated 
Amiodarone 
Felodipine 
Azithromycin 
Itraconazole 
Captopril 
Ketoconazole 
Carvedilol 
Lopinavir 
Clarithromycin 
Ritonavir 
Conivaptan 
Quercetin 
Cyclosporine 
Quinidine 
Diltiazem 
Ranolazine 
Dronedarone 
Ticagrelor 
Erythromycin 
Verapamil 
 
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Protocol AG348-C-003, Amendment 2, Protocol Version 3.0 
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Table 11:  
Sensitive CYP3A4 Substrates 
Sensitive CYP3A4 Substrates:  Substitute or Use with Caution 
Macrolide 
antibiotics: 
Antihistamines: 
Miscellaneous: 
 
 
Erythromycin  
Chlorpheniramine 
Alfentanil 
Finasteride 
Salmeterol 
 
Aprepitant 
Gleevec 
Sildenafil 
Benzodiazepines: 
Calcium Channel 
Blockers: 
Aripiprazole 
Haloperidol 
Sirolimus 
Alprazolam 
Amlodipine 
Boceprevir 
Irinotecan 
Sorafenib 
Diazepam→3OH 
Lercanidipine 
Buspirone 
LAAM 
Sunitinib 
Midazolam 
Nifedipine 
Cafergot 
Lidocaine 
Tamoxifen 
Triazolam 
Nisoldipine 
Caffeine→TMU 
Methadone 
Taxol 
Nitrendipine 
Cilostazol 
Nateglinide 
Telaprevir 
Immune Modulators: 
 
Cocaine 
Nevirapine 
Terfenadine 
Tacrolimus (FK506) 
HMG CoA 
Reductase 
Inhibitors: 
Codeine-N-
demethylation 
Ondansetron 
Torisel 
 
Atorvastatin 
Dapsone 
Pimozide 
Trazodone 
Steroid 6beta-OH: 
Cerivastatin  
Dextromethorphan 
Propranolol 
Vemurafenib 
Estradiol 
Lovastatin 
Docetaxel 
Quetiapine 
Vincristine 
Hydrocortisone (and 
other glucocorticoids) 
Simvastatin 
Domperidone 
Quinine 
Zaleplon 
Progesterone 
 
Eplerenone 
Risperidone 
Ziprasidone 
Testosterone 
 
Fentanyl 
Romidepsin 
Zolpidem 
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15.5. 
Declaration of Helsinki 
WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI: 
Recommendations Guiding Medical Doctors in Biomedical Research Involving Human Subjects 
Adopted by the 18th World Medical Association (WMA) General Assembly, Helsinki, Finland, 
June 1964 and amended by the 29th WMA General Assembly, Tokyo, Japan, October 1975, 35th 
WMA General Assembly, Venice, Italy, October 1983, and the 41st WMA General Assembly, 
Hong Kong, September 1989, the 48th WMA General Assembly, Somerset West, Republic of 
South Africa, October 1996; 52nd WMA General Assembly, Edinburgh, Scotland, October 2000; 
53rd WMA General Assembly, Washington 2002 (Note of Clarification on paragraph 29 added); 
55th WMA General Assembly, Tokyo 2004 (Note of Clarification on Paragraph 30 added); and 
59th WMA General Assembly, Seoul, October 2008. 
A. INTRODUCTION  
1. The World Medical Association (WMA) has developed the Declaration of Helsinki as a 
statement of ethical principles for medical research involving human subjects, including 
research on identifiable human material and data.  
The Declaration is intended to be read as a whole and each of its constituent paragraphs 
should not be applied without consideration of all other relevant paragraphs.  
2. Although the Declaration is addressed primarily to physicians, the WMA encourages 
other participants in medical research involving human subjects to adopt these principles.  
3. It is the duty of the physician to promote and safeguard the health of subjects, including 
those who are involved in medical research. The physician's knowledge and conscience 
are dedicated to the fulfilment of this duty.  
4. The Declaration of Geneva of the WMA binds the physician with the words, "The health 
of my subject will be my first consideration," and the International Code of Medical 
Ethics declares that, "A physician shall act in the subject's best interest when providing 
medical care."  
5. Medical progress is based on research that ultimately must include studies involving 
human subjects. Populations that are underrepresented in medical research should be 
provided appropriate access to participation in research.  
6. In medical research involving human subjects, the well-being of the individual research 
subject must take precedence over all other interests.  
7. The primary purpose of medical research involving human subjects is to understand the 
causes, development and effects of diseases and improve preventive, diagnostic and 
therapeutic interventions (methods, procedures and treatments). Even the best current 
interventions must be evaluated continually through research for their safety, 
effectiveness, efficiency, accessibility and quality.  
8. In medical practice and in medical research, most interventions involve risks and 
burdens.  
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Protocol AG348-C-003, Amendment 2, Protocol Version 3.0 
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9. Medical research is subject to ethical standards that promote respect for all human 
subjects and protect their health and rights. Some research populations are particularly 
vulnerable and need special protection. These include those who cannot give or refuse 
consent for themselves and those who may be vulnerable to coercion or undue influence.  
10. Physicians should consider the ethical, legal and regulatory norms and standards for 
research involving human subjects in their own countries as well as applicable 
international norms and standards. No national or international ethical, legal or regulatory 
requirement should reduce or eliminate any of the protections for research subjects set 
forth in this Declaration.  
B. BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH  
11. It is the duty of physicians who participate in medical research to protect the life, health, 
dignity, integrity, right to self-determination, privacy, and confidentiality of personal 
information of research subjects.  
12. Medical research involving human subjects must conform to generally accepted scientific 
principles, be based on a thorough knowledge of the scientific literature, other relevant 
sources of information, and adequate laboratory and, as appropriate, animal 
experimentation. The welfare of animals used for research must be respected.  
13. Appropriate caution must be exercised in the conduct of medical research that may harm 
the environment.  
14. The design and performance of each research study involving human subjects must be 
clearly described in a research protocol. The protocol should contain a statement of the 
ethical considerations involved and should indicate how the principles in this Declaration 
have been addressed. The protocol should include information regarding funding, 
Sponsors, institutional affiliations, other potential conflicts of interest, incentives for 
subjects and provisions for treating and/or compensating subjects who are harmed as a 
consequence of participation in the research study. The protocol should describe 
arrangements for post-study access by study subjects to interventions identified as 
beneficial in the study or access to other appropriate care or benefits.  
15. The research protocol must be submitted for consideration, comment, guidance and 
approval to a research ethics committee before the study begins. This committee must be 
independent of the researcher, the Sponsor and any other undue influence. It must take 
into consideration the laws and regulations of the country or countries in which the 
research is to be performed as well as applicable international norms and standards but 
these must not be allowed to reduce or eliminate any of the protections for research 
subjects set forth in this Declaration. The committee must have the right to monitor 
ongoing studies. The researcher must provide monitoring information to the committee, 
especially information about any SAEs. No change to the protocol may be made without 
consideration and approval by the committee.  
16. Medical research involving human subjects must be conducted only by individuals with 
the appropriate scientific training and qualifications. Research on subjects or healthy 
volunteers requires the supervision of a competent and appropriately qualified physician 
or other health care professional. The responsibility for the protection of research subjects 
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Protocol AG348-C-003, Amendment 2, Protocol Version 3.0 
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must always rest with the physician or other health care professional and never the 
research subjects, even though they have given consent.  
17. Medical research involving a disadvantaged or vulnerable population or community is 
only justified if the research is responsive to the health needs and priorities of this 
population or community and if there is a reasonable likelihood that this population or 
community stands to benefit from the results of the research.  
18. Every medical research study involving human subjects must be preceded by careful 
assessment of predictable risks and burdens to the individuals and communities involved 
in the research in comparison with foreseeable benefits to them and to other individuals 
or communities affected by the condition under investigation.  
19. Every clinical trial must be registered in a publicly accessible database before recruitment 
of the first subject.  
20. Physicians may not participate in a research study involving human subjects unless they 
are confident that the risks involved have been adequately assessed and can be 
satisfactorily managed. Physicians must immediately stop a study when the risks are 
found to outweigh the potential benefits or when there is conclusive proof of positive and 
beneficial results.  
21. Medical research involving human subjects may only be conducted if the importance of 
the objective outweighs the inherent risks and burdens to the research subjects.  
22. Participation by competent individuals as subjects in medical research must be voluntary. 
Although it may be appropriate to consult family members or community leaders, no 
competent individual may be enrolled in a research study unless he or she freely agrees.  
23. Every precaution must be taken to protect the privacy of research subjects and the 
confidentiality of their personal information and to minimize the impact of the study on 
their physical, mental and social integrity.  
24. In medical research involving competent human subjects, each potential subject must be 
adequately informed of the aims, methods, sources of funding, any possible conflicts of 
interest, institutional affiliations of the researcher, the anticipated benefits and potential 
risks of the study and the discomfort it may entail, and any other relevant aspects of the 
study. The potential subject must be informed of the right to refuse to participate in the 
study or to withdraw consent to participate at any time without reprisal. Special attention 
should be given to the specific information needs of individual potential subjects as well 
as to the methods used to deliver the information. After ensuring that the potential subject 
has understood the information, the physician or another appropriately qualified 
individual must then seek the potential subject's freely-given informed consent, 
preferably in writing. If the consent cannot be expressed in writing, the non-written 
consent must be formally documented and witnessed.  
25. For medical research using identifiable human material or data, physicians must normally 
seek consent for the collection, analysis, storage and/or reuse. There may be situations 
where consent would be impossible or impractical to obtain for such research or would 
pose a threat to the validity of the research. In such situations the research may be done 
only after consideration and approval of a research ethics committee.  
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Protocol AG348-C-003, Amendment 2, Protocol Version 3.0 
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26. When seeking informed consent for participation in a research study the physician should 
be particularly cautious if the potential subject is in a dependent relationship with the 
physician or may consent under duress. In such situations the informed consent should be 
sought by an appropriately qualified individual who is completely independent of this 
relationship.  
27. For a potential research subject who is incompetent, the physician must seek informed 
consent from the legally authorized representative. These individuals must not be 
included in a research study that has no likelihood of benefit for them unless it is intended 
to promote the health of the population represented by the potential subject, the research 
cannot instead be performed with competent persons, and the research entails only 
minimal risk and minimal burden.  
28. When a potential research subject who is deemed incompetent is able to give assent to 
decisions about participation in research, the physician must seek that assent in addition 
to the consent of the legally authorized representative. The potential subject's dissent 
should be respected.  
29. Research involving subjects who are physically or mentally incapable of giving consent, 
for example, unconscious subjects, may be done only if the physical or mental condition 
that prevents giving informed consent is a necessary characteristic of the research 
population. In such circumstances the physician should seek informed consent from the 
legally authorized representative. If no such representative is available and if the research 
cannot be delayed, the study may proceed without informed consent provided that the 
specific reasons for involving subjects with a condition that renders them unable to give 
informed consent have been stated in the research protocol and the study has been 
approved by a research ethics committee. Consent to remain in the research should be 
obtained as soon as possible from the subject or a legally authorized representative.  
30. Authors, editors and publishers all have ethical obligations with regard to the publication 
of the results of research. Authors have a duty to make publicly available the results of 
their research on human subjects and are accountable for the completeness and accuracy 
of their reports. They should adhere to accepted guidelines for ethical reporting. Negative 
and inconclusive as well as positive results should be published or otherwise made 
publicly available. Sources of funding, institutional affiliations and conflicts of interest 
should be declared in the publication. Reports of research not in accordance with the 
principles of this Declaration should not be accepted for publication.  
C. ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH COMBINED WITH 
MEDICAL CARE  
31. The physician may combine medical research with medical care only to the extent that 
the research is justified by its potential preventive, diagnostic or therapeutic value and if 
the physician has good reason to believe that participation in the research study will not 
adversely affect the health of the subjects who serve as research subjects.  
96 
 


Protocol AG348-C-003, Amendment 2, Protocol Version 3.0 
05 August 2015 
 
32. The benefits, risks, burdens and effectiveness of a new intervention must be tested 
against those of the best current proven intervention, except in the following 
circumstances:  
• The use of placebo, or no treatment, is acceptable in studies where no current proven 
intervention exists; or  
• Where for compelling and scientifically sound methodological reasons the use of 
placebo is necessary to determine the efficacy or safety of an intervention and the 
subjects who receive placebo or no treatment will not be subject to any risk of serious 
or irreversible harm. Extreme care must be taken to avoid abuse of this option.  
33. At the conclusion of the study, subjects entered into the study are entitled to be informed 
about the outcome of the study and to share any benefits that result from it, for example, 
access to interventions identified as beneficial in the study or to other appropriate care or 
benefits.  
34. The physician must fully inform the subject which aspects of the care are related to the 
research. The refusal of a subject to participate in a study or the subject's decision to 
withdraw from the study must never interfere with the subject-physician relationship.  
35. In the treatment of a subject, where proven interventions do not exist or have been 
ineffective, the physician, after seeking expert advice, with informed consent from the 
subject or a legally authorized representative, may use an unproven intervention if in the 
physician's judgement it offers hope of saving life, re-establishing health or alleviating 
suffering. Where possible, this intervention should be made the object of research, 
designed to evaluate its safety and efficacy. In all cases, new information should be 
recorded and, where appropriate, made publicly available. 
97 
 


 
1. 
TITLE PAGE 
Clinical Study Protocol AG348-C-003 
EudraCT No. 2015-000484-13 
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, 
Pharmacokinetic and Pharmacodynamic Study of AG-348 in Adult Patients 
with Pyruvate Kinase Deficiency 
Study Sponsor: 
Agios Pharmaceuticals, Inc. 
88 Sidney Street 
Cambridge, MA 02139-4169 
Phone:  617-649-8600 
Fax:  617-649-8618 
Responsible Medical Officer: 
, MD 
 
Agios Pharmaceuticals, Inc. 
Mobile Phone: 
 
Office Phone: 
 
Email: 
 
Study Medical Monitor 
, MD 
 
On behalf of Agios Pharmaceuticals, Inc. 
Mobile Phone: 
 
Office Phone: 
 
Email: 
  
Document Version (Date): 
Revised 
Version 1.0 (05 January 2015) 
Amendment 1, Protocol Version 2.0 (02 February 2015) Final 
This study will be conducted according to the protocol and in compliance with Good Clinical 
Practice, the ethical principles stated in the Declaration of Helsinki, and other applicable 
regulatory requirements. 
CONFIDENTIALITY NOTE: 
The information contained in this document is confidential and proprietary to Agios Pharmaceuticals, Inc. Any 
distribution, copying, or disclosure is strictly prohibited unless such disclosure is required by federal regulations or 
state law. Persons to whom the information is disclosed must know that it is confidential and that it may not be 
further disclosed by them. 
1 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
INVESTIGATOR’S AGREEMENT 
 
I understand that all documentation provided to me by Agios 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). No changes will be made to the study protocol without the 
prior written approval of Agios and the IRB, except where necessary to eliminate an immediate 
hazard to the patient. 
I have read, understood, and agree to conduct this study as outlined in the protocol and in 
accordance with the guidelines and all applicable government regulations. 
 
 
Investigator Name (printed) 
 
Investigator Signature 
 
Date 
 
 
 
 
Investigational site or name of institution and location (printed) 
2 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
2. 
SYNOPSIS 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Title of Study:  
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, Pharmacokinetic and 
Pharmacodynamic Study of AG-348 in Adult Patients with Pyruvate Kinase Deficiency 
Study Center(s): 
This study will be conducted at multiple study centers. 
Phase of Development: 2 
Objectives: 
Primary: 
• 
Evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in patients with 
pyruvate kinase deficiency (PK deficiency). 
Secondary: 
• 
Evaluate the pharmacokinetics (PK) of AG-348 and the metabolite AGI-8702. 
• 
Evaluate the pharmacodynamic (PD) response of adenosine triphosphate (ATP) and 
2,3-diphosphoglycerate (2,3-DPG) after administration of AG-348. 
• 
Evaluate indicators of clinical activity of AG-348 in patients with PK deficiency, including changes 
in hemoglobin (Hb), hematocrit (HCT), reticulocyte count, haptoglobin (Hp), carboxyhemoglobin 
(COHb), lactate dehydrogenase (LDH), total and indirect bilirubin, erythropoietin (EPO), ferritin, 
and transferrin saturation (serum iron/iron binding capacity). 
Methodology: 
Study AG348-C-003 is a Phase 2, open label, two arm, multicenter, randomized, dose-ranging study 
during which adult patients with PK deficiency will receive multiple doses of AG-348 for up to 
24 weeks. Patients with PK deficiency confirmed by red blood cell PK enzymatic assay performed at 
Screening will be eligible to participate in this study. At Week 25, patients who safely tolerate AG-348 
and demonstrate clinical activity of AG-348 may be eligible to immediately roll over to a separate safety 
extension study for continued treatment. Patients who finish treatment at the end of 24 weeks or sooner 
will undergo follow-up assessment 4 weeks after the last dose of study drug. Patients with toxicity 
suspected to be related to study drug will continue follow-up until the adverse event (AE) resolves, is 
declared chronic by the Investigator, or the patient is lost to follow-up.  
3 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Initially, up to 25 patients will be randomized on an open-label 1:1 basis to each of two twice-daily 
(BID) doses of AG-348 (up to 50 patients total; see Figure 1, Study Schema). The dose of Arm 1 is 
300 mg of AG-348 administered orally every 12 hours (q12h, BID). The dose of Arm 2 is 50 mg of 
AG-348 administered orally q12h (BID). Randomization will be stratified by PKR mutation in order to 
maintain balance as much as possible across the dose arms for the specific mutations expected to be 
most frequently enrolled. The PKR mutation stratification factor will consist of 4 levels (R510Q, 
R486W, and R479H) and all other mutations (“other”). Mutation status is defined by the presence of at 
least one of the indicated mutations; patients with more than one stratified mutation will be assigned 
based on Sponsor’s discretion.  
The doses for each arm have been selected from the forerunner AG348-C-001 single ascending dose 
(SAD) and AG348-C-002 multiple ascending dose (MAD) studies in healthy adult volunteers to 
represent the range of doses/exposures that were safely tolerated and resulted in maximal or near-
maximal PD effects on 2,3-DPG and ATP. 
Because PK deficiency is a rare disease with a limited eligible patient population and because the 
underlying pathophysiology and clinical phenotype of affected patients is heterogenous due to the wide 
variety of mutations in PKR that cause the disease, it is important to focus closely on dose findings in 
this first-in-patient study. Therefore, in addition to initiating this study with 2 different doses of AG-348 
administered q12h, a Data Review Team (DRT) will be established to review study data on a regular 
basis and adapt the study design, dose and schedule of AG-348. 
The DRT will monitor safety on an on-going basis and meet at regular intervals (approximately every 
6 weeks), or ad hoc, as necessary, to review AEs, vital signs (VS), clinical laboratory (hematology, 
clinical chemistry, coagulation, and urinalysis), and electrocardiograms (ECGs) on enrolled patients. 
The DRT will also review available PK/PD data and indicators of clinical activity (e.g., changes from 
baseline in Hb). The DRT will be comprised of the study Coordinating Investigator, treating 
Investigators, Medical Monitor, Clinical Pharmacologist, Statistician, and Sponsor’s Responsible 
Medical Officer.  
Approximately every 6 weeks beginning six weeks after the first patient is dosed or ad hoc as necessary, 
the DRT will review cumulative safety data, available PK/PD data, and clinical activity data. Based on 
the DRT’s recurring 6 week reviews, the DRT may exercise one or more of the following options: 
• 
Continue treatment and enrollment in existing arms without change. 
• 
Add 1 new dose arm (Arm 3) to enroll up to 25 patients at a dose to be determined; the dose for 
Arm 3 may be lower or higher than Arm 1 and Arm 2 doses, but will not exceed 360 mg q12h; 
and the dose regimen may be less frequent than q12h. 
• 
Terminate or suspend enrollment to allow further review of clinical data in Arm 1 and/or Arm 2 
(and/or potential Arm 3). Enrollment in an arm could be terminated or suspended to allow 
further review, for example, for unacceptable safety/tolerability, poor PD response, or lack of 
signs of clinical activity. 
• 
Re-assign patients’ doses and schedule in a terminated arm to match the dose and schedule of 
another arm of the study. In this case, the patients in the terminated arm will remain in their 
original arm, i.e., they will not count towards the enrollment quota of the arm whose dose and 
4 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
schedule is being adopted. 
• 
Implement specific genotype restrictions to enrollment in one or more arms to ensure 
representation of patients with genotypes of greatest clinical relevance. 
The DRT will perform these evaluations on a recurring 6-week basis. The data that the DRT will review 
to make these decisions is expected to include, but are not necessarily limited to, the following:  
• 
Safety Observations: all AEs; VS, clinical laboratory (hematology, clinical chemistry, 
coagulation, and urinalysis), and ECG;  
• 
PK and PD Observations: including changes in 2,3-DPG and ATP; 
• 
Indicators of Clinical Activity: including changes in Hb, HCT, reticulocyte count, Hp, COHb, 
LDH, EPO, total and indirect bilirubin, ferritin, and transferrin saturation.  
If a third dose arm is implemented, the dose of AG-348 selected will not exceed 360 mg BID, as this 
was the highest dose that demonstrated acceptable safety and tolerance in the 14-day multiple BID 
dosing study in healthy volunteers. The PK/PD sampling schedule in a potential third dosing arm will be 
determined by Sponsor’s discretion and may follow either the extensive or limited PK/PD sampling 
schedules as specified. 
The DRT will monitor the data in an ongoing manner as described and may make a decision to terminate 
enrollment in an arm if unacceptable AEs are observed, PD activity is limited, and/or markers of clinical 
activity indicate no effect. Unacceptable AEs are defined as ≥ Grade 3 AEs (using National Cancer 
Institute Common Terminology Criteria for Adverse Events [NCI CTCAEv4.03]) that are assessed as at 
least possibly related to AG-348, with the exception of Grade 3 headache, hot flash/flushing, nausea, 
vomiting, and/or diarrhea that is transient in duration (< 24 hours) or able to be medically managed to 
≤ Grade 2 within 24 hours. The DRT may also exercise discretion to terminate enrollment in an arm if it 
observes a frequency of Grade 2 AEs that would make it difficult to support long-term dosing. 
Due to the potential for AG-348-mediated aromatase inhibition, dual-energy x-ray absorptiometry 
(DXA) scan (hip and spine) will be performed at Screening (if patient has not had prior DXA scan 
within 3 months of Day 1) to obtain T and Z scores. These data are intended to serve as a baseline 
measure of bone mineral density for all enrolling patients, and are deemed of particular importance for 
those who may enter the longer term safety extension study after completing the current study. All 
patients will have a second DXA scan in the interval between Weeks 24 and 28. The safety extension 
study will separately address additional follow-up DXA scanning. 
As the number of enrolling arms changes in the study (for example, from 2 to 3), the randomization 
scheme will adjust to enable balanced randomization into each actively accruing arm. Randomization 
and stratification will cease in the event that only a single arm is left enrolling. 
Depending on possible early termination of 1 or both of the initial 2 arms, or the addition of a third arm, 
the study could enroll up to a maximum of 75 patients. 
5 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Figure 1:  Study Schema 
 
Abbreviations:  BID (q12h) = twice-daily (every 12 hours); DRT = data review team; PKR = pyruvate kinase red blood cell 
isoform; TBD= to be determined; w = week(s). 
 
Visit Schedule 
Screening assessments will occur within 28 days prior to the first dose of study treatment. During the 
Treatment period, patients will attend visits at baseline (Day 1), weekly through Week 3 (Days 8, 15, 
and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12), and monthly through 
Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate AG-348 and demonstrate evidence of 
clinical activity of AG-348 through Week 24 may be eligible to immediately enter a separate extension 
study for continued treatment. For patients who finish treatment at the end of 24 weeks or sooner, or 
who elect not to enter the extension trial, study discharge will occur 4 weeks (Week 28 or earlier) 
following the last dose of study treatment at the final follow-up assessment. 
Dose Modifications for Safety and/or Increase in Hb Level 
The Investigator will monitor all patients for safety and tolerability. Modification of an individual 
patient’s dose of AG-348 will be based on AEs and/or observed changes in Hb level as described in 
Section 9.7.1 and Section 9.7.2. 
6 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Number of patients (planned): Up to approximately 75 patients. 
Diagnosis and main criteria for inclusion: 
Inclusion criteria: 
For entry into the study, patients must meet all of the following criteria during the Screening or other 
specified period: 
1. Signed written informed consent obtained prior to performing any study procedure, including 
screening procedures.  
2. Male or female, aged 18 years and older. 
3. Known medical history of PK deficiency.  
4. All patients must have documented clinical laboratory confirmation of PK deficiency by red 
blood cell pyruvate kinase enzymatic assay performed at Screening by a designated central 
laboratory. Patients with prior documentation of PK deficiency by red blood cell (RBC) 
enzymatic assay will have a reconfirmation of this result during Screening as a condition of 
enrollment. 
a. In the event that a patient’s screening pyruvate kinase enzymatic assay is negative (i.e., 
shows normal pyruvate kinase activity), the patient will be eligible for enrollment if the 
genotyping shows a mutant genotype that has been previously documented in the 
literature to be associated with pyruvate kinase deficiency. If the genotyping shows a 
previously undescribed mutation in the PKR gene, then the eligibility for enrollment 
will be determined on a case-by-case basis by the Coordinating Investigator and 
Medical Monitor in discussion with the Investigator. If no mutation is defined, then the 
patient will not be eligible. 
5. All patients must have genotypic characterization of the mutant PKR gene performed by a 
designated central laboratory at Screening, unless genotype is available from the patient’s 
participation in the Pyruvate Kinase Deficiency Natural History Study (NCT02053480). 
6. All patients must have genotypic characterization of the UGT1A1 gene performed by a 
designated central laboratory to document whether they may have underlying Gilbert's Disease. 
Patients with Gilbert’s Disease are eligible to enroll. 
7. Males must have Hb ≤ 12.0 g/dL; females must have Hb ≤ 11.0 g/dL.  
8. All patients must be considered transfusion independent as defined by: no greater than 3 units of 
RBCs transfused in the 12-month period up to the first day of study dosing and no transfusions 
within 4 months of first day of study dosing. Patients who have received more transfusion 
support than described above will evaluated for eligibility on a case-by-case basis by the 
7 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Medical Monitor. 
9. Splenectomized patients: 
a. Must have undergone their procedure at least 6 months prior to Screening. 
b. Must be current in their vaccinations for Pneumococcal Conjugate (PCV13), 
Pneumococcal Polysaccharide (PPSV23), Quadrivalent Meningococcal vaccine, and 
Haemophilus influenzae type b (Hib) as recommended by Centers for Disease Control 
and Prevention's (CDC) Advisory Committee on Immunization Practices (ACIP) or 
immunization advisory groups in Canada and the European Union (for patients enrolled 
in Canada and the EU). [http://www.cdc.gov/vaccines/schedules/downloads/adult/adult-
schedule.pdf] [Any missing vaccinations may be administered during the screening 
period.] 
10. Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2. 
11. Patients must be taking at least 1 mg of folic acid daily for at least 21 days prior to first dose and 
continue daily during study participation. 
12. Adequate organ function, defined as: 
a. Serum aspartate transaminase (AST) and alanine aminotransferase (ALT) ≤ 1.5 × upper 
limit of normal (ULN) (unless the increased AST is assessed by the Investigator as due 
to hemolysis). 
b. Normal or elevated levels of serum bilirubin. In patients with serum bilirubin > ULN, 
the elevation must be attributed to hemolysis with or without Gilbert's syndrome and 
must not be choledocholithiasis, cholecystitis, biliary obstruction, or hepatocellular 
disease. 
c. Serum creatinine ≤ 1.25 × ULN. If serum creatinine > 1.25 × ULN, then 24-hour 
measured or calculated (Cockcroft-Gault) glomerular filtration rate (GFR) ≥ 60 mL/min. 
d. Absolute neutrophil count (ANC) > 1.0 × 109/L. 
e. Platelet count ≥ 100 × 109/L. 
f. Activated partial thromboplastin time (aPTT) and international normalized ratio (INR) 
≤ 1.25 × ULN, unless the patient is receiving therapeutic anticoagulants. 
13. Women of childbearing potential (WOCBP) must agree to abstain from sexual intercourse or to 
use an acceptable/effective method of contraception (i.e., condom plus spermicide, condom plus 
oral contraceptive, condom plus intrauterine device [IUD], condom plus diaphragm with 
spermicide) from as soon as feasible during the Screening period until 30 days following the last 
dose of AG-348.  
a. WOCBP include any female who has experienced menarche and who has not undergone 
successful surgical sterilization (hysterectomy, bilateral tubal ligation, and/or bilateral 
oophorectomy) or is not post-menopausal. Post-menopausal is defined as: 
i. 
Amenorrhea ≥ 12 consecutive months without another cause, and a documented 
8 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
serum follicle stimulating hormone (FSH) level > 35 mIU/mL; 
ii. 
Amenorrhea ≥ 12 consecutive months in women ≥ 62 years old (FSH testing is 
not required). 
14. WOCBP must have a negative serum or urine pregnancy test within 72 hours before start of 
AG-348 dosing. 
15. Women must not be breastfeeding.  
16. Male patients, with the exception of those who have undergone vasectomy at least 6 months 
prior to Screening, must agree to abstain from sexual intercourse or, if sexually active, to use a 
condom with spermicide as contraception (regardless of their female partner's childbearing 
potential or their partner's use of their own contraception) from Day 1 of dosing until 30 days 
following the last dose of AG-348. 
Exclusion criteria: 
Patients who meet any of the following criteria at Screening or prior to dosing on Day 1 will not be 
enrolled in the study: 
1. Hemoglobin level > 12.0 g/dL if male; Hb > 11.0 g/dL if female. 
2. Additional diagnosis of any other congenital or acquired blood disorder, including 
glucose-6-phosphate-dehydrogenase (G6PD) deficiency, or any other hemolytic anemia 
process except for mild allo-immunization as a consequence of transfusion therapy. 
3. Iron overload (hemosiderosis or concurrent hemochromatosis) sufficiently severe to result in a 
clinical diagnosis by the Investigator of cardiac, hepatic, or pancreatic insufficiency. 
4. Prior bone marrow or stem cell transplant. 
5. Clinically symptomatic cholelithiasis or cholecystitis. (Prior cholecystectomy is not 
exclusionary. Patients with symptomatic cholelithiasis or cholecystitis may be rescreened once 
the disorder has been treated and clinical symptoms have resolved.) 
6. Currently enrolled in another therapeutic clinical trial involving on-going therapy with any 
investigational or marketed product or placebo. Concurrent participation in the Pyruvate Kinase 
Deficiency Natural History Study (NCT02053480) is permitted. 
7. Exposure to any investigational drug, device, or procedure within 28 days prior to Screening. 
8. Concurrent medical condition that could compromise participation in the study such as: 
a. Poorly controlled hypertension (defined as systolic blood pressure (BP) > 150 mm Hg 
or diastolic BP > 90 mm Hg) refractory to medical management. 
b. History of recent (within < 6 months from Screening date) congestive heart failure; 
9 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
myocardial infarction or unstable angina pectoris; or hemorrhagic, embolic, or 
thrombotic stroke; deep venous thrombosis; or pulmonary or arterial embolism.  
c. Currently active infection requiring the use of parenteral anti-microbial agents or that is 
greater than Grade 3 (CTCAEv4.03) within 6 months of first dose. 
d. A pattern or frequency of post-splenectomy sepsis that in the assessment of the 
Investigator could reasonably be expected to interfere with the ability of the patient to 
complete the 24 week study participation. 
e. Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) 
antibody with signs of active Hepatitis B or C virus infection.  
f. Positive test for human immunodeficiency virus (HIV) 1 or 2 antibody. 
g. Diabetes mellitus judged to be in poor control by the Investigator or requiring > 3 anti-
diabetic agents counting insulin (all insulins are considered one agent); use of insulin 
per se is not exclusionary.  
h. History of any primary malignancy with the exception of: curatively treated non-
melanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; or 
other primary tumor treated with curative intent and no known active disease present 
and no treatment administered during the last 3 years. 
9. Undergone major surgery within 6 months of first dose.  
10. Current or recent history of psychiatric disorder that in the opinion of the Investigator or 
Medical Monitor could compromise the ability of the patient to cooperate with study visits and 
procedures. 
11. Use of any of the restricted list of products known to strongly inhibit CYP3A4 drug metabolism 
(Appendix 15.3, Table 7) within 5 days prior to Day 1 dosing; or to strongly induce CYP3A4 
metabolism (Appendix 15.3, Table 8) within 28 days prior to Day 1 dosing; or to strongly inhibit 
P-glycoprotein (P-gp) transporter (Appendix 15.3, Table 9) within 5 days prior to Day 1 dosing; 
or digoxin within 5 days prior to Day 1 dosing. 
12. Serum bilirubin > ULN attributable to factors other than hemolysis and/or Gilbert's syndrome. 
13. Male patients with heart-rate corrected QT (Fridericia's correction factor) QTcF interval 
> 450 msec, or female patients with QTcF interval > 470 msec with the exception of patients 
with a left bundle branch block (LBBB). Medical Monitor approval needed in patients with a 
LBBB. 
14. Cardiac dysrhythmias judged as clinically significant by the Investigator or requiring therapy 
with drugs that are primarily substrates of CYP3A4. 
10 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
15. History of allergy to sulfonamides if characterized by acute hemolytic anemia, anaphylaxis, or 
rash of erythema multiforme type or Stevens-Johnson syndrome. 
16. Any other medical or psychological condition deemed by the Investigator to be likely to 
interfere with a patient's ability to understand and sign informed consent; cooperate with study 
visits, tests, and procedures; or otherwise safely and reliably participate in the study. 
Investigational product, dosage, and mode of administration: 
AG-348 sulfate hydrate capsules will be provided as 25 mg or 100 mg (free-base equivalent) of AG-348 
sulfate hydrate without excipients in Swedish orange (25 mg) or white opaque (100 mg) gelatin capsules 
(size 2 capsules for all dose strengths). AG-348 will be administered orally BID. The number of capsules 
per dose will vary by assigned dose group. Patients will receive multiple oral (PO) doses of AG-348 
over a 24-week treatment period. AG-348 will be administered with water and may be administered with 
or without food. 
Reference therapy, dosage and mode of administration:  
Not applicable. 
Duration of treatment:  
The duration of treatment for all patients on this study will be up to 24 weeks. Patients who safely 
tolerate and demonstrate one or more indicators of clinical activity of AG-348 may be eligible to 
immediately roll over to a separate safety extension study for continued treatment. 
Criteria for evaluation: 
Safety: 
Monitoring of AEs, including determination of serious adverse events (SAEs) and AEs leading to 
discontinuation; safety laboratory parameters (hematology, chemistry, urinalysis, coagulation); physical 
examination findings; VS; 12-lead ECGs, and DXA scans. Adverse events will be graded using CTCAE, 
Version 4.03. Serum sex hormone levels (testosterone, estrone, and estradiol), bone turnover markers 
(serum N-terminal telopeptide [NTX] and serum C-terminal telopeptide [CTX]), total cholesterol, high-
density lipoprotein-C (HDL-C), and triglycerides will be monitored for evidence of potential inhibition 
of aromatase by AG-348 
Indicators of Clinical Activity: 
Monitoring of potential indicators of clinical activity will include evaluating changes in Hb, HCT, 
reticulocyte count, COHb, LDH, total and indirect bilirubin, EPO, ferritin, and transferrin saturation.  
Pharmacokinetics: 
Approximately the first 10 patients treated, contingent on clinical site feasibility, will undergo extensive 
PK sampling as detailed in Appendix 15.1, Table 5. The remainder of treated patients will undergo 
limited PK sampling as detailed in Appendix 15.1, Table 6. Serial blood sampling for determination of 
concentration-time profiles of AG-348 and its metabolite AGI-8702 will be conducted following the first 
dose and the morning Day 15 dose, and additional trough levels of AG-348 and AGI-8702 will be 
obtained. AG-348 and AGI-8702 will be analyzed using qualified assays to determine concentrations in 
11 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
plasma. Pharmacokinetic parameters on Day 1 and Day 15 will be computed using standard non-
compartmental methods based on observed plasma AG-348 and AGI-8702 concentrations. 
Pharmacodynamics: 
Pharmacodynamic assessments will include 2,3-DPG, ATP (secondary objectives), 
 
.
 
 Approximately 
the first 10 patients treated will undergo extensive PD sampling as detailed in Appendix 15.1, Table 5. 
The remainder of treated patients will undergo limited PD sampling as detailed in Appendix 15.1, 
Table 6. Serial blood sampling for determination of levels of ATP and, 2,3-DPG will be conducted 
following the first dose and the morning Day 15 dose, and additional trough levels of ATP and 2,3-DPG 
will be obtained. Adenosine triphosphate and 2,3 DPG will be analyzed using qualified assays to 
determine concentrations in whole blood. Pharmacodynamic parameters on Day 1 and Day 15 will be 
computed based on observed whole blood ATP and 2,3-DPG concentrations.  
Statistical methods: 
The primary objective of this Phase 2 study is to evaluate the safety and tolerability of up to 24 weeks of 
AG-348 in patients with PK deficiency. Therefore, analyses will be primarily descriptive in nature; no 
formal hypothesis testing will be conducted. All analyses will be conducted separately within each dose 
arm, or pooled when appropriate. 
Summaries will be produced for disposition, baseline disease characteristics and demographic data 
including genotype, safety measurements, PK, PD parameters and indicators of clinical activity. Data 
from each AG-348 dose group will be analyzed separately, and pooled across all dose groups where 
appropriate. All data will also be listed by individual patient. Categorical variables will be summarized 
by frequency distributions (number and percentages) and continuous variables will be summarized by 
descriptive statistics (mean, standard deviation [SD], median, minimum, and maximum). 
Populations for analysis will include a Safety Analysis Set, a PK Analysis Set, and an Efficacy Analysis 
Set. The Safety Analysis set will include all patients who are enrolled and receive any dose of study 
treatment; this population will be the primary set for the analysis of safety data and the default analysis 
set for all data analyses unless otherwise stated. Patients will be classified according to treatment 
received, where treatment received is defined as the assigned treatment if it is received at least once, or 
as the first treatment received if assigned treatment is never received. The PK Analysis Set will include 
all patients in the Safety Analysis Set with sufficient plasma sample data to assess PK parameters. 
Results of the potential PD activity of AG-348 will also be based on the PK analysis set. The Efficacy 
12 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Analysis Set will include all patients who are enrolled and achieve at least 50% compliance with their 
assigned dose intensity for at least 4 weeks of continued dosing. Additional efficacy analyses may be 
performed on subsets of patients with other degrees of compliance with their assigned dose intensity 
and/or duration of dosing. The Efficacy Analysis Set will be the primary set for the analysis of 
preliminary clinical activity data. Patients will be classified according to assigned treatment. 
 
 
 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) and the 
incidence of treatment-emergent AEs (TEAEs) (new or worsening from baseline) will be summarized by 
primary system organ class (SOC), preferred term (PT), CTCAE Version 4.03 severity, outcome, action 
taken with study drug, and relationship to study drug by dose group. Separate summaries will be 
produced for all TEAEs, treatment-related AEs (those considered by the Investigator as at least possibly 
drug related), SAEs, discontinuations due to AEs, and AEs ≥ Grade 3 severity. Individual patient listings 
will be provided for any deaths, SAEs, AEs leading to interruption and/or reduction of study drug dose, 
and AEs leading to discontinuation of treatment. Analyses of AEs will be performed for those events 
that are considered treatment-emergent, where treatment-emergent is defined as any AE with onset 
beginning at the day of first administration of study drug, throughout the treatment period until 30 days 
after the last dose of study drug, or any event that was present at baseline but worsened in intensity or 
was subsequently considered drug-related by the Investigator through the end of the study. 
Descriptive statistics will be provided for clinical laboratory values (e.g., hematology, serum chemistry, 
coagulation studies, urinalysis) and VS data, presented as both actual values and changes from baseline 
relative to each on-study evaluation. Shift analyses will be conducted for selected laboratory parameters 
based on the baseline CTCAE grade to maximum CTCAE grade. Where applicable CTCAE terms do 
not exist, a grading system based on the upper and/or lower limits of normal will be used to classify the 
results. 
Electrocardiogram analyses will include individual patient listings and summaries of abnormal and 
clinically significant ECG results. Actual values and changes from baseline in PR, QRS, QTc intervals 
will be summarized by visit and dose arm.  
Concomitant medications will be coded using the WHO Drug Dictionary. Concomitant medications 
prior to and after the start of the study drug will be listed by patient, and summarized by ATC term and 
dose arm. 
Descriptive statistics will be used to summarize PK parameters for the parent compound AG-348 and the 
metabolite AGI-8702 for each dose group, and where appropriate, for the entire population. 
Pharmacokinetic parameters will be summarized using the following descriptive statistics: n, mean, 
standard deviation (SD), coefficient of variation %, median, minimum, and maximum, geometric mean, 
and geometric coefficient of variation.  
Descriptive statistics will be used to summarize Pharmacodynamic parameters for 2,3-DPG and ATP for 
each dose group, and where appropriate for the entire population. Pharmacodynamic parameters will be 
summarized using the following descriptive statistics: n, mean, SD, coefficient of variation %, median, 
minimum, and maximum, geometric mean, and geometric coefficient of variation %. 
13 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Analyses of serum sex hormones will use appropriate graphic displays and statistical analyses to 
evaluate patient change from baseline for each parameter. These analyses will include summaries of 
actual values and change from baseline using appropriate descriptive statistics (mean, SD, median, min 
and max) as described above.  
Analyses evaluating indicators of potential clinical activity of AG-348 in patients with PK deficiency 
will include changes in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, 
ferritin, and transferrin saturation (serum iron/iron binding capacity). Characterization of Hb response 
(e.g., % of patients whose Hb increases by a certain amount), as well as time to Hb response, and 
duration of Hb response will be explored, among others. 
Interim Review 
No formal statistical analysis will be conducted. Safety data will be reviewed on an ongoing basis by the 
DRT, who will meet to review safety, PK, PD, and preliminary clinical activity data at regular intervals 
(approximately every 6 weeks) throughout the duration of the study. The composition of the DRT, its 
meeting schedule, the data to be reviewed, and the decisions it is empowered to make have been 
described previously. 
Additional interim reviews of data may be conducted to support decision making concerning the current 
clinical study, the sponsor’s development programs in general, or in case of any safety concerns.  
Sample Size 
Due to the rare disease setting, the minimal sample size may be determined by feasibility. Depending on 
possible early termination of 1 or both of the initial 2 arms or the addition of a 3rd dose arm, the study 
could enroll up to a maximum of 75 patients. 
14 
 


Protocol AG348-C-003, Amendment 1, Version 2.0 
02 February 2015 
 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF 
FIGURES 
TABLE OF CONTENTS 
1. 
TITLE PAGE ................................................................................................................1 
2. 
SYNOPSIS ...................................................................................................................3 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES ...............15 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS.............................20 
5. 
INTRODUCTION ......................................................................................................24 
5.1. 
Pyruvate Kinase Deficiency .......................................................................................24 
5.2. 
AG-348 .......................................................................................................................25 
5.2.1. 
Summary of Nonclinical Data ....................................................................................25 
5.2.1.1. 
Pharmacology .............................................................................................................25 
5.2.1.2. 
Safety Pharmacology ..................................................................................................26 
5.2.1.3. 
Pharmacokinetics ........................................................................................................26 
5.2.1.4. 
Toxicology ..................................................................................................................27 
5.2.2. 
Summary of Clinical Data ..........................................................................................29 
5.2.2.1. 
Pharmacokinetics ........................................................................................................29 
5.2.2.2. 
Pharmacodynamics .....................................................................................................30 
5.2.2.3. 
Safety ..........................................................................................................................30 
5.3. 
Study Rationale ...........................................................................................................31 
5.3.1. 
Summary of Overall Safety Management Plan ..........................................................32 
6. 
TRIAL OBJECTIVES AND ENDPOINTS ...............................................................33 
6.1. 
Primary Objective .......................................................................................................33 
6.2. 
Secondary Objectives .................................................................................................33 
 
33 
6.4. 
Study Measures and Endpoints ...................................................................................33 
6.4.1. 
Safety Measures and Endpoints ..................................................................................33 
6.4.2. 
Clinical Activity Measures and Endpoints .................................................................34 
6.4.3. 
Pharmacokinetic and Pharmacodynamic Measures and Endpoints ............................34 
 
34 
7. 
INVESTIGATIONAL PLAN .....................................................................................35 
7.1. 
Overall Study Design ..................................................................................................35 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
02 February 2015 
 
7.2. 
Justification of the Study Design ................................................................................38 
7.3. 
Rationale for the Starting Dose and Dose Range .......................................................39 
7.4. 
Criteria for Study Termination ...................................................................................40 
8. 
STUDY POPULATION .............................................................................................41 
8.1. 
Number of Patients .....................................................................................................41 
8.2. 
Inclusion Criteria ........................................................................................................41 
8.3. 
Exclusion Criteria .......................................................................................................43 
8.4. 
Patient Identification and Registration .......................................................................44 
8.5. 
Patient Randomization ................................................................................................45 
8.6. 
Patient Withdrawal Criteria ........................................................................................45 
8.7. 
Replacement of Patients .............................................................................................46 
9. 
STUDY TREATMENT ..............................................................................................47 
9.1. 
Description of Study Drug ..........................................................................................47 
9.2. 
Study Drug Packaging and Labeling ..........................................................................47 
9.3. 
Study Drug Storage .....................................................................................................47 
9.4. 
Method of Assigning Patients to Treatment ...............................................................47 
9.5. 
Blinding ......................................................................................................................47 
9.6. 
Study Drug Preparation and Administration ..............................................................47 
9.7. 
Criteria for Dose Escalation, Dose Modification, or Discontinuation of Study 
Drug ............................................................................................................................48 
9.7.1. 
Dose Modification for Safety .....................................................................................48 
9.7.2. 
Dose Modification for Increase in Hemoglobin Level ...............................................49 
9.7.3. 
Stopping Criteria .........................................................................................................50 
9.8. 
Duration of Patient Participation ................................................................................50 
9.9. 
Treatment Compliance ................................................................................................50 
9.10. 
Study Drug Accountability .........................................................................................50 
9.11. 
Prior and Concomitant Medications and Treatments .................................................51 
9.11.1. 
Prior Medications and Procedures ..............................................................................51 
9.11.2. 
Prohibited Concomitant Therapy ................................................................................51 
9.11.3. 
Allowed Concomitant Therapy ...................................................................................52 
9.11.4. 
Potential for Phototoxicity ..........................................................................................52 
9.11.5. 
Management of Nausea, Vomiting, and Diarrhea ......................................................53 
9.11.6. 
Other Restrictions and Precautions .............................................................................53 
16 
 


Protocol AG348-C-003, Amendment 1, Version 2.0 
02 February 2015 
 
10. 
STUDY ASSESSMENTS ..........................................................................................54 
10.1. 
Schedule of Assessments ............................................................................................54 
10.2. 
Informed Consent and Confirmation of Eligibility ....................................................54 
10.3. 
Demographic Data, Medical and Medication History ................................................54 
10.4. 
PKR Enzymatic Assay and PKR Genotyping ............................................................55 
10.5. 
Safety Assessments .....................................................................................................55 
10.5.1. 
Physical Examination, Height, and Weight ................................................................55 
10.5.2. 
Vital Signs ..................................................................................................................55 
10.5.3. 
Electrocardiogram .......................................................................................................55 
10.5.4. 
DXA Bone Scans ........................................................................................................55 
10.5.5. 
Safety Laboratory Assessments ..................................................................................56 
10.5.5.1. 
Hematology, Serum Chemistry, Coagulation Studies, and Urinalysis .......................56 
10.5.5.2. 
Screening Serology .....................................................................................................57 
10.5.6. 
Adverse Events ...........................................................................................................57 
10.6. 
Pharmacokinetic Assessments ....................................................................................57 
10.6.1. 
Blood Sample Collection and Pharmacokinetic Measurements During Dose 
Escalation ....................................................................................................................57 
10.7. 
Pharmacodynamic Assessments .................................................................................58 
10.8. 
Ordering of Blood Sample Collection ........................................................................59 
10.9. 
Sample Processing, Storage, and Shipment ................................................................59 
11. 
ADVERSE EVENTS..................................................................................................60 
11.1. 
Definition of Adverse Events .....................................................................................60 
11.1.1. 
Adverse Event (AE) ....................................................................................................60 
11.1.2. 
Suspected Adverse Reaction .......................................................................................60 
11.1.3. 
Unexpected Adverse Event .........................................................................................60 
11.1.4. 
Serious Adverse Event ................................................................................................60 
11.1.4.1. 
Potential Severe Drug-Induced Liver Injury ..............................................................61 
11.2. 
Procedures for Reporting Adverse Events and Serious Adverse Events ....................61 
11.3. 
Pregnancy Reporting ..................................................................................................63 
12. 
STATISTICAL METHODS .......................................................................................65 
12.1. 
Sample Size Estimation ..............................................................................................65 
12.2. 
Populations for Analysis .............................................................................................65 
12.3. 
Procedures for Handling Missing, Unused, and Spurious Data .................................66 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
02 February 2015 
 
12.4. 
Interim Analysis ..........................................................................................................66 
12.5. 
Statistical Methodology ..............................................................................................66 
12.5.1. 
General Methods .........................................................................................................66 
12.5.2. 
Disposition ..................................................................................................................67 
12.5.3. 
Exposure and Safety Analyses ....................................................................................67 
12.5.4. 
Pharmacokinetic Analyses ..........................................................................................67 
12.5.5. 
Pharmacodynamic Analyses .......................................................................................68 
12.5.6. 
Aromatase Hormone Analysis ....................................................................................68 
12.5.7. 
Clinical Activity ..........................................................................................................68 
 
68 
12.6. 
Procedures for Reporting Deviations to Original Statistical Analysis Plan ...............68 
13. 
ADMINSTRATIVE REQUIREMENTS ....................................................................69 
13.1. 
Good Clinical Practice ................................................................................................69 
13.2. 
Ethical Considerations ................................................................................................69 
13.3. 
Patient Information and Informed Consent ................................................................69 
13.4. 
Patient Confidentiality ................................................................................................70 
13.5. 
Protocol Compliance ..................................................................................................70 
13.6. 
Data Management .......................................................................................................70 
13.7. 
Source Document/Case Report Form Completion .....................................................71 
13.8. 
Direct Access to Source Data .....................................................................................71 
13.9. 
Record Retention ........................................................................................................71 
13.10. 
Liability and Insurance ...............................................................................................72 
13.11. 
Publication of Study Findings and Use of Information ..............................................72 
14. 
LIST OF REFERENCES ............................................................................................73 
15. 
APPENDICES ............................................................................................................74 
15.1. 
Schedules of Assessments ..........................................................................................75 
15.2. 
National Cancer Institute Common Terminology Criteria for Adverse Events .........81 
15.3. 
Potential Drug Interactions .........................................................................................82 
15.4. 
Declaration of Helsinki ...............................................................................................85 
A. 
INTRODUCTION ......................................................................................................85 
B. 
BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH ......................................86 
C 
ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH COMBINED 
WITH MEDICAL CARE ...........................................................................................88 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
02 February 2015 
 
LIST OF TABLES 
Table 1: 
Dose Modification for Adverse Events Deemed at Least Possibly Related to 
AG-348 .......................................................................................................................49 
Table 2: 
Dose Reduction Table (by Dosing Arm) ....................................................................50 
Table 3: 
Sample Size Estimation ..............................................................................................65 
Table 4: 
Schedule of Assessments: ...........................................................................................75 
Table 5: 
Schedule of Assessments: Extensive PK/PD Sampling .............................................79 
Table 6: 
Schedule of Assessments: Limited PK/PD Sampling ................................................80 
Table 7: 
Strong and Moderate CYP3A4 Inhibitors ..................................................................82 
Table 8: 
Strong CYP3A4 Inducers ...........................................................................................83 
Table 9: 
Strong P-glycoprotein Inhibitors ................................................................................83 
Table 10:  Sensitive CYP3A4 Substrates.....................................................................................84 
 
LIST OF FIGURES 
Figure 1:  Study Schema ...............................................................................................................6 
Figure 2: 
Red Cell Glycolysis ....................................................................................................24 
Figure 3: 
Study Schema .............................................................................................................37 
Figure 4: 
PKR Enzymatic Reaction ...........................................................................................58 
 
19 
 


Protocol AG348-C-003, Amendment 1, Version 2.0 
02 February 2015 
 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation or Specialist Term 
Explanation 
2,3-DPG 
2,3-diphosphoglycerate 
ACIP 
Advisory Committee on Immunization Practices 
ADME 
Absorption, distribution, metabolism, excretion 
ADP 
Adenosine diphosphate 
AE 
Adverse event 
ALC 
Absolute lymphocyte count 
ALP 
Alkaline phosphatase 
ALT 
Alanine aminotransferase 
ANC 
Absolute neutrophil count 
aPTT 
Activated partial thromboplastin time 
AST 
Aspartate aminotransferase 
ATP 
Adenosine triphosphate 
AUC 
Area under the curve 
AUC0-12hr 
Area under the plasma concentration versus time curve from 0 to 12 
hours 
AUC0 24hr 
Area under the plasma concentration versus time curve from 0 to 
24 hours 
AUC0-∞ 
Area under the plasma concentration versus time curve from 0 to 
infinity 
BCRP 
Breast cancer resistance protein 
BID 
Twice-daily 
BMI 
Body mass index 
BP 
Blood pressure 
BUN 
Blood urea nitrogen 
CDC 
Centers for Disease Control and Prevention 
CFR 
Code of Federal Regulations 
CLP 
Total body plasma clearance 
Cmax 
Maximum plasma concentration 
CO2 
Carbon dioxide 
COHb 
Carboxyhemoglobin 
CTCAE 
Common Terminology Criteria for Adverse Events 
20 
 


Protocol AG348-C-003, Amendment 1, Version 2.0 
02 February 2015 
 
Abbreviation or Specialist Term 
Explanation 
CTX 
Serum C-terminal telopeptide 
CV 
Cardiovascular 
DDI 
Drug-drug interaction 
CYP 
Cytochrome P450 
DILI 
Drug-induced liver injury 
DLT 
Dose-limiting toxicity 
DRT 
Data review team 
DXA 
Dual-energy x-ray absorptiometry 
ECG 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic case report form 
EDC 
Electronic Data Capture 
EPO 
Erythropoietin 
F 
Oral bioavailability 
FDA 
Food and Drug Administration 
FSH 
Follicle stimulating hormone 
G6PD 
Glucose-6-phosphate-dehydrogenase 
GCP 
Good Clinical Practice 
GFR 
Glomerular filtration rate 
GLP 
Good Laboratory Practice 
Hb 
Hemoglobin 
HBsAg 
Hepatitis B surface antigen 
HCT 
Hematocrit 
HCV 
Hepatitis C virus 
HDL-C 
High-density lipoprotein-C 
HDPE 
High density polyethylene 
hERG 
Human ether à-go-go related gene 
Hib 
Haemophilus influenzae type b 
HIV 
Human immunodeficiency virus 
Hp 
Haptoglobin 
IC50 
Concentration of drug that achieved half-maximal inhibition 
ICH 
International Conference on Harmonization 
21 
 


Protocol AG348-C-003, Amendment 1, Version 2.0 
02 February 2015 
 
Abbreviation or Specialist Term 
Explanation 
INR 
International normalized ratio 
IRB 
Institutional Review Board 
IUD 
Intrauterine device 
IV 
Intravenous 
LDH 
Lactate dehydrogenase 
MAD 
Multiple ascending dose 
MCH 
Mean corpuscular hemoglobin 
MCHC 
Mean corpuscular hemoglobin concentration 
MCV 
Mean corpuscular volume 
MedDRA 
Medical Dictionary for Regulatory Activities 
mPKR 
PKR mutants 
MTD 
Maximum tolerated dose 
NCI 
National Cancer Institute 
NOEL 
No-observed-effect-level 
NOAEL 
No-observed-adverse-effect-level 
NTX 
Serum N-terminal telopeptide 
P-gp 
P-glycoprotein 
PCV13 
Pneumococcal Conjugate 
PD 
Pharmacodynamic 
PEP 
Phosphoenolpyruvate 
PK 
Pharmacokinetic 
PK deficiency 
Pyruvate kinase deficiency 
PKR 
Pyruvate kinase isoform R 
PO 
Oral 
PPSV23 
Pneumococcal polysaccharide 
PR 
The portion of the ECG wave from the beginning of the P wave to 
the beginning of the QRS complex 
PT 
Preferred term 
q12h 
Every 12 hours 
q24h 
Every 24 hours 
QD 
Once-daily 
QRS 
QRS interval on an electrocardiogram 
QTc 
Heart-rate corrected QT interval 
22 
 


Protocol AG348-C-003, Amendment 1, Version 2.0 
02 February 2015 
 
Abbreviation or Specialist Term 
Explanation 
QTcB 
Corrected QT interval - Bazett correction formula 
QTcF 
Corrected QT interval - Fridericia’s method 
RBC 
Red blood cell (count) 
RDW 
Red cell distribution width 
SAD 
Single ascending dose 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SD 
Standard deviation 
SOC 
System organ class 
SOP(s) 
Standard Operating Procedure(s) 
t½ 
Apparent terminal half-life 
TIBC 
Total iron-binding capacity 
Tmax 
Time to maximum concentration 
TEAE 
Treatment-emergent adverse event 
ULN 
Upper limit of normal 
VS 
Vital signs 
Vss 
Volume of distribution at steady-state 
Vz/F 
Mean apparent volume of distribution 
WBC 
White blood cell  
WMA 
World Medical Association 
WOCBP 
Women of childbearing potential 
WT 
Wild type 
23 
 


Protocol AG348-C-003, Amendment 1, Version 2.0 
02 February 2015 
 
5. 
INTRODUCTION 
5.1. 
Pyruvate Kinase Deficiency 
Pyruvate kinase deficiency (PK deficiency) is a glycolytic enzymopathy that results in non-
spherocytic hemolytic anemia with a variable clinical presentation, ranging from mild or fully 
compensated forms to life-threatening neonatal anemia and life-long chronic hemolytic anemia 
associated with severe, debilitating co-morbidities. PK deficiency is caused by mutations in the 
PKLR gene, which results in defective pyruvate kinase isoform R (PKR) in the red blood cell 
(RBC). As shown in the diagram below, PKR catalyzes the final and irreversible step in 
glycolysis, converting phosphoenolpyruvate (PEP) to pyruvate, and formation of adenosine 
triphosphate (ATP) from adenosine diphosphate (ADP). Mature RBCs rely almost exclusively on 
the process of glycolysis to generate the energy carrier molecule ATP. PKR is thus a key enzyme 
for maintaining energy homeostasis in erythrocytes, and it has been proposed that ATP levels are 
critical for optimally maintaining RBC membrane integrity (van Wijk and van Solinge 2005). 
Figure 2: 
Red Cell Glycolysis 
 
Abbreviations:  1,3 DPG = 1,3 diphosphoglycerate; 2,3 DPG = 2,3 diphosphoglycerate; 3-PG = 3-phosphoglycerate; 
ADP = adenosine diphosphate; ATP = adenosine triphosphate; FBP = fructose 1,6-bisphosphate; PEP = 
phosphoenolpyruvate; PKR = red blood cell-specific form of pyruvate kinase 
Note: Not all steps in glycolysis are shown. 
The majority of mutations in PKR that have been described have a deleterious effect on PKR 
catalytic activity, protein stability, and/or protein expression. PK deficiency is associated with 
reduced RBC survival as well as impaired red cell maturation. Mature erythrocytes lack 
mitochondria relying predominantly on glycolysis to generate ATP. It is hypothesized that 
insufficient energy production promotes erythrocyte hemolysis due to impaired maintenance of 
cellular membrane homeostasis. 
PK deficiency is an autosomal recessive disease, in which both homozygotes and compound 
heterozygotes develop hemolytic anemia. More than 200 different mutations in the PKLR gene 
have been identified in patients with PK deficiency to date, and the majority of these mutations 
are single nucleotide missense mutations (Zanella, et al. 2005). The prevalence of PK deficiency, 
based on the most common pyruvate kinase mutation in Southern Europe, is estimated at 51 
cases per million in the Caucasian population (Beutler and Gelbart 2000). 
Some patients with PK deficiency have a clinically severe disease course that can present as a 
severe hemolytic anemia in early infancy, requiring immediate care which typically includes 
blood transfusion support (Christensen, et al. 2011; Ghidini, et al. 1991; Hennekam, et al. 1990). 
Infants with PK deficiency may have erythroblastosis fetalis with hepatosplenomegaly, 
extramedullary hematopoiesis, and severe indirect hyperbilirubinemia, which is associated with a 
high risk of kernicterus. Hyperbilirubinemia associated with the hemolytic state often requires 
Glucose
Pyruvate
PKR
`
ATP
FBP
1,3-DPG
2,3-DPG
3-PG
PEP
ADP
24 
 


Protocol AG348-C-003, Amendment 1, Version 2.0 
02 February 2015 
 
supportive treatment with intensive phototherapy. For other infants, the hyperbilirubinemia 
caused by hemolysis can require exchange transfusion, as well as supportive transfusion to keep 
their hematocrit (HCT) levels >60%. 
Adults with PK deficiency are reported to have life-long hemolysis and the subsequent 
associated co-morbidities including anemia and transfusion dependence. Other co-morbidities 
include frequent miscarriages, aplastic crises, as well as symptoms associated with an acute on 
chronic hemolytic anemia (Rider, et al. 2011). Since unconjugated bilirubin is often chronically 
elevated, pigmented gallstones are common in children and adults. Severe and sometimes life-
threatening iron deposition occurs and is typically progressive. 
The current therapeutic options for patients with PK deficiency are supportive. Most require life-
long treatment, including blood transfusions at a frequency depending on the disease state. Long-
term surveillance for systemic iron overload, even in transfusion-independent patients, is 
standard as is the use of chelation therapy. Case reports of cure by allogeneic bone marrow 
transplant have been published (Tanphaichitr, et al. 2000) but have been infrequently performed. 
Splenectomy, while not curative, can reduce the need for supportive blood transfusions and 
decrease bilirubin levels in some patients. However, this intervention is associated with 
significant morbidity including infection risk, and possible increased risk of thrombosis. 
5.2. 
AG-348 
AG-348 is an orally available, allosteric activator of PKR. It is hypothesized that drug 
intervention with AG-348 restores glycolytic pathway activity and normalizes red cell 
metabolism in vivo. Biochemical experiments demonstrate that AG-348 is a potent, pan-activator 
of many PKR alleles associated with PK deficiency. PK deficiency red cells and their 
progenitors are characterized by changes in metabolism associated with defective glycolysis, 
including a build-up of PEP and 2,3-diphosphoglycerate (2,3-DPG), and lowered ATP levels. 
Treatment of PK deficiency patient red cells ex vivo with AG-348 results in increased ATP 
levels, and reductions in PEP and 2,3-DPG, consistent with pharmacological activation of PKR 
enzyme activity. In vitro studies against wild type (WT) PKR enzymes and ex vivo studies in 
blood from mice, monkeys and humans indicate that AG-348 activates WT PKR with a similar 
potency and efficacy as it does with the mutant versions. In vivo studies in mice confirm the 
potency of AG-348 in increasing WT PKR enzyme activity and modulating the levels of 
downstream markers such as ATP and 2,3-DPG. This therapeutic approach may be an effective 
way to correct the underlying pathology of PK deficiency and, importantly, provide clinical 
benefit to patients. 
5.2.1. 
Summary of Nonclinical Data 
A series of exploratory pharmacology studies were conducted to characterize the ability of 
AG-348 to activate WT PKR and anemia-associated PKR mutants (mPKR) in vitro, ex vivo, and 
in vivo. Additional details regarding nonclinical data can be found in the AG-348 Investigator’s 
Brochure 
5.2.1.1. 
Pharmacology 
AG-348 is a potent, broad-spectrum activator of PKR with low nM potency against both WT and 
mutant enzymes. AG-348 is also able to effectively stabilize mPKR enzymes that are 
25 
 


Protocol AG348-C-003, Amendment 1, Version 2.0 
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hypersensitive to thermal denaturation, suggesting that AG-348 may be able to increase PKR 
pathway activity by preventing destabilization and degradation of mPKR enzymes. Its major 
metabolite, AGI-8702, has low potency mixed activity against PKR and mutant isoforms and can 
increase the thermostability of some mPKR, albeit with significantly weaker potency than that of 
AG-348. 
The effect of AG-348 and AGI-8702 on PKR activity and a number of downstream pathway 
markers was evaluated in human RBCs. AG-348 dose-response curves in human whole blood 
showed potent activation of PKR, while AGI-8702 is a weak activator of the PKR enzyme. In 
whole blood from mice treated with AG-348, 13C-glucose labeling through the PKR reaction was 
increased by 80% demonstrating AG-348-mediated activation of the WT PKR enzyme. AG-348 
treatment of RBCs from healthy human donors potently increased levels of the PKR reaction 
product ATP consistent with activation of PKR.  
The effects of AG-348 on PKR activity and RBC metabolism also were assessed in blood 
samples from patients with PK deficiency. AG-348 activated PKR and induced metabolic 
changes (increased ATP levels and decreased 2,3-DPG levels) consistent with increased 
glycolytic pathway activity in RBCs from PK deficiency patients with different mutations in the 
PKR enzyme.  
Assessments of binding inhibition potential and enzymatic activity were evaluated in a panel of 
89 receptors, ion channels, and enzymes. Although AG-348 is a histamine H3 antagonist/inverse 
agonist and an aromatase inhibitor, no effects of histamine H3 modulation have been observed in 
safety pharmacology or toxicology studies. Effects consistent with aromatase inhibition were 
seen in toxicology studies (Section 5.2.1.4). 
5.2.1.2. 
Safety Pharmacology 
Safety pharmacology studies demonstrated that neither AG-348 nor AGI-8702 exhibited 
significant inhibition of the human ether-à-go-go related gene (hERG) current (concentration of 
drug that achieved half-maximal inhibition [IC50] 29.4 μM and > 10 μM, respectively). 
Additionally, in the monkey cardiovascular (CV) study, no CV effects were seen at any dose 
level. No effects were seen in AG-348 Good Laboratory Practice (GLP)-compliant Irwin and 
respiratory safety pharmacology studies in Sprague-Dawley rats. AG-348 administered via oral 
gavage had a dose-dependent emetic activity in the ferret at ≥ 30 mg/kg. 
5.2.1.3. 
Pharmacokinetics 
Absorption, distribution, metabolism, and excretion (ADME) studies of AG-348 were performed 
in Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys. Both oral (PO) and intravenous 
(IV) routes were evaluated at doses ranging from 1 to 50 mg/kg. A dose of 200 mg (50 mg/kg) 
was administered PO in the food-effect evaluation in monkeys. The PK of AG-348 in animal 
species is characterized by rapid oral absorption, medium to high total body plasma clearance 
(CLp), and high volume of distribution at steady-state (Vss) in rats, dogs, and monkeys. 
AG-348 has a medium to long apparent terminal half-life (t1/2) in the rat, dog, and monkey. 
Medium to high oral bioavailability (F) was observed for AG-348 in animals. A high-fat diet led 
to lower oral absorption of AG-348 in monkeys. 
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AG-348 showed low brain penetration in rats (brain/plasma ratio of 5.7% to 9.1%) based on area 
under the plasma concentration versus time curve from 0 to 24 hours (AUC0 24hr) following 
single and repeated-dose administration, respectively. Metabolism appears to be the major 
elimination pathway for AG-348 with minimal biliary or urinary excretion.  
Both in vitro and in vivo, the predominant metabolite was the N-dealkylated product, AGI-8702. 
There were no metabolites unique to human liver microsomes and no direct Phase 2 metabolites. 
The plasma metabolite profile of AG-348 did not change with repeat or ascending dosing, 
suggesting that AG-348 is not susceptible to metabolic switching with increasing or prolonged 
dose administration. 
AG-348 is a weak direct inhibitor of cytochrome P450 (CYP) 2C8, CYP2C9, CYP2C19, 
CYP2D6, and CYP3A4/5 (testosterone 6β-hydroxylation). There was no direct inhibition of 
CYP1A2, CYP2B6, or CYP3A4/5 (midazolam 1′-hydroxylation) by AG-348. There was 
evidence of metabolism-dependent inhibition of CYP2C19 (largely reversible) and CYP3A4 
(largely irreversible) by AG-348. AG-348 is considered a weak time-dependent CYP3A4 
inhibitor. AGI-8702 is a weak direct inhibitor of CYP2C9 and CYP2C19. AG-348 has the 
potential to cause an induction-related drug-drug interaction (DDI) with sensitive CYP2B6 and 
CYP3A4 substrates. 
The routes of metabolism for AG-348 are via multiple CYPs with CYP3A4 contributing > 70% 
of the total. CYP1A2, CYP2C9, and CYP2C8 contribute approximately 6%, 10%, and 7% to the 
remaining metabolism of AG-348; other isoforms contribute < 4% each. 
AG-348 is a substrate for P-glycoprotein (P-gp), but not breast cancer resistance protein (BCRP). 
AG-348 is an inhibitor of P-gp (91% and 99% inhibition at 41 and 411 µM, respectively), but 
does not inhibit BCRP. AGI-8702 is not an inhibitor of P-gp or BCRP under the concentrations 
tested (5 and 100 μM). 
5.2.1.4. 
Toxicology 
In rats, the no-observed-effect level/no-observed-adverse-effect level (NOEL/NOAEL), 
determined as 2000 mg/kg, was associated with area under the plasma concentration versus time 
curve from 0 to 12 hours (AUC0-12hr) values 223- to 526-fold the projected human efficacious 
AUC0-12hr value. In dogs, clinical observations consistent with anaphylactoid reactions were seen, 
and the maximum tolerated dose (MTD) was 62.5 mg/kg, which was associated with an 
AUC0-12hr value 5.7-fold the projected efficacious AUC0-12hr value. The NOEL/NOAEL in dogs 
was 10 mg/kg, which was associated with an AUC0-12hr value 0.8-fold the projected efficacious 
AUC0-12hr value. In monkeys, the NOAEL was 1000 mg/kg, with only non-adverse emesis and 
body weight loss seen; this dose was associated with an AUC0-12hr value 70-fold the projected 
efficacious AUC0 12hr value. Based on the results of single-dose studies, the rat and the monkey 
were chosen as the most appropriate species for further evaluation in toxicology studies.  
Dose-limiting toxicity (DLT) in cynomolgus monkeys was defined in non-GLP 5-day and 
14-day repeat-dose studies as emesis, inappetence, and weight loss. These toxicities became dose 
limiting at AUC0-12hr values 27- to 34-fold the projected efficacious AUC0-12hr value, and 
precluded meaningful evaluation of other toxicities at this exposure level. Potential other effects 
at this exposure level were observed in a number hematology and serum chemistry parameters as 
well as in lymphoid organs. Additionally, minimal potential effects in kidneys (renal 
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tubulointerstitial nephritis) and heart (myocardial degeneration), which could not be 
differentiated from spontaneous background lesions, were seen when monkeys were exposed to 
AG-348 AUC0-12hr values ≥ 27- to 34-fold the projected human efficacious AUC0-12hr value.  
In GLP-compliant 28-day monkey study, the dose of 150 mg/kg/day (75 mg/kg/dose twice daily 
[BID]) was the NOAEL. Effects were limited to increased liver weights without serum chemistry 
or microscopic correlate. At this dosage level, the Day 27 AUC0-12hr values were 8.9- and 
8.5-fold the projected efficacious AUC0-12hr value in males and females respectively. In the same 
study, the low dosage of 20 mg/kg/day (10 mg/kg/dose) resulted in AUC0-12hr values that 
approximated the efficacious AUC0-12hr value, and there were no test article-related effects seen. 
The next highest dose of 50 mg/kg/day (25 mg/kg/dose) was the NOEL and was associated with 
AUC0-12hr values 3.1- and 2.6-fold the projected efficacious AUC0-12hr value in males and females 
respectively.  
In the GLP-compliant 28-day rat study, the NOAEL in males was the highest dose tested, 
600 mg/kg/day (300 mg/kg/dose), and the NOAEL in females was the lowest dose tested, 
20 mg/kg/day (10 mg/kg/dose). At the 600 mg/kg/day dosage level in males, AG-348-related 
findings were limited to mild effects on hematology, serum chemistry, and urinalysis parameters, 
and microscopic findings in the adrenal gland (minimal to mild vacuolation of the adrenal zona 
glomerulosa and decreased thickness of the zona fasciculate), liver (minimal to mild 
hepatocellular hypertrophy), kidney (minimal tubular vacuolation), pancreas (minimal to 
moderate decreased zymogen granules), heart (minimal myocardial vacuolation), and prostate 
(minimal to mild decreased secretion). All findings were fully reversible over the 14-day 
recovery period with the exception of decreased serum glucose levels and decreased prostate 
secretion. In females, the highest dosage tested was 200 mg/kg/day (100 mg/kg/dose); adverse 
effects observed were similar to those observed in the 600 mg/kg/day males, with the exception 
that in females, fewer effects in hematology and serum chemistry parameters were seen, and also 
in females, adverse effects in the reproductive organs consistent with aromatase inhibition were 
observed.  
In the 13-week repeat-dose rat study, adverse effects in males were identified in the testes and 
consisted of seminiferous tubular degeneration, spermatid retention, and Leydig cell 
hypertrophy. These effects were defined as adverse at dose levels at which they are expected to 
impair fertility. These effects were either not present or present with incidence and severity 
similar to that of the vehicle group in lower dose levels. Adverse effects in females included 
uterine atrophy and increased folding of the luminal surface; these effects were defined as 
adverse at the dose level at which they are expected to impair fertility  
In the 13-week repeat-dose study in monkeys, no adverse effects were identified, and no new 
effects were identified when compared to the 4-week repeat-dose study. Similar to what occurred 
on the 4-week study, inappetence and emesis during the initial 1-2 weeks of dosing occurred, 
precluding evaluation of higher doses.  
AG-348 inhibits human aromatase activity with an IC50 of 2050 nM (based on human placental 
microsomes) and rat aromatase with an IC50 of 493 nM (based on rat ovarian microsomes). In the 
GLP-compliant 28-day rat study, histologic effects consistent with aromatase inhibition were 
seen in the female reproductive tract at the mid- and high-dosage levels (100 and 200 mg/kg/day) 
and included incomplete corpora lutea; ovarian follicular cysts; ovarian cystic, luteinized 
follicles; uterine atrophy; vaginal mucification; and altered cyclicity. Although these findings 
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were minimal to mild and were fully reversible (over 14 days), they were considered adverse and 
the next lower dosage evaluated, 20 mg/kg/day (10 mg/kg/dose BID) was the NOAEL in 
females. The Day 27 AUC0-12hr value associated with this dosage level was 6.9-fold the projected 
human efficacious AUC0-12hr value. The potential for aromatase inhibition effects occurring in 
female rats at AUC0-12hr values > 6.9-fold and < 53-fold the projected efficacious AUC0-12hr value 
has been addressed in a 13-week rat study. In this study using doses between the NOAEL and 
LOAEL in the 28-day study, the NOAEL for histologic lesions of the uterus that may be 
associated with aromatase inhibition resulted in an AUC0-12hr that was 26-fold the projected 
efficacious value. Notably, due to the potency difference of AG-348 against rat versus human 
aromatase inhibition, there is potential for a 4-fold wider margin for aromatase inhibition in 
humans versus rats. AGI-8702 is not an aromatase inhibitor. 
5.2.2. 
Summary of Clinical Data 
To date, 72 healthy adult volunteers have been exposed to AG-348 in 2 clinical studies, a single 
ascending dose (SAD) study and a multiple ascending dose (MAD) study, with 31 of these 
subjects exposed to a single AG-348 dose under fasted conditions; 5 exposed to 2 doses of 
AG-348 under fasted and fed conditions; and 36 exposed to repeated doses of AG-348 for up to 
14 days1. The following discussion of clinical data refers only to healthy adult volunteer 
subjects, as this is the first clinical trial in which patients with PK deficiency will be treated with 
AG-348. 
5.2.2.1. 
Pharmacokinetics 
The PK of AG-348 showed low to moderate variability between subjects. After single doses of 
AG-348 from 30 to 2500 mg, AG-348 was rapidly absorbed as indicated by the short time to 
maximum plasma concentration (Tmax). There appeared to be some lengthening of the period of 
absorption at higher doses as indicated by higher Tmax and a less than proportional increase in 
maximum concentration (Cmax). Dose-normalized area under the curve (AUC) generally 
remained constant over the dose range studied, suggesting that AG-348 total exposure increased 
in a dose-proportional manner. The mean t1/2 ranged from 17.8 to 20.4 hours when samples were 
collected through 72 hours and from 50.3 to 79.3 when samples were collected through 
120 hours. However, this terminal elimination phase contributed little to overall exposure of 
AG-348, as indicated by the small difference between AUC0-12 and area under the plasma 
concentration versus time curve from 0 to infinity (AUC0-∞), suggesting a shorter effective half-
life of approximately 3 to 6 hours. AG-348 was extensively distributed (mean apparent volume 
of distribution [Vz/F] range of 271 to 1148 L) and had a moderate rate of clearance (geometric 
mean clearance [CL/F] range of 10.3 to 13.9 L/hr). The fraction of AG-348 excreted in urine 
ranged from 0.0145 to 0.0209 across the dose levels suggesting that renal excretion plays a 
minor role in the systemic elimination of AG-348. 
The preliminary repeat-dose PK of AG-348 at doses ranging from 15 mg every 12 hours (q12h) 
to 700 mg q12h also showed an increase in AG-348 exposure in a dose-proportional manner 
after the first dose. AG-348 exposure observed after repeated dosing for 14 days at doses of 
1 At the time of this document, results from ongoing Study AG348-C-002 in healthy volunteers are blinded. Based 
on the randomization scheme, 36 subjects were assigned to AG-348 and 12 subjects were assigned to placebo. 
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120 mg every 24 hours (q24h) to 700 mg q12h were lower than observed on Day 1 after the first 
dose. The magnitude of the effect was greater at higher doses; lower doses of 15 mg q12h and 
60 mg q12h did not show this effect. This observation may be related to the nonclinical finding 
that AG-348 is a potential inducer of human CYP3A4, an enzyme which biotransforms AG-348. 
It is possible that multiple doses of AG-348 leads to an increased rate of its own metabolism. 
Evaluation of the effect of food on the PK of a single 700 mg dose of AG-348 in 5 subjects who 
were administered the drug fasting and then, after an appropriate wash-out period, re-
administered the drug following ingestion of a standard US Food and Drug Administration 
(FDA) high fat meal, showed that food likely has a minimal effect on the PK of AG-348. 
5.2.2.2. 
Pharmacodynamics 
After a single dose of AG-348, a decrease in the concentration of 2,3-DPG was observed at 
3 hours post-dose, decreased in a dose-dependent manner to a minimum at 24-hour post-dose, 
and then returned to values similar to baseline by 72 to 120 hours post-dose. The mean decrease 
at 24 hours was approximately 300 µg/mL at the 700 through 2500 mg dose levels. Similar 
decreases were observed after the first dose of multiple doses and prior to doses in the midst of 
the MAD study. After the final dose of multiple doses, the concentration of 2,3-DPG returned to 
values similar to baseline between 72 and 120 hours post- dose. 
After a single dose of AG-348, a minimal increase in the concentration of ATP was observed at 
24 to 120 hours post-dose. In the MAD study, no increase was observed at 12 hours after the first 
dose; the concentration of ATP was increased on Day 8 to concentrations greater than in the 
single dose study, continued to trend upward on Day 11, and remained at a similar level through 
Day 14. The concentration of ATP remained elevated through 120 hours after the last dose on 
Day 14. The magnitude of the increase in ATP was similar across the dose range from 60 mg 
BID to 700 mg BID. 
5.2.2.3. 
Safety 
Overall, AG-348 has been well-tolerated among healthy volunteers. 
After a single AG-348 dose, treatment-emergent adverse events (TEAEs) reported by > 1 subject 
at any time on study (either under fasted or fed conditions) included headache (22%), nausea 
(14%), and contact dermatitis and vomiting (each 6%). After repeated dosing of AG-348 for 14 
days, based on blinded data, TEAEs that occurred in > 2 subjects within any cohort and therefore 
must have been experienced by at least 1 AG-348-treated subject included dizziness, feeling hot, 
headache, hyperhidrosis, nausea, restlessness, and vomiting. 
A dose-relationship was apparent with regard to the incidence of gastrointestinal events, 
primarily nausea and vomiting, with the incidence of such events increasing with increasing 
dose. Nausea and/or vomiting were observed only at doses ≥ 1400 mg in the single dose study 
and only at doses ≥ 700 BID in the MAD study. Nausea and vomiting were not observed at any 
dose ≤ 360 mg in either the single or multiple dose studies. 
All but 1 TEAE reported to date has been mild or moderate (Grade 1 or 2) in intensity. The only 
Grade 3 TEAE was elevated liver transaminases (alanine aminotransferase [ALT]; aspartate 
aminotransferase [AST]) in a single subject in the MAD study treated with 700 mg of blinded 
study drug q12h BID. The treatment this subject received (AG-348 versus placebo) remains 
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blinded, and the events were considered to be study drug-related and led to study drug 
discontinuation, following which the elevated liver transaminases resolved. No AG-348-treated 
subject discontinued in the SAD study due to an adverse event (AE). Four subjects, at doses of 
700 mg BID, have discontinued study drug in the MAD study due to at least 1 TEAE, with 
nausea and vomiting contributing to study drug discontinuation in 3 of these 4 subjects. The 
fourth subject discontinued for a cutaneous rash. 
No deaths or other serious adverse events (SAEs) have been reported in any clinical study of 
AG-348. Furthermore, no DLTs have been declared after a single administration of AG-348 at 
doses of 30 mg to 2500 mg or after repeat administration of AG-348 for 14 days at daily doses of 
120 mg to 1400 mg (although the event of Grade 3 elevated liver function tests described above 
in the MAD study may be assessed as DLT after the subject’s treatment assignment is 
unblinded). 
Due to preclinical observations pertaining to the potential for inhibition of the aromatase enzyme 
(see Section 5.2.1.4), the AG348-C-002 study included assessment of baseline and serial 
measures of free and total serum testosterone and serum estradiol and estrone. The data are still 
blinded, and therefore, no quantitative statistical analyses have yet been performed. Upon 
qualitative review of the serum hormone data, at least some subjects in the 120 mg BID cohort 
and higher dose cohorts demonstrate modest increases in androgenic hormones and decreases in 
estrogens, compatible with a potential signal of aromatase inhibition. Additional analyses will be 
performed after the study is completed and the data are locked and unblinded. 
5.3. 
Study Rationale 
Study AG348-C-003 is the first study that will be conducted in patients with PK deficiency. This 
study is primarily intended to evaluate the safety and tolerability and potential indicators of 
clinical activity of AG-348 administered for up to 24 weeks. This study will also evaluate the PK 
profile of AG-348 and its metabolite AGI-8702, the PD responses in ATP and 2,3-DPG 
following administration of AG-348, and the clinical activity of AG-348 in PK deficiency 
patients. Two previous double-blind, placebo-controlled clinical trials of AG-348 conducted in 
healthy adult male and female volunteers (AG348-C-001, a SAD study; and AG348-C-002, a 
MAD study) have established an acceptable safety and tolerability profile for AG-348 for up to 
14 days of both once-daily (QD) and BID dosing at exposures that result in significant 
pharmacodynamic (PD) changes in whole blood levels of the glycolytic metabolites 2,3-DPG 
and ATP. Due to the potential for AG-348-mediated aromatase inhibition, dual-energy x-ray 
absorptiometry (DXA) scan (hip and spine) will be performed at Screening (if patient has not 
had prior DXA scan within 3 months of Day 1) to obtain T and Z scores. These data will serve as 
baseline measures of bone mineral density for all enrolling patients (see Section 7.1 for more 
details). 
The target population of this study consists of adult males and females with a diagnosis of PK 
deficiency, who are anemic but non-transfusion dependent. Non-transfusion dependent patients 
are preferred for this study in order to reduce any potential confounding effect of transfusion 
therapies on evaluation of potential indicators of clinical activity and PD responses. The safety, 
tolerability, and PK/PD findings in this study will form the basis for subsequent clinical 
development of AG-348. 
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5.3.1. 
Summary of Overall Safety Management Plan 
Measures to minimize the risks to patients enrolled in this study have been taken with respect to 
the following study design elements: 
• The initial doses for Arms 1 and 2 have been selected on the basis of safety and 
tolerability observations already carefully made in two predecessor clinical trials in 
adult healthy male and female volunteers;  
• The specified inclusion/exclusion criteria have been carefully considered to avoid 
enrollment of subjects for whom exposure to the study drug might pose a hazard; 
• A designated Data Review Team (DRT) consisting of the overall study Coordinating 
Investigator, treating Investigators, Medical Monitor, Clinical Pharmacologist, 
Statistician, and Sponsor’s Responsible Medical Officer has been established to meet 
regularly at approximately 6-week intervals throughout the duration of the study to 
review the accumulating study data and will exercise options to suspend enrollment to 
one or both of the initial two study dose arms, discontinue enrollment to one or both 
of the initial two study dose arms, adjust the dose of patients in one or both of the 
initial two study arms, and/or implement one new study dose arm. If one new dosing 
arm is implemented, the dose selected will not exceed 360 mg BID, the highest dose 
that demonstrated acceptable safety and tolerance in the 14-day multiple BID dosing 
study in healthy volunteers. Group cohort stopping rules for terminating enrollment 
into an arm based on the severity (CTCAEv4.03 grade) and frequency of AEs are 
defined;  
• Dose modification and stopping rules are defined for individual patients; 
• Guidance for permitted, prohibited, and cautionary concomitant medications is 
specified based on the estimated potential for drug-drug interactions from hepatic 
cytochrome enzyme interactions with AG-348. 
• Due to the potential for AG-348-mediated aromatase inhibition, bone mineral density 
will be monitored using DXA scans (hip and spine) at Baseline (if patient has not had 
prior DXA scan within 3 months of Day 1) and between Week 24 and Week 28. 
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6. 
TRIAL OBJECTIVES AND ENDPOINTS 
6.1. 
Primary Objective 
The primary objective of the study is to: 
• Evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in 
patients with PK deficiency. 
6.2. 
Secondary Objectives 
The secondary objectives of the study are to: 
• Evaluate the pharmacokinetics (PK) of AG-348 and the metabolite AGI-8702. 
• Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348. 
• Evaluate indicators of clinical activity of AG-348 in patients with PK deficiency, 
including changes in hemoglobin (Hb), HCT, reticulocyte count, haptoglobin (Hp), 
carboxyhemoglobin (COHb), lactate dehydrogenase (LDH), total and indirect 
bilirubin, erythropoietin (EPO), ferritin, and transferrin saturation (serum iron/iron 
binding capacity). 
6.4. 
Study Measures and Endpoints 
6.4.1. 
Safety Measures and Endpoints 
Safety will be evaluated by: 
• Monitoring of AEs, including determination of SAEs and AEs leading to 
discontinuation; safety laboratory parameters (hematology, chemistry, urinalysis, 
coagulation); physical examination findings; vital signs (VS); 12 lead 
electrocardiograms (ECGs); and DXA scans. Adverse events will be graded using 
Common Toxicity Criteria for Adverse Events (CTCAE), Version 4.03. Serum sex 
hormone levels (testosterone, estrone, and estradiol), bone turnover markers (serum 
N-terminal telopeptide [NTX] and serum C-terminal telopeptide [CTX]), total 
cholesterol, high-density lipoprotein-C (HDL-C), and triglycerides will be monitored 
for evidence of potential inhibition of aromatase by AG-348. 
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6.4.2. 
Clinical Activity Measures and Endpoints 
• Monitoring of potential indicators of clinical activity will include evaluating changes 
in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, 
ferritin, and transferrin saturation. 
6.4.3. 
Pharmacokinetic and Pharmacodynamic Measures and Endpoints 
The PK and PD profile of AG-348 will be evaluated by: 
• Approximately the first 10 patients treated, contingent on clinical site feasibility, will 
undergo extensive PK sampling as detailed in Appendix 15.1, Table 5. The remainder 
of treated patients will undergo limited PK sampling as detailed in Appendix 15.1, 
Table 6. Serial blood sampling for determination of concentration-time profiles of 
AG-348 and its metabolite AGI-8702 will be conducted following the first dose and 
the morning Day 15 dose, and additional trough levels of AG-348 and AGI-8702 will 
be obtained. AG-348 and AGI-8702 will be analyzed using qualified assays to 
determine concentrations in plasma. Pharmacokinetic parameters on Day 1 and 
Day 15 will be computed using standard non-compartmental methods based on 
observed plasma AG-348 and AGI-8702 concentrations. 
• Pharmacodynamic assessments will include 2,3-DPG, ATP (secondary objectives), 
 
 
Approximately the first 10 patients 
treated will undergo extensive PD sampling as detailed in Appendix 15.1, Table 5. 
The remainder of treated patients will undergo limited PD sampling as detailed in 
Appendix 15.1, Table 6. Serial blood sampling for determination of levels of ATP 
and, 2,3-DPG will be conducted following the first dose and the morning Day15 dose, 
and additional trough levels of ATP and 2,3-DPG will be obtained. ATP and 2,3 DPG 
will be analyzed using qualified assays to determine concentrations in whole blood. 
PD parameters on Day 1 and Day 15 will be computed based on observed whole 
blood ATP and 2,3-DPG concentrations.  
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7. 
INVESTIGATIONAL PLAN 
7.1. 
Overall Study Design 
Study AG348-C-003 is a Phase 2, open-label, two-arm, multicenter, randomized, dose-ranging 
study during which adult patients with PK deficiency will receive multiple doses of AG-348 for 
up to 24 weeks. Patients with PK deficiency confirmed by red blood cell PK enzymatic assay 
performed at Screening will be eligible to participate in this study. At Week 25, patients who 
safely tolerate AG-348 and demonstrate clinical activity of AG-348 may be eligible to 
immediately roll over to a separate safety extension study for continued treatment. Patients who 
finish treatment at the end of 24 weeks or sooner will undergo follow-up assessment 4 weeks 
after the last dose of study drug. Patients with toxicity suspected to be related to study drug will 
continue follow-up until the AE resolves, is declared chronic by the Investigator, or the patient is 
lost to follow-up. 
Initially, up to 25 patients will be randomized on an open-label, 1:1 basis to each of two BID 
doses of AG-348 (up to 50 patients; see Figure 3, Study Schema). The dose of Arm 1 is 300 mg 
of AG-348 administered orally q12h (BID). The dose of Arm 2 is 50 mg of AG-348 
administered orally q12h (BID) for 12 weeks. Randomization will be stratified by PKR mutation 
in order to maintain balance as much as possible across the dose arms for the specific mutations 
expected to be most frequently enrolled. The PKR mutation stratification factor will consist of 
4 levels (R510Q, R486W, and R479H) and all other mutations (“other”). Mutation status is 
defined by the presence of at least one of the indicated mutations; patients with more than one 
stratified mutation will be assigned based on Sponsor’s discretion. 
The doses for each arm have been selected from the forerunner AG348-C-001 SAD study and 
AG348-C-002 MAD studies in healthy adult volunteers to represent the range of doses/exposures 
that were safely tolerated and resulted in maximal or near maximal PD effects on 2,3-DPG and 
ATP. 
Because PK deficiency is a rare disease with a limited eligible patient population and because the 
underlying pathophysiology and clinical phenotype of affected patients is heterogenous due to 
the wide variety of mutations in PKR that cause the disease, it is deemed important to focus 
closely on dose findings in this first-in-patient study. Therefore, in addition to initiating this 
study with 2 different doses of AG-348 administered q12h, a DRT will be established to review 
study data on a frequent basis and adapt the study design, dose and schedule of AG-348 as 
indicated.  
The DRT will monitor safety on an on-going basis and meet at regular intervals (approximately 
every 6 weeks), or ad hoc as necessary, to review AEs, VS, clinical laboratory (hematology, 
clinical chemistry, coagulation, and urinalysis), and ECGs on enrolled patients. The DRT will 
also review available PK/PD data and indicators of clinical activity (e.g., changes from baseline 
in Hb). The DRT will be comprised of the study Coordinating Investigator, treating 
Investigators, Medical Monitor, Clinical Pharmacologist, Statistician, and Sponsor’s Responsible 
Medical Officer. 
Approximately every 6 weeks, beginning 6 weeks after the first patient is doses or ad hoc as 
necessary, the DRT will review cumulative safety data, available PK/PD data, and clinical 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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activity data. Based on the DRT’s recurring 6-week reviews, the DRT may exercise one or more 
of the following options: 
• Continue treatment and enrollment in existing arms without change. 
• Add 1 new dose arm (Arm 3) to enroll up to 25 patients at a dose to be determined; 
the dose for Arm 3 may be lower or higher than Arm 1 and Arm 2 doses, but will not 
exceed 360 mg q12h; and the dose regimen may be less frequent than q12h. 
• Terminate or suspend enrollment to allow further review of clinical data in Arm 1 
and/or Arm 2 (and/or potential Arm 3). Enrollment in an arm could be terminated or 
suspended to allow further review, for example, for unacceptable safety/tolerability, 
poor PD response, or lack of signs of clinical activity. 
• Re-assign patient’s doses and schedule in a terminated arm to match the dose and 
schedule of another arm of the study. In this case, the patients in the terminated arm 
will remain in their original arm, i.e., they will not count towards the enrollment 
quota of the arm whose dose and schedule is being adopted. 
• Implement specific genotype restrictions to enrollment in one or more arms to ensure 
representation of patients with genotypes of greatest clinical relevance. 
The DRT will perform these evaluations on a recurring 6-week basis. The data that the DRT will 
review to make these decisions is expected to include, but are not necessarily limited to, the 
following:  
• Safety Observations: all AEs; VS, clinical laboratory (hematology, clinical chemistry, 
coagulation, and urinalysis), and ECGs;  
• PK and PD Observations: including changes in 2,3-DPG and ATP; 
• Indicators of Clinical Activity: including changes in Hb, HCT, reticulocyte count, Hp, 
COHb, LDH, EPO, total and indirect bilirubin, ferritin, and transferrin saturation.  
If a third dose arm is implemented, the dose of AG-348 selected will not exceed 360 mg BID, as 
this was the highest dose that demonstrated acceptable safety and tolerance in the 14-day 
multiple BID dosing study in healthy volunteers. The PK/PD sampling schedule in a potential 
third dosing arm will be determined by Sponsor’s discretion and may follow either the extensive 
or limited PK/PD sampling schedules as specified. 
The DRT will monitor the safety data in an ongoing manner as described and may make a 
decision to terminate enrollment in an arm if unacceptable AEs are observed, PD activity is 
limited, and/or markers of clinical activity indicate no effect. Unacceptable AEs are defined as 
≥ Grade 3 AEs (using National Cancer Institute Common Terminology Criteria for Adverse 
Events [NCI CTCAEv4.03]) that are assessed as at least possibly related to AG-348, with the 
exception of Grade 3 headache, hot flash/flushing, nausea, vomiting, and/or diarrhea that is 
transient in duration (< 24 hours) or able to be medically managed to ≤ Grade 2 within 24 hours. 
The DRT may also exercise discretion to terminate enrollment in an arm if it observes a 
frequency of Grade 2 AEs that would make it difficult to support long-term dosing. 
Due to the potential for AG-348-mediated aromatase inhibition, dual-energy x-ray 
absorptiometry (DXA) scan (hip and spine) will be performed at Screening (if patient has not 
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had prior DXA scan within 3 months of Day 1) to obtain T and Z scores. These data are intended 
to serve as a baseline measure of bone mineral density for all enrolling patients, and are deemed 
of particular importance for those who may enter the longer term safety extension study after 
completing the current study. All patients will have a second DXA scan in the interval between 
Weeks 24 and 28. The safety extension study will separately address additional follow-up DXA 
scanning. 
As the number of enrolling arms changes in the study (for example, from 2 to 3), the 
randomization scheme will adjust to enable balanced randomization into each actively accruing 
arm. Randomization and stratification will cease in the event that only a single arm is left 
enrolling. 
Depending on possible early termination of 1 or both of the initial 2 arms, or the addition of a 
third arm, the study could enroll up to a maximum of 75 patients. 
Figure 3: 
Study Schema 
 
Abbreviations:  BID (q12h) = twice-daily (every 12 hours); DRT = data review team; PKR = pyruvate kinase red 
blood cell isoform; TBD = to be determined; w = weeks.  
The Investigator will monitor all patients for safety and tolerability. Modification of an 
individual patient’s dose of AG-348 will be based on AEs and observed changes in Hb levels as 
detailed in Section 9.7.1 and Section 9.7.2. 
Screening assessments will occur within 28 days prior to the first dose of study treatment. During 
the Treatment period, patients will attend visits at baseline (Day 1), weekly through Week 3 
(Days 8, 15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12), and 
monthly through Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate AG 348 and 
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demonstrate evidence of clinical activity of AG-348 through Week 24 may be eligible to 
immediately enter a separate extension study for continued treatment. For patients who finish 
treatment at the end of 24 weeks or sooner, or who elect not to enter the extension trial, study 
discharge will occur 4 weeks (Week 28 or earlier) following the last dose of study treatment at 
the final follow-up assessment. 
Safety assessments will include monitoring of AEs, including determination of SAEs and AEs 
leading to discontinuation; safety laboratory parameters (e.g., hematology, serum chemistry, 
coagulation studies, and urinalysis); physical examination findings; VS; 12 lead ECGs, and DXA 
scans. Additional safety assessments will include monitoring of sex hormone levels (testosterone 
[total and free], estrone, and estradiol), and bone turnover markers (NTX and CTX). Follow-up 
assessments will be conducted on Day 197 (Week 28) and will include physical examination, 
weight, performance status, VS, 12-lead ECGs, laboratory evaluations (hematology, Hp, EPO 
levels, serum chemistry, coagulation studies, urinalysis; lipids, hormonal testing), AEs, and 
transfusion record.  
Pharmacokinetic assessments will include serial blood sampling for PK profiles of AG-348 and 
its metabolite AGI-8702. Pharmacodynamic evaluations will include serial blood sampling for 
determination of levels of ATP and 2,3 DPG. Extensive PK/PD sampling will be conducted on 
the first approximately 10 patients total treated in Arms 1 and 2 (see Appendix 15.1, Table 5) 
while more limited PK/PD sampling will be conducted on additional patients treated if 
enrollment in Arm 1 or Arm 2 is expanded or if an alternate dosing arm is added (see 
Appendix 15.1, Table 6).  
 
 
 
 
7.2. 
Justification of the Study Design 
The primary and secondary objectives of this study are to evaluate the safety, tolerability, PK 
and PD, and indicators of clinical activity of AG-348 in patients with PK deficiency. The choice 
of dose and schedule of administration of AG -348 for Arms 1 and 2 was based on the highest 
safely tolerated dose (Arm 1: 300 mg BID) and the lowest dose with potentially relevant PD 
activity (Arm 2: 50 mg BID) from the forerunner AG348-C-002 MAD study in healthy 
volunteers. Decisions regarding continuing enrollment and treatment in these initial dose arms 
and/or implementation of an additional dose arm will be based on DRT review of safety, PK and 
PD data, and indicators of clinical activity collected from all patients treated in Arm 1 and 
Arm 2. This design was chosen to minimize risk to patients while allowing evaluation of safe 
and pharmacologically active dose levels of AG-348, and to allow the necessary flexibility to 
adjust dose and schedule should the safety, tolerability, PK, and/or PD be different in patients 
with PK deficiency compared with healthy volunteers.  
Additional safety measures intended to minimize risk to patients include monitoring of AEs by 
the DRT and specified provisions for individual patient dose modification as needed for safety 
and (potentially) large increases in Hb level (Section 9.7.1 and Section 9.7.2). Measures intended 
to maximize the opportunity for patients with demonstrated safety and tolerability to continue to 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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derive benefit from any observed clinical activity of AG-348 include the option for continued 
treatment in a separate extension study.  
A comprehensive series of safety evaluations, including laboratory parameters, physical 
examinations, VS, 12-lead ECGs, and monitoring for SAEs and AEs, will be conducted to 
evaluate the safety profile of AG-348 and to aid in the determination of the recommended dose 
for continued development. 
The study includes serial blood sampling across at least two different doses of the study drug to 
assess its PK and PD profiles.  
Consistent with the design of many Phase 2 studies, preliminary evaluation of the potential PD 
and clinical activity of AG-348 are secondary objectives of this study. The latter will include 
assessments of the PK/PD relationship between AG-348 and the biomarkers ATP and 2,3-DPG, 
 
7.3. 
Rationale for the Starting Dose and Dose Range 
Prior to execution of this study, Agios conducted two clinical studies of AG-348 in healthy 
volunteers, including a SAD study (AG48-C-001) and a MAD (14 day q12h) study 
(AG348-C-002). Available details of these studies are discussed in the current Investigator’s 
Brochure (IB). Between these two studies, 72 healthy human subjects have been dosed with 
AG-348. In vitro investigations, also reported in the IB, had previously demonstrated that 
AG-348 increased the activity of wild-type PKR approximately to the same extent as it did a 
series a recombinant mPKRs. Therefore it was deemed reasonable to study the safety, 
tolerability, PK, and PD of AG-348 in healthy subjects in a controlled Phase 1 setting as a more 
efficient means of obtaining information than in the actual rare disease population of patients 
with PK deficiency.  
The MAD study demonstrated that the exposures produced by AG-348 doses from 60 mg q12h 
to 360 mg q12h (including 120 mg q24h) resulted in maximal changes from baseline for the PD 
markers 2,3-DPG (reduction from baseline) and ATP (increase from baseline). The exposures 
resulting from doses less than 60 mg q12h were of lesser magnitude and the exposures resulting 
from doses greater than 360 mg q12h were of no greater magnitude than the aforementioned 
range. Therefore the starting doses for this first dose ranging study in patients with PK deficiency 
were selected to be 300 mg q12h (Arm 1) and 50 mg q12h (Arm 2). These doses were 
demonstrated to be safe and tolerable in the healthy volunteer studies. The availability of ATP is 
proposed as being critical for optimally maintaining RBC membrane integrity (see Section 5.1). 
The dose ranges from 50 mg q12h to 300 mg q12h may result in clinically effective modulation 
of PKR in PK deficiency patients if the mutated enzyme is responsive to AG-348 in a similar 
manner to the wild-type enzyme in healthy subjects. However, there are many different 
mutations in PKR that result in PK deficiency, and these mutations produce variable effects on 
the enzyme in terms of catalytic activity and thermal stability. It is not known if different 
mutations will respond clinically in a similar manner to the same exposure to AG-348. 
Therefore, it is prudent to study the range of safe and pharmacodynamically relevant doses as 
specified in this study, and to allow flexibility for the DRT to analyze the evolving study data to 
adapt the dose and schedule of administration of AG-348 to produce the optimal combination of 
safety, tolerability, and PD, and, potentially, clinical response. 
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7.4. 
Criteria for Study Termination 
This study may be prematurely terminated if, in the opinion of the Sponsor, there is sufficient 
reasonable cause. In the event of such action, written notification documenting the reason for 
study termination will be provided to each Investigator. 
Circumstances that may warrant termination include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to patients; 
• Insufficient adherence to protocol requirements; 
• Plans to modify, suspend, or discontinue the development of the study drug; 
• Other administrative reasons. 
Should the study be closed prematurely, all study materials must be returned to the Sponsor or 
the Sponsor’s designee. 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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8. 
STUDY POPULATION 
8.1. 
Number of Patients 
Up to approximately 75 patients may be enrolled in this study. 
8.2. 
Inclusion Criteria 
For entry into the study, patients must meet all of the following criteria during the Screening or 
other specified period: 
1. Signed written informed consent obtained prior to performing any study procedure, 
including screening procedures.  
2. Male or female, aged 18 years and older. 
3. Known medical history of PK deficiency. 
4. All patients must have documented clinical laboratory confirmation of PK deficiency by 
RBC pyruvate kinase enzymatic assay performed at Screening by a designated central 
laboratory. Patients with prior documentation of PK deficiency by RBC enzymatic assay 
will have a reconfirmation of this result during Screening as a condition of enrollment. 
a. In the event that a patient’s screening pyruvate kinase enzymatic assay is negative 
(i.e., shows normal pyruvate kinase activity), the patient will be eligible for 
enrollment if the genotyping shows a mutant genotype that has been previously 
documented in the literature to be associated with pyruvate kinase deficiency. If 
the genotyping shows a previously undescribed mutation in the PKR gene, then 
the eligibility for enrollment will be determined on a case-by-case basis by the 
Coordinating Investigator and Medical Monitor in discussion with the 
Investigator. If no mutation is defined, then the patient will not be eligible. 
5. All patients must have genotypic characterization of the mutant PKR gene performed by 
a designated central laboratory at Screening, unless genotype is available from the 
patient's participation in the Pyruvate Kinase Deficiency Natural History Study 
(NCT02053480). 
6. All patients must have genotypic characterization of the UGT1A1 gene performed by a 
designated central laboratory to document whether they may have underlying Gilbert's 
Disease. Patients with Gilbert’s Disease are eligible to enroll. 
7. Males must have Hb ≤ 12.0 g/dL; females must have Hb ≤ 11.0 g/dL. 
8. All patients must be considered transfusion independent as defined by: no greater than 
3 units of RBCs transfused in the 12-month period up to the first day of study dosing and 
no transfusions within 4 months of first day of study dosing. Patients who have received 
more transfusion support than described above will evaluated for eligibility on a case-by-
case basis by the Medical Monitor. 
9. Splenectomized patients: 
a. Must have undergone their procedure at least 6 months prior to Screening. 
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b. Must be current in their vaccinations for Pneumococcal Conjugate (PCV13), 
Pneumococcal Polysaccharide (PPSV23), Quadrivalent Meningococcal vaccine, 
and Haemophilus influenzae type b (Hib) as recommended by Centers for Disease 
Control and Prevention's (CDC) Advisory Committee on Immunization Practices 
(ACIP) or immunization advisory groups in Canada and the European Union (for 
patients enrolled in Canada and the EU). 
[http://www.cdc.gov/vaccines/schedules/downloads/adult/adult-schedule.pdf] 
[Any missing vaccinations may be administered during the screening period.] 
10. Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2. 
11. Patients must be taking at least 1 mg of folic acid daily for at least 21 days prior to first 
dose and continued daily during study participation. 
12. Adequate organ function, defined as: 
a. Serum AST and ALT ≤ 1.5 × upper limit of normal (ULN) (unless the increased 
AST is assessed by the Investigator as due to hemolysis). 
b. Normal or elevated levels of serum bilirubin. In patients with serum bilirubin 
> ULN, the elevation must be attributed to hemolysis with or without Gilbert's 
syndrome and must not be choledocholithiasis, cholecystitis, biliary obstruction, 
or hepatocellular disease. 
c. Serum creatinine ≤ 1.25 × ULN. If serum creatinine > 1.25 × ULN, then 24-hour 
measured or calculated (Cockcroft-Gault) glomerular filtration rate 
(GFR) ≥ 60 mL/min. 
d. Absolute neutrophil count (ANC) > 1.0 × 109/L. 
e. Platelet count ≥ 100 × 109/L. 
f. Activated partial thromboplastin time (aPTT) and international normalized ratio 
(INR) ≤ 1.25 × ULN, unless the patient is receiving therapeutic anticoagulants. 
13. Women of childbearing potential (WOCBP) must agree to abstain from sexual 
intercourse or to use an acceptable/effective method of contraception (i.e., condom plus 
spermicide, condom plus oral contraceptive, condom plus intrauterine device [IUD], 
condom plus diaphragm with spermicide) from as soon as feasible during the Screening 
period until 30 days following the last dose of AG-348. 
a. WOCBP include any female who has experienced menarche and who has not 
undergone successful surgical sterilization (hysterectomy, bilateral tubal ligation, 
and/or bilateral oophorectomy) or is not post-menopausal. Post-menopausal is 
defined as: 
i. Amenorrhea ≥ 12 consecutive months without another cause, and a 
documented serum follicle stimulating hormone (FSH) level 
> 35 mIU/mL; 
ii. Amenorrhea≥ 12 consecutive months in women ≥ 62 years old (FSH 
testing is not required). 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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14. WOCBP must have a negative serum or urine pregnancy test within 72 hours before start 
of AG-348 dosing. 
15. Women must not be breastfeeding.  
16. Male patients, with the exception of those who have undergone vasectomy at least 
6 months prior to Screening, must agree to abstain from sexual intercourse or, if sexually 
active, to use a condom with spermicide as contraception (regardless of their female 
partner's childbearing potential or their partner's use of their own contraception) from 
Day 1 of dosing until 30 days following the last dose of AG-348. 
8.3. 
Exclusion Criteria 
Patients who meet any of the following criteria at Screening or prior to dosing on Day 1 will not 
be enrolled in the study: 
1. Hemoglobin level > 12.0 g/dL if male; Hb > 11.0 g/dL if female. 
2. Additional diagnosis of any other congenital or acquired blood disorder, including 
glucose-6-phosphate-dehydrogenase (G6PD) deficiency, or any other hemolytic anemia 
process except for mild allo-immunization as a consequence of transfusion therapy. 
3. Iron overload (hemosiderosis or concurrent hemochromatosis) sufficiently severe to 
result in a clinical diagnosis by the Investigator of cardiac, hepatic, or pancreatic 
insufficiency. 
4. Prior bone marrow or stem cell transplant. 
5. Clinically symptomatic cholelithiasis or cholecystitis. (Prior cholecystectomy is not 
exclusionary. Patients with symptomatic cholelithiasis or cholecystitis may be rescreened 
once the disorder has been treated and clinical symptoms have resolved.) 
6. Currently enrolled in another therapeutic clinical trial involving on-going therapy with 
any investigational or marketed product or placebo. Concurrent participation in the 
Pyruvate Kinase Deficiency Natural History Study (NCT02053480) is permitted. 
7. Exposure to any investigational drug, device, or procedure within 28 days prior to 
Screening. 
8. Concurrent medical condition that could compromise participation in the study such as: 
a. Poorly controlled hypertension (defined as systolic blood pressure (BP) 
> 150 mm Hg or diastolic BP > 90 mm Hg) refractory to medical management. 
b. History of recent (within < 6 months from Screening date) congestive heart 
failure; myocardial infarction or unstable angina pectoris; or hemorrhagic, 
embolic, or thrombotic stroke; deep venous thrombosis; or pulmonary or arterial 
embolism. 
c. Currently active infection requiring the use of parenteral anti-microbial agents or 
that is ≥ Grade 3 (CTCAEv4.03) within 6 months of first dose. 
d. A pattern or frequency of post-splenectomy sepsis that in the assessment of the 
Investigator could reasonably be expected to interfere with the ability of the 
patient to complete the 24 week study participation. 
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e. Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) 
antibody with signs of active Hepatitis B or C virus infection.  
f. Positive test for human immunodeficiency virus (HIV) 1 or 2 antibody. 
g. Diabetes mellitus judged to be in poor control by the Investigator or requiring 
> 3 anti-diabetic agents counting insulin; use of insulin per se is not exclusionary.  
h. History of any primary malignancy with the exception of: curatively treated non-
melanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; 
or other primary tumor treated with curative intent and no known active disease 
present and no treatment administered during the last 3 years. 
9. Undergone major surgery within 6 months of first dose.  
10. Current or recent history of psychiatric disorder that in the opinion of the Investigator or 
Medical Monitor could compromise the ability of the patient to cooperate with study 
visits and procedures. 
11. Use of any of the restricted list of products known to strongly inhibit CYP3A4 
metabolism (Appendix 15.3, Table 7) within 5 days prior to Day 1 dosing; or to strongly 
induce CYP3A4 metabolism (Appendix 15.3, Table 8) within 28 days prior to Day 1 
dosing; or to strongly inhibit P-gp transporter (Appendix 15.3, Table 9) within 5 days 
prior to Day 1 dosing; or digoxin within 5 days prior to Day 1 dosing. 
12. Serum bilirubin > ULN attributable to factors other than hemolysis and/or Gilbert's 
syndrome. 
13. Male patients with heart-rate corrected QT (Fridericia's correction factor) QTcF 
interval > 450 msec, or female patients with QTcF interval > 470 msec with the 
exception of patients with a left bundle branch block (LBBB). Medical Monitor approval 
needed in patients with a LBBB. 
14. Cardiac dysrhythmias judged as clinically significant by the Investigator or requiring 
therapy with drugs that are primarily substrates of CYP3A4. 
15. History of allergy to sulfonamides if characterized by acute hemolytic anemia, 
anaphylaxis, or rash of erythema multiforme type or Stevens-Johnson syndrome. 
16. Any other medical or psychological condition deemed by the Investigator to be likely to 
interfere with a patient’s ability to understand and sign informed consent; cooperate with 
study visits, tests, and procedures; or otherwise safely and reliably participate in the 
study. 
8.4. 
Patient Identification and Registration 
Patients who are candidates for enrollment into the study will be evaluated for eligibility by the 
Investigator to ensure that the inclusion and exclusion criteria (see Section 8.2 and Section 8.3, 
respectively) have been satisfied and that the patient is eligible for participation in this clinical 
study. The site will submit to the Sponsor an Eligibility form for each eligible patient and the 
Medical Monitor will confirm eligibility for all patients prior to receipt of the first dose of 
AG-348. 
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8.5. 
Patient Randomization 
Patients who have been confirmed as eligible will be randomized in an equal ratio to a treatment 
arm (e.g., 1:1 or 1:1:1 depending on which arms are open). A randomization number will be 
assigned. The site will provide a request for randomization form (including the patient’s 
confirmed genotype) to a central randomization center. The randomization will be stratified by 
PKR mutation in order to maintain balance across the dose arms for the specific mutations 
expected to be most frequently enrolled. The PKR mutation stratification factor will consist of 
4 levels (R510Q, R486W, and R479H) and all other mutations (“other”). Since this is an open 
label study, randomization will not be blinded. 
Please refer to the study manual for the randomization procedure. 
8.6. 
Patient Withdrawal Criteria 
Patients have the right to withdraw from the study at any time for any reason. Patients may 
withdraw or be withdrawn from study-related procedures and treatments under the following 
conditions: 
• Withdrawal of consent; 
• Experiences unacceptable toxicity; 
• Development of an intercurrent medical condition that precludes further participation 
in the trial; 
• Patient requires use of a prohibited concomitant medication (Section 9.11.2); 
• Investigator decision; 
• Protocol violation: non-adherence to protocol requirements; 
• Pregnancy; 
• Lost to follow-up. 
Should a patient decide to withdraw, all efforts will be made to complete and report the protocol-
defined study observations up to the time of the patient’s withdrawal as completely as possible 
and to determine the reason for withdrawal. 
In the event a patient is withdrawn from the study, the Medical Monitor must be informed. If 
there is a medical reason for withdrawal, the patient will remain under the supervision of the 
Investigator until satisfactory health is returned. 
When a patient withdraws from the study, the primary reason for discontinuation must be 
recorded in the appropriate section of the electronic case report form (eCRF) and all efforts will 
be made to complete and report final study observations as thoroughly as possible. 
All AEs should be followed until resolution or for a period of 30 days from the last dose of study 
drug, whichever is shorter. Patients with toxicity suspected to be related to study drug will 
continue follow-up until the AE resolves, is declared chronic by the Investigator, or the patient is 
lost to follow-up. 
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8.7. 
Replacement of Patients 
Patients who drop out of the study prior to completing the first 12 weeks of assigned dosing for 
reasons other than AEs may be replaced at the Sponsor’s discretion. 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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9. 
STUDY TREATMENT 
9.1. 
Description of Study Drug 
AG-348 sulfate hydrate capsules will be provided as 25 mg or 100 mg (free-base equivalent) of 
AG-348 sulfate hydrate without excipients in Swedish orange (25 mg) or white opaque (100 mg) 
gelatin capsules (size 2 capsules for all dose strengths). 
All study drugs are for investigational use only and are to be used only within the context of this 
study. All study drug products will be supplied by the Sponsor. Please see the Investigator’s 
Brochure for further details regarding study drug. 
9.2. 
Study Drug Packaging and Labeling 
AG-348 sulfate hydrate capsules are packaged in white, high-density polyethylene (HDPE) 
induction sealed bottles with a child-resistant screw cap. 
Packaging and labeling will be prepared to meet all regulatory requirements. 
9.3. 
Study Drug Storage 
AG-348 sulfate hydrate drug capsules must be stored at room temperature of 15 to 30°C 
(59 - 86°F). 
All study drug products must be stored in a secure, limited-access location and may be dispensed 
only by the Investigator or by a member of the staff specifically authorized by the Investigator. 
9.4. 
Method of Assigning Patients to Treatment 
Up to a maximum of 25 patients will be randomized to any one of the dosing arms in this study. 
Randomization will be balanced 1:1 or 1:1:1, depending on whether 2 or potentially 3 arms are 
open, and will be stratified by PKR mutation (see Section 8.5). The dose and schedule of 
AG-348 each patient receives will be dependent upon which dose arm is open for enrollment 
when the patient qualifies for and is randomized into the study.  
9.5. 
Blinding 
This is an open-label study; no blinding methods will be used. 
9.6. 
Study Drug Preparation and Administration 
For the initial two treatment arms, (Arm 1 and Arm 2), AG-348 will be administered orally BID 
(approximately every 12 hours with a minimum of 10 hours between doses) over a 24 week 
treatment period. Starting with Day 1, dosing will be continuous; there will be no rest periods. 
Patients who do not meet any of the treatment withdrawal criteria (see Section 8.5) may continue 
treatment for the entire 24-week treatment period. 
Patients will be dispensed the appropriate number of Sponsor-packaged, labeled bottles to allow 
for 28 days of dosing until the next scheduled visit.  
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Patients will be given a dosing diary to be used for each 28-day dosing period. They should 
record relevant information regarding their study drug in the diary (e.g., confirmation that each 
daily dose was taken, reasons for missed doses). 
Treatment compliance will be assessed based on return of unused drug and the dosing diary (see 
Section 9.9). 
Patients should be instructed to take their daily dose at approximately the same times each day 
except for dosing on in-clinic visiting days.  
Patients who undergo extensive PK/PD sampling (see Appendix 15.1, Table 5) should be 
instructed from Week 3 on to bring the AM dose with them for in-clinic visits and to ingest the 
dose following PK/PD blood draws.  
Patients receiving limited PK/PD sampling (see Appendix 15.1, Table 6) should be instructed to 
bring the AM dose with them for all in-clinic visits and to take the AM dose following PK/PD 
blood draws.  
Patients receiving extensive PK/PD sampling on Day 1 and 15 will also have limited PKPD on 
other visit days. As a general rule, regardless of extensive or limited schedule, patients will bring 
in the AM dose for all visits and take this dose following PK/PD blood draws.  
A minimum of 10 hours between the AM and PM dose will be required on those dosing days. 
Each dose should be taken with a glass of water and consumed over as short a time as possible. 
AG-348 may be taken with or without food. Patients should be instructed to swallow capsules 
whole and to not chew the capsules. For patients who have difficulty swallowing tablet(s), the 
Medical Monitor should be contacted to discuss administration. 
Patients will receive their first dose of AG -348 in the clinic on Day 1 and then may take the 
remaining doses on an outpatient basis. 
9.7. 
Criteria for Dose Escalation, Dose Modification, or Discontinuation 
of Study Drug 
No intra-patient dose escalations will be permitted in this study unless the DRT decides to re-
assign patients’ doses and schedule in a terminated arm to match the dose and schedule of 
another arm of the study. In this case, the patients in the terminated arm will remain in their 
originally assigned arm; i.e., they will not count towards the enrollment quota of the arm whose 
dose and schedule is being adopted. All dosing modifications, as outlined below, will be 
implemented following discussions with the Medical Monitor. 
9.7.1. 
Dose Modification for Safety 
The Investigator will monitor all patients for safety and tolerability. Modification of the patient’s 
dose of AG-348 will be based on AEs and observed changes in Hb levels (see Section 9.7.2). 
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Table 1: 
Dose Modification for Adverse Events Deemed at Least Possibly Related to 
AG-348 
Adverse Events(s) 
AG-348 Dose Adjustment 
Grade 1 
None required. 
Grade 2 
None required; Investigator and Medical Monitor judgment to manage as for 
Grade 3. 
Grade 3 
Suspend dosing;  
If event resolves to Grade 1 or baseline within approximately 14 days of 
suspension, resume dosing with 1 dose level reduction (see Table 2 below).  
If event does not resolve to Grade 1 or baseline within approximately 14 days of 
suspension, permanently discontinue dosing, unless the benefits outweigh the 
risks of resuming treatment and are approved by the Medical Monitor. 
Grade 4 
Permanently discontinue dosing, unless the benefits outweigh the risks of 
resuming treatment and are approved by the Medical Monitor. 
Dose modifications for Grade 2 and 3 AEs should be maintained long enough for the 
Investigator to be confident that the reduced dose is being well tolerated before considering a 
request of the Medical Monitor to permit subsequent re-escalation to the former dose level. 
Dosing for an individual patient will be discontinued permanently for Grade 3 and Grade 4 AEs 
that do not resolve to Grade 1 or baseline within approximately 14 days of suspension of dosing, 
unless the benefits outweigh the risks of resuming treatment and are approved by the Medical 
Monitor. No patients may be re-escalated to their former dose level after a dose modification 
without discussion with the Medical Monitor.   
It should be noted that if the initial dose of 300 mg q12h BID selected for Arm 1 demonstrates an 
unacceptable safety profile resulting in multiple subjects undergoing dose modifications, the 
DRT may exercise its option to re-assign these patients’ dose and schedule to match the dose and 
schedule of another study arm (for example, Arm 2 of the study, or to match the dose and 
schedule of a [potential] Arm 3, if implemented). 
9.7.2. 
Dose Modification for Increase in Hemoglobin Level 
It is presently unknown to what magnitude, how rapidly, or even whether, AG-348 will result in 
increased Hb levels in patients with PK deficiency across the variety of potential PKR mutations 
that may be encountered during this study. Moreover, it is unknown whether any potential 
increases in Hb experienced by patients in this study may be a safety risk. As a conservative 
measure, this study incorporates guidance for dose modification based on potential large 
increases in Hb levels that exceed the midpoint of the typical normal range by gender. The 
Investigator will monitor all patients for changes in Hb levels and should adjust the dose of 
AG-348 as outlined in Table 2 per the following guidelines and with Medical Monitor approval. 
• Males: If Hb > 15 g/dL and confirmed with a second test, suspend dosing until Hgb 
≤ 13.5 g/dL; then resume dosing with a 1 dose level reduction.  
• Females: If Hb > 13.5 g/dL and confirmed with a second test, suspend dosing until 
Hb ≤ 12.5 g/dL; then resume with a 1 dose level reduction.  
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• The treating Investigator will discuss with the Medical Monitor questions relating to 
dose modifications on an as needed basis.  
Table 2: 
Dose Reduction Table (by Dosing Arm) 
Dose Group 
Starting Dose 
1st Dose Reduction 
2nd Dose Reduction 
Arm 1 
300 mg BID 
200 mg BID 
100 mg BID 
Arm 2 
50 mg BID 
25 mg BID 
TBD1 
Potential Arm 3 
TBD 
To approximately 
50-66% of initial dose 
To approximately 
25-33% of initial dose 
1 Dose to be determined by Medical Monitor. 
9.7.3. 
Stopping Criteria 
Dosing for an individual patient will be discontinued permanently for Grade 3 AEs that do not 
resolve to Grade 1 or baseline within approximately 14 days of suspension of dosing, and for 
Grade 4 AEs, unless the benefits outweigh the risks of resuming treatment and are approved by 
the Medical Monitor (Section 9.7.1). Other reasons for treatment termination are provided in 
Section 8.5. 
9.8. 
Duration of Patient Participation 
The duration of treatment for all patients on this study will be up to 24 weeks. Patients who 
safely tolerate and demonstrate one or more indicators of clinical activity of AG-348 may be 
eligible to immediately roll over to a separate safety extension study for continued treatment. 
9.9. 
Treatment Compliance 
During in-clinic visits, doses of AG-348 will be ingested by the patient under the supervision of 
clinical facility personnel. For at-home dosing, patients will be given a dosing diary to be used 
for the duration of the 24-week treatment period. Patients should record relevant information 
regarding their study drug in the diary (e.g., confirmation that each daily dose was taken, reasons 
for missed doses) and return the diary at each study visit. 
9.10. 
Study Drug Accountability 
Accountability for the study drug at the clinical facility is the responsibility of the Investigator. 
The Investigator will ensure that the study drug is used only in accordance with this protocol. 
Where allowed, the Investigator may choose to assign drug accountability responsibilities to a 
pharmacist or other appropriate individual. 
The Investigator or delegate will maintain accurate drug accountability records indicating the 
drug’s delivery date to the site, inventory at the site, use by each patient, and return to Agios or 
its designee (or disposal of the drug, if approved by Agios). These records will adequately 
document that the patients were provided the doses as specified in the protocol and should 
reconcile all study drug received from Agios. Accountability records will include dates, 
quantities, batch/serial numbers, expiration dates (if applicable), and patient numbers. An 
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unblinded monitor will review drug accountability at the site on a schedule agreed to by the 
Sponsor. 
Study drug must not be used for any purpose other than the present study.  
All unused and used study drug will be retained at the site until it is inventoried by the Study 
Monitor after database lock has occurred. All used, unused or expired study drug will be returned 
to Agios or its designee or, if authorized, disposed of at the study site per the site’s Standard 
Operating Procedures (SOPs) and documented. All material containing AG-348 will be treated 
and disposed of as hazardous waste in accordance with governing regulations. 
9.11. 
Prior and Concomitant Medications and Treatments 
9.11.1. 
Prior Medications and Procedures 
All medications administered and procedures conducted within 28 days prior to the first day of 
study drug administration are to be recorded on the source documentation and included in the 
eCRF. 
9.11.2. 
Prohibited Concomitant Therapy 
All concomitant medications and procedures administered from 28 days before administration of 
study drug through the last Follow-up Visit (Week 28/Day 197) must be recorded in the 
appropriate section of the source documentation and eCRF along with dosage information, dates 
of administration, and reason for use.  
The following are prohibited at all times during participation in this study: 
• Investigational drugs must be discontinued 28 days prior to the first dose of study 
drug; 
• Products known to strongly inhibit CYP3A4 metabolism (listed in Appendix 15.3, 
Table 7) must be discontinued within 5 days prior to Day 1 dosing; 
• Products known to strongly induce CYP3A4 metabolism (listed in Appendix 15.3, 
Table 8) must be discontinued within 28 days prior to Day 1 dosing; 
• Products known to strongly inhibit P-gp transporter (listed in Appendix 15.3, Table 9) 
must be discontinued within 5 days prior to Day 1 dosing; 
• Digoxin must be discontinued within 5 days prior to Day 1 dosing; 
• Hematopoietic stimulating agents (erythropoietins, granulocyte colony stimulating 
factors, thrombopoietins, etc) must be discontinued no less than 28 days prior to the 
first dose of study drug. [Folic acid 1 mg orally per day is required for all patients. 
B12 injections are permitted for patients with a prior diagnosis of B12 deficiency 
syndromes, but must be repleted to stability of the Hb and mean corpuscular volume  
(MCV) prior to enrollment in the study]; 
• Anabolic steroids, including testosterone preparations, administered for anemia must 
be discontinued no less than 28 days prior to the first dose of study drug;  
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• As the target population for this study consists of transfusion independent patients 
and transfusion of blood products could confound key endpoints of the study, blood 
transfusions of any type must be strictly avoided except in cases of compelling 
medical need. If medical circumstances permit, the Medical Monitor should be 
contacted for discussion before any transfusions are administered. 
• Other drugs that displace unconjugated bilirubin from albumin. 
Potential Drug Interactions 
Strong inhibitors of CYP3A4 (listed in Appendix 15.3, Table 7) are not permitted for use with 
AG-348. Based on modeling of AG-348 metabolism and tolerability data from the Phase 1 MAD 
study, moderate inhibitors of CYP3A4 do not appear to pose a risk to patients dosed with 
AG-348. 
In-vivo data from the Phase 1 MAD show AG-348 induces CYP3A4 and its own metabolism, 
presumably via CYP3A4. The use of strong CYP3A4 inducers in combination with AG-348 is 
expected to reduce AG-348’s efficacy. Therefore, administration of strong CYP3A4 inducers 
(listed in Appendix 15.3, Table 8) is not permitted with AG-348.  
Strong inhibitors of drug transport (listed in Appendix 15.3, Table 9) are not permitted for use 
with AG-348. 
Digoxin is not permitted for use with AG-348. 
Induction of CYP3A4 by AG-348 is expected to reduce the efficacy of certain sensitive co-
medications. Co-administration of AG-348 with the drugs in Appendix 15.3, Table 10 should be 
done with caution, as their efficacy may be reduced.  
Of note, women in the trial utilizing oral contraception must utilize barrier methods as per the 
Inclusion Criteria 14 (Section 8.2) while taking AG-348. 
The expected patient co-medications deferoxamine, deferasirox, and oral penicillin are not 
expected to interact with AG-348. 
9.11.3. 
Allowed Concomitant Therapy 
Medications and treatments other than those specified above are permitted during the study. All 
intercurrent medical conditions will be treated at the discretion of the Investigator according to 
acceptable local standards of medical care. Subjects may receive analgesics, antiemetics, anti-
infectives (including penicillins), and antipyretics as medically indicated and consistent with the 
guidance in the previous two sections. Patients may continue iron chelation therapy with 
deferoxamine and deferasirox. Patients must continue taking 1 mg of folic acid for the duration 
of the study. 
All concomitant medications, including transfusions of blood products, procedures performed 
during the study, including those used to treat AEs, will be recorded on the eCRF.  
9.11.4. 
Potential for Phototoxicity 
AG-348 may cause sensitivity to direct and indirect sunlight. Patients should be warned to avoid 
direct sun exposure. When exposure to sunlight is anticipated for longer than 15 minutes, the 
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patient should be instructed to apply factor 30 or higher sunscreen to exposed areas and wear 
protective clothing and sunglasses. 
9.11.5. 
Management of Nausea, Vomiting, and Diarrhea 
As the primary objective of this study is to assess the safety and tolerability of up to 24 weeks of 
AG-348, routine use of prophylactic anti-emetic and antidiarrheal or other classes of medications 
is prohibited. However, if patients experience AEs relating to nausea, vomiting, or diarrhea these 
may be treated at the Investigator's clinical discretion with recommended medications as follows: 
• Nausea/vomiting: allowed agents include standard clinical dosing with palonosetron 
(Aloxi), dolasetron (Anzemet), or promethazine (Phenergen). Promethazine is a 
substrate for CYP2B6, and it is presently unknown if the potential for 2B6 induction 
after AG-348 dosing could be sufficient to reduce the therapeutic effect of 
promethazine. Not recommended are aprepitant (Emend; CYP3A4 inhibitor), 
ondansetron (Zofran; CYP3A4 inducer), chlorpromazine (Thorazine; CYP3A4 
inducer), prochlorperazine (Compazine; CYP3A4 substrate, QT prolongation has 
been reported), and granisetron (Kytril; CYP3A4 substrate, QT prolongation has been 
reported);  
• Diarrhea: recommended management includes standard clinical dosing with 
Kaopectate or other non-absorbable anti-diarrheals, diphenoxylate/atropine (Lomotil), 
or loperamide (Imodium). Loperamide is the least preferred choice because it is both 
a substrate and inhibitor for CYP3A4, a substrate for CYP2B6, and a substrate for 
P-gp. 
• For the use of any medications not specifically mentioned above the Investigator may 
confer with the Sponsor’s Medical Monitor. 
9.11.6. 
Other Restrictions and Precautions 
Patients should be advised to refrain from altering their normal exercise routine for the first 
28 days of treatment with AG-348 as symptoms of anemia could theoretically temporarily 
worsen before the eventual potential beneficial effect of AG-348 on the Hb level becomes 
evident. The elevated blood levels of 2,3-DPG commonly seen in patients with PK deficiency 
may produce a right shift in the Hb-O2 dissociation curve. The effect of this is to enhance 
oxygen delivery at the tissue level, thus counteracting to some extent the physiologic 
consequences of the anemia. AG-348 was shown to produce rapid decreases (within the first 
12 hours following a single dose) in 2,3-DPG in two prior clinical trials with healthy adult male 
and female volunteers. In patients with PK deficiency who have elevated 2,3-DPG levels, it is 
theoretically possible that decreases in 2,3-DPG that precede correction of the anemia could 
result in a temporary decrease of oxygen delivery at the tissue level resulting in clinical 
symptoms (e.g., increased fatigue). 
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10. 
STUDY ASSESSMENTS 
10.1. 
Schedule of Assessments 
The Schedules of Assessments for this study are provided in Appendix 15.1.  
After obtaining written informed consent, patients will undergo screening evaluations. The 
Screening visit is to be conducted within approximately 28 days prior to first dose of study 
treatment.  
During the Treatment period, patients will attend visits at baseline (Day 1), weekly through 
Week 3 (Days 8, 15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 
12) and monthly through Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate and 
demonstrate one or more indicators of clinical activity of AG-348 through Week 24 may be 
eligible to immediately enter a separate extension study for continued treatment upon agreement 
of the treating Investigator and the Medical Monitor. For patients who finish treatment, Study 
Discharge will occur 4 weeks (Week 28 or earlier) following the last dose of study treatment at 
the final follow-up assessment. 
Whenever more than one assessment is scheduled for the same nominal time, the assessments 
should be performed in the order of least invasive to most invasive assessment (e.g., VS first, 
ECG, blood draw). The timing of these assessments should allow the PK blood draw to occur at 
the exact nominal time. The order of procedures may be revised with prior discussion between 
Sponsor and Site. 
Minor adjustments to the timing, number of planned safety monitoring procedures (e.g., VS, 
ECG, blood draw), and PK/PD assessments may be made during the course of the study based on 
collected data to ensure appropriate safety monitoring and will not require a protocol 
amendment. These minor changes will require prior approval from the Sponsor’s Medical 
Monitor (or Responsible Medical Officer) as well as appropriate documentation in the study 
records. The addition of new safety monitoring procedures or other assessments will require a 
protocol amendment. 
10.2. 
Informed Consent and Confirmation of Eligibility 
A complete description of the study is to be presented to each potential patient and a signed and 
dated informed consent is to be obtained before any study specific procedures are performed.  
Patient’s eligibility will be confirmed at Screening and within 24 hours prior to study treatment 
dose. If a patient is determined to be ineligible, the patient will be excluded from participation. 
10.3. 
Demographic Data, Medical and Medication History 
Patient demographic data, including gender, date of birth, age, race, and ethnicity, will be 
obtained at Screening. Collection of demographic data will be modified by country regulatory 
requirements, as appropriate.  
Medication history, including all relevant prior medical history and current medical conditions, 
will be obtained at the Screening assessment and on Day -1; all concomitant medications 
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administered and procedures conducted within 28 days prior to Day 1 should be reported in the 
source documentation and eCRF. Investigators will be asked to provide information on the 
patient’s history of any medical diagnoses (e.g., iron overload) and surgical procedures (e.g., 
splenectomy, cholecystectomy) pertaining to their diagnosis of PK deficiency and prior available 
complete blood counts (CBCs) over the preceding 6 months and transfusion history over the 
preceding 12 months prior to the date of signing informed consent.  
10.4. 
PKR Enzymatic Assay and PKR Genotyping 
Assessments for PKR enzymatic activity and PKR genotyping will be performed at Screening 
only for confirmation of study eligibility. PKR enzymatic assays will be conducted at 
 
. PKR genotyping will be conducted at 
 
. 
10.5. 
Safety Assessments 
10.5.1. 
Physical Examination, Height, and Weight 
A complete physical examination (genital and rectal examinations will be performed at the 
discretion of the Investigator) will be obtained at Screening, Baseline, Week 12, and the Follow 
up Visit (Week 28). For patients rolling over into extension study, the last physical examination 
will occur at Week 24. Limited focused physical examinations will be performed at all other 
visits. Any findings will be recorded on the eCRF. Height will be collected at Screening only. 
10.5.2. 
Vital Signs 
Vital signs, including systolic and diastolic BP, heart rate, respiratory rate, and temperature, will 
be obtained according to the according to the Schedule of Assessments (Appendix 15.1). Vital 
sign measurements will be repeated if clinically significant or machine/equipment errors occur. 
Out of range BP or heart rate measurements will be repeated at the Investigator’s discretion. Any 
confirmed, clinically significant vital sign measurements will be recorded as AEs. 
10.5.3. 
Electrocardiogram  
A 12-lead ECG will be obtained according to the Schedule of Assessments (Appendix 15.1). The 
ECGs will be measured using an ECG machine that calculates the heart rate and measures PR, 
QRS, QT, QTcB (Bazett correction formula), and QTcF (Frederica’s correction). Only QTcF 
(not QTcB) will be used for determination of eligibility. 
The 12-lead ECGs should be obtained following 5 minutes of recumbency. The Screening ECG 
will be performed at least 7 days prior to Day 1 dosing. ECGs will be repeated if clinically 
significant abnormalities are observed, if artifacts are present, or if machine/equipment errors 
occur. Any confirmed, clinically significant ECG findings will be recorded as AEs. 
10.5.4. 
DXA Scans 
DXA scans (hip and spine) will be performed at Screening to obtain T and Z scores that will 
serve as a baseline measure of bone mineral density for all enrolling patients. A second DXA 
scan will be conducted in the interval between Week 24 and Week 28 as indicated in the 
Schedule of Assessments (Appendix 15.1). 
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10.5.5. 
Safety Laboratory Assessments 
10.5.5.1. Hematology, Serum Chemistry, Coagulation Studies, and Urinalysis 
Laboratory values obtained prior to Screening and RBC antibodies obtained at Screening will be 
performed at a local laboratory. On-study clinical laboratory evaluations are to be performed by 
a central laboratory. If Investigators believe that it is clinically indicated to obtain safety 
laboratory results from their own local laboratories on the day of the subject's visit, and before 
the results are returned from the designated central laboratory, they are free to exercise their 
discretion to do so. Investigators should be aware that since the Hb level is a secondary endpoint 
of the study, it behooves them to minimize blood volumes drawn, and samples for the central lab 
must still be collected, since it will serve as the official lab result for this study. 
Clinical laboratory evaluations are to be collected according to the Schedule of Assessments 
(Appendix 15.1). In addition, all clinically significant laboratory abnormalities noted on testing 
will be followed by repeat testing and further investigated according to the judgment of the 
Investigator. Please note that serum estradiol and free and total testosterone will be collected in 
the AM at 2 time points during Screening at least 2 days apart in addition to Baseline/Day 1 
(total of 3 time points prior to Day 1 dosing).  
The following safety laboratory parameters are to be determined: 
Hematology: 
HCT, Hb, RBC count, absolute reticulocyte count, percent 
reticulocyte count, MCV, mean corpuscular Hb (MCH), mean 
corpuscular Hb concentration (MCHC), red cell distribution width 
(RDW), white blood cell (WBC) count with manual differential, 
ANC, and absolute lymphocyte count (ALC), and platelet count. 
G6PD and RBC antibody screen will be performed at Screening only 
Other 
EPO, Hp, COHb 
Serum Chemistry: 
alkaline phosphatase (ALP), sodium, potassium, chloride, calcium, 
magnesium, phosphorus, carbon dioxide (CO2), albumin, total 
protein, glucose, blood urea nitrogen (BUN), creatinine, uric acid, 
LDH, ALT, AST, total bilirubin, and indirect bilirubin. 
Sex Hormones: 
testosterone (total and free), estrone, and estradiol. FSH will only be 
performed at Screening for female patients only for confirmation of 
post-menopausal status. 
Bone Turnover 
serum NTX and CTX. 
Lipids 
total cholesterol, HDL-C, triglycerides. 
Iron Panel 
iron (Fe), total iron-binding capacity (TIBC), percent saturation, 
ferritin. 
Coagulation Studies: 
fibrinogen, aPTT, INR. 
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Urinalysis: 
color and appearance, pH, specific gravity, ketones, protein, glucose, 
bilirubin, nitrite, urobilinogen, pregnancy screen (dipstick), occult 
blood. Microscopic inspection of sediment will only be performed 
for cause or to investigate an abnormal dipstick finding per the 
Investigator’s discretion. 
10.5.5.2. Screening Serology 
A blood sample for serology, including HBsAg, HCV Ab screen, and HIV1 and HIV2 Ab, is to 
be collected from all patients at Screening.  
10.5.6. 
Adverse Events 
Each patient will be carefully monitored for the development of any AEs throughout the study 
from signing of the informed consent through all scheduled study follow-up visits, or withdrawal 
of consent, whichever occurs first. In addition, SAEs that are assessed as possibly or probably 
related to study treatment that occur > 30 days post-treatment also are to be reported. 
AEs will be evaluated by the Investigator and recorded as described in the Schedule of 
Assessments. On dosing visits, all AEs (elicited and spontaneously reported) will be 
continuously evaluated by the Investigator and recorded. At any non-dosing day visit, AEs will 
be evaluated by the Investigator and recorded. Any AEs already documented at a previous 
assessment and designated as ongoing will be reviewed at subsequent visits or assessment time 
points as necessary. If these AEs have resolved, this will be documented. 
All AEs will be graded using the CTCAE grading system (Appendix 15.2 ). 
Complete details on AE monitoring are provided in Section 11. 
10.6. 
Pharmacokinetic Assessments 
10.6.1. 
Blood Sample Collection and Pharmacokinetic Measurements During Dose 
Escalation 
The first approximately 10 patients treated, contingent on clinical site feasibility, will undergo 
extensive PK sampling as detailed in Appendix 15.1, Table 5. The remainder of treated patients 
will undergo limited PK sampling as detailed in Appendix 15.1, Table 6. In-clinic visits on 
Day 8 and Day 22 may be performed by the patient’s primary care physician if necessary and 
must be approved by the Sponsor on a case by case basis; in these instances PK sampling would 
not be required. 
The collection times for post-dose PK samples will start from the time that dosing is completed. 
(For example, a PK draw at 30 minutes will be collected 30 minutes after the last capsule has 
been ingested.) The completion time of each dose will be recorded. 
Procedures for sample collection and processing will be provided in a separate study manual. 
The actual time point of each sample collection will be recorded. 
Samples for PK and PD assessments may be retained for up to 2 years from collection.  
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10.7. 
Pharmacodynamic Assessments 
The first approximately 10 patients treated, contingent on clinical site feasibility, will undergo 
extensive PD sampling for 2,3-DPG and ATP as detailed in Appendix 15.1, Table 5. The 
remainder of treated patients will undergo limited PD for 2,3-DPG and ATP sampling as detailed 
in Appendix 15.3, Table 6. 
The collection times for post-dose PD samples will start from the time that dosing is completed. 
(For example, a PD draw at 30 minutes will be collected 30 minutes after the last capsule has 
been ingested.) The completion time of each dose will be recorded. 
Procedures for sample collection and processing will be provided in a separate study manual. 
The actual time point of each sample collection will be recorded. 
Pharmacodynamic assessments will include 2,3-DPG, ATP, 
 
 
 
 In-clinic visits on Day 8 and Day 22 may be 
performed by the patient’s primary care physician if necessary and must be approved by the 
Sponsor on a case by case basis; in these instances PK sampling would not be required. 
Figure 4 provides a brief schematic outlining the PKR reaction and how each of these PD 
assessments fits into a complete mechanistic understanding of the action of AG-348. 
Figure 4: 
PKR Enzymatic Reaction 
 
The PKR enzyme catalyzes the PEP to pyruvate reaction, with concomitant formation of ATP. 
17. Binding of AG-348 to the PKR tetramer can be assessed through an ex-vivo biochemical 
assay of cell lysates from AG-348 treated patients. Because WBCs contain a high level of 
pyruvate kinase from a non-PKR pyruvate kinase isoform, WBCs are first removed by 
filtration before the purified red cells are frozen.
 
 
 
18. 
 
 
 
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19. AG-348 target engagement 
 has been 
shown in preclinical models and healthy volunteer clinical studies to result in 
accumulation of ATP and depletion of the upstream metabolite 2,3-DPG. Therefore, 
levels of these metabolites will be measured by mass spectrometry in frozen whole blood 
samples. 
The first approximately 10 patients treated, contingent on clinical site feasibility, will undergo 
extensive PD sampling as detailed in Appendix 15.1, Table 5. The remainder of treated patients 
will undergo limited PD sampling as detailed in Appendix 15.1, Table 6. The collection times for 
post-dose PD samples will start from the time that dosing is completed. (For example, a PD draw 
at 30 minutes will be collected 30 minutes after the last capsule has been ingested.) The 
completion time of each dose will be recorded. 
Blood samples will be stored at the site and regularly transported at -80°C ±10 C to the 
bioanalytical laboratory for analysis. Procedures for sample collection and processing will be 
provided in a separate study manual. 
10.8. 
Ordering of Blood Sample Collection 
When more than 1 blood sample is collected at the same nominal time, the samples will be 
collected in the following order: 
1. Safety laboratory assessments 
2. PK  
3. PD (2,3 DPG, ATP) 
 
 
 
10.9. 
Sample Processing, Storage, and Shipment 
Instructions for the processing, storage and shipment of all study samples for central analysis 
will be provided in a separate study manual. 
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11. 
ADVERSE EVENTS 
Monitoring of AEs will be conducted throughout the study. Adverse events and SAEs will be 
recorded in the source documentation and eCRF from time of the signing informed consent 
through the Follow-up Visits. All AEs should be monitored until they are resolved or are clearly 
determined to be due to a patient’s stable or chronic condition or intercurrent illness(es). 
All SAEs will be followed through 30 days after the last dose of study treatment or until the SAE 
has resolved. Any SAEs that are assessed as possibly or probably related to study treatment that 
occur > 30 days post-treatment also are to be reported. 
11.1. 
Definition of Adverse Events 
11.1.1. 
Adverse Event (AE) 
An AE is any untoward medical occurrence associated with the use of a drug in humans, whether 
or not considered drug related. An AE (also referred to as an adverse experience) can be any 
unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a drug, without any judgment about causality. An AE can 
arise from any use of the drug (e.g., off-label use, use in combination with another drug) and 
from any route of administration, formulation, or dose, including an overdose. 
11.1.2. 
Suspected Adverse Reaction 
A suspected adverse reaction is any AE for which there is a reasonable possibility that the drug 
caused the AE. For the purposes of expedited safety reporting, ‘reasonable possibility’ means 
that there is evidence to suggest a causal relationship between the drug and the AE.  
11.1.3. 
Unexpected Adverse Event 
An unexpected AE is one for which the nature or severity of the event is not consistent with the 
applicable product information, e.g., the Investigator’s Brochure. 
11.1.4. 
Serious Adverse Event 
An AE or suspected adverse reaction is considered serious (SAE) if, in the view of either the 
Investigator or Sponsor, it results in any of the following outcomes: 
• Death; 
• Life-threatening. Life-threatening means that the patient was at immediate risk of 
death from the reaction as it occurred, i.e., it does not include a reaction which 
hypothetically might have caused death had it occurred in a more severe form; 
• In-patient hospitalization or prolongation of existing hospitalization. Hospitalization 
admissions and/or surgical operations scheduled to occur during the study period, but 
planned prior to study entry are not considered AEs if the illness or disease existed 
before the patient was enrolled in the study, provided that it did not deteriorate in an 
unexpected manner during the study (e.g., surgery performed earlier than planned); 
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• A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions; 
• Congenital anomaly/birth defect; 
• Important medical event. An important medical event is an event that may not result 
in death, be life-threatening, or require hospitalization but may be considered an SAE 
when, based upon appropriate medical judgment, it may jeopardize the patient or 
patient and 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 convulsions that do not result in in-patient hospitalization, 
or the development of drug dependency or drug abuse. 
11.1.4.1. Potential Severe Drug-Induced Liver Injury 
The document entitled FDA Guidance for Industry, Drug-Induced Liver Injury: Premarketing 
Clinical Evaluation (FDA 2009) provides guidance on how the measurement of various 
laboratory parameters may be used to assess a given drug’s potential to cause severe liver injury 
(i.e., irreversible liver failure that is fatal or requires liver transplantation). Such cases are 
suggested by the following three components: 
1. The drug causes hepatocellular injury, generally shown by a higher incidence of 3-fold or 
greater elevations above the ULN of ALT or AST than the (non-hepatotoxic) control 
drug or placebo; 
2. Among trial patients showing such aminotransferase elevations, often with 
aminotransferases much greater than 3×ULN, one or more also show elevation of serum 
total bilirubin to > 2×ULN, without initial findings of cholestasis (elevated serum 
elevated serum ALP); 
3. No other reason can be found to explain the combination of increased aminotransferases 
and total bilirubin, such as viral hepatitis A, B, or C; pre-existing acute liver disease; or 
another drug capable of causing the observed injury.  
Clinical safety laboratory results compatible with the definition of drug-induced liver injury 
(DILI) stated above must be repeated for confirmation as soon as possible, and if confirmed, will 
be scored as an unacceptable AE and reported to FDA as a serious unexpected AE. 
11.2. 
Procedures for Reporting Adverse Events and Serious Adverse 
Events  
Each patient must be carefully monitored for the development of any AEs. This information 
should be obtained in the form of non-leading questions (e.g., “How are you feeling?”) and from 
signs and symptoms detected during each examination, observations of study personnel, and 
spontaneous reports from patients. 
All AEs (serious and non-serious) spontaneously reported by the patient and/or in response to an 
open question from study personnel or revealed by observation, physical examination, or other 
diagnostic procedures will be recorded in the source documentation and eCRF. Any clinically 
relevant deterioration in laboratory assessments or other clinical findings is considered an AE 
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and must be recorded in the appropriate source documentation and eCRF. Treatment-emergent 
abnormal clinical laboratory results should generally be reported as AEs if there are 
accompanying symptoms; if additional diagnostic evaluations or medical (including drug 
therapy) or surgical interventions are undertaken; if a change in study drug dosing or study drug 
discontinuation is required; or, if the laboratory result is considered clinically significant by the 
Investigator. When possible, signs and symptoms indicating a common underlying pathology 
should be noted as one comprehensive event. 
All SAEs that occur during the course of the study must be promptly reported by the Investigator 
to 
 Global Safety and Pharmacovigilance (see below). Deaths and AEs assessed 
as life-threatening are to be reported immediately and SAEs that meet other criteria are to be 
reported within 24 hours from the point in time when the Investigator becomes aware of the 
SAE. All SAEs must be reported whether or not they are considered causally related to AG-348. 
Serious adverse event forms will be completed and the information collected will include subject 
number, a narrative description of the event, and an assessment by the Investigator as to the 
severity of the event and relatedness to study drug. Follow-up information on the SAE may be 
requested by the Sponsor or Medical Monitor. 
Contact Information: 
 Safety Management 
SAE Hotline (United States): 
 
SAE Fax (United States): 
 
SAE Hotline (France): 
 
SAE Fax (France): 
  
SAE Hotline (Netherlands): 
 
SAE Fax (Netherlands): 
 
SAE Hotline (Italy): 
 
SAE Fax (Italy): 
 
SAE Hotline (United Kingdom): 
 
SAE Fax (United Kingdom): 
 
E-mail: 
 
If there are serious, unexpected adverse drug reactions associated with the use of AG-348, the 
Sponsor will notify the appropriate regulatory agency(ies) and all participating Investigators on 
an expedited basis. The local 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. 
All AEs, whether serious or not, will be described in the source documents in the database. All 
new events, as well as those that worsen in intensity or frequency relative to baseline, which 
occur after signing the informed consent through the final Follow-up Visit (Day 29 ± 3 days) 
must be recorded. Adverse events that are ongoing at the time of treatment discontinuation 
should be followed up to 30 days after the last dose of study treatment. All SAEs will be 
followed up to 30 days after the last dose of study treatment, or until the SAE has resolved.  
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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); 
• The date of onset of the event; 
• The date of resolution of the event; 
• Whether the event is serious or not; 
• Intensity of the event (see below for definitions); 
• Relationship of the event to study treatment (see below for definitions); 
• Action taken: none; change in the study drug administration (e.g., temporary 
interruption in dosing); drug treatment required; non-drug treatment required; 
hospitalization or prolongation of hospitalization required (complete SAE page); 
diagnostic procedure performed; patient discontinued from the study (complete End 
of Study visit); 
• Outcome: patient recovered without sequelae; patient recovered with sequelae; event 
ongoing; patient died (notify the Medical Monitor immediately, and complete the 
SAE form). 
Intensity of all AEs will be graded according to the NCI CTCAE Version 4.03 (Appendix 15.1). 
Relationship to study drug administration will be determined by the Investigator according to 
the following criteria: 
• Not Related:  Exposure to the study treatment did not occur, or the occurrence of the 
AE is not reasonably related in time, or the AE is considered unlikely to be related to 
the study treatment. 
• Possibly Related:  The study treatment and the AE were reasonably related in time, 
and the AE could be explained equally well by causes other than exposure to the 
study treatment. An attribution of possibly related means that there are facts in 
evidence to suggest a possible relationship. 
• Probably Related:  The study treatment and the AE were reasonably related in time, 
and the AE was more likely explained by exposure to the study treatment than by 
other causes, or the study treatment was the most likely cause of the AE. An 
attribution of probably related means that there are facts in evidence to suggest a 
probable relationship. 
For the purpose of safety analyses related to final database review, all AEs that are classified as 
possible or probable will be considered treatment-related AEs. 
11.3. 
Pregnancy Reporting 
Pregnancy is neither an AE nor an SAE, unless a complication relating to the pregnancy occurs 
(e.g., spontaneous abortion, which may qualify as an SAE). However, any pregnancy in a 
participating female patient or a female partner of a participating male patient that occurs during 
this study or within 30 days following the last dose of AG-348 must be reported to the Sponsor 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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or Medical Monitor within 30 days of being notified of the pregnancy. The Investigator must 
follow up and document the course and outcome of all pregnancies even if the patient was 
discontinued from the study or if the study has finished. The female patient or partner of a male 
patient should receive any necessary counseling regarding the risks of continuing the pregnancy 
and the possible effects on the fetus. Monitoring should continue until conclusion of the 
pregnancy. 
All outcomes of pregnancy must be reported by the Investigator to the Sponsor or Sponsor’s 
designee on a Pregnancy Outcome Report form within 30 days after he/she has gained 
knowledge of the delivery or elective abortion. 
Any SAE that occurs during pregnancy must be recorded on the SAE report form (e.g., maternal 
serious complications, spontaneous or therapeutic abortion, ectopic pregnancy, stillbirth, 
neonatal death, congenital anomaly, or birth defect) and reported within 24 hours in accordance 
with the procedure for reporting SAEs. 
All patients, male and female, must agree to use effective contraception during the entire study 
and for 30 days following the last dose of AG-348. 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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12. 
STATISTICAL METHODS 
The primary objective of this Phase 2 study is to evaluate the safety and tolerability of up to 
24 weeks of AG-348 in patients with PK deficiency. Therefore, analyses will be primarily 
descriptive in nature; no formal hypothesis testing will be conducted. All analyses will be 
conducted separately within each dose arm, or pooled where appropriate. 
12.1. 
Sample Size Estimation 
Due to the rare disease setting, the minimal sample size in each dose arm may be determined by 
feasibility. In order to evaluate the primary objective of safety and tolerability of AG-348 in 
Arm 1 and Arm 2, up to a maximum of 25 patients may be randomized onto each arm. The 
actual number of patients enrolled into Arms 1 and 2 will depend on the safety reviews and 
decisions made by the DRT. In addition, up to 25 additional patients may be enrolled to evaluate 
an additional dose arm (Arm 3; see Section 7.1). As for Arms 1 and 2, the actual enrollment in a 
potential Arm 3 will depend on the safety reviews and decisions made by the DRT. Therefore, up 
to approximately 75 total patients may be enrolled in this study across 2 to 3 dose arms. 
Table 3 provides the probability within a dose arm of detecting 1 or more AEs with varying 
sample size and the true underlying AE rates. 
Table 3: 
Sample Size Estimation 
Sample Size 
True Underlying AE Rate 
15% 
10% 
5% 
5 
56% 
41% 
23% 
10 
80% 
65% 
40% 
15 
91% 
79% 
54% 
25 
98% 
93% 
72% 
 
12.2. 
Populations for Analysis 
The following patient populations (i.e., analysis sets) will be evaluated and used for presentation 
of the data: 
• Safety Analysis Set: All patients who are enrolled and receive any dose of study 
treatment. The Safety Analysis Set will be the primary set for the analysis of safety 
data. Patients will be classified according to treatment received, where treatment 
received is defined as the assigned treatment if it is received at least once, or as the 
first treatment received if assigned treatment is never received. Unless otherwise 
stated, the Safety Analysis Set will be the default analysis set for all data analyses. 
• Pharmacokinetic (PK) Analysis Set: All patients in the Safety Analysis Set with 
sufficient plasma sample data to assess PK parameters. Results of the potential PD 
activity of AG-348 will also be based on the PK analysis set.  
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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• Efficacy Analysis Set: All patients who are enrolled and achieve at least 50% 
compliance with their assigned dose intensity for at least 4 weeks of continuous 
dosing. Additional efficacy analyses may be performed on subsets of patients with 
other degrees of compliance with their assigned dose intensity and/or duration of 
dosing. The Efficacy Analysis Set will be the primary set for the analysis of 
preliminary clinical activity data. Patients will be classified according to assigned 
treatment. 
 
 
 If such analyses are performed, they will be 
described in a separate PK Statistical Analysis Plan (SAP) and may be reported separately in a 
stand-alone report. 
12.3. 
Procedures for Handling Missing, Unused, and Spurious Data 
No imputation will be performed for missing data elements. When summarizing AE data, partial 
dates will be imputed as described in the SAP. Additional rules addressing the handling of 
missing data will be detailed in the SAP. 
12.4. 
Interim Analysis 
No formal statistical interim analysis will be conducted. Safety data will be reviewed on an 
ongoing basis by the DRT, who will meet to review safety, PK, PD, and preliminary clinical 
activity data at regular intervals (approximately every 6 weeks) throughout the duration of the 
study. The DRT’s decisions to suspend, terminate, or open a potential third dosing arm, or re-
assign patients’ dosing in a terminated arm to match the dose and schedule of another arm of the 
study will be based on the totality of the data including, safety, PK, PD, and preliminary clinical 
activity (e.g., changes in Hb levels). 
Additional interim reviews of data may be conducted to support decision making concerning the 
current clinical study, the sponsor’s development programs in general, or in case of any safety 
concerns. 
12.5. 
Statistical Methodology 
12.5.1. 
General Methods 
This study will be primarily descriptive in nature; therefore, there will be no formal hypothesis 
testing. Summaries will be produced for disposition, baseline disease characteristics and 
demographic data including genotype, safety measurements, PK, PD parameters and indicators 
of clinical activity. Data from each AG-348 dose group will be analyzed separately, and pooled 
across all dose groups where appropriate. All data will also be listed by individual patient. 
Categorical variables will be summarized by frequency distributions (number and percentages) 
and continuous variables will be summarized by descriptive statistics (mean, standard deviation 
[SD], median, minimum, and maximum). 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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12.5.2. 
Disposition 
A summary of the disposition of patients will be presented, including the number enrolled, the 
number treated, and the reasons for study discontinuation. Entry criteria and protocol deviations 
will be listed. 
12.5.3. 
Exposure and Safety Analyses 
Patients will receive multiple PO doses of AG-348 over a 24-week treatment period. The actual 
dose and duration in days of AG-348, as well as the dose intensity (computed as the ratio of 
actual dose received and actual duration) and the relative dose intensity (computed as the ratio of 
dose intensity and planned dose received/planned duration) will be listed and summarized using 
descriptive statistics by dose arm. 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) 
and the incidence of TEAEs (new or worsening from baseline) will be summarized by primary 
system organ class (SOC), preferred term (PT), CTCAE Version 4.03 severity, outcome, action 
taken with study drug, and relationship to the study drug by dose group. Separate summaries will 
be produced for all TEAEs, treatment-related AEs (those considered by the Investigator as at 
least possibly drug related), SAEs, discontinuations due to AEs, and AEs ≥ Grade 3 severity. 
Individual patient listings will be provided for deaths, SAEs, AEs leading to interruption and/or 
reduction of study drug dose, and AEs leading to discontinuation of treatment. 
Analyses of AEs will be performed for those events that are considered treatment-emergent, 
where treatment-emergent is defined as any AE with onset beginning at the day of first 
administration of study drug, throughout the treatment period until 30 days after the last dose of 
study drug, or any event that was present at baseline but worsened in intensity or was 
subsequently considered drug-related by the Investigator through the end of the study. 
Descriptive statistics will be provided for clinical laboratory values (e.g., hematology, serum 
chemistry, coagulation studies, urinalysis) and VS data, presented as both actual values and 
changes from baseline relative to each on-study evaluation. Shift analyses will be conducted for 
selected laboratory parameters based on the baseline CTCAE grade to maximum CTCAE grade. 
Where applicable CTCAE terms do not exist, a grading system based on the upper and/or lower 
limits of normal will be used to classify the results. 
No formal hypothesis-testing analysis of AE incidence rates will be performed. 
Electrocardiogram analyses will include individual patient listings and summaries of abnormal 
and clinically significant ECG results. Actual values and changes from baseline in PR, QRS, and 
heart-rate corrected QT interval (QTc) intervals will be summarized by visit and dose arm. Full 
details of the QTc analysis including correction methods used will be described in the SAP. 
Concomitant medications will be coded using the WHO Drug Dictionary. Concomitant 
medications prior to and after the start of the study drug will be listed by patient, and 
summarized by ATC term and dose arm. 
12.5.4. 
Pharmacokinetic Analyses 
Descriptive statistics will be used to summarize PK parameters for the parent compound AG-348 
and the metabolite AGI-8702 for each dose group, and where appropriate, for the entire 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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population. Pharmacokinetic parameters will be summarized using the following descriptive 
statistics: n, mean, SD, coefficient of variation %, median, minimum and maximum, geometric 
mean, and geometric coefficient of variation. PK analyses will be described in a separate PK 
SAP. 
12.5.5. 
Pharmacodynamic Analyses 
Descriptive statistics will be used to summarize PD parameters for 2,3-DPG and ATP for each 
dose group, and where appropriate for the entire population. Pharmacodynamic parameters will 
be summarized using the following descriptive statistics: n, mean, SD, coefficient of variation %, 
median, minimum, and maximum, geometric mean, and geometric coefficient of variation %. PD 
analyses will be described in a separate PD SAP. 
12.5.6. 
Aromatase Hormone Analysis  
The analyses of serum sex hormones will use appropriate graphic displays and statistical 
analyses to evaluate patient change from baseline for each parameter. These analyses will 
include summaries of actual values and change from baseline using appropriate descriptive 
statistics (mean, SD, median, min and max) as described above.  
 
These analyses will present information by each dose arm, and analyses of a pooled 
AG-348 cohort. Additional details regarding these analyses will be provided in the SAP. 
12.5.7. 
Clinical Activity 
Details on analyses to evaluate indicators of potential clinical activity of AG-348 in patients with 
PK deficiency will be described in the SAP. These will include changes in Hb, HCT, reticulocyte 
count, Hp, COHb, LDH, total and indirect bilirubin, EPO, ferritin, and transferrin saturation 
(serum iron/iron binding capacity). Characterization of Hb response which will include, but is 
not limited to percent of patients with increase in Hb, time to Hb response, and duration of Hb 
response will be explored. 
12.6. 
Procedures for Reporting Deviations to Original Statistical Analysis 
Plan 
All deviations from the original SAP will be provided in the final clinical study report. 
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13. 
ADMINSTRATIVE REQUIREMENTS 
13.1. 
Good Clinical Practice 
The study will be conducted in accordance with the International Conference on Harmonization 
(ICH) for Good Clinical Practice (GCP) and the appropriate regulatory requirement(s). The 
Investigator will be thoroughly familiar with the appropriate use of the study drug as described in 
the protocol and Investigator’s Brochure. Essential clinical documents will be maintained to 
demonstrate the validity of the study and the integrity of the data collected. Master files should 
be established at the beginning of the study, maintained for the duration of the study, and 
retained according to the appropriate regulations. 
13.2. 
Ethical Considerations 
The study will be conducted in accordance with ethical principles founded in the Declaration of 
Helsinki (Appendix 15.4). 
The Investigator must obtain IRB approval for the investigation and must submit written 
documentation of the approval to the Sponsor before he or she can enroll any patient into the 
study. The IRB will review all appropriate study documentation in order to safeguard the rights, 
safety, and well-being of the patients. The study will only be conducted at sites where IRB 
approval has been obtained. The protocol, Investigator’s Brochure, informed consent, 
advertisements (if applicable), written information given to the patients, signed Dose Escalation 
Interim Safety Reports, annual progress reports, and any revisions to these documents will be 
provided to the IRB. The IRB is to be notified of any amendment to the protocol in accordance 
with local requirements. Progress reports and notifications of serious unexpected adverse drug 
reactions are to be provided to the IRB according to local regulations and guidelines. 
13.3. 
Patient Information and Informed Consent 
The Investigator or trained designee will ensure that the patient is given full and adequate oral 
and written information about the nature, purpose, possible risk, and benefit of the study. Patients 
must also be notified that they are free to discontinue from the study at any time. The patient 
should be given the opportunity to ask questions and allowed time to consider the information 
provided. 
After the study has been fully explained, written informed consent will be obtained from the 
patient prior to study participation.  
The patient’s signed and dated informed consent must be obtained before conducting any study-
related procedures. The Investigator must maintain the original, signed consent form. A copy of 
the signed form must be given to the patient. 
The method of obtaining and documenting the informed consent and the contents of the consent 
will comply with ICH-GCP and all applicable regulatory requirement(s). 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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13.4. 
Patient Confidentiality 
In order to maintain patient privacy, all source documents, study drug accountability records, 
study reports and communications will identify the patient by the assigned patient number. The 
Investigator will grant monitor(s) and auditor(s) from the Sponsor or its designee and regulatory 
authority(ies) access to the patient’s original medical records for verification of data gathered on 
the source documents and to audit the data collection process. The patient’s confidentiality will 
be maintained and will not be made publicly available to the extent permitted by the applicable 
laws and regulations. 
13.5. 
Protocol Compliance 
The Investigator will conduct the study in compliance with the protocol. Modifications to the 
protocol should not be made without agreement of both the Investigator and the Sponsor. 
Changes to the protocol will require written IRB approval/favorable opinion prior to 
implementation, except when the modification is needed to eliminate an immediate hazard(s) to 
patients. The IRB may provide, if applicable, where regulatory authority(ies) permit, expedited 
review and approval/favorable opinion for minor change(s) in ongoing studies that have the 
approval/favorable opinion of the IRB. The Sponsor or designee will submit all protocol 
modifications to the regulatory authority(ies) in accordance with the governing regulations.  
When immediate deviation from the protocol is required to eliminate an immediate hazard(s) to 
patients, the Investigator will contact the Sponsor’s Medical Monitor (or Responsible Medical 
Officer), if circumstances permit, to discuss the planned course of action. Any departures from 
the protocol must be fully documented in the source documents/database. 
13.6. 
Data Management 
A paper or eCRF will be completed for each patient or an electronic data capture system will be 
used. The electronic data capture system (EDC) (Medidata Rave®) is a software tool designed to 
ensure quality assurance and facilitate data capture during clinical trials. Through a system 
regulated workflow that includes barcode scanning and interfaces to medical equipment to avoid 
manual data entry, study operations performance is controlled and captured in real time. The 
system is fully Code of Federal Regulations (CFR) 21 Part 11 compliant. Source documentation 
supporting the data should indicate participation in the study and should document the dates and 
details of study procedures, AEs, and patient status. The Investigator, or trained designee should 
complete and the Investigator should verify the source documents as the information is collected. 
Any outstanding entries must be completed immediately after the final examination. An 
explanation should be given for all missing data. The Investigator will ensure the accuracy, 
completeness, and timeliness of the data reported to the Sponsor. Data collection processes and 
procedures will be reviewed and validated to ensure completeness, accuracy, reliability, and 
consistency. A complete audit trail will be maintained of all data changes. The Investigator or 
designee will cooperate with the Sponsor’s representative(s) for the periodic review of study 
documents to ensure the accuracy and completeness of the data capture system at each scheduled 
monitoring visit. Electronic consistency checks and manual review will be used to identify any 
errors or inconsistencies in the data. This information will be provided to the respective study 
sites by means of electronic or manual queries. The Investigator or designee will prepare and 
maintain adequate and accurate study documents (medical records, ECGs, AE, and concomitant 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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medication reporting, raw data collection forms, etc.) designed to record all observations and 
other pertinent data for each patient receiving study treatment. The Investigator will allow 
Sponsor representatives, contract designees, authorized regulatory authority inspectors, and the 
IRB to have direct access to all documents pertaining to the study. 
13.7. 
Source Document/Case Report Form Completion 
Source documents will be completed for each study patient. It is the Investigator’s responsibility 
to ensure the accuracy, completeness, and timeliness of the data reported in the patient’s source 
document. The source document should indicate the patient’s participation in the study and 
should document the dates and details of study procedures, AEs and patient status. 
The Investigator, or designated representative, should complete the source document as soon as 
possible after information is collected, preferably on the same day that a study patient is seen for 
an examination, treatment, or any other study procedure. Any outstanding entries must be 
completed immediately after the final examination. An explanation should be given for all 
missing data. 
The Investigator will sign and date each required assessment for all study patients. The 
Investigator will retain all completed source documents. 
13.8. 
Direct Access to Source Data 
The study will be monitored by the Sponsor or its designee. Monitoring will be done by personal 
visits from a representative of the Sponsor (site monitor) and will include on-site review of the 
source documents for completeness and clarity, cross-checking with source documents, and 
clarification of administrative matters will be performed. The review of medical records will be 
performed in a manner to ensure that patient confidentiality is maintained. 
The site monitor will ensure that the investigation is conducted according to protocol design and 
regulatory requirements by frequent communications (letter, telephone, e-mail, and fax). 
All unused study drug and other study materials are to be returned to the Sponsor or designee 
after the study has been completed and the database has been locked. 
Regulatory authorities, the IRB, and/or the Sponsor’s clinical quality assurance group or 
designee may request access to all source documents, database, and any other applicable study 
documentation for an on-site audit or inspection. Direct access to these documents must be 
guaranteed by the Investigator, who must provide support at all times for these activities. 
13.9. 
Record Retention 
The Investigator will maintain all study records according to ICH-GCP and applicable regulatory 
requirement(s). Records will be retained for at least 2 years after the last marketing application 
approval or 2 years after formal discontinuation of the clinical development of the investigational 
product or according to applicable regulatory requirement(s). If the Investigator withdraws from 
the responsibility of keeping the study records, custody must be transferred to a person willing to 
accept the responsibility. The Sponsor must be notified in writing if a custodial change occurs. 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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13.10. 
Liability and Insurance 
The Sponsor has subscribed to an insurance policy covering, in its terms and provisions, its legal 
liability for injuries caused to participating persons and arising out of this research performed 
strictly in accordance with the scientific protocol as well as with applicable law and professional 
standards.  
13.11. 
Publication of Study Findings and Use of Information 
All information regarding AG-348 supplied by the Sponsor or designee to the Investigator is 
privileged and confidential information. The Investigator agrees to use this information to 
accomplish the study and will not use it for other purposes without consent from the Sponsor. It 
is understood that there is an obligation to provide the Sponsor with complete data obtained 
during the study. The information obtained from the clinical study will be used towards the 
development of AG-348 and may be disclosed to regulatory authority(ies), other Investigators, 
corporate partners, or consultants as required. 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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14. 
LIST OF REFERENCES 
 
Beutler E, and Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene 
frequency in the general white population. Blood. 2000;95(11):3585-8. 
Christensen RD, Yaish HM, Johnson CB, Bianchi P, and Zanella A. Six children with pyruvate 
kinase deficiency from one small town: molecular characterization of the PK-LR gene. J Pediatr. 
2011;159(4):695-7. 
FDA. Guidance for Industry: Drug-Induced Liver Injury: Premarketing Clinical Evaluation. 
2009. 
Ghidini A, Sirtori M, Romero R, Yarkoni S, Solomon L, and Hobbins JC. Hepatosplenomegaly 
as the only prenatal finding in a fetus with pyruvate kinase deficiency anemia. Am J Perinatol. 
1991;8(1):44-6. 
Hennekam RC, Beemer FA, Cats BP, Jansen G, and Staal GE. Hydrops fetalis associated with 
red cell pyruvate kinase deficiency. Genet Couns. 1990;1(1):75-9. 
Rider NL, Strauss KA, Brown K, Finkenstedt A, Puffenberger EG, Hendrickson CL, et al. 
Erythrocyte pyruvate kinase deficiency in an old-order Amish cohort: longitudinal risk and 
disease management. Am J Hematol. 2011;86(10):827-34. 
Tanphaichitr VS, Suvatte V, Issaragrisil S, Mahasandana C, Veerakul G, Chongkolwatana V, et 
al. Successful bone marrow transplantation in a child with red blood cell pyruvate kinase 
deficiency. Bone Marrow Transplant. 2000;26(6):689-90. 
van Wijk R, and van Solinge WW. The energy-less red blood cell is lost: erythrocyte enzyme 
abnormalities of glycolysis. Blood. 2005;106(13):4034-42. 
Zanella A, Fermo E, Bianchi P, and Valentini G. Red cell pyruvate kinase deficiency: molecular 
and clinical aspects. Br J Haematol. 2005;130(1):11-25. 
 
 
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Protocol AG348-C-003, Amendment 1, Version 2.0 
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15. 
APPENDICES 
 
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Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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15.1. 
Schedules of Assessments 
Table 4: 
Schedule of Assessments:  
Timing 
Pre-
Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow Up 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
1 
81 
15 
221 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Written Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
PK enzyme assay 
(confirmation of PK 
deficiency) 
X 
 
 
 
 
 
 
 
 
 
 
 
PKR Genotype 
(for randomization) 
X 
 
 
 
 
 
 
 
 
 
 
 
UGT1A1 Genotype 
X 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical/Surgical History 
(General and PK 
deficiency-specific)2 
X 
 
 
 
 
 
 
 
 
 
 
 
Medication History 
X 
 
 
 
 
 
 
 
 
 
 
 
Transfusion History 
X 
 
 
 
 
 
 
 
 
 
 
 
Confirmation of 
Vaccinations 
(Splenectomized Patients) 
X 
 
 
 
 
 
 
 
 
 
 
 
Physical Examination3/ 
Height3 and Weight 
X 
X 
 
 
X 
 
X 
X 
X 
X 
X 
X 
Performance Status 
X 
X 
 
 
X 
 
X 
X 
X 
X 
X 
X 
Vital signs  
(BP, HR, RR, T)4 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG5 
X 
X 
X 
 
X 
 
 
 
 
 
X 
X 
DXA Scan6 
X 
 
 
 
 
 
 
 
 
 
X7 
 
Laboratory Evaluations8: 
 
 
 
 
 
 
 
 
 
 
 
 
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Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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Timing 
Pre-
Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow Up 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
1 
81 
15 
221 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
HBsAg, HCV Ab, HIV1 
and 2 Ab 
X 
 
 
 
 
 
 
 
 
 
 
 
RBC antibody Screen  
X 
 
 
 
 
 
 
 
 
 
 
 
Hematology (CBC)9 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin10 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
EPO levels11 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
G6PD screen 
X 
 
 
 
 
 
 
 
 
 
 
 
Serum Chemistry12 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Iron Panel13 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Carboxyhemoglobin 
(COHb) 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
 
Coagulation Studies14 
X 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Urinalysis15 
X 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Serum or Urine 
Pregnancy16 
X 
X 
 
 
 
 
 
 
 
 
 
 
Lipids17 
 
X 
 
 
 
X 
 
X 
 
 
X 
X 
Hormonal Testing18 
X 
X 
 
 
 
 
 
X 
 
 
X 
X 
Serum NTX and CTX 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Randomization19 
X 
 
 
 
 
 
 
 
 
 
 
 
Study Drug Administration 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Dispense Study Drug20 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
PK blood sampling21 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
PD Assessments21 
 
 
 
 
 
 
 
 
 
 
 
 
2,3 DPG/ATP 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
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Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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Timing 
Pre-
Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow Up 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
1 
81 
15 
221 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Adverse Events23 
 
Continuous 
X 
Transfusion Record 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant Medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Rollover to extension study 
 
 
 
 
 
 
 
 
 
 
X 
 
Abbreviations:  Ab = antibody; ATP = adenosine triphosphate; BP = blood pressure; CBC= complete blood count; COHb = carboxyhemoglobin; CTX = C-
terminal telopeptide; D = day; DPG = diphosphoglycerate; DXA = Dual-energy x-ray absorptiometry; ECG = electrocardiogram; EPO = erythropoietin; 
FSH = follicle stimulating hormone; G6PD = glucose-6-phosphate-dehydrogenase; HBsAg = hepatitis B surface antigen; HCV Ab = hepatitis C virus antibody; 
HDL-C = high-density lipoprotein-C; HIV = human immunodeficiency virus; HR = heart rate; NTX = N-terminal telopeptide; PD = pharmacodynamic; PK = 
pharmacokinetic; PK deficiencyD = pyruvate kinase deficiency; PKR = pyruvate kinase isoform R; RR = resting rate; W = week. 
Whenever more than one assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive 
assessment (e.g., VS, ECG, blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time. The order of 
procedures may be revised with prior discussion between Sponsor and site. 
 
1 In-clinic visits on Day 8 and Day 22 may be performed by the patient’s primary care physician if necessary and must be approved by the Sponsor on a case by 
case basis; in these instances PK/PD sampling would not be required and dispensing of study medication would not be performed. 
2 Medical history, including all relevant prior medical history, current medical conditions, and hematology profile (CBCs) over prior 6 months, will be obtained 
at the Screening assessment. 
3 A complete physical examinations (genital and rectal examinations will be performed at the discretion of the Investigator) will be obtained at Screening, 
Baseline, Week 12, and the Follow up Visit (Week 28), or Week 24 for patients rolling over into extension study. Limited focused physical examinations will 
be performed at all other specified visits. Height to be collected at Screening only. 
4 Vital signs will include systolic and diastolic blood pressure, heart rate, respiratory rate, and temperature.  
5 12-lead ECGs are to be conducted after 5 minutes of recumbency. Screening ECG will be performed at least 7 days prior to Day 1 dosing. 
6 If a DXA scan of the hip and spine with T and Z scores has been performed within 3 months preceding the first day of dosing, this may be used to meet this 
requirement.  
7 Week 24 DXA scan may be performed anytime between Weeks 24 and 28 and must be performed at the same imaging center on the same DXA machine as the 
original Screening DXA scan. 
77 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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8 Laboratory evaluations (hematology, serum chemistry, coagulation studies, and urinalysis) are to be collected in the morning. These should be collected 
following an overnight fast on Baseline Day 1 Week 6 (Day 43), Week 12 (Day 85), Week 24 (Day 169), and Follow-up Week 28 (Day 197), when the lipid 
samples are also included. 
9 Complete blood count (CBC) to be performed at Screening and prior to dosing on Day 1 and will include HCT, Hb, red blood cell (RBC) count, absolute 
reticulocyte count, percent reticulocyte count, mean corpuscular volume (MCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), 
red cell distribution width (RDW), white blood cell (WBC) count with manual differential, absolute neutrophil count (ANC), absolute lymphocyte count 
(ALC), and platelet count 
10 Haptoglobin will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28. 
11 Erythropoietin (EPO) levels will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 
28. 
12Alkaline phosphatase, sodium, potassium, chloride, calcium, magnesium, phosphorus, carbon dioxide (CO2), albumin, total protein, glucose, blood urea 
nitrogen (BUN), creatinine, uric acid, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin and 
indirect bilirubin. 
13 Iron (Fe), total iron-binding capacity (TIBC), percent saturation, and ferritin will be performed prior to dosing on Day 1, at the end of Week 12 and at the end 
of Week 24. 
14 Fibrinogen, activated partial thromboplastin time (aPTT), and international normalized ratio (INR) will be performed at Screening, prior to dosing on Day 1, at 
the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28. 
15 Color, appearance, pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite, urobilinogen, and occult blood. Microscopic inspection of sediment should 
only be performed for cause or to investigate an abnormal dipstick finding per the Investigator’s discretion. Urinalysis will be performed at Screening, prior to 
dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28. 
16 Must be repeated at any point throughout the study period if pregnancy is clinically suspected. 
17 Total cholesterol, HDL-C, and triglyceride samples will be collected in the morning following an overnight fast. 
18 Three Screening/Baseline samples will be collected for estradiol and testosterone (total and free). Samples will be collected in the AM on 3 different days; the 
samples collected on Baseline/Day 1 may comprise one of these. Serum estrone, estradiol, and free and total testosterone will then follow the schedule 
indicated on Weeks 12, 24, and 28. FSH will only be performed at Screening for female patients only for confirmation of post-menopausal status. 
19 Randomization will be performed following PKR genotyping and prior to dosing on Day 1. 
20 Study drug will be dispensed on a 28-day schedule, or on an alternate schedule (< 28 days) as needed to accommodate patient visit schedule and dose 
modifications. 
21 For the first 10 patients treated, extensive PK/PD sampling will be conducted on Days 1 and 15 (see Appendix 15.1, Table 5 for details), followed by limited 
PK/PD sampling from Week 3 to Week 24 (see Appendix 15.1, Table 6 for details). Limited PK/PD sampling will be conducted on the remainder of patients 
treated (see Appendix 15.1 , Table 6). See Section 10.6.1, Section 10.7, and Section 10.9 for details on blood sampling for PK and PD assessments, 
respectively, and guidelines on sample processing and storage.  
 
  
23 All patients will be evaluated for AEs from the time they sign informed consent until they complete all scheduled study follow-up visits or withdraw consent, 
whichever occurs first.  
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Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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Table 5: 
Schedule of Assessments: Extensive PK/PD Sampling 
Sample Timing/Interval 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Visit 
Baseline / D1 
W2 / D15 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
Study Day 
1/15 
22 
43 
64 
85 
113 
141 
169 
Visit Window 
± 2 D (D15) 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Timing 
Pre-
dose1 
30 
min2 
1 hr2 
2 hr2 
4 hr3 
8 hr3 
12 
hr3 
Pre-
dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
PK blood sample 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
2,3 DPG/ATP 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; D = day; DPG = diphosphoglycerate; PD = pharmacodynamic; PK = pharmacokinetic; PKR = pyruvate kinase 
isoform R; W = week. 
 
1 The acceptable time window will be within 60 minutes prior to study treatment dose administration for the pre-dose PK/PD sample. 
2 The acceptable time window will be within ± 5 minutes of the scheduled collection time for the 30 minute, 1 and 2 hour PK/PD samples. 
3 The acceptable time window will be within ± 30 minutes of the scheduled collection time for the 4, 8, and 12 hour PK/PD samples. 
 
  
5 If the 12 hour time point cannot be collected at site, an 8 hour time point may be collected instead. 
79 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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Table 6: 
Schedule of Assessments: Limited PK/PD Sampling 
Sample Timing/Interval 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Visit 
Baseline / 
D1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
Study Day 
1 
15 
22 
43 
64 
85 
113 
141 
169 
Visit Window 
- 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Timing 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
PK blood sample 
X 
X 
X 
X 
X 
X 
X 
X 
X 
2,3 DPG/ATP 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; D = day; DPG = diphosphoglycerate; PD = pharmacodynamic; PK = pharmacokinetic; PKR = pyruvate kinase 
isoform R; W = week. 
 
1 The predose blood sample for plasma PK/PD analysis should be collected within 60 minutes prior to study treatment dose administration. 
 
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Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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15.2. 
National Cancer Institute Common Terminology Criteria for 
Adverse Events 
 
The NCI CTCAE, Version 4.03, can be accessed using the following link:   
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_8.5x11.pdf 
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Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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15.3. 
Potential Drug Interactions 
Strong inhibitors of CYP3A4 (listed in Table 7) are not permitted for use with AG-348. Based on 
modeling of AG-348 metabolism and tolerability data from the Phase 1 MAD study, it is thought 
that moderate inhibitors of CYP3A4 do not pose a risk to patients dosed with AG-348. 
In-vivo data from the Phase 1 MAD show AG-348 induces CYP3A4 and induces its own 
metabolism, presumably via CYP3A4. The use of strong CYP3A4 inducers in combination with 
AG-348 is expected to reduce AG-348’s efficacy. Therefore, administration of strong CYP3A4 
inducers (listed in Table 8) with AG-348 is not permitted. Strong inhibitors of drug transport 
(listed in Table 9) are not permitted for use with AG-348. 
Digoxin is not permitted for use with AG-348. 
Induction of CYP3A4 by AG-348 is expected to reduce the efficacy of certain sensitive co-
medications. Co-administration of AG-348 with the drugs in Table 10 should be done with 
caution, as their efficacy may be reduced.  
Of note, in accordance with Inclusion Criteria 14, women in the trial utilizing oral contraception 
must utilize barrier methods while taking AG-348. 
The expected patient co-medications deferoxamine, deferasirox and oral penicillin are not 
expected to interact with AG-348. 
Table 7: 
Strong and Moderate CYP3A4 Inhibitors 
Strong CYP3A4 Inhibitors: Contraindicated 
Moderate CYP3A4 Inhibitors: No Action 
Indinavir 
Aprepitant 
Nelfinavir 
Erythromycin 
Ritonavir 
Fluconazole 
Clarithromycin 
Grapefruit juice1 
Itraconazole 
Verapamil 
Ketoconazole 
Diltiazem 
Nefazodone 
 
Saquinavir 
 
Suboxone 
 
Telithromycin 
 
Strong Inhibitor; > 5 fold increase in AUC 
Moderate Inhibitor; > 2 fold, < 5 fold increase in AUC 
1 Although classified as a moderate CYP3A4 inhibitor, grapefruit and grapefruit juice are prohibited. 
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Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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Table 8: 
Strong CYP3A4 Inducers 
Strong CYP3A4 Inducers:  Contraindicated 
Efavirenz 
Phenytoin 
Nevirapine 
Pioglitazone 
Carbamazepine 
Rifabutin 
Glucocorticoids 
Rifampin 
Modafinil 
St. John's Wort 
Oxcarbazepine 
Troglitazone 
Phenobarbital 
 
 
Table 9: 
Strong P-glycoprotein Inhibitors 
Strong P-gp Inhibitors:  Contraindicated 
Amiodarone 
Felodipine 
Azithromycin 
Itraconazole 
Captopril 
Ketoconazole 
Carvedilol 
Lopinavir 
Clarithromycin 
Ritonavir 
Conivaptan 
Quercetin 
Cyclosporine 
Quinidine 
Diltiazem 
Ranolazine 
Dronedarone 
Ticagrelor 
Erythromycin 
Verapamil 
 
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Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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Table 10:  
Sensitive CYP3A4 Substrates 
Sensitive CYP3A4 Substrates:  Substitute or Use with Caution 
Macrolide 
antibiotics: 
Prokinetic: 
Steroid 6beta-OH: 
 
 
Clarithromycin 
Cisapride 
Estradiol 
Finasteride 
Terfenadine 
Erythromycin  
 
hydrocortisone 
Gleevec 
Torisel 
Telithromycin 
Antihistamines: 
progesterone 
Haloperidol 
Trazodone 
Astemizole 
Testosterone 
Irinotecan 
Vemurafenib 
Anti-arrhythmics: 
Chlorpheniramine 
 
LAAM 
Vincristine 
Quinidine→3-OH  
Terfenadine 
Miscellaneous: 
Lidocaine 
Zaleplon 
 
 
Alfentanil 
Methadone 
Ziprasidone 
Benzodiazepines: 
Calcium Channel 
Blockers: 
Aprepitant 
Nateglinide 
Zolpidem 
Alprazolam 
Amlodipine 
Aripiprazole 
Nevirapine 
 
Diazepam→3OH 
Diltiazem 
Boceprevir 
Ondansetron 
 
Midazolam 
Felodipine 
Buspirone 
Pimozide 
 
Triazolam 
Lercanidipine 
Carbamazepine 
Propranolol 
 
 
Nifedipine 
Cafergot 
Quetiapine 
 
Immune Modulators: 
Nisoldipine 
Caffeine→TMU 
Quinine 
 
Cyclosporine 
Nitrendipine 
Cilostazol 
Risperidone 
 
Tacrolimus (FK506) 
Verapamil 
Cocaine 
Romidepsin 
 
 
 
Codeine-N-
demethylation 
Salmeterol 
 
HIV Antivirals: 
HMG CoA 
Reductase 
Inhibitors: 
Dapsone 
Sildenafil 
 
Indinavir 
Atorvastatin 
Dexamethasone 
Sirolimus 
 
Nelfinavir 
Cerivastatin  
Dextromethorphan 
Sorafenib 
 
Ritonavir 
Lovastatin 
Docetaxel 
Sunitinib 
 
Saquinavir 
Simvastatin 
Domperidone 
Tamoxifen 
 
 
 
Eplerenone 
Taxol 
 
 
 
Fentanyl 
Telaprevir 
 
 
84 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
 
15.4. 
Declaration of Helsinki 
WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI: 
Recommendations Guiding Medical Doctors in Biomedical Research Involving Human Subjects 
Adopted by the 18th World Medical Association (WMA) General Assembly, Helsinki, Finland, 
June 1964 and amended by the 29th WMA General Assembly, Tokyo, Japan, October 1975, 35th 
WMA General Assembly, Venice, Italy, October 1983, and the 41st WMA General Assembly, 
Hong Kong, September 1989, the 48th WMA General Assembly, Somerset West, Republic of 
South Africa, October 1996; 52nd WMA General Assembly, Edinburgh, Scotland, October 2000; 
53rd WMA General Assembly, Washington 2002 (Note of Clarification on paragraph 29 added); 
55th WMA General Assembly, Tokyo 2004 (Note of Clarification on Paragraph 30 added); and 
59th WMA General Assembly, Seoul, October 2008. 
A. 
INTRODUCTION  
1. The World Medical Association (WMA) has developed the Declaration of Helsinki as a 
statement of ethical principles for medical research involving human subjects, including 
research on identifiable human material and data.  
The Declaration is intended to be read as a whole and each of its constituent paragraphs 
should not be applied without consideration of all other relevant paragraphs.  
2. Although the Declaration is addressed primarily to physicians, the WMA encourages 
other participants in medical research involving human subjects to adopt these principles.  
3. It is the duty of the physician to promote and safeguard the health of subjects, including 
those who are involved in medical research. The physician's knowledge and conscience 
are dedicated to the fulfilment of this duty.  
4. The Declaration of Geneva of the WMA binds the physician with the words, "The health 
of my subject will be my first consideration," and the International Code of Medical 
Ethics declares that, "A physician shall act in the subject's best interest when providing 
medical care."  
5. Medical progress is based on research that ultimately must include studies involving 
human subjects. Populations that are underrepresented in medical research should be 
provided appropriate access to participation in research.  
6. In medical research involving human subjects, the well-being of the individual research 
subject must take precedence over all other interests.  
7. The primary purpose of medical research involving human subjects is to understand the 
causes, development and effects of diseases and improve preventive, diagnostic and 
therapeutic interventions (methods, procedures and treatments). Even the best current 
interventions must be evaluated continually through research for their safety, 
effectiveness, efficiency, accessibility and quality.  
8. In medical practice and in medical research, most interventions involve risks and 
burdens.  
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Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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9. Medical research is subject to ethical standards that promote respect for all human 
subjects and protect their health and rights. Some research populations are particularly 
vulnerable and need special protection. These include those who cannot give or refuse 
consent for themselves and those who may be vulnerable to coercion or undue influence.  
10. Physicians should consider the ethical, legal and regulatory norms and standards for 
research involving human subjects in their own countries as well as applicable 
international norms and standards. No national or international ethical, legal or regulatory 
requirement should reduce or eliminate any of the protections for research subjects set 
forth in this Declaration.  
B. 
BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH  
11. It is the duty of physicians who participate in medical research to protect the life, health, 
dignity, integrity, right to self-determination, privacy, and confidentiality of personal 
information of research subjects.  
12. Medical research involving human subjects must conform to generally accepted scientific 
principles, be based on a thorough knowledge of the scientific literature, other relevant 
sources of information, and adequate laboratory and, as appropriate, animal 
experimentation. The welfare of animals used for research must be respected.  
13. Appropriate caution must be exercised in the conduct of medical research that may harm 
the environment.  
14. The design and performance of each research study involving human subjects must be 
clearly described in a research protocol. The protocol should contain a statement of the 
ethical considerations involved and should indicate how the principles in this Declaration 
have been addressed. The protocol should include information regarding funding, 
Sponsors, institutional affiliations, other potential conflicts of interest, incentives for 
subjects and provisions for treating and/or compensating subjects who are harmed as a 
consequence of participation in the research study. The protocol should describe 
arrangements for post-study access by study subjects to interventions identified as 
beneficial in the study or access to other appropriate care or benefits.  
15. The research protocol must be submitted for consideration, comment, guidance and 
approval to a research ethics committee before the study begins. This committee must be 
independent of the researcher, the Sponsor and any other undue influence. It must take 
into consideration the laws and regulations of the country or countries in which the 
research is to be performed as well as applicable international norms and standards but 
these must not be allowed to reduce or eliminate any of the protections for research 
subjects set forth in this Declaration. The committee must have the right to monitor 
ongoing studies. The researcher must provide monitoring information to the committee, 
especially information about any SAEs. No change to the protocol may be made without 
consideration and approval by the committee.  
16. Medical research involving human subjects must be conducted only by individuals with 
the appropriate scientific training and qualifications. Research on subjects or healthy 
volunteers requires the supervision of a competent and appropriately qualified physician 
or other health care professional. The responsibility for the protection of research subjects 
86 
 


Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
02 February 2015 
 
must always rest with the physician or other health care professional and never the 
research subjects, even though they have given consent.  
17. Medical research involving a disadvantaged or vulnerable population or community is 
only justified if the research is responsive to the health needs and priorities of this 
population or community and if there is a reasonable likelihood that this population or 
community stands to benefit from the results of the research.  
18. Every medical research study involving human subjects must be preceded by careful 
assessment of predictable risks and burdens to the individuals and communities involved 
in the research in comparison with foreseeable benefits to them and to other individuals 
or communities affected by the condition under investigation.  
19. Every clinical trial must be registered in a publicly accessible database before recruitment 
of the first subject.  
20. Physicians may not participate in a research study involving human subjects unless they 
are confident that the risks involved have been adequately assessed and can be 
satisfactorily managed. Physicians must immediately stop a study when the risks are 
found to outweigh the potential benefits or when there is conclusive proof of positive and 
beneficial results.  
21. Medical research involving human subjects may only be conducted if the importance of 
the objective outweighs the inherent risks and burdens to the research subjects.  
22. Participation by competent individuals as subjects in medical research must be voluntary. 
Although it may be appropriate to consult family members or community leaders, no 
competent individual may be enrolled in a research study unless he or she freely agrees.  
23. Every precaution must be taken to protect the privacy of research subjects and the 
confidentiality of their personal information and to minimize the impact of the study on 
their physical, mental and social integrity.  
24. In medical research involving competent human subjects, each potential subject must be 
adequately informed of the aims, methods, sources of funding, any possible conflicts of 
interest, institutional affiliations of the researcher, the anticipated benefits and potential 
risks of the study and the discomfort it may entail, and any other relevant aspects of the 
study. The potential subject must be informed of the right to refuse to participate in the 
study or to withdraw consent to participate at any time without reprisal. Special attention 
should be given to the specific information needs of individual potential subjects as well 
as to the methods used to deliver the information. After ensuring that the potential subject 
has understood the information, the physician or another appropriately qualified 
individual must then seek the potential subject's freely-given informed consent, 
preferably in writing. If the consent cannot be expressed in writing, the non-written 
consent must be formally documented and witnessed.  
25. For medical research using identifiable human material or data, physicians must normally 
seek consent for the collection, analysis, storage and/or reuse. There may be situations 
where consent would be impossible or impractical to obtain for such research or would 
pose a threat to the validity of the research. In such situations the research may be done 
only after consideration and approval of a research ethics committee.  
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Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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26. When seeking informed consent for participation in a research study the physician should 
be particularly cautious if the potential subject is in a dependent relationship with the 
physician or may consent under duress. In such situations the informed consent should be 
sought by an appropriately qualified individual who is completely independent of this 
relationship.  
27. For a potential research subject who is incompetent, the physician must seek informed 
consent from the legally authorized representative. These individuals must not be 
included in a research study that has no likelihood of benefit for them unless it is intended 
to promote the health of the population represented by the potential subject, the research 
cannot instead be performed with competent persons, and the research entails only 
minimal risk and minimal burden.  
28. When a potential research subject who is deemed incompetent is able to give assent to 
decisions about participation in research, the physician must seek that assent in addition 
to the consent of the legally authorized representative. The potential subject's dissent 
should be respected.  
29. Research involving subjects who are physically or mentally incapable of giving consent, 
for example, unconscious subjects, may be done only if the physical or mental condition 
that prevents giving informed consent is a necessary characteristic of the research 
population. In such circumstances the physician should seek informed consent from the 
legally authorized representative. If no such representative is available and if the research 
cannot be delayed, the study may proceed without informed consent provided that the 
specific reasons for involving subjects with a condition that renders them unable to give 
informed consent have been stated in the research protocol and the study has been 
approved by a research ethics committee. Consent to remain in the research should be 
obtained as soon as possible from the subject or a legally authorized representative.  
30. Authors, editors and publishers all have ethical obligations with regard to the publication 
of the results of research. Authors have a duty to make publicly available the results of 
their research on human subjects and are accountable for the completeness and accuracy 
of their reports. They should adhere to accepted guidelines for ethical reporting. Negative 
and inconclusive as well as positive results should be published or otherwise made 
publicly available. Sources of funding, institutional affiliations and conflicts of interest 
should be declared in the publication. Reports of research not in accordance with the 
principles of this Declaration should not be accepted for publication.  
C 
ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH 
COMBINED WITH MEDICAL CARE  
31. The physician may combine medical research with medical care only to the extent that 
the research is justified by its potential preventive, diagnostic or therapeutic value and if 
the physician has good reason to believe that participation in the research study will not 
adversely affect the health of the subjects who serve as research subjects.  
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Protocol AG348-C-003, Amendment 1, Protocol Version 2.0 
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32. The benefits, risks, burdens and effectiveness of a new intervention must be tested 
against those of the best current proven intervention, except in the following 
circumstances:  
• The use of placebo, or no treatment, is acceptable in studies where no current proven 
intervention exists; or  
• Where for compelling and scientifically sound methodological reasons the use of 
placebo is necessary to determine the efficacy or safety of an intervention and the 
subjects who receive placebo or no treatment will not be subject to any risk of serious 
or irreversible harm. Extreme care must be taken to avoid abuse of this option.  
33. At the conclusion of the study, subjects entered into the study are entitled to be informed 
about the outcome of the study and to share any benefits that result from it, for example, 
access to interventions identified as beneficial in the study or to other appropriate care or 
benefits.  
34. The physician must fully inform the subject which aspects of the care are related to the 
research. The refusal of a subject to participate in a study or the subject's decision to 
withdraw from the study must never interfere with the subject-physician relationship.  
35. In the treatment of a subject, where proven interventions do not exist or have been 
ineffective, the physician, after seeking expert advice, with informed consent from the 
subject or a legally authorized representative, may use an unproven intervention if in the 
physician's judgement it offers hope of saving life, re-establishing health or alleviating 
suffering. Where possible, this intervention should be made the object of research, 
designed to evaluate its safety and efficacy. In all cases, new information should be 
recorded and, where appropriate, made publicly available. 
 
 
89 
 


 
1. 
TITLE PAGE 
Clinical Study Protocol AG348-C-003 
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, 
Pharmacokinetic and Pharmacodynamic Study of AG-348 in Adult Patients 
with Pyruvate Kinase Deficiency 
Study Sponsor: 
Agios Pharmaceuticals, Inc. 
88 Sidney Street, Suite 250 
Cambridge, MA 02139-4169 
Phone:  617-649-8600 
Fax:  617-649-8618 
Responsible Medical Officer: 
, MD, MPH & TM 
 
Agios Pharmaceuticals, Inc. 
Mobile Phone: 
 
Office Phone: 
 
Email: 
 
Study Medical Monitor 
, MD 
 
On behalf of Agios Pharmaceuticals, Inc. 
Mobile Phone: 
 
Office Phone: 
 
Email: 
  
Document Version (Date): 
Version 1.0 (05 January 2015) 
This study will be conducted according to the protocol and in compliance with Good Clinical 
Practice, the ethical principles stated in the Declaration of Helsinki, and other applicable 
regulatory requirements. 
CONFIDENTIALITY NOTE: 
The information contained in this document is confidential and proprietary to Agios Pharmaceuticals, Inc. Any 
distribution, copying, or disclosure is strictly prohibited unless such disclosure is required by federal regulations or 
state law. Persons to whom the information is disclosed must know that it is confidential and that it may not be 
further disclosed by them. 
1 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
INVESTIGATOR’S AGREEMENT 
 
I understand that all documentation provided to me by Agios 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). No changes will be made to the study protocol without the 
prior written approval of Agios and the IRB, except where necessary to eliminate an immediate 
hazard to the patient. 
I have read, understood, and agree to conduct this study as outlined in the protocol and in 
accordance with the guidelines and all applicable government regulations. 
 
 
Investigator Name (printed) 
 
Investigator Signature 
 
Date 
 
 
 
 
Investigational site or name of institution and location (printed) 
2 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
2. 
SYNOPSIS 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Title of Study:  
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, Pharmacokinetic and 
Pharmacodynamic Study of AG-348 in Adult Patients with Pyruvate Kinase Deficiency 
Study Center(s): 
This study will be conducted at multiple study centers. 
Phase of Development: 2 
Objectives: 
Primary: 
• 
Evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in patients with 
pyruvate kinase deficiency (PK Deficiency). 
Secondary: 
• 
Evaluate the pharmacokinetics (PK) of AG-348 and the metabolite AGI-8702. 
• 
Evaluate the pharmacodynamic (PD) response of adenosine triphosphate (ATP) and 
2,3-diphosphoglycerate (2,3-DPG) after administration of AG-348. 
• 
Evaluate indicators of clinical activity of AG-348 in patients with PK Deficiency, including changes 
in hemoglobin (Hb), hematocrit (HCT), reticulocyte count, haptoglobin (Hp), carboxyhemoglobin 
(COHb), lactate dehydrogenase (LDH), total and indirect bilirubin, erythropoietin (EPO), ferritin, 
and transferrin saturation (serum iron/iron binding capacity). 
Methodology: 
Study AG348-C-003 is a Phase 2, open label, two arm, multicenter, randomized, dose-ranging study 
during which adult patients with PK Deficiency will receive multiple doses of AG-348 for up to 
24 weeks. Patients with PK Deficiency confirmed by red blood cell PK enzymatic assay performed at 
Screening will be eligible to participate in this study. At Week 25, patients who safely tolerate AG-348 
and demonstrate clinical activity of AG-348 may be eligible to immediately roll over to a separate safety 
extension study for continued treatment. Patients who finish treatment at the end of 24 weeks or sooner 
will undergo follow-up assessment 4 weeks after the last dose of study drug. Patients with toxicity 
suspected to be related to study drug will continue follow-up until the adverse event (AE) resolves, is 
declared chronic by the Investigator, or the patient is lost to follow-up. 
3 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Initially, up to 25 patients will be randomized on an open-label 1:1 basis to each of two twice-daily 
(BID) doses of AG-348 (up to 50 patients total; see Figure 1, Study Schema). The dose of Arm 1 is 
300 mg of AG-348 administered orally every 12 hours (q12h, BID). The dose of Arm 2 is 50 mg of 
AG-348 administered orally q12h (BID). Randomization will be stratified by PKR mutation in order to 
maintain balance as much as possible across the dose arms for the specific mutations expected to be 
most frequently enrolled. The PKR mutation stratification factor will consist of 4 levels (R510Q, 
R486W, and R479H) and all other mutations (“other”). Mutation status is defined by the presence of at 
least one of the indicated mutations; patients with more than one stratified mutation will be assigned 
based on Sponsor’s discretion.  
The doses for each arm have been selected from the forerunner AG348-C-001 single ascending dose 
(SAD) and AG348-C-002 multiple ascending dose (MAD) studies in healthy adult volunteers to 
represent the range of doses/exposures that were safely tolerated and resulted in maximal or near-
maximal PD effects on 2,3-DPG and ATP. 
Because PK Deficiency is a rare disease with a limited eligible patient population and because the 
underlying pathophysiology and clinical phenotype of affected patients is heterogenous due to the wide 
variety of mutations in PKR that cause the disease, it is important to focus closely on dose findings in 
this first-in-patient study. Therefore, in addition to initiating this study with 2 different doses of AG-348 
administered q12h, a Data Review Team (DRT) will be established to review study data on a regular 
basis and adapt the study design, dose and schedule of AG-348. 
The DRT will monitor safety on an on-going basis and meet at regular intervals (approximately every 
6 weeks), or ad hoc, as necessary, to review AEs, vital signs (VS), clinical laboratory (hematology, 
clinical chemistry, coagulation, and urinalysis), and electrocardiograms (ECGs) on enrolled patients. 
The DRT will also review available PK/PD data and indicators of clinical activity (e.g., changes from 
baseline in Hb). The DRT will be comprised of the study Coordinating Investigator, treating 
Investigators, Medical Monitor, Clinical Pharmacologist, Statistician, and Sponsor’s Responsible 
Medical Officer.  
Approximately every 6 weeks beginning six weeks after the first patient is dosed or ad hoc as necessary, 
the DRT will review cumulative safety data, available PK/PD data, and clinical activity data. Based on 
the DRT’s recurring 6 week reviews, the DRT may exercise one or more of the following options: 
• 
Continue treatment and enrollment in existing arms without change. 
• 
Add 1 new dose arm (Arm 3) to enroll up to 25 patients at a dose to be determined; the dose for 
Arm 3 may be lower or higher than Arm 1 and Arm 2 doses, but will not exceed 360 mg q12h; 
and the dose regimen may be less frequent than q12h. 
• 
Terminate or suspend enrollment to allow further review of clinical data in Arm 1 and/or Arm 2 
(and/or potential Arm 3). Enrollment in an arm could be terminated or suspended to allow 
further review, for example, for unacceptable safety/tolerability, poor PD response, or lack of 
signs of clinical activity. 
• 
Re-assign patients’ doses and schedule in a terminated arm to match the dose and schedule of 
another arm of the study. In this case, the patients in the terminated arm will remain in their 
original arm, i.e., they will not count towards the enrollment quota of the arm whose dose and 
4 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
schedule is being adopted. 
• 
Implement specific genotype restrictions to enrollment in one or more arms to ensure 
representation of patients with genotypes of greatest clinical relevance. 
The DRT will perform these evaluations on a recurring 6-week basis. The data that the DRT will review 
to make these decisions is expected to include, but are not necessarily limited to, the following:  
• 
Safety Observations: all AEs; VS, clinical laboratory (hematology, clinical chemistry, 
coagulation, and urinalysis), and ECG;  
• 
PK and PD Observations: including changes in 2,3-DPG and ATP; 
• 
Indicators of Clinical Activity: including changes in Hb, HCT, reticulocyte count, Hp, COHb, 
LDH, EPO, total and indirect bilirubin, ferritin, and transferrin saturation.  
If a third dose arm is implemented, the dose of AG-348 selected will not exceed 360 mg BID, as this 
was the highest dose that demonstrated acceptable safety and tolerance in the 14-day multiple BID 
dosing study in healthy volunteers. The PK/PD sampling schedule in a potential third dosing arm will be 
determined by Sponsor’s discretion and may follow either the extensive or limited PK/PD sampling 
schedules as specified. 
The DRT will monitor the data in an ongoing manner as described and may make a decision to terminate 
enrollment in an arm if unacceptable AEs are observed, PD activity is limited, and/or markers of clinical 
activity indicate no effect. Unacceptable AEs are defined as ≥ Grade 3 AEs (using National Cancer 
Institute Common Terminology Criteria for Adverse Events [NCI CTCAEv4.03]) that are assessed as at 
least possibly related to AG-348, with the exception of Grade 3 headache, hot flash/flushing, nausea, 
vomiting, and/or diarrhea that is transient in duration (< 24 hours) or able to be medically managed to 
≤ Grade 2 within 24 hours. The DRT may also exercise discretion to terminate enrollment in an arm if it 
observes a frequency of Grade 2 AEs that would make it difficult to support long-term dosing. 
As the number of enrolling arms changes in the study (for example, from 2 to 3), the randomization 
scheme will adjust to enable balanced randomization into each actively accruing arm. Randomization 
and stratification will cease in the event that only a single arm is left enrolling. 
Depending on possible early termination of 1 or both of the initial 2 arms, or the addition of a third arm, 
the study could enroll up to a maximum of 75 patients. 
5 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Figure 1:  Study Schema 
 
Abbreviations:  BID (q12h) = twice-daily (every 12 hours); DRT = data review team; PKR = pyruvate kinase red blood cell 
isoform; TBD= to be determined; w = week(s). 
 
Visit Schedule 
Screening assessments will occur within 28 days prior to the first dose of study treatment. During the 
Treatment period, patients will attend visits at baseline (Day 1), weekly through Week 3 (Days 8, 15, 
and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12), and monthly through 
Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate AG-348 and demonstrate evidence of 
clinical activity of AG-348 through Week 24 may be eligible to immediately enter a separate extension 
study for continued treatment. For patients who finish treatment at the end of 24 weeks or sooner, or 
who elect not to enter the extension trial, study discharge will occur 4 weeks (Week 28 or earlier) 
following the last dose of study treatment at the final follow-up assessment. 
Dose Modifications for Safety and/or Increase in Hb Level 
The Investigator will monitor all patients for safety and tolerability. Modification of an individual 
patient’s dose of AG-348 will be based on AEs and/or observed changes in Hb level as described in 
Section 9.7.1 and Section 9.7.2. 
6 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
Number of patients (planned): Up to approximately 75 patients. 
Diagnosis and main criteria for inclusion: 
Inclusion criteria: 
For entry into the study, patients must meet all of the following criteria during the Screening or other 
specified period: 
1. Signed written informed consent obtained prior to performing any study procedure, including 
screening procedures.  
2. Male or female, aged 18 years and older. 
3. Known medical history of PK Deficiency.  
4. All patients must have documented clinical laboratory confirmation of PKD by red blood cell 
pyruvate kinase enzymatic assay performed at Screening by a designated central laboratory. 
Patients with prior documentation of PK Deficiency by red blood cell (RBC) enzymatic assay 
will have a reconfirmation of this result during Screening as a condition of enrollment. 
a. In the event that a patient’s screening pyruvate kinase enzymatic assay is negative (i.e., 
shows normal pyruvate kinase activity), the patient will be eligible for enrollment if the 
genotyping shows a mutant genotype that has been previously documented in the 
literature to be associated with pyruvate kinase deficiency. If the genotyping shows a 
previously undescribed mutation in the PKR gene, then the eligibility for enrollment 
will be determined on a case-by-case basis by the Coordinating Investigator and 
Medical Monitor in discussion with the Investigator. If no mutation is defined, then the 
patient will not be eligible. 
5. All patients must have genotypic characterization of the mutant PKR gene performed by a 
designated central laboratory at Screening, unless genotype is available from the patient’s 
participation in the Pyruvate Kinase Deficiency Natural History Study (NCT02053480). 
6. All patients must have genotypic characterization of the UGT1A1 gene performed by a 
designated central laboratory to document whether they may have underlying Gilbert's Disease. 
Patients with Gilbert’s Disease are eligible to enroll. 
7. Males must have Hb ≤ 12.0 g/dL; females must have Hb ≤ 11.0 g/dL.  
8. All patients must be considered transfusion independent as defined by: no greater than 3 units of 
RBCs transfused in the 12-month period up to the first day of study dosing and no transfusions 
within 4 months of first day of study dosing. Patients who have received more transfusion 
support than described above will evaluated for eligibility on a case-by-case basis by the 
Medical Monitor. 
7 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
9. Splenectomized patients: 
a. Must have undergone their procedure at least 6 months prior to Screening. 
b. Must be current in their vaccinations for Pneumococcal Conjugate (PCV13), 
Pneumococcal Polysaccharide (PPSV23), Quadrivalent Meningococcal vaccine, and 
Haemophilus influenzae type b (Hib) as recommended by Centers for Disease Control 
and Prevention's (CDC) Advisory Committee on Immunization Practices (ACIP) or 
immunization advisory groups in Canada and the European Union (for patients enrolled 
in Canada and the EU). [http://www.cdc.gov/vaccines/schedules/downloads/adult/adult-
schedule.pdf] [Any missing vaccinations may be administered during the screening 
period.] 
10. Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2. 
11. Patients must be taking at least 1 mg of folic acid daily for at least 21 days prior to first dose and 
continue daily during study participation. 
12. Adequate organ function, defined as: 
a. Serum aspartate transaminase (AST) and alanine aminotransferase (ALT) ≤ 1.5 × upper 
limit of normal (ULN) (unless the increased AST is assessed by the Investigator as due 
to hemolysis). 
b. Normal or elevated levels of serum bilirubin. In patients with serum bilirubin > ULN, 
the elevation must be attributed to hemolysis with or without Gilbert's syndrome and 
must not be choledocholithiasis, cholecystitis, biliary obstruction, or hepatocellular 
disease. 
c. Serum creatinine ≤ 1.25 × ULN. If serum creatinine > 1.25 × ULN, then 24-hour 
measured or calculated (Cockcroft-Gault) glomerular filtration rate (GFR) ≥ 60 mL/min. 
d. Absolute neutrophil count (ANC) > 1.0 × 109/L. 
e. Platelet count ≥ 100 × 109/L. 
f. Activated partial thromboplastin time (aPTT) and international normalized ratio (INR) 
≤ 1.25 × ULN, unless the patient is receiving therapeutic anticoagulants. 
13. Women of childbearing potential (WOCBP) must agree to abstain from sexual intercourse or to 
use an acceptable/effective method of contraception (i.e., condom plus spermicide, condom plus 
oral contraceptive, condom plus intrauterine device [IUD], condom plus diaphragm with 
spermicide) from as soon as feasible during the Screening period until 30 days following the last 
dose of AG-348.  
a. WOCBP include any female who has experienced menarche and who has not undergone 
successful surgical sterilization (hysterectomy, bilateral tubal ligation, and/or bilateral 
oophorectomy) or is not post-menopausal. Post-menopausal is defined as: 
i. 
Amenorrhea ≥ 12 consecutive months without another cause, and a documented 
serum follicle stimulating hormone (FSH) level > 35 mIU/mL; 
ii. 
Amenorrhea ≥ 12 consecutive months in women ≥ 62 years old (FSH testing is 
8 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
not required). 
14. WOCBP must have a negative serum or urine pregnancy test within 72 hours before start of 
AG-348 dosing. 
15. Women must not be breastfeeding.  
16. Male patients, with the exception of those who have undergone vasectomy at least 6 months 
prior to Screening, must agree to abstain from sexual intercourse or, if sexually active, to use a 
condom with spermicide as contraception (regardless of their female partner's childbearing 
potential or their partner's use of their own contraception) from Day 1 of dosing until 30 days 
following the last dose of AG-348. 
Exclusion criteria: 
Patients who meet any of the following criteria at Screening or prior to dosing on Day 1 will not be 
enrolled in the study: 
1. Hemoglobin level > 12.0 g/dL if male; Hb > 11.0 g/dL if female. 
2. Additional diagnosis of any other congenital or acquired blood disorder, including 
glucose-6-phosphate-dehydrogenase (G6PD) deficiency, or any other hemolytic anemia 
process except for mild allo-immunization as a consequence of transfusion therapy. 
3. Iron overload (hemosiderosis or concurrent hemochromatosis) sufficiently severe to result in a 
clinical diagnosis by the Investigator of cardiac, hepatic, or pancreatic insufficiency. 
4. Prior bone marrow or stem cell transplant. 
5. Clinically symptomatic cholelithiasis or cholecystitis. (Prior cholecystectomy is not 
exclusionary. Patients with symptomatic cholelithiasis or cholecystitis may be rescreened once 
the disorder has been treated and clinical symptoms have resolved.) 
6. Currently enrolled in another therapeutic clinical trial involving on-going therapy with any 
investigational or marketed product or placebo. Concurrent participation in the Pyruvate Kinase 
Deficiency Natural History Study (NCT02053480) is permitted. 
7. Exposure to any investigational drug, device, or procedure within 28 days prior to Screening. 
8. Concurrent medical condition that could compromise participation in the study such as: 
a. Poorly controlled hypertension (defined as systolic blood pressure (BP) > 150 mm Hg 
or diastolic BP > 90 mm Hg) refractory to medical management. 
b. History of recent (within < 6 months from Screening date) congestive heart failure; 
myocardial infarction or unstable angina pectoris; or hemorrhagic, embolic, or 
9 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
thrombotic stroke; deep venous thrombosis; or pulmonary or arterial embolism.  
c. Currently active infection requiring the use of parenteral anti-microbial agents or that is 
greater than Grade 3 (CTCAEv4.03) within 6 months of first dose. 
d. A pattern or frequency of post-splenectomy sepsis that in the assessment of the 
Investigator could reasonably be expected to interfere with the ability of the patient to 
complete the 24 week study participation. 
e. Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) 
antibody with signs of active Hepatitis B or C virus infection.  
f. Positive test for human immunodeficiency virus (HIV) 1 or 2 antibody. 
g. Diabetes mellitus judged to be in poor control by the Investigator or requiring > 3 anti-
diabetic agents counting insulin (all insulins are considered one agent); use of insulin 
per se is not exclusionary.  
h. History of any primary malignancy with the exception of: curatively treated non-
melanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; or 
other primary tumor treated with curative intent and no known active disease present 
and no treatment administered during the last 3 years. 
9. Undergone major surgery within 6 months of first dose.  
10. Current or recent history of psychiatric disorder that in the opinion of the Investigator or 
Medical Monitor could compromise the ability of the patient to cooperate with study visits and 
procedures. 
11. Use of any of the restricted list of products known to strongly inhibit CYP3A4 drug metabolism 
(Appendix 15.3, Table 7) within 5 days prior to Day 1 dosing; or to strongly induce CYP3A4 
metabolism (Appendix 15.3, Table 8) within 28 days prior to Day 1 dosing; or to strongly inhibit 
P-glycoprotein (P-gp) transporter (Appendix 15.3, Table 9) within 5 days prior to Day 1 dosing; 
or digoxin within 5 days prior to Day 1 dosing. 
12. Serum bilirubin > ULN attributable to factors other than hemolysis and/or Gilbert's syndrome. 
13. Male patients with heart-rate corrected QT (Fridericia's correction factor) QTcF interval 
> 450 msec, or female patients with QTcF interval > 470 msec with the exception of patients 
with a left bundle branch block (LBBB). Medical Monitor approval needed in patients with a 
LBBB. 
14. Cardiac dysrhythmias judged as clinically significant by the Investigator or requiring therapy 
with drugs that are primarily substrates of CYP3A4. 
15. History of allergy to sulfonamides if characterized by acute hemolytic anemia, anaphylaxis, or 
10 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
rash of erythema multiforme type or Stevens-Johnson syndrome. 
16. Any other medical or psychological condition deemed by the Investigator to be likely to 
interfere with a patient's ability to understand and sign informed consent; cooperate with study 
visits, tests, and procedures; or otherwise safely and reliably participate in the study. 
Investigational product, dosage, and mode of administration: 
AG-348 sulfate hydrate capsules will be provided as 25 mg or 100 mg (free-base equivalent) of AG-348 
sulfate hydrate without excipients in Swedish orange (25 mg) or white opaque (100 mg) gelatin capsules 
(size 2 capsules for all dose strengths). AG-348 will be administered orally BID. The number of capsules 
per dose will vary by assigned dose group. Patients will receive multiple oral (PO) doses of AG-348 
over a 24-week treatment period. AG-348 will be administered with water and may be administered with 
or without food. 
Reference therapy, dosage and mode of administration:  
Not applicable. 
Duration of treatment:  
The duration of treatment for all patients on this study will be up to 24 weeks. Patients who safely 
tolerate and demonstrate one or more indicators of clinical activity of AG-348 may be eligible to 
immediately roll over to a separate safety extension study for continued treatment. 
Criteria for evaluation: 
Safety: 
Monitoring of AEs, including determination of serious adverse events (SAEs) and AEs leading to 
discontinuation; safety laboratory parameters (hematology, chemistry, urinalysis, coagulation); physical 
examination findings; VS; and 12-lead ECGs. Adverse events will be graded using CTCAE, Version 
4.03. Serum sex hormone levels (testosterone, estrone, and estradiol), bone turnover markers (serum N-
terminal telopeptide [NTX] and serum C-terminal telopeptide [CTX]), total cholesterol, high-density 
lipoprotein-C (HDL-C), and triglycerides will be monitored for evidence of potential inhibition of 
aromatase by AG-348 
Indicators of Clinical Activity: 
Monitoring of potential indicators of clinical activity will include evaluating changes in Hb, HCT, 
reticulocyte count, COHb, LDH, total and indirect bilirubin, EPO, ferritin, and transferrin saturation.  
Pharmacokinetics: 
Approximately the first 10 patients treated, contingent on clinical site feasibility, will undergo extensive 
PK sampling as detailed in Appendix 15.1, Table 5. The remainder of treated patients will undergo 
limited PK sampling as detailed in Appendix 15.1, Table 6. Serial blood sampling for determination of 
concentration-time profiles of AG-348 and its metabolite AGI-8702 will be conducted following the first 
dose and the morning Day 15 dose, and additional trough levels of AG-348 and AGI-8702 will be 
obtained. AG-348 and AGI-8702 will be analyzed using qualified assays to determine concentrations in 
plasma. Pharmacokinetic parameters on Day 1 and Day 15 will be computed using standard non-
11 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
compartmental methods based on observed plasma AG-348 and AGI-8702 concentrations. 
Pharmacodynamics: 
Pharmacodynamic assessments will include 2,3-DPG, ATP (secondary objectives), 
 
 
. Approximately 
the first 10 patients treated will undergo extensive PD sampling as detailed in Appendix 15.1, Table 5. 
The remainder of treated patients will undergo limited PD sampling as detailed in Appendix 15.1, 
Table 6. Serial blood sampling for determination of levels of ATP and, 2,3-DPG will be conducted 
following the first dose and the morning Day 15 dose, and additional trough levels of ATP and 2,3-DPG 
will be obtained. Adenosine triphosphate and 2,3 DPG will be analyzed using qualified assays to 
determine concentrations in whole blood. Pharmacodynamic parameters on Day 1 and Day 15 will be 
computed based on observed whole blood ATP and 2,3-DPG concentrations.  
Statistical methods: 
The primary objective of this Phase 2 study is to evaluate the safety and tolerability of up to 24 weeks of 
AG-348 in patients with PK Deficiency. Therefore, analyses will be primarily descriptive in nature; no 
formal hypothesis testing will be conducted. All analyses will be conducted separately within each dose 
arm, or pooled when appropriate. 
Summaries will be produced for disposition, baseline disease characteristics and demographic data 
including genotype, safety measurements, PK, PD parameters and indicators of clinical activity. Data 
from each AG-348 dose group will be analyzed separately, and pooled across all dose groups where 
appropriate. All data will also be listed by individual patient. Categorical variables will be summarized 
by frequency distributions (number and percentages) and continuous variables will be summarized by 
descriptive statistics (mean, standard deviation [SD], median, minimum, and maximum). 
Populations for analysis will include a Safety Analysis Set, a PK Analysis Set, and an Efficacy Analysis 
Set. The Safety Analysis set will include all patients who are enrolled and receive any dose of study 
treatment; this population will be the primary set for the analysis of safety data and the default analysis 
set for all data analyses unless otherwise stated. Patients will be classified according to treatment 
received, where treatment received is defined as the assigned treatment if it is received at least once, or 
as the first treatment received if assigned treatment is never received. The PK Analysis Set will include 
all patients in the Safety Analysis Set with sufficient plasma sample data to assess PK parameters. 
Results of the potential PD activity of AG-348 will also be based on the PK analysis set. The Efficacy 
Analysis Set will include all patients who are enrolled and achieve at least 50% compliance with their 
12 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
assigned dose intensity for at least 4 weeks of continued dosing. Additional efficacy analyses may be 
performed on subsets of patients with other degrees of compliance with their assigned dose intensity 
and/or duration of dosing. The Efficacy Analysis Set will be the primary set for the analysis of 
preliminary clinical activity data. Patients will be classified according to assigned treatment. 
 
 
 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) and the 
incidence of treatment-emergent AEs (TEAEs) (new or worsening from baseline) will be summarized by 
primary system organ class (SOC), preferred term (PT), CTCAE Version 4.03 severity, outcome, action 
taken with study drug, and relationship to study drug by dose group. Separate summaries will be 
produced for all TEAEs, treatment-related AEs (those considered by the Investigator as at least possibly 
drug related), SAEs, discontinuations due to AEs, and AEs ≥ Grade 3 severity. Individual patient listings 
will be provided for any deaths, SAEs, AEs leading to interruption and/or reduction of study drug dose, 
and AEs leading to discontinuation of treatment. Analyses of AEs will be performed for those events 
that are considered treatment-emergent, where treatment-emergent is defined as any AE with onset 
beginning at the day of first administration of study drug, throughout the treatment period until 30 days 
after the last dose of study drug, or any event that was present at baseline but worsened in intensity or 
was subsequently considered drug-related by the Investigator through the end of the study. 
Descriptive statistics will be provided for clinical laboratory values (e.g., hematology, serum chemistry, 
coagulation studies, urinalysis) and VS data, presented as both actual values and changes from baseline 
relative to each on-study evaluation. Shift analyses will be conducted for selected laboratory parameters 
based on the baseline CTCAE grade to maximum CTCAE grade. Where applicable CTCAE terms do 
not exist, a grading system based on the upper and/or lower limits of normal will be used to classify the 
results. 
Electrocardiogram analyses will include individual patient listings and summaries of abnormal and 
clinically significant ECG results. Actual values and changes from baseline in PR, QRS, QTc intervals 
will be summarized by visit and dose arm.  
Concomitant medications will be coded using the WHO Drug Dictionary. Concomitant medications 
prior to and after the start of the study drug will be listed by patient, and summarized by ATC term and 
dose arm. 
Descriptive statistics will be used to summarize PK parameters for the parent compound AG-348 and the 
metabolite AGI-8702 for each dose group, and where appropriate, for the entire population. 
Pharmacokinetic parameters will be summarized using the following descriptive statistics: n, mean, 
standard deviation (SD), coefficient of variation %, median, minimum, and maximum, geometric mean, 
and geometric coefficient of variation.  
Descriptive statistics will be used to summarize Pharmacodynamic parameters for 2,3-DPG and ATP for 
each dose group, and where appropriate for the entire population. Pharmacodynamic parameters will be 
summarized using the following descriptive statistics: n, mean, SD, coefficient of variation %, median, 
minimum, and maximum, geometric mean, and geometric coefficient of variation %. 
Analyses of serum sex hormones will use appropriate graphic displays and statistical analyses to 
13 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
Name of Sponsor/Company: 
Agios Pharmaceuticals, Inc. 
Name of Investigational Product: 
AG-348 
Name of Active Ingredient: 
AG-348 sulfate hydrate 
evaluate patient change from baseline for each parameter. These analyses will include summaries of 
actual values and change from baseline using appropriate descriptive statistics (mean, SD, median, min 
and max) as described above.  
Analyses evaluating indicators of potential clinical activity of AG-348 in patients with PK Deficiency 
will include changes in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, 
ferritin, and transferrin saturation (serum iron/iron binding capacity). Characterization of Hb response 
(e.g., % of patients whose Hb increases by a certain amount), as well as time to Hb response, and 
duration of Hb response will be explored, among others. 
Interim Review 
No formal statistical analysis will be conducted. Safety data will be reviewed on an ongoing basis by the 
DRT, who will meet to review safety, PK, PD, and preliminary clinical activity data at regular intervals 
(approximately every 6 weeks) throughout the duration of the study. The composition of the DRT, its 
meeting schedule, the data to be reviewed, and the decisions it is empowered to make have been 
described previously. 
Additional interim reviews of data may be conducted to support decision making concerning the current 
clinical study, the sponsor’s development programs in general, or in case of any safety concerns.  
Sample Size 
Due to the rare disease setting, the minimal sample size may be determined by feasibility. Depending on 
possible early termination of 1 or both of the initial 2 arms or the addition of a 3rd dose arm, the study 
could enroll up to a maximum of 75 patients. 
14 
 


Protocol AG348-C-003, Version 1.0 
05 January 2015 
 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF 
FIGURES 
TABLE OF CONTENTS 
1. 
TITLE PAGE ................................................................................................................1 
2. 
SYNOPSIS ...................................................................................................................3 
3. 
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES ...............15 
4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS.............................20 
5. 
INTRODUCTION ......................................................................................................24 
5.1. 
Pyruvate Kinase Deficiency .......................................................................................24 
5.2. 
AG-348 .......................................................................................................................25 
5.2.1. 
Summary of Nonclinical Data ....................................................................................25 
5.2.1.1. 
Pharmacology .............................................................................................................25 
5.2.1.2. 
Safety Pharmacology ..................................................................................................26 
5.2.1.3. 
Pharmacokinetics ........................................................................................................26 
5.2.1.4. 
Toxicology ..................................................................................................................27 
5.2.2. 
Summary of Clinical Data ..........................................................................................29 
5.2.2.1. 
Pharmacokinetics ........................................................................................................29 
5.2.2.2. 
Pharmacodynamics .....................................................................................................30 
5.2.2.3. 
Safety ..........................................................................................................................30 
5.3. 
Study Rationale ...........................................................................................................31 
5.3.1. 
Summary of Overall Safety Management Plan ..........................................................31 
6. 
TRIAL OBJECTIVES AND ENDPOINTS ...............................................................33 
6.1. 
Primary Objective .......................................................................................................33 
6.2. 
Secondary Objectives .................................................................................................33 
 
33 
6.4. 
Study Measures and Endpoints ...................................................................................33 
6.4.1. 
Safety Measures and Endpoints ..................................................................................33 
6.4.2. 
Clinical Activity Measures and Endpoints .................................................................34 
6.4.3. 
Pharmacokinetic and Pharmacodynamic Measures and Endpoints ............................34 
 
34 
7. 
INVESTIGATIONAL PLAN .....................................................................................35 
7.1. 
Overall Study Design ..................................................................................................35 
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7.2. 
Justification of the Study Design ................................................................................38 
7.3. 
Rationale for the Starting Dose and Dose Range .......................................................39 
7.4. 
Criteria for Study Termination ...................................................................................39 
8. 
STUDY POPULATION .............................................................................................41 
8.1. 
Number of Patients .....................................................................................................41 
8.2. 
Inclusion Criteria ........................................................................................................41 
8.3. 
Exclusion Criteria .......................................................................................................43 
8.4. 
Patient Identification and Registration .......................................................................44 
8.5. 
Patient Randomization ................................................................................................45 
8.6. 
Patient Withdrawal Criteria ........................................................................................45 
8.7. 
Replacement of Patients .............................................................................................46 
9. 
STUDY TREATMENT ..............................................................................................47 
9.1. 
Description of Study Drug ..........................................................................................47 
9.2. 
Study Drug Packaging and Labeling ..........................................................................47 
9.3. 
Study Drug Storage .....................................................................................................47 
9.4. 
Method of Assigning Patients to Treatment ...............................................................47 
9.5. 
Blinding ......................................................................................................................47 
9.6. 
Study Drug Preparation and Administration ..............................................................47 
9.7. 
Criteria for Dose Escalation, Dose Modification, or Discontinuation of Study 
Drug ............................................................................................................................48 
9.7.1. 
Dose Modification for Safety .....................................................................................48 
9.7.2. 
Dose Modification for Increase in Hemoglobin Level ...............................................49 
9.7.3. 
Stopping Criteria .........................................................................................................50 
9.8. 
Duration of Patient Participation ................................................................................50 
9.9. 
Treatment Compliance ................................................................................................50 
9.10. 
Study Drug Accountability .........................................................................................50 
9.11. 
Prior and Concomitant Medications and Treatments .................................................51 
9.11.1. 
Prior Medications and Procedures ..............................................................................51 
9.11.2. 
Prohibited Concomitant Therapy ................................................................................51 
9.11.3. 
Allowed Concomitant Therapy ...................................................................................52 
9.11.4. 
Potential for Phototoxicity ..........................................................................................52 
9.11.5. 
Management of Nausea, Vomiting, and Diarrhea ......................................................53 
9.11.6. 
Other Restrictions and Precautions .............................................................................53 
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10. 
STUDY ASSESSMENTS ..........................................................................................54 
10.1. 
Schedule of Assessments ............................................................................................54 
10.2. 
Informed Consent and Confirmation of Eligibility ....................................................54 
10.3. 
Demographic Data, Medical and Medication History ................................................54 
10.4. 
PKR Enzymatic Assay and PKR Genotyping ............................................................55 
10.5. 
Safety Assessments .....................................................................................................55 
10.5.1. 
Physical Examination, Height, and Weight ................................................................55 
10.5.2. 
Vital Signs ..................................................................................................................55 
10.5.3. 
Electrocardiogram .......................................................................................................55 
10.5.4. 
Safety Laboratory Assessments ..................................................................................55 
10.5.4.1. 
Hematology, Serum Chemistry, Coagulation Studies, and Urinalysis .......................55 
10.5.4.2. 
Screening Serology .....................................................................................................57 
10.5.5. 
Adverse Events ...........................................................................................................57 
10.6. 
Pharmacokinetic Assessments ....................................................................................57 
10.6.1. 
Blood Sample Collection and Pharmacokinetic Measurements During Dose 
Escalation ....................................................................................................................57 
10.7. 
Pharmacodynamic Assessments .................................................................................57 
10.8. 
Ordering of Blood Sample Collection ........................................................................59 
10.9. 
Sample Processing, Storage, and Shipment ................................................................59 
11. 
ADVERSE EVENTS..................................................................................................60 
11.1. 
Definition of Adverse Events .....................................................................................60 
11.1.1. 
Adverse Event (AE) ....................................................................................................60 
11.1.2. 
Suspected Adverse Reaction .......................................................................................60 
11.1.3. 
Unexpected Adverse Event .........................................................................................60 
11.1.4. 
Serious Adverse Event ................................................................................................60 
11.1.4.1. 
Potential Severe Drug-Induced Liver Injury ..............................................................61 
11.2. 
Procedures for Reporting Adverse Events and Serious Adverse Events ....................61 
11.3. 
Pregnancy Reporting ..................................................................................................63 
12. 
STATISTICAL METHODS .......................................................................................65 
12.1. 
Sample Size Estimation ..............................................................................................65 
12.2. 
Populations for Analysis .............................................................................................65 
12.3. 
Procedures for Handling Missing, Unused, and Spurious Data .................................66 
12.4. 
Interim Analysis ..........................................................................................................66 
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12.5. 
Statistical Methodology ..............................................................................................66 
12.5.1. 
General Methods .........................................................................................................66 
12.5.2. 
Disposition ..................................................................................................................67 
12.5.3. 
Exposure and Safety Analyses ....................................................................................67 
12.5.4. 
Pharmacokinetic Analyses ..........................................................................................67 
12.5.5. 
Pharmacodynamic Analyses .......................................................................................68 
12.5.6. 
Aromatase Hormone Analysis ....................................................................................68 
12.5.7. 
Clinical Activity ..........................................................................................................68 
 
68 
12.6. 
Procedures for Reporting Deviations to Original Statistical Analysis Plan ...............68 
13. 
ADMINSTRATIVE REQUIREMENTS ....................................................................69 
13.1. 
Good Clinical Practice ................................................................................................69 
13.2. 
Ethical Considerations ................................................................................................69 
13.3. 
Patient Information and Informed Consent ................................................................69 
13.4. 
Patient Confidentiality ................................................................................................70 
13.5. 
Protocol Compliance ..................................................................................................70 
13.6. 
Data Management .......................................................................................................70 
13.7. 
Source Document/Case Report Form Completion .....................................................71 
13.8. 
Direct Access to Source Data .....................................................................................71 
13.9. 
Record Retention ........................................................................................................71 
13.10. 
Liability and Insurance ...............................................................................................72 
13.11. 
Publication of Study Findings and Use of Information ..............................................72 
14. 
LIST OF REFERENCES ............................................................................................73 
15. 
APPENDICES ............................................................................................................74 
15.1. 
Schedules of Assessments ..........................................................................................75 
15.2. 
National Cancer Institute Common Terminology Criteria for Adverse Events .........81 
15.3. 
Potential Drug Interactions .........................................................................................82 
15.4. 
Declaration of Helsinki ...............................................................................................85 
A. 
INTRODUCTION ......................................................................................................85 
B. 
BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH ......................................86 
C 
ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH COMBINED 
WITH MEDICAL CARE ...........................................................................................88 
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LIST OF TABLES 
Table 1: 
Dose Modification for Adverse Events Deemed at Least Possibly Related to 
AG-348 .......................................................................................................................49 
Table 2: 
Dose Reduction Table (by Dosing Arm) ....................................................................50 
Table 3: 
Sample Size Estimation ..............................................................................................65 
Table 4: 
Schedule of Assessments: ...........................................................................................75 
Table 5: 
Schedule of Assessments: Extensive PK/PD Sampling .............................................79 
Table 6: 
Schedule of Assessments: Limited PK/PD Sampling ................................................80 
Table 7: 
Strong and Moderate CYP3A4 Inhibitors ..................................................................82 
Table 8: 
Strong CYP3A4 Inducers ...........................................................................................83 
Table 9: 
Strong P-glycoprotein Inhibitors ................................................................................83 
Table 10:  Sensitive CYP3A4 Substrates.....................................................................................84 
 
LIST OF FIGURES 
Figure 1:  Study Schema ...............................................................................................................6 
Figure 2: 
Red Cell Glycolysis ....................................................................................................24 
Figure 3: 
Study Schema .............................................................................................................37 
Figure 4: 
PKR Enzymatic Reaction ...........................................................................................58 
 
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4. 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation or Specialist Term 
Explanation 
2,3-DPG 
2,3-diphosphoglycerate 
ACIP 
Advisory Committee on Immunization Practices 
ADME 
Absorption, distribution, metabolism, excretion 
ADP 
Adenosine diphosphate 
AE 
Adverse event 
ALC 
Absolute lymphocyte count 
ALP 
Alkaline phosphatase 
ALT 
Alanine aminotransferase 
ANC 
Absolute neutrophil count 
aPTT 
Activated partial thromboplastin time 
AST 
Aspartate aminotransferase 
ATP 
Adenosine triphosphate 
AUC 
Area under the curve 
AUC0-12hr 
Area under the plasma concentration versus time curve from 0 to 12 
hours 
AUC0 24hr 
Area under the plasma concentration versus time curve from 0 to 
24 hours 
AUC0-∞ 
Area under the plasma concentration versus time curve from 0 to 
infinity 
BCRP 
Breast cancer resistance protein 
BID 
Twice-daily 
BMI 
Body mass index 
BP 
Blood pressure 
BUN 
Blood urea nitrogen 
CDC 
Centers for Disease Control and Prevention 
CFR 
Code of Federal Regulations 
CLP 
Total body plasma clearance 
Cmax 
Maximum plasma concentration 
CO2 
Carbon dioxide 
COHb 
Carboxyhemoglobin 
CTCAE 
Common Terminology Criteria for Adverse Events 
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Abbreviation or Specialist Term 
Explanation 
CTX 
Serum C-terminal telopeptide 
CV 
Cardiovascular 
DDI 
Drug-drug interaction 
CYP 
Cytochrome P450 
DILI 
Drug-induced liver injury 
DLT 
Dose-limiting toxicity 
DRT 
Data review team 
ECG 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic case report form 
EDC 
Electronic Data Capture 
EPO 
Erythropoietin 
F 
Oral bioavailability 
FDA 
Food and Drug Administration 
FSH 
Follicle stimulating hormone 
G6PD 
Glucose-6-phosphate-dehydrogenase 
GCP 
Good Clinical Practice 
GFR 
Glomerular filtration rate 
GLP 
Good Laboratory Practice 
Hb 
Hemoglobin 
HBsAg 
Hepatitis B surface antigen 
HCT 
Hematocrit 
HCV 
Hepatitis C virus 
HDL-C 
High-density lipoprotein-C 
HDPE 
High density polyethylene 
hERG 
Human ether à-go-go related gene 
Hib 
Haemophilus influenzae type b 
HIV 
Human immunodeficiency virus 
Hp 
Haptoglobin 
IC50 
Concentration of drug that achieved half-maximal inhibition 
ICH 
International Conference on Harmonization 
INR 
International normalized ratio 
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Abbreviation or Specialist Term 
Explanation 
IRB 
Institutional Review Board 
IUD 
Intrauterine device 
IV 
Intravenous 
LDH 
Lactate dehydrogenase 
MAD 
Multiple ascending dose 
MCH 
Mean corpuscular hemoglobin 
MCHC 
Mean corpuscular hemoglobin concentration 
MCV 
Mean corpuscular volume 
MedDRA 
Medical Dictionary for Regulatory Activities 
mPKR 
PKR mutants 
MTD 
Maximum tolerated dose 
NCI 
National Cancer Institute 
NOEL 
No-observed-effect-level 
NOAEL 
No-observed-adverse-effect-level 
NTX 
Serum N-terminal telopeptide 
P-gp 
P-glycoprotein 
PCV13 
Pneumococcal Conjugate 
PD 
Pharmacodynamic 
PEP 
Phosphoenolpyruvate 
PK 
Pharmacokinetic 
PK Deficiency 
Pyruvate kinase deficiency 
PKR 
Pyruvate kinase isoform R 
PO 
Oral 
PPSV23 
Pneumococcal polysaccharide 
PR 
The portion of the ECG wave from the beginning of the P wave to 
the beginning of the QRS complex 
PT 
Preferred term 
q12h 
Every 12 hours 
q24h 
Every 24 hours 
QD 
Once-daily 
QRS 
QRS interval on an electrocardiogram 
QTc 
Heart-rate corrected QT interval 
QTcB 
Corrected QT interval - Bazett correction formula 
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Abbreviation or Specialist Term 
Explanation 
QTcF 
Corrected QT interval - Fridericia’s method 
RBC 
Red blood cell (count) 
RDW 
Red cell distribution width 
SAD 
Single ascending dose 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SD 
Standard deviation 
SOC 
System organ class 
SOP(s) 
Standard Operating Procedure(s) 
t½ 
Apparent terminal half-life 
TIBC 
Total iron-binding capacity 
Tmax 
Time to maximum concentration 
TEAE 
Treatment-emergent adverse event 
ULN 
Upper limit of normal 
VS 
Vital signs 
Vss 
Volume of distribution at steady-state 
Vz/F 
Mean apparent volume of distribution 
WBC 
White blood cell  
WMA 
World Medical Association 
WOCBP 
Women of childbearing potential 
WT 
Wild type 
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5. 
INTRODUCTION 
5.1. 
Pyruvate Kinase Deficiency 
Pyruvate kinase deficiency (PK Deficiency) is a glycolytic enzymopathy that results in non-
spherocytic hemolytic anemia with a variable clinical presentation, ranging from mild or fully 
compensated forms to life-threatening neonatal anemia and life-long chronic hemolytic anemia 
associated with severe, debilitating co-morbidities. PK Deficiency is caused by mutations in the 
PKLR gene, which results in defective pyruvate kinase isoform R (PKR) in the red blood cell 
(RBC). As shown in the diagram below, PKR catalyzes the final and irreversible step in 
glycolysis, converting phosphoenolpyruvate (PEP) to pyruvate, and formation of adenosine 
triphosphate (ATP) from adenosine diphosphate (ADP). Mature RBCs rely almost exclusively on 
the process of glycolysis to generate the energy carrier molecule ATP. PKR is thus a key enzyme 
for maintaining energy homeostasis in erythrocytes, and it has been proposed that ATP levels are 
critical for optimally maintaining RBC membrane integrity (van Wijk and van Solinge 2005). 
Figure 2: 
Red Cell Glycolysis 
 
Abbreviations:  1,3 DPG = 1,3 diphosphoglycerate; 2,3 DPG = 2,3 diphosphoglycerate; 3-PG = 3-phosphoglycerate; 
ADP = adenosine diphosphate; ATP = adenosine triphosphate; FBP = fructose 1,6-bisphosphate; PEP = 
phosphoenolpyruvate; PKR = red blood cell-specific form of pyruvate kinase 
Note: Not all steps in glycolysis are shown. 
The majority of mutations in PKR that have been described have a deleterious effect on PKR 
catalytic activity, protein stability, and/or protein expression. PK Deficiency is associated with 
reduced RBC survival as well as impaired red cell maturation. Mature erythrocytes lack 
mitochondria relying predominantly on glycolysis to generate ATP. It is hypothesized that 
insufficient energy production promotes erythrocyte hemolysis due to impaired maintenance of 
cellular membrane homeostasis. 
PK Deficiency is an autosomal recessive disease, in which both homozygotes and compound 
heterozygotes develop hemolytic anemia. More than 200 different mutations in the PKLR gene 
have been identified in patients with PK Deficiency to date, and the majority of these mutations 
are single nucleotide missense mutations (Zanella, et al. 2005). The prevalence of PK 
Deficiency, based on the most common pyruvate kinase mutation in Southern Europe, is 
estimated at 51 cases per million in the Caucasian population (Beutler and Gelbart 2000). 
Some patients with PK Deficiency have a clinically severe disease course that can present as a 
severe hemolytic anemia in early infancy, requiring immediate care which typically includes 
blood transfusion support (Christensen, et al. 2011; Ghidini, et al. 1991; Hennekam, et al. 1990). 
Infants with PK Deficiency may have erythroblastosis fetalis with hepatosplenomegaly, 
extramedullary hematopoiesis, and severe indirect hyperbilirubinemia, which is associated with a 
high risk of kernicterus. Hyperbilirubinemia associated with the hemolytic state often requires 
Glucose
Pyruvate
PKR
`
ATP
FBP
1,3-DPG
2,3-DPG
3-PG
PEP
ADP
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supportive treatment with intensive phototherapy. For other infants, the hyperbilirubinemia 
caused by hemolysis can require exchange transfusion, as well as supportive transfusion to keep 
their hematocrit (HCT) levels >60%. 
Adults with PK Deficiency are reported to have life-long hemolysis and the subsequent 
associated co-morbidities including anemia and transfusion dependence. Other co-morbidities 
include frequent miscarriages, aplastic crises, as well as symptoms associated with an acute on 
chronic hemolytic anemia (Rider, et al. 2011). Since unconjugated bilirubin is often chronically 
elevated, pigmented gallstones are common in children and adults. Severe and sometimes life-
threatening iron deposition occurs and is typically progressive. 
The current therapeutic options for patients with PK Deficiency are supportive. Most require life-
long treatment, including blood transfusions at a frequency depending on the disease state. Long-
term surveillance for systemic iron overload, even in transfusion-independent patients, is 
standard as is the use of chelation therapy. Case reports of cure by allogeneic bone marrow 
transplant have been published (Tanphaichitr, et al. 2000) but have been infrequently performed. 
Splenectomy, while not curative, can reduce the need for supportive blood transfusions and 
decrease bilirubin levels in some patients. However, this intervention is associated with 
significant morbidity including infection risk, and possible increased risk of thrombosis. 
5.2. 
AG-348 
AG-348 is an orally available, allosteric activator of PKR. It is hypothesized that drug 
intervention with AG-348 restores glycolytic pathway activity and normalizes red cell 
metabolism in vivo. Biochemical experiments demonstrate that AG-348 is a potent, pan-activator 
of many PKR alleles associated with PK Deficiency. PK Deficiency red cells and their 
progenitors are characterized by changes in metabolism associated with defective glycolysis, 
including a build-up of PEP and 2,3-diphosphoglycerate (2,3-DPG), and lowered ATP levels. 
Treatment of PK Deficiency patient red cells ex vivo with AG-348 results in increased ATP 
levels, and reductions in PEP and 2,3-DPG, consistent with pharmacological activation of PKR 
enzyme activity. In vitro studies against wild type (WT) PKR enzymes and ex vivo studies in 
blood from mice, monkeys and humans indicate that AG-348 activates WT PKR with a similar 
potency and efficacy as it does with the mutant versions. In vivo studies in mice confirm the 
potency of AG-348 in increasing WT PKR enzyme activity and modulating the levels of 
downstream markers such as ATP and 2,3-DPG. This therapeutic approach may be an effective 
way to correct the underlying pathology of PK Deficiency and, importantly, provide clinical 
benefit to patients. 
5.2.1. 
Summary of Nonclinical Data 
A series of exploratory pharmacology studies were conducted to characterize the ability of 
AG-348 to activate WT PKR and anemia-associated PKR mutants (mPKR) in vitro, ex vivo, and 
in vivo. Additional details regarding nonclinical data can be found in the AG-348 Investigator’s 
Brochure 
5.2.1.1. 
Pharmacology 
AG-348 is a potent, broad-spectrum activator of PKR with low nM potency against both WT and 
mutant enzymes. AG-348 is also able to effectively stabilize mPKR enzymes that are 
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hypersensitive to thermal denaturation, suggesting that AG-348 may be able to increase PKR 
pathway activity by preventing destabilization and degradation of mPKR enzymes. Its major 
metabolite, AGI-8702, has low potency mixed activity against PKR and mutant isoforms and can 
increase the thermostability of some mPKR, albeit with significantly weaker potency than that of 
AG-348. 
The effect of AG-348 and AGI-8702 on PKR activity and a number of downstream pathway 
markers was evaluated in human RBCs. AG-348 dose-response curves in human whole blood 
showed potent activation of PKR, while AGI-8702 is a weak activator of the PKR enzyme. In 
whole blood from mice treated with AG-348, 13C-glucose labeling through the PKR reaction was 
increased by 80% demonstrating AG-348-mediated activation of the WT PKR enzyme. AG-348 
treatment of RBCs from healthy human donors potently increased levels of the PKR reaction 
product ATP consistent with activation of PKR.  
The effects of AG-348 on PKR activity and RBC metabolism also were assessed in blood 
samples from patients with PK Deficiency. AG-348 activated PKR and induced metabolic 
changes (increased ATP levels and decreased 2,3-DPG levels) consistent with increased 
glycolytic pathway activity in RBCs from PK Deficiency patients with different mutations in the 
PKR enzyme.  
Assessments of binding inhibition potential and enzymatic activity were evaluated in a panel of 
89 receptors, ion channels, and enzymes. Although AG-348 is a histamine H3 antagonist/inverse 
agonist and an aromatase inhibitor, no effects of histamine H3 modulation have been observed in 
safety pharmacology or toxicology studies. Effects consistent with aromatase inhibition were 
seen in toxicology studies (Section 5.2.1.4). 
5.2.1.2. 
Safety Pharmacology 
Safety pharmacology studies demonstrated that neither AG-348 nor AGI-8702 exhibited 
significant inhibition of the human ether-à-go-go related gene (hERG) current (concentration of 
drug that achieved half-maximal inhibition [IC50] 29.4 μM and > 10 μM, respectively). 
Additionally, in the monkey cardiovascular (CV) study, no CV effects were seen at any dose 
level. No effects were seen in AG-348 Good Laboratory Practice (GLP)-compliant Irwin and 
respiratory safety pharmacology studies in Sprague-Dawley rats. AG-348 administered via oral 
gavage had a dose-dependent emetic activity in the ferret at ≥ 30 mg/kg. 
5.2.1.3. 
Pharmacokinetics 
Absorption, distribution, metabolism, and excretion (ADME) studies of AG-348 were performed 
in Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys. Both oral (PO) and intravenous 
(IV) routes were evaluated at doses ranging from 1 to 50 mg/kg. A dose of 200 mg (50 mg/kg) 
was administered PO in the food-effect evaluation in monkeys. The PK of AG-348 in animal 
species is characterized by rapid oral absorption, medium to high total body plasma clearance 
(CLp), and high volume of distribution at steady-state (Vss) in rats, dogs, and monkeys. 
AG-348 has a medium to long apparent terminal half-life (t1/2) in the rat, dog, and monkey. 
Medium to high oral bioavailability (F) was observed for AG-348 in animals. A high-fat diet led 
to lower oral absorption of AG-348 in monkeys. 
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AG-348 showed low brain penetration in rats (brain/plasma ratio of 5.7% to 9.1%) based on area 
under the plasma concentration versus time curve from 0 to 24 hours (AUC0 24hr) following 
single and repeated-dose administration, respectively. Metabolism appears to be the major 
elimination pathway for AG-348 with minimal biliary or urinary excretion.  
Both in vitro and in vivo, the predominant metabolite was the N-dealkylated product, AGI-8702. 
There were no metabolites unique to human liver microsomes and no direct Phase 2 metabolites. 
The plasma metabolite profile of AG-348 did not change with repeat or ascending dosing, 
suggesting that AG-348 is not susceptible to metabolic switching with increasing or prolonged 
dose administration. 
AG-348 is a weak direct inhibitor of cytochrome P450 (CYP) 2C8, CYP2C9, CYP2C19, 
CYP2D6, and CYP3A4/5 (testosterone 6β-hydroxylation). There was no direct inhibition of 
CYP1A2, CYP2B6, or CYP3A4/5 (midazolam 1′-hydroxylation) by AG-348. There was 
evidence of metabolism-dependent inhibition of CYP2C19 (largely reversible) and CYP3A4 
(largely irreversible) by AG-348. AG-348 is considered a weak time-dependent CYP3A4 
inhibitor. AGI-8702 is a weak direct inhibitor of CYP2C9 and CYP2C19. AG-348 has the 
potential to cause an induction-related drug-drug interaction (DDI) with sensitive CYP2B6 and 
CYP3A4 substrates. 
The routes of metabolism for AG-348 are via multiple CYPs with CYP3A4 contributing > 70% 
of the total. CYP1A2, CYP2C9, and CYP2C8 contribute approximately 6%, 10%, and 7% to the 
remaining metabolism of AG-348; other isoforms contribute < 4% each. 
AG-348 is a substrate for P-glycoprotein (P-gp), but not breast cancer resistance protein (BCRP). 
AG-348 is an inhibitor of P-gp (91% and 99% inhibition at 41 and 411 µM, respectively), but 
does not inhibit BCRP. AGI-8702 is not an inhibitor of P-gp or BCRP under the concentrations 
tested (5 and 100 μM). 
5.2.1.4. 
Toxicology 
In rats, the no-observed-effect level/no-observed-adverse-effect level (NOEL/NOAEL), 
determined as 2000 mg/kg, was associated with area under the plasma concentration versus time 
curve from 0 to 12 hours (AUC0-12hr) values 223- to 526-fold the projected human efficacious 
AUC0-12hr value. In dogs, clinical observations consistent with anaphylactoid reactions were seen, 
and the maximum tolerated dose (MTD) was 62.5 mg/kg, which was associated with an 
AUC0-12hr value 5.7-fold the projected efficacious AUC0-12hr value. The NOEL/NOAEL in dogs 
was 10 mg/kg, which was associated with an AUC0-12hr value 0.8-fold the projected efficacious 
AUC0-12hr value. In monkeys, the NOAEL was 1000 mg/kg, with only non-adverse emesis and 
body weight loss seen; this dose was associated with an AUC0-12hr value 70-fold the projected 
efficacious AUC0 12hr value. Based on the results of single-dose studies, the rat and the monkey 
were chosen as the most appropriate species for further evaluation in toxicology studies.  
Dose-limiting toxicity (DLT) in cynomolgus monkeys was defined in non-GLP 5-day and 
14-day repeat-dose studies as emesis, inappetence, and weight loss. These toxicities became dose 
limiting at AUC0-12hr values 27- to 34-fold the projected efficacious AUC0-12hr value, and 
precluded meaningful evaluation of other toxicities at this exposure level. Potential other effects 
at this exposure level were observed in a number hematology and serum chemistry parameters as 
well as in lymphoid organs. Additionally, minimal potential effects in kidneys (renal 
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Protocol AG348-C-003, Version 1.0 
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tubulointerstitial nephritis) and heart (myocardial degeneration), which could not be 
differentiated from spontaneous background lesions, were seen when monkeys were exposed to 
AG-348 AUC0-12hr values ≥ 27- to 34-fold the projected human efficacious AUC0-12hr value.  
In GLP-compliant 28-day monkey study, the dose of 150 mg/kg/day (75 mg/kg/dose twice daily 
[BID]) was the NOAEL. Effects were limited to increased liver weights without serum chemistry 
or microscopic correlate. At this dosage level, the Day 27 AUC0-12hr values were 8.9- and 
8.5-fold the projected efficacious AUC0-12hr value in males and females respectively. In the same 
study, the low dosage of 20 mg/kg/day (10 mg/kg/dose) resulted in AUC0-12hr values that 
approximated the efficacious AUC0-12hr value, and there were no test article-related effects seen. 
The next highest dose of 50 mg/kg/day (25 mg/kg/dose) was the NOEL and was associated with 
AUC0-12hr values 3.1- and 2.6-fold the projected efficacious AUC0-12hr value in males and females 
respectively.  
In the GLP-compliant 28-day rat study, the NOAEL in males was the highest dose tested, 
600 mg/kg/day (300 mg/kg/dose), and the NOAEL in females was the lowest dose tested, 
20 mg/kg/day (10 mg/kg/dose). At the 600 mg/kg/day dosage level in males, AG-348-related 
findings were limited to mild effects on hematology, serum chemistry, and urinalysis parameters, 
and microscopic findings in the adrenal gland (minimal to mild vacuolation of the adrenal zona 
glomerulosa and decreased thickness of the zona fasciculate), liver (minimal to mild 
hepatocellular hypertrophy), kidney (minimal tubular vacuolation), pancreas (minimal to 
moderate decreased zymogen granules), heart (minimal myocardial vacuolation), and prostate 
(minimal to mild decreased secretion). All findings were fully reversible over the 14-day 
recovery period with the exception of decreased serum glucose levels and decreased prostate 
secretion. In females, the highest dosage tested was 200 mg/kg/day (100 mg/kg/dose); adverse 
effects observed were similar to those observed in the 600 mg/kg/day males, with the exception 
that in females, fewer effects in hematology and serum chemistry parameters were seen, and also 
in females, adverse effects in the reproductive organs consistent with aromatase inhibition were 
observed.  
In the 13-week repeat-dose rat study, adverse effects in males were identified in the testes and 
consisted of seminiferous tubular degeneration, spermatid retention, and Leydig cell 
hypertrophy. These effects were defined as adverse at dose levels at which they are expected to 
impair fertility. These effects were either not present or present with incidence and severity 
similar to that of the vehicle group in lower dose levels. Adverse effects in females included 
uterine atrophy and increased folding of the luminal surface; these effects were defined as 
adverse at the dose level at which they are expected to impair fertility  
In the 13-week repeat-dose study in monkeys, no adverse effects were identified, and no new 
effects were identified when compared to the 4-week repeat-dose study. Similar to what occurred 
on the 4-week study, inappetence and emesis during the initial 1-2 weeks of dosing occurred, 
precluding evaluation of higher doses.  
AG-348 inhibits human aromatase activity with an IC50 of 2050 nM (based on human placental 
microsomes) and rat aromatase with an IC50 of 493 nM (based on rat ovarian microsomes). In the 
GLP-compliant 28-day rat study, histologic effects consistent with aromatase inhibition were 
seen in the female reproductive tract at the mid- and high-dosage levels (100 and 200 mg/kg/day) 
and included incomplete corpora lutea; ovarian follicular cysts; ovarian cystic, luteinized 
follicles; uterine atrophy; vaginal mucification; and altered cyclicity. Although these findings 
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were minimal to mild and were fully reversible (over 14 days), they were considered adverse and 
the next lower dosage evaluated, 20 mg/kg/day (10 mg/kg/dose BID) was the NOAEL in 
females. The Day 27 AUC0-12hr value associated with this dosage level was 6.9-fold the projected 
human efficacious AUC0-12hr value. The potential for aromatase inhibition effects occurring in 
female rats at AUC0-12hr values > 6.9-fold and < 53-fold the projected efficacious AUC0-12hr value 
has been addressed in a 13-week rat study. In this study using doses between the NOAEL and 
LOAEL in the 28-day study, the NOAEL for histologic lesions of the uterus that may be 
associated with aromatase inhibition resulted in an AUC0-12hr that was 26-fold the projected 
efficacious value. Notably, due to the potency difference of AG-348 against rat versus human 
aromatase inhibition, there is potential for a 4-fold wider margin for aromatase inhibition in 
humans versus rats. AGI-8702 is not an aromatase inhibitor. 
5.2.2. 
Summary of Clinical Data 
To date, 72 healthy adult volunteers have been exposed to AG-348 in 2 clinical studies, a single 
ascending dose (SAD) study and a multiple ascending dose (MAD) study, with 31 of these 
subjects exposed to a single AG-348 dose under fasted conditions; 5 exposed to 2 doses of 
AG-348 under fasted and fed conditions; and 36 exposed to repeated doses of AG-348 for up to 
14 days1. The following discussion of clinical data refers only to healthy adult volunteer 
subjects, as this is the first clinical trial in which patients with PK Deficiency will be treated with 
AG-348. 
5.2.2.1. 
Pharmacokinetics 
The PK of AG-348 showed low to moderate variability between subjects. After single doses of 
AG-348 from 30 to 2500 mg, AG-348 was rapidly absorbed as indicated by the short time to 
maximum plasma concentration (Tmax). There appeared to be some lengthening of the period of 
absorption at higher doses as indicated by higher Tmax and a less than proportional increase in 
maximum concentration (Cmax). Dose-normalized area under the curve (AUC) generally 
remained constant over the dose range studied, suggesting that AG-348 total exposure increased 
in a dose-proportional manner. The mean t1/2 ranged from 17.8 to 20.4 hours when samples were 
collected through 72 hours and from 50.3 to 79.3 when samples were collected through 
120 hours. However, this terminal elimination phase contributed little to overall exposure of 
AG-348, as indicated by the small difference between AUC0-12 and area under the plasma 
concentration versus time curve from 0 to infinity (AUC0-∞), suggesting a shorter effective half-
life of approximately 3 to 6 hours. AG-348 was extensively distributed (mean apparent volume 
of distribution [Vz/F] range of 271 to 1148 L) and had a moderate rate of clearance (geometric 
mean clearance [CL/F] range of 10.3 to 13.9 L/hr). The fraction of AG-348 excreted in urine 
ranged from 0.0145 to 0.0209 across the dose levels suggesting that renal excretion plays a 
minor role in the systemic elimination of AG-348. 
The preliminary repeat-dose PK of AG-348 at doses ranging from 15 mg every 12 hours (q12h) 
to 700 mg q12h also showed an increase in AG-348 exposure in a dose-proportional manner 
after the first dose. AG-348 exposure observed after repeated dosing for 14 days at doses of 
1 At the time of this document, results from ongoing Study AG348-C-002 in healthy volunteers are blinded. Based 
on the randomization scheme, 36 subjects were assigned to AG-348 and 12 subjects were assigned to placebo. 
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120 mg every 24 hours (q24h) to 700 mg q12h were lower than observed on Day 1 after the first 
dose. The magnitude of the effect was greater at higher doses; lower doses of 15 mg q12h and 
60 mg q12h did not show this effect. This observation may be related to the nonclinical finding 
that AG-348 is a potential inducer of human CYP3A4, an enzyme which biotransforms AG-348. 
It is possible that multiple doses of AG-348 leads to an increased rate of its own metabolism. 
Evaluation of the effect of food on the PK of a single 700 mg dose of AG-348 in 5 subjects who 
were administered the drug fasting and then, after an appropriate wash-out period, re-
administered the drug following ingestion of a standard US Food and Drug Administration 
(FDA) high fat meal, showed that food likely has a minimal effect on the PK of AG-348. 
5.2.2.2. 
Pharmacodynamics 
After a single dose of AG-348, a decrease in the concentration of 2,3-DPG was observed at 
3 hours post-dose, decreased in a dose-dependent manner to a minimum at 24-hour post-dose, 
and then returned to values similar to baseline by 72 to 120 hours post-dose. The mean decrease 
at 24 hours was approximately 300 µg/mL at the 700 through 2500 mg dose levels. Similar 
decreases were observed after the first dose of multiple doses and prior to doses in the midst of 
the MAD study. After the final dose of multiple doses, the concentration of 2,3-DPG returned to 
values similar to baseline between 72 and 120 hours post- dose. 
After a single dose of AG-348, a minimal increase in the concentration of ATP was observed at 
24 to 120 hours post-dose. In the MAD study, no increase was observed at 12 hours after the first 
dose; the concentration of ATP was increased on Day 8 to concentrations greater than in the 
single dose study, continued to trend upward on Day 11, and remained at a similar level through 
Day 14. The concentration of ATP remained elevated through 120 hours after the last dose on 
Day 14. The magnitude of the increase in ATP was similar across the dose range from 60 mg 
BID to 700 mg BID. 
5.2.2.3. 
Safety 
Overall, AG-348 has been well-tolerated among healthy volunteers. 
After a single AG-348 dose, treatment-emergent adverse events (TEAEs) reported by > 1 subject 
at any time on study (either under fasted or fed conditions) included nausea (14%), headache 
(14%), and upper respiratory tract infection and vomiting (each 6%). After repeated dosing of 
AG-348 for 14 days, based on blinded data, TEAEs that occurred in > 2 subjects within any 
cohort and therefore must have been experienced by at least 1 AG-348-treated subject included 
dizziness, feeling hot, headache, hyperhidrosis, nausea, restlessness, and vomiting. 
A dose-relationship was apparent with regard to the incidence of gastrointestinal events, 
primarily nausea and vomiting, with the incidence of such events increasing with increasing 
dose. Nausea and/or vomiting were observed only at doses ≥ 1400 mg in the single dose study 
and only at doses ≥ 700 BID in the MAD study. Nausea and vomiting were not observed at any 
dose ≤ 360 mg in either the single or multiple dose studies. 
All but 1 TEAE reported to date has been mild or moderate (Grade 1 or 2) in intensity. The only 
Grade 3 TEAE was elevated liver transaminases (alanine aminotransferase [ALT]; aspartate 
aminotransferase [AST]) in a single subject in the MAD study treated with 700 mg of blinded 
study drug q12h BID. The treatment this subject received (AG-348 versus placebo) remains 
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blinded, and the events were considered to be study drug-related and led to study drug 
discontinuation, following which the elevated liver transaminases resolved. No subject 
discontinued in the SAD study due to an adverse event (AE). Four subjects, at doses of 700 mg 
BID, have discontinued study drug in the MAD study due to at least 1 TEAE, with nausea and 
vomiting contributing to study drug discontinuation in 3 of these 4 subjects. The fourth subject 
discontinued for a cutaneous rash. 
No deaths or other serious adverse events (SAEs) have been reported in any clinical study of 
AG-348. Furthermore, no DLTs have been declared after a single administration of AG-348 at 
doses of 30 mg to 2500 mg or after repeat administration of AG-348 for 14 days at daily doses of 
120 mg to 1400 mg (although the event of Grade 3 elevated liver function tests described above 
in the MAD study may be assessed as DLT after the subject’s treatment assignment is 
unblinded). 
Due to preclinical observations pertaining to the potential for inhibition of the aromatase enzyme 
(see Section 5.2.1.4), the AG348-C-002 study included assessment of baseline and serial 
measures of free and total serum testosterone and serum estradiol and estrone. The data are still 
blinded, and therefore, no quantitative statistical analyses have yet been performed. Upon 
qualitative review of the serum hormone data, at least some subjects in the 120 mg BID cohort 
and higher dose cohorts demonstrate modest increases in androgenic hormones and decreases in 
estrogens, compatible with a potential signal of aromatase inhibition. Additional analyses will be 
performed after the study is completed and the data are locked and unblinded. 
5.3. 
Study Rationale 
Study AG348-C-003 is the first study that will be conducted in patients with PK Deficiency. This 
study is primarily intended to evaluate the safety and tolerability and potential indicators of 
clinical activity of AG-348 administered for up to 24 weeks. This study will also evaluate the PK 
profile of AG-348 and its metabolite AGI-8702, the PD responses in ATP and 2,3-DPG 
following administration of AG-348, and the clinical activity of AG-348 in PK Deficiency 
patients. Two previous double-blind, placebo-controlled clinical trials of AG-348 conducted in 
healthy adult male and female volunteers (AG348-C-001, a SAD study; and AG348-C-002, a 
MAD study) have established an acceptable safety and tolerability profile for AG-348 for up to 
14 days of both once-daily (QD) and BID dosing at exposures that result in significant 
pharmacodynamic (PD) changes in whole blood levels of the glycolytic metabolites 2,3-DPG 
and ATP. The target population of this study consists of adult males and females with a 
diagnosis of PK Deficiency, who are anemic but non-transfusion dependent. Non-transfusion 
dependent patients are preferred for this study in order to reduce any potential confounding effect 
of transfusion therapies on evaluation of potential indicators of clinical activity and PD 
responses. The safety, tolerability, and PK/PD findings in this study will form the basis for 
subsequent clinical development of AG-348. 
5.3.1. 
Summary of Overall Safety Management Plan 
Measures to minimize the risks to patients enrolled in this study have been taken with respect to 
the following study design elements: 
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• The initial doses for Arms 1 and 2 have been selected on the basis of safety and 
tolerability observations already carefully made in two predecessor clinical trials in 
adult healthy male and female volunteers;  
• The specified inclusion/exclusion criteria have been carefully considered to avoid 
enrollment of subjects for whom exposure to the study drug might pose a hazard; 
• A designated Data Review Team (DRT) consisting of the overall study Coordinating 
Investigator, treating Investigators, Medical Monitor, Clinical Pharmacologist, 
Statistician, and Sponsor’s Responsible Medical Officer has been established to meet 
regularly at approximately 6-week intervals throughout the duration of the study to 
review the accumulating study data and will exercise options to suspend enrollment to 
one or both of the initial two study dose arms, discontinue enrollment to one or both 
of the initial two study dose arms, adjust the dose of patients in one or both of the 
initial two study arms, and/or implement one new study dose arm. If one new dosing 
arm is implemented, the dose selected will not exceed 360 mg BID, the highest dose 
that demonstrated acceptable safety and tolerance in the 14-day multiple BID dosing 
study in healthy volunteers. Group cohort stopping rules for terminating enrollment 
into an arm based on the severity (CTCAEv4.03 grade) and frequency of AEs are 
defined;  
• Dose modification and stopping rules are defined for individual patients; 
• Guidance for permitted, prohibited, and cautionary concomitant medications is 
specified based on the estimated potential for drug-drug interactions from hepatic 
cytochrome enzyme interactions with AG-348. 
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6. 
TRIAL OBJECTIVES AND ENDPOINTS 
6.1. 
Primary Objective 
The primary objective of the study is to: 
• Evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in 
patients with PK Deficiency. 
6.2. 
Secondary Objectives 
The secondary objectives of the study are to: 
• Evaluate the pharmacokinetics (PK) of AG-348 and the metabolite AGI-8702. 
• Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348. 
• Evaluate indicators of clinical activity of AG-348 in patients with PK Deficiency, 
including changes in hemoglobin (Hb), HCT, reticulocyte count, haptoglobin (Hp), 
carboxyhemoglobin (COHb), lactate dehydrogenase (LDH), total and indirect 
bilirubin, erythropoietin (EPO), ferritin, and transferrin saturation (serum iron/iron 
binding capacity). 
6.4. 
Study Measures and Endpoints 
6.4.1. 
Safety Measures and Endpoints 
Safety will be evaluated by: 
• Monitoring of AEs, including determination of SAEs and AEs leading to 
discontinuation; safety laboratory parameters (hematology, chemistry, urinalysis, 
coagulation); physical examination findings; vital signs (VS); and 12 lead 
electrocardiograms (ECGs). Adverse events will be graded using Common Toxicity 
Criteria for Adverse Events (CTCAE), Version 4.03. Serum sex hormone levels 
(testosterone, estrone, and estradiol), bone turnover markers (serum N-terminal 
telopeptide [NTX] and serum C-terminal telopeptide [CTX]), total cholesterol, high-
density lipoprotein-C (HDL-C), and triglycerides will be monitored for evidence of 
potential inhibition of aromatase by AG-348. 
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6.4.2. 
Clinical Activity Measures and Endpoints 
• Monitoring of potential indicators of clinical activity will include evaluating changes 
in Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, 
ferritin, and transferrin saturation. 
6.4.3. 
Pharmacokinetic and Pharmacodynamic Measures and Endpoints 
The PK and PD profile of AG-348 will be evaluated by: 
• Approximately the first 10 patients treated, contingent on clinical site feasibility, will 
undergo extensive PK sampling as detailed in Appendix 15.1, Table 5. The remainder 
of treated patients will undergo limited PK sampling as detailed in Appendix 15.1, 
Table 6. Serial blood sampling for determination of concentration-time profiles of 
AG-348 and its metabolite AGI-8702 will be conducted following the first dose and 
the morning Day 15 dose, and additional trough levels of AG-348 and AGI-8702 will 
be obtained. AG-348 and AGI-8702 will be analyzed using qualified assays to 
determine concentrations in plasma. Pharmacokinetic parameters on Day 1 and 
Day 15 will be computed using standard non-compartmental methods based on 
observed plasma AG-348 and AGI-8702 concentrations. 
• Pharmacodynamic assessments will include 2,3-DPG, ATP (secondary objectives), 
 
 
Approximately the first 10 patients 
treated will undergo extensive PD sampling as detailed in Appendix 15.1, Table 5. 
The remainder of treated patients will undergo limited PD sampling as detailed in 
Appendix 15.1, Table 6. Serial blood sampling for determination of levels of ATP 
and, 2,3-DPG will be conducted following the first dose and the morning Day15 dose, 
and additional trough levels of ATP and 2,3-DPG will be obtained. ATP and 2,3 DPG 
will be analyzed using qualified assays to determine concentrations in whole blood. 
PD parameters on Day 1 and Day 15 will be computed based on observed whole 
blood ATP and 2,3-DPG concentrations.  
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7. 
INVESTIGATIONAL PLAN 
7.1. 
Overall Study Design 
Study AG348-C-003 is a Phase 2, open-label, two-arm, multicenter, randomized, dose-ranging 
study during which adult patients with PK Deficiency will receive multiple doses of AG-348 for 
up to 24 weeks. Patients with PK Deficiency confirmed by red blood cell PK enzymatic assay 
performed at Screening will be eligible to participate in this study. At Week 25, patients who 
safely tolerate AG-348 and demonstrate clinical activity of AG-348 may be eligible to 
immediately roll over to a separate safety extension study for continued treatment. Patients who 
finish treatment at the end of 24 weeks or sooner will undergo follow-up assessment 4 weeks 
after the last dose of study drug. Patients with toxicity suspected to be related to study drug will 
continue follow-up until the AE resolves, is declared chronic by the Investigator, or the patient is 
lost to follow-up. 
Initially, up to 25 patients will be randomized on an open-label, 1:1 basis to each of two BID 
doses of AG-348 (up to 50 patients; see Figure 3, Study Schema). The dose of Arm 1 is 300 mg 
of AG-348 administered orally q12h (BID). The dose of Arm 2 is 50 mg of AG-348 
administered orally q12h (BID) for 12 weeks. Randomization will be stratified by PKR mutation 
in order to maintain balance as much as possible across the dose arms for the specific mutations 
expected to be most frequently enrolled. The PKR mutation stratification factor will consist of 
4 levels (R510Q, R486W, and R479H) and all other mutations (“other”). Mutation status is 
defined by the presence of at least one of the indicated mutations; patients with more than one 
stratified mutation will be assigned based on Sponsor’s discretion. 
The doses for each arm have been selected from the forerunner AG348-C-001 SAD study and 
AG348-C-002 MAD studies in healthy adult volunteers to represent the range of doses/exposures 
that were safely tolerated and resulted in maximal or near maximal PD effects on 2,3-DPG and 
ATP. 
Because PK Deficiency is a rare disease with a limited eligible patient population and because 
the underlying pathophysiology and clinical phenotype of affected patients is heterogenous due 
to the wide variety of mutations in PKR that cause the disease, it is deemed important to focus 
closely on dose findings in this first-in-patient study. Therefore, in addition to initiating this 
study with 2 different doses of AG-348 administered q12h, a DRT will be established to review 
study data on a frequent basis and adapt the study design, dose and schedule of AG-348 as 
indicated.  
The DRT will monitor safety on an on-going basis and meet at regular intervals (approximately 
every 6 weeks), or ad hoc as necessary, to review AEs, VS, clinical laboratory (hematology, 
clinical chemistry, coagulation, and urinalysis), and ECGs on enrolled patients. The DRT will 
also review available PK/PD data and indicators of clinical activity (e.g., changes from baseline 
in Hb). The DRT will be comprised of the study Coordinating Investigator, treating 
Investigators, Medical Monitor, Clinical Pharmacologist, Statistician, and Sponsor’s Responsible 
Medical Officer. 
Approximately every 6 weeks, beginning 6 weeks after the first patient is doses or ad hoc as 
necessary, the DRT will review cumulative safety data, available PK/PD data, and clinical 
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activity data. Based on the DRT’s recurring 6-week reviews, the DRT may exercise one or more 
of the following options: 
• Continue treatment and enrollment in existing arms without change. 
• Add 1 new dose arm (Arm 3) to enroll up to 25 patients at a dose to be determined; 
the dose for Arm 3 may be lower or higher than Arm 1 and Arm 2 doses, but will not 
exceed 360 mg q12h; and the dose regimen may be less frequent than q12h. 
• Terminate or suspend enrollment to allow further review of clinical data in Arm 1 
and/or Arm 2 (and/or potential Arm 3). Enrollment in an arm could be terminated or 
suspended to allow further review, for example, for unacceptable safety/tolerability, 
poor PD response, or lack of signs of clinical activity. 
• Re-assign patient’s doses and schedule in a terminated arm to match the dose and 
schedule of another arm of the study. In this case, the patients in the terminated arm 
will remain in their original arm, i.e., they will not count towards the enrollment 
quota of the arm whose dose and schedule is being adopted. 
• Implement specific genotype restrictions to enrollment in one or more arms to ensure 
representation of patients with genotypes of greatest clinical relevance. 
The DRT will perform these evaluations on a recurring 6-week basis. The data that the DRT will 
review to make these decisions is expected to include, but are not necessarily limited to, the 
following:  
• Safety Observations: all AEs; VS, clinical laboratory (hematology, clinical chemistry, 
coagulation, and urinalysis), and ECGs;  
• PK and PD Observations: including changes in 2,3-DPG and ATP; 
• Indicators of Clinical Activity: including changes in Hb, HCT, reticulocyte count, Hp, 
COHb, LDH, EPO, total and indirect bilirubin, ferritin, and transferrin saturation.  
If a third dose arm is implemented, the dose of AG-348 selected will not exceed 360 mg BID, as 
this was the highest dose that demonstrated acceptable safety and tolerance in the 14-day 
multiple BID dosing study in healthy volunteers. The PK/PD sampling schedule in a potential 
third dosing arm will be determined by Sponsor’s discretion and may follow either the extensive 
or limited PK/PD sampling schedules as specified. 
The DRT will monitor the safety data in an ongoing manner as described and may make a 
decision to terminate enrollment in an arm if unacceptable AEs are observed, PD activity is 
limited, and/or markers of clinical activity indicate no effect. Unacceptable AEs are defined as 
≥ Grade 3 AEs (using National Cancer Institute Common Terminology Criteria for Adverse 
Events [NCI CTCAEv4.03]) that are assessed as at least possibly related to AG-348, with the 
exception of Grade 3 headache, hot flash/flushing, nausea, vomiting, and/or diarrhea that is 
transient in duration (< 24 hours) or able to be medically managed to ≤ Grade 2 within 24 hours. 
The DRT may also exercise discretion to terminate enrollment in an arm if it observes a 
frequency of Grade 2 AEs that would make it difficult to support long-term dosing. 
As the number of enrolling arms changes in the study (for example, from 2 to 3), the 
randomization scheme will adjust to enable balanced randomization into each actively accruing 
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arm. Randomization and stratification will cease in the event that only a single arm is left 
enrolling. 
Depending on possible early termination of 1 or both of the initial 2 arms, or the addition of a 
third arm, the study could enroll up to a maximum of 75 patients. 
Figure 3: 
Study Schema 
 
Abbreviations:  BID (q12h) = twice-daily (every 12 hours); DRT = data review team; PKR = pyruvate kinase red 
blood cell isoform; TBD = to be determined; w = weeks.  
The Investigator will monitor all patients for safety and tolerability. Modification of an 
individual patient’s dose of AG-348 will be based on AEs and observed changes in Hb levels as 
detailed in Section 9.7.1 and Section 9.7.2. 
Screening assessments will occur within 28 days prior to the first dose of study treatment. During 
the Treatment period, patients will attend visits at baseline (Day 1), weekly through Week 3 
(Days 8, 15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 12), and 
monthly through Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate AG 348 and 
demonstrate evidence of clinical activity of AG-348 through Week 24 may be eligible to 
immediately enter a separate extension study for continued treatment. For patients who finish 
treatment at the end of 24 weeks or sooner, or who elect not to enter the extension trial, study 
discharge will occur 4 weeks (Week 28 or earlier) following the last dose of study treatment at 
the final follow-up assessment. 
Safety assessments will include monitoring of AEs, including determination of SAEs and AEs 
leading to discontinuation; safety laboratory parameters (e.g., hematology, serum chemistry, 
coagulation studies, and urinalysis); physical examination findings; VS; and 12 lead ECGs. 
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Additional safety assessments will include monitoring of sex hormone levels (testosterone [total 
and free], estrone, and estradiol), and bone turnover markers (NTX and CTX). Follow-up 
assessments will be conducted on Day 197 (Week 28) and will include physical examination, 
weight, performance status, VS, 12-lead ECGs, laboratory evaluations (hematology, Hp, EPO 
levels, serum chemistry, coagulation studies, urinalysis; lipids, hormonal testing), AEs, and 
transfusion record.  
Pharmacokinetic assessments will include serial blood sampling for PK profiles of AG-348 and 
its metabolite AGI-8702. Pharmacodynamic evaluations will include serial blood sampling for 
determination of levels of ATP and 2,3 DPG. Extensive PK/PD sampling will be conducted on 
the first approximately 10 patients total treated in Arms 1 and 2 (see Appendix 15.1, Table 5) 
while more limited PK/PD sampling will be conducted on additional patients treated if 
enrollment in Arm 1 or Arm 2 is expanded or if an alternate dosing arm is added (see 
Appendix 15.1, Table 6).  
 
 
 
 
7.2. 
Justification of the Study Design 
The primary and secondary objectives of this study are to evaluate the safety, tolerability, PK 
and PD, and indicators of clinical activity of AG-348 in patients with PK Deficiency. The choice 
of dose and schedule of administration of AG -348 for Arms 1 and 2 was based on the highest 
safely tolerated dose (Arm 1: 300 mg BID) and the lowest dose with potentially relevant PD 
activity (Arm 2: 50 mg BID) from the forerunner AG348-C-002 MAD study in healthy 
volunteers. Decisions regarding continuing enrollment and treatment in these initial dose arms 
and/or implementation of an additional dose arm will be based on DRT review of safety, PK and 
PD data, and indicators of clinical activity collected from all patients treated in Arm 1 and 
Arm 2. This design was chosen to minimize risk to patients while allowing evaluation of safe 
and pharmacologically active dose levels of AG-348, and to allow the necessary flexibility to 
adjust dose and schedule should the safety, tolerability, PK, and/or PD be different in patients 
with PK Deficiency compared with healthy volunteers.  
Additional safety measures intended to minimize risk to patients include monitoring of AEs by 
the DRT and specified provisions for individual patient dose modification as needed for safety 
and (potentially) large increases in Hb level (Section 9.7.1 and Section 9.7.2). Measures intended 
to maximize the opportunity for patients with demonstrated safety and tolerability to continue to 
derive benefit from any observed clinical activity of AG-348 include the option for continued 
treatment in a separate extension study.  
A comprehensive series of safety evaluations, including laboratory parameters, physical 
examinations, VS, 12-lead ECGs, and monitoring for SAEs and AEs, will be conducted to 
evaluate the safety profile of AG-348 and to aid in the determination of the recommended dose 
for continued development. 
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The study includes serial blood sampling across at least two different doses of the study drug to 
assess its PK and PD profiles.  
Consistent with the design of many Phase 2 studies, preliminary evaluation of the potential PD 
and clinical activity of AG-348 are secondary objectives of this study. The latter will include 
assessments of the PK/PD relationship between AG-348 and the biomarkers ATP and 2,3-DPG, 
 
7.3. 
Rationale for the Starting Dose and Dose Range 
Prior to execution of this study, Agios conducted two clinical studies of AG-348 in healthy 
volunteers, including a SAD study (AG48-C-001) and a MAD (14 day q12h) study 
(AG348-C-002). Available details of these studies are discussed in the current Investigator’s 
Brochure (IB). Between these two studies, 72 healthy human subjects have been dosed with 
AG-348. In vitro investigations, also reported in the IB, had previously demonstrated that 
AG-348 increased the activity of wild-type PKR approximately to the same extent as it did a 
series a recombinant mPKRs. Therefore it was deemed reasonable to study the safety, 
tolerability, PK, and PD of AG-348 in healthy subjects in a controlled Phase 1 setting as a more 
efficient means of obtaining information than in the actual rare disease population of patients 
with PK Deficiency.  
The MAD study demonstrated that the exposures produced by AG-348 doses from 60 mg q12h 
to 360 mg q12h (including 120 mg q24h) resulted in maximal changes from baseline for the PD 
markers 2,3-DPG (reduction from baseline) and ATP (increase from baseline). The exposures 
resulting from doses less than 60 mg q12h were of lesser magnitude and the exposures resulting 
from doses greater than 360 mg q12h were of no greater magnitude than the aforementioned 
range. Therefore the starting doses for this first dose ranging study in patients with PK 
Deficiency were selected to be 300 mg q12h (Arm 1) and 50 mg q12h (Arm 2). These doses 
were demonstrated to be safe and tolerable in the healthy volunteer studies. The availability of 
ATP is proposed as being critical for optimally maintaining RBC membrane integrity (see 
Section 5.1). The dose ranges from 50 mg q12h to 300 mg q12h may result in clinically effective 
modulation of PKR in PK Deficiency patients if the mutated enzyme is responsive to AG-348 in 
a similar manner to the wild-type enzyme in healthy subjects. However, there are many different 
mutations in PKR that result in PK Deficiency, and these mutations produce variable effects on 
the enzyme in terms of catalytic activity and thermal stability. It is not known if different 
mutations will respond clinically in a similar manner to the same exposure to AG-348. 
Therefore, it is prudent to study the range of safe and pharmacodynamically relevant doses as 
specified in this study, and to allow flexibility for the DRT to analyze the evolving study data to 
adapt the dose and schedule of administration of AG-348 to produce the optimal combination of 
safety, tolerability, and PD, and, potentially, clinical response. 
7.4. 
Criteria for Study Termination 
This study may be prematurely terminated if, in the opinion of the Sponsor, there is sufficient 
reasonable cause. In the event of such action, written notification documenting the reason for 
study termination will be provided to each Investigator. 
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Circumstances that may warrant termination include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to patients; 
• Insufficient adherence to protocol requirements; 
• Plans to modify, suspend, or discontinue the development of the study drug; 
• Other administrative reasons. 
Should the study be closed prematurely, all study materials must be returned to the Sponsor or 
the Sponsor’s designee. 
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8. 
STUDY POPULATION 
8.1. 
Number of Patients 
Up to approximately 75 patients may be enrolled in this study. 
8.2. 
Inclusion Criteria 
For entry into the study, patients must meet all of the following criteria during the Screening or 
other specified period: 
1. Signed written informed consent obtained prior to performing any study procedure, 
including screening procedures.  
2. Male or female, aged 18 years and older. 
3. Known medical history of PK Deficiency. 
4. All patients must have documented clinical laboratory confirmation of PKD by RBC 
pyruvate kinase enzymatic assay performed at Screening by a designated central 
laboratory. Patients with prior documentation of PK Deficiency by RBC enzymatic assay 
will have a reconfirmation of this result during Screening as a condition of enrollment. 
a. In the event that a patient’s screening pyruvate kinase enzymatic assay is negative 
(i.e., shows normal pyruvate kinase activity), the patient will be eligible for 
enrollment if the genotyping shows a mutant genotype that has been previously 
documented in the literature to be associated with pyruvate kinase deficiency. If 
the genotyping shows a previously undescribed mutation in the PKR gene, then 
the eligibility for enrollment will be determined on a case-by-case basis by the 
Coordinating Investigator and Medical Monitor in discussion with the 
Investigator. If no mutation is defined, then the patient will not be eligible. 
5. All patients must have genotypic characterization of the mutant PKR gene performed by 
a designated central laboratory at Screening, unless genotype is available from the 
patient's participation in the Pyruvate Kinase Deficiency Natural History Study 
(NCT02053480). 
6. All patients must have genotypic characterization of the UGT1A1 gene performed by a 
designated central laboratory to document whether they may have underlying Gilbert's 
Disease. Patients with Gilbert’s Disease are eligible to enroll. 
7. Males must have Hb ≤ 12.0 g/dL; females must have Hb ≤ 11.0 g/dL. 
8. All patients must be considered transfusion independent as defined by: no greater than 
3 units of RBCs transfused in the 12-month period up to the first day of study dosing and 
no transfusions within 4 months of first day of study dosing. Patients who have received 
more transfusion support than described above will evaluated for eligibility on a case-by-
case basis by the Medical Monitor. 
9. Splenectomized patients: 
a. Must have undergone their procedure at least 6 months prior to Screening. 
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b. Must be current in their vaccinations for Pneumococcal Conjugate (PCV13), 
Pneumococcal Polysaccharide (PPSV23), Quadrivalent Meningococcal vaccine, 
and Haemophilus influenzae type b (Hib) as recommended by Centers for Disease 
Control and Prevention's (CDC) Advisory Committee on Immunization Practices 
(ACIP) or immunization advisory groups in Canada and the European Union (for 
patients enrolled in Canada and the EU). 
[http://www.cdc.gov/vaccines/schedules/downloads/adult/adult-schedule.pdf] 
[Any missing vaccinations may be administered during the screening period.] 
10. Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 2. 
11. Patients must be taking at least 1 mg of folic acid daily for at least 21 days prior to first 
dose and continued daily during study participation. 
12. Adequate organ function, defined as: 
a. Serum AST and ALT ≤ 1.5 × upper limit of normal (ULN) (unless the increased 
AST is assessed by the Investigator as due to hemolysis). 
b. Normal or elevated levels of serum bilirubin. In patients with serum bilirubin 
> ULN, the elevation must be attributed to hemolysis with or without Gilbert's 
syndrome and must not be choledocholithiasis, cholecystitis, biliary obstruction, 
or hepatocellular disease. 
c. Serum creatinine ≤ 1.25 × ULN. If serum creatinine > 1.25 × ULN, then 24-hour 
measured or calculated (Cockcroft-Gault) glomerular filtration rate 
(GFR) ≥ 60 mL/min. 
d. Absolute neutrophil count (ANC) > 1.0 × 109/L. 
e. Platelet count ≥ 100 × 109/L. 
f. Activated partial thromboplastin time (aPTT) and international normalized ratio 
(INR) ≤ 1.25 × ULN, unless the patient is receiving therapeutic anticoagulants. 
13. Women of childbearing potential (WOCBP) must agree to abstain from sexual 
intercourse or to use an acceptable/effective method of contraception (i.e., condom plus 
spermicide, condom plus oral contraceptive, condom plus intrauterine device [IUD], 
condom plus diaphragm with spermicide) from as soon as feasible during the Screening 
period until 30 days following the last dose of AG-348. 
a. WOCBP include any female who has experienced menarche and who has not 
undergone successful surgical sterilization (hysterectomy, bilateral tubal ligation, 
and/or bilateral oophorectomy) or is not post-menopausal. Post-menopausal is 
defined as: 
i. Amenorrhea ≥ 12 consecutive months without another cause, and a 
documented serum follicle stimulating hormone (FSH) level 
> 35 mIU/mL; 
ii. Amenorrhea≥ 12 consecutive months in women ≥ 62 years old (FSH 
testing is not required). 
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14. WOCBP must have a negative serum or urine pregnancy test within 72 hours before start 
of AG-348 dosing. 
15. Women must not be breastfeeding.  
16. Male patients, with the exception of those who have undergone vasectomy at least 
6 months prior to Screening, must agree to abstain from sexual intercourse or, if sexually 
active, to use a condom with spermicide as contraception (regardless of their female 
partner's childbearing potential or their partner's use of their own contraception) from 
Day 1 of dosing until 30 days following the last dose of AG-348. 
8.3. 
Exclusion Criteria 
Patients who meet any of the following criteria at Screening or prior to dosing on Day 1 will not 
be enrolled in the study: 
1. Hemoglobin level > 12.0 g/dL if male; Hb > 11.0 g/dL if female. 
2. Additional diagnosis of any other congenital or acquired blood disorder, including 
glucose-6-phosphate-dehydrogenase (G6PD) deficiency, or any other hemolytic anemia 
process except for mild allo-immunization as a consequence of transfusion therapy. 
3. Iron overload (hemosiderosis or concurrent hemochromatosis) sufficiently severe to 
result in a clinical diagnosis by the Investigator of cardiac, hepatic, or pancreatic 
insufficiency. 
4. Prior bone marrow or stem cell transplant. 
5. Clinically symptomatic cholelithiasis or cholecystitis. (Prior cholecystectomy is not 
exclusionary. Patients with symptomatic cholelithiasis or cholecystitis may be rescreened 
once the disorder has been treated and clinical symptoms have resolved.) 
6. Currently enrolled in another therapeutic clinical trial involving on-going therapy with 
any investigational or marketed product or placebo. Concurrent participation in the 
Pyruvate Kinase Deficiency Natural History Study (NCT02053480) is permitted. 
7. Exposure to any investigational drug, device, or procedure within 28 days prior to 
Screening. 
8. Concurrent medical condition that could compromise participation in the study such as: 
a. Poorly controlled hypertension (defined as systolic blood pressure (BP) 
> 150 mm Hg or diastolic BP > 90 mm Hg) refractory to medical management. 
b. History of recent (within < 6 months from Screening date) congestive heart 
failure; myocardial infarction or unstable angina pectoris; or hemorrhagic, 
embolic, or thrombotic stroke; deep venous thrombosis; or pulmonary or arterial 
embolism. 
c. Currently active infection requiring the use of parenteral anti-microbial agents or 
that is ≥ Grade 3 (CTCAEv4.03) within 6 months of first dose. 
d. A pattern or frequency of post-splenectomy sepsis that in the assessment of the 
Investigator could reasonably be expected to interfere with the ability of the 
patient to complete the 24 week study participation. 
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e. Positive test for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) 
antibody with signs of active Hepatitis B or C virus infection.  
f. Positive test for human immunodeficiency virus (HIV) 1 or 2 antibody. 
g. Diabetes mellitus judged to be in poor control by the Investigator or requiring 
> 3 anti-diabetic agents counting insulin; use of insulin per se is not exclusionary.  
h. History of any primary malignancy with the exception of: curatively treated non-
melanomatous skin cancer; curatively treated cervical or breast carcinoma in situ; 
or other primary tumor treated with curative intent and no known active disease 
present and no treatment administered during the last 3 years. 
9. Undergone major surgery within 6 months of first dose.  
10. Current or recent history of psychiatric disorder that in the opinion of the Investigator or 
Medical Monitor could compromise the ability of the patient to cooperate with study 
visits and procedures. 
11. Use of any of the restricted list of products known to strongly inhibit CYP3A4 
metabolism (Appendix 15.3, Table 7) within 5 days prior to Day 1 dosing; or to strongly 
induce CYP3A4 metabolism (Appendix 15.3, Table 8) within 28 days prior to Day 1 
dosing; or to strongly inhibit P-gp transporter (Appendix 15.3, Table 9) within 5 days 
prior to Day 1 dosing; or digoxin within 5 days prior to Day 1 dosing. 
12. Serum bilirubin > ULN attributable to factors other than hemolysis and/or Gilbert's 
syndrome. 
13. Male patients with heart-rate corrected QT (Fridericia's correction factor) QTcF 
interval > 450 msec, or female patients with QTcF interval > 470 msec with the 
exception of patients with a left bundle branch block (LBBB). Medical Monitor approval 
needed in patients with a LBBB. 
14. Cardiac dysrhythmias judged as clinically significant by the Investigator or requiring 
therapy with drugs that are primarily substrates of CYP3A4. 
15. History of allergy to sulfonamides if characterized by acute hemolytic anemia, 
anaphylaxis, or rash of erythema multiforme type or Stevens-Johnson syndrome. 
16. Any other medical or psychological condition deemed by the Investigator to be likely to 
interfere with a patient’s ability to understand and sign informed consent; cooperate with 
study visits, tests, and procedures; or otherwise safely and reliably participate in the 
study. 
8.4. 
Patient Identification and Registration 
Patients who are candidates for enrollment into the study will be evaluated for eligibility by the 
Investigator to ensure that the inclusion and exclusion criteria (see Section 8.2 and Section 8.3, 
respectively) have been satisfied and that the patient is eligible for participation in this clinical 
study. The site will submit to the Sponsor an Eligibility form for each eligible patient and the 
Medical Monitor will confirm eligibility for all patients prior to receipt of the first dose of 
AG-348. 
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8.5. 
Patient Randomization 
Patients who have been confirmed as eligible will be randomized in an equal ratio to a treatment 
arm (e.g., 1:1 or 1:1:1 depending on which arms are open). A randomization number will be 
assigned. The site will provide a request for randomization form (including the patient’s 
confirmed genotype) to a central randomization center. The randomization will be stratified by 
PKR mutation in order to maintain balance across the dose arms for the specific mutations 
expected to be most frequently enrolled. The PKR mutation stratification factor will consist of 
4 levels (R510Q, R486W, and R479H) and all other mutations (“other”). Since this is an open 
label study, randomization will not be blinded. 
Please refer to the study manual for the randomization procedure. 
8.6. 
Patient Withdrawal Criteria 
Patients have the right to withdraw from the study at any time for any reason. Patients may 
withdraw or be withdrawn from study-related procedures and treatments under the following 
conditions: 
• Withdrawal of consent; 
• Experiences unacceptable toxicity; 
• Development of an intercurrent medical condition that precludes further participation 
in the trial; 
• Patient requires use of a prohibited concomitant medication (Section 9.11.2); 
• Investigator decision; 
• Protocol violation: non-adherence to protocol requirements; 
• Pregnancy; 
• Lost to follow-up. 
Should a patient decide to withdraw, all efforts will be made to complete and report the protocol-
defined study observations up to the time of the patient’s withdrawal as completely as possible 
and to determine the reason for withdrawal. 
In the event a patient is withdrawn from the study, the Medical Monitor must be informed. If 
there is a medical reason for withdrawal, the patient will remain under the supervision of the 
Investigator until satisfactory health is returned. 
When a patient withdraws from the study, the primary reason for discontinuation must be 
recorded in the appropriate section of the electronic case report form (eCRF) and all efforts will 
be made to complete and report final study observations as thoroughly as possible. 
All AEs should be followed until resolution or for a period of 30 days from the last dose of study 
drug, whichever is shorter. Patients with toxicity suspected to be related to study drug will 
continue follow-up until the AE resolves, is declared chronic by the Investigator, or the patient is 
lost to follow-up. 
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8.7. 
Replacement of Patients 
Patients who drop out of the study prior to completing the first 12 weeks of assigned dosing for 
reasons other than AEs may be replaced at the Sponsor’s discretion. 
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9. 
STUDY TREATMENT 
9.1. 
Description of Study Drug 
AG-348 sulfate hydrate capsules will be provided as 25 mg or 100 mg (free-base equivalent) of 
AG-348 sulfate hydrate without excipients in Swedish orange (25 mg) or white opaque (100 mg) 
gelatin capsules (size 2 capsules for all dose strengths). 
All study drugs are for investigational use only and are to be used only within the context of this 
study. All study drug products will be supplied by the Sponsor. Please see the Investigator’s 
Brochure for further details regarding study drug. 
9.2. 
Study Drug Packaging and Labeling 
AG-348 sulfate hydrate capsules are packaged in white, high-density polyethylene (HDPE) 
induction sealed bottles with a child-resistant screw cap. 
Packaging and labeling will be prepared to meet all regulatory requirements. 
9.3. 
Study Drug Storage 
AG-348 sulfate hydrate drug capsules must be stored at room temperature of 15 to 30° C 
(59 - 86 °F). 
All study drug products must be stored in a secure, limited-access location and may be dispensed 
only by the Investigator or by a member of the staff specifically authorized by the Investigator. 
9.4. 
Method of Assigning Patients to Treatment 
Up to a maximum of 25 patients will be randomized to any one of the dosing arms in this study. 
Randomization will be balanced 1:1 or 1:1:1, depending on whether 2 or potentially 3 arms are 
open, and will be stratified by PKR mutation (see Section 8.5). The dose and schedule of 
AG-348 each patient receives will be dependent upon which dose arm is open for enrollment 
when the patient qualifies for and is randomized into the study.  
9.5. 
Blinding 
This is an open-label study; no blinding methods will be used. 
9.6. 
Study Drug Preparation and Administration 
For the initial two treatment arms, (Arm 1 and Arm 2), AG-348 will be administered orally BID 
(approximately every 12 hours with a minimum of 10 hours between doses) over a 24 week 
treatment period. Starting with Day 1, dosing will be continuous; there will be no rest periods. 
Patients who do not meet any of the treatment withdrawal criteria (see Section 8.5) may continue 
treatment for the entire 24-week treatment period. 
Patients will be dispensed the appropriate number of Sponsor-packaged, labeled bottles to allow 
for 28 days of dosing until the next scheduled visit.  
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Patients will be given a dosing diary to be used for each 28-day dosing period. They should 
record relevant information regarding their study drug in the diary (e.g., confirmation that each 
daily dose was taken, reasons for missed doses). 
Treatment compliance will be assessed based on return of unused drug and the dosing diary (see 
Section 9.9). 
Patients should be instructed to take their daily dose at approximately the same times each day 
except for dosing on in-clinic visiting days.  
Patients who undergo extensive PK/PD sampling (see Appendix 15.1, Table 5) should be 
instructed from Week 3 on to bring the AM dose with them for in-clinic visits and to ingest the 
dose following PK/PD blood draws.  
Patients receiving limited PK/PD sampling (see Appendix 15.1, Table 6) should be instructed to 
bring the AM dose with them for all in-clinic visits and to take the AM dose following PK/PD 
blood draws.  
Patients receiving extensive PK/PD sampling on Day 1 and 15 will also have limited PKPD on 
other visit days. As a general rule, regardless of extensive or limited schedule, patients will bring 
in the AM dose for all visits and take this dose following PK/PD blood draws.  
A minimum of 10 hours between the AM and PM dose will be required on those dosing days. 
Each dose should be taken with a glass of water and consumed over as short a time as possible. 
AG-348 may be taken with or without food. Patients should be instructed to swallow capsules 
whole and to not chew the capsules. For patients who have difficulty swallowing tablet(s), the 
Medical Monitor should be contacted to discuss administration. 
Patients will receive their first dose of AG -348 in the clinic on Day 1 and then may take the 
remaining doses on an outpatient basis. 
9.7. 
Criteria for Dose Escalation, Dose Modification, or Discontinuation 
of Study Drug 
No intra-patient dose escalations will be permitted in this study unless the DRT decides to re-
assign patients’ doses and schedule in a terminated arm to match the dose and schedule of 
another arm of the study. In this case, the patients in the terminated arm will remain in their 
originally assigned arm; i.e., they will not count towards the enrollment quota of the arm whose 
dose and schedule is being adopted. All dosing modifications, as outlined below, will be 
implemented following discussions with the Medical Monitor. 
9.7.1. 
Dose Modification for Safety 
The Investigator will monitor all patients for safety and tolerability. Modification of the patient’s 
dose of AG-348 will be based on AEs and observed changes in Hb levels (see Section 9.7.2). 
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Table 1: 
Dose Modification for Adverse Events Deemed at Least Possibly Related to 
AG-348 
Adverse Events(s) 
AG-348 Dose Adjustment 
Grade 1 
None required. 
Grade 2 
None required; Investigator and Medical Monitor judgment to manage as for 
Grade 3. 
Grade 3 
Suspend dosing;  
If event resolves to Grade 1 or baseline within approximately 14 days of 
suspension, resume dosing with 1 dose level reduction (see Table 2 below).  
If event does not resolve to Grade 1 or baseline within approximately 14 days of 
suspension, permanently discontinue dosing, unless the benefits outweigh the 
risks of resuming treatment and are approved by the Medical Monitor. 
Grade 4 
Permanently discontinue dosing, unless the benefits outweigh the risks of 
resuming treatment and are approved by the Medical Monitor. 
Dose modifications for Grade 2 and 3 AEs should be maintained long enough for the 
Investigator to be confident that the reduced dose is being well tolerated before considering a 
request of the Medical Monitor to permit subsequent re-escalation to the former dose level. 
Dosing for an individual patient will be discontinued permanently for Grade 3 and Grade 4 AEs 
that do not resolve to Grade 1 or baseline within approximately 14 days of suspension of dosing, 
unless the benefits outweigh the risks of resuming treatment and are approved by the Medical 
Monitor. No patients may be re-escalated to their former dose level after a dose modification 
without discussion with the Medical Monitor.   
It should be noted that if the initial dose of 300 mg q12h BID selected for Arm 1 demonstrates an 
unacceptable safety profile resulting in multiple subjects undergoing dose modifications, the 
DRT may exercise its option to re-assign these patients’ dose and schedule to match the dose and 
schedule of another study arm (for example, Arm 2 of the study, or to match the dose and 
schedule of a [potential] Arm 3, if implemented). 
9.7.2. 
Dose Modification for Increase in Hemoglobin Level 
It is presently unknown to what magnitude, how rapidly, or even whether, AG-348 will result in 
increased Hb levels in patients with PK Deficiency across the variety of potential PKR mutations 
that may be encountered during this study. Moreover, it is unknown whether any potential 
increases in Hb experienced by patients in this study may be a safety risk. As a conservative 
measure, this study incorporates guidance for dose modification based on potential large 
increases in Hb levels that exceed the midpoint of the typical normal range by gender. The 
Investigator will monitor all patients for changes in Hb levels and should adjust the dose of 
AG-348 as outlined in Table 2 per the following guidelines and with Medical Monitor approval. 
• Males: If Hb > 15 g/dL and confirmed with a second test, suspend dosing until Hgb 
≤ 13.5 g/dL; then resume dosing with a 1 dose level reduction.  
• Females: If Hb > 13.5 g/dL and confirmed with a second test, suspend dosing until 
Hb ≤ 12.5 g/dL; then resume with a 1 dose level reduction.  
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• The treating Investigator will discuss with the Medical Monitor questions relating to 
dose modifications on an as needed basis.  
Table 2: 
Dose Reduction Table (by Dosing Arm) 
Dose Group 
Starting Dose 
1st Dose Reduction 
2nd Dose Reduction 
Arm 1 
300 mg BID 
200 mg BID 
100 mg BID 
Arm 2 
50 mg BID 
25 mg BID 
TBD1 
Potential Arm 3 
TBD 
To approximately 
50-66% of initial dose 
To approximately 
25-33% of initial dose 
1 Dose to be determined by Medical Monitor. 
9.7.3. 
Stopping Criteria 
Dosing for an individual patient will be discontinued permanently for Grade 3 AEs that do not 
resolve to Grade 1 or baseline within approximately 14 days of suspension of dosing, and for 
Grade 4 AEs, unless the benefits outweigh the risks of resuming treatment and are approved by 
the Medical Monitor (Section 9.7.1). Other reasons for treatment termination are provided in 
Section 8.5. 
9.8. 
Duration of Patient Participation 
The duration of treatment for all patients on this study will be up to 24 weeks. Patients who 
safely tolerate and demonstrate one or more indicators of clinical activity of AG-348 may be 
eligible to immediately roll over to a separate safety extension study for continued treatment. 
9.9. 
Treatment Compliance 
During in-clinic visits, doses of AG-348 will be ingested by the patient under the supervision of 
clinical facility personnel. For at-home dosing, patients will be given a dosing diary to be used 
for the duration of the 24-week treatment period. Patients should record relevant information 
regarding their study drug in the diary (e.g., confirmation that each daily dose was taken, reasons 
for missed doses) and return the diary at each study visit. 
9.10. 
Study Drug Accountability 
Accountability for the study drug at the clinical facility is the responsibility of the Investigator. 
The Investigator will ensure that the study drug is used only in accordance with this protocol. 
Where allowed, the Investigator may choose to assign drug accountability responsibilities to a 
pharmacist or other appropriate individual. 
The Investigator or delegate will maintain accurate drug accountability records indicating the 
drug’s delivery date to the site, inventory at the site, use by each patient, and return to Agios or 
its designee (or disposal of the drug, if approved by Agios). These records will adequately 
document that the patients were provided the doses as specified in the protocol and should 
reconcile all study drug received from Agios. Accountability records will include dates, 
quantities, batch/serial numbers, expiration dates (if applicable), and patient numbers. An 
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unblinded monitor will review drug accountability at the site on a schedule agreed to by the 
Sponsor. 
Study drug must not be used for any purpose other than the present study.  
All unused and used study drug will be retained at the site until it is inventoried by the Study 
Monitor after database lock has occurred. All used, unused or expired study drug will be returned 
to Agios or its designee or, if authorized, disposed of at the study site per the site’s Standard 
Operating Procedures (SOPs) and documented. All material containing AG-348 will be treated 
and disposed of as hazardous waste in accordance with governing regulations. 
9.11. 
Prior and Concomitant Medications and Treatments 
9.11.1. 
Prior Medications and Procedures 
All medications administered and procedures conducted within 28 days prior to the first day of 
study drug administration are to be recorded on the source documentation and included in the 
eCRF. 
9.11.2. 
Prohibited Concomitant Therapy 
All concomitant medications and procedures administered from 28 days before administration of 
study drug through the last Follow-up Visit (Week 28/Day 197) must be recorded in the 
appropriate section of the source documentation and eCRF along with dosage information, dates 
of administration, and reason for use.  
The following are prohibited at all times during participation in this study: 
• Investigational drugs must be discontinued 28 days prior to the first dose of study 
drug; 
• Products known to strongly inhibit CYP3A4 metabolism (listed in Appendix 15.3, 
Table 7) must be discontinued within 5 days prior to Day 1 dosing; 
• Products known to strongly induce CYP3A4 metabolism (listed in Appendix 15.3, 
Table 8) must be discontinued within 28 days prior to Day 1 dosing; 
• Products known to strongly inhibit P-gp transporter (listed in Appendix 15.3, Table 9) 
must be discontinued within 5 days prior to Day 1 dosing; 
• Digoxin must be discontinued within 5 days prior to Day 1 dosing; 
• Hematopoietic stimulating agents (erythropoietins, granulocyte colony stimulating 
factors, thrombopoietins, etc) must be discontinued no less than 28 days prior to the 
first dose of study drug. [Folic acid 1 mg orally per day is required for all patients. 
B12 injections are permitted for patients with a prior diagnosis of B12 deficiency 
syndromes, but must be repleted to stability of the Hb and mean corpuscular volume  
(MCV) prior to enrollment in the study]; 
• Anabolic steroids, including testosterone preparations, administered for anemia must 
be discontinued no less than 28 days prior to the first dose of study drug;  
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• As the target population for this study consists of transfusion independent patients 
and transfusion of blood products could confound key endpoints of the study, blood 
transfusions of any type must be strictly avoided except in cases of compelling 
medical need. If medical circumstances permit, the Medical Monitor should be 
contacted for discussion before any transfusions are administered. 
• Other drugs that displace unconjugated bilirubin from albumin. 
Potential Drug Interactions 
Strong inhibitors of CYP3A4 (listed in Appendix 15.3, Table 7) are not permitted for use with 
AG-348. Based on modeling of AG-348 metabolism and tolerability data from the Phase 1 MAD 
study, moderate inhibitors of CYP3A4 do not appear to pose a risk to patients dosed with 
AG-348. 
In-vivo data from the Phase 1 MAD show AG-348 induces CYP3A4 and its own metabolism, 
presumably via CYP3A4. The use of strong CYP3A4 inducers in combination with AG-348 is 
expected to reduce AG-348’s efficacy. Therefore, administration of strong CYP3A4 inducers 
(listed in Appendix 15.3, Table 8) is not permitted with AG-348.  
Strong inhibitors of drug transport (listed in Appendix 15.3, Table 9) are not permitted for use 
with AG-348. 
Digoxin is not permitted for use with AG-348. 
Induction of CYP3A4 by AG-348 is expected to reduce the efficacy of certain sensitive co-
medications. Co-administration of AG-348 with the drugs in Appendix 15.3, Table 10 should be 
done with caution, as their efficacy may be reduced.  
Of note, women in the trial utilizing oral contraception must utilize barrier methods as per the 
Inclusion Criteria 14 (Section 8.2) while taking AG-348. 
The expected patient co-medications deferoxamine, deferasirox, and oral penicillin are not 
expected to interact with AG-348. 
9.11.3. 
Allowed Concomitant Therapy 
Medications and treatments other than those specified above are permitted during the study. All 
intercurrent medical conditions will be treated at the discretion of the Investigator according to 
acceptable local standards of medical care. Subjects may receive analgesics, antiemetics, anti-
infectives (including penicillins), and antipyretics as medically indicated and consistent with the 
guidance in the previous two sections. Patients may continue iron chelation therapy with 
deferoxamine and deferasirox. Patients must continue taking 1 mg of folic acid for the duration 
of the study. 
All concomitant medications, including transfusions of blood products, procedures performed 
during the study, including those used to treat AEs, will be recorded on the eCRF.  
9.11.4. 
Potential for Phototoxicity 
AG-348 may cause sensitivity to direct and indirect sunlight. Patients should be warned to avoid 
direct sun exposure. When exposure to sunlight is anticipated for longer than 15 minutes, the 
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patient should be instructed to apply factor 30 or higher sunscreen to exposed areas and wear 
protective clothing and sunglasses. 
9.11.5. 
Management of Nausea, Vomiting, and Diarrhea 
As the primary objective of this study is to assess the safety and tolerability of up to 24 weeks of 
AG-348, routine use of prophylactic anti-emetic and antidiarrheal or other classes of medications 
is prohibited. However, if patients experience AEs relating to nausea, vomiting, or diarrhea these 
may be treated at the Investigator's clinical discretion with recommended medications as follows: 
• Nausea/vomiting: allowed agents include standard clinical dosing with palonosetron 
(Aloxi), dolasetron (Anzemet), or promethazine (Phenergen). Promethazine is a 
substrate for CYP2B6, and it is presently unknown if the potential for 2B6 induction 
after AG-348 dosing could be sufficient to reduce the therapeutic effect of 
promethazine. Not recommended are aprepitant (Emend; CYP3A4 inhibitor), 
ondansetron (Zofran; CYP3A4 inducer), chlorpromazine (Thorazine; CYP3A4 
inducer), prochlorperazine (Compazine; CYP3A4 substrate, QT prolongation has 
been reported), and granisetron (Kytril; CYP3A4 substrate, QT prolongation has been 
reported);  
• Diarrhea: recommended management includes standard clinical dosing with 
Kaopectate or other non-absorbable anti-diarrheals, diphenoxylate/atropine (Lomotil), 
or loperamide (Imodium). Loperamide is the least preferred choice because it is both 
a substrate and inhibitor for CYP3A4, a substrate for CYP2B6, and a substrate for 
P-gp. 
• For the use of any medications not specifically mentioned above the Investigator may 
confer with the Sponsor’s Medical Monitor. 
9.11.6. 
Other Restrictions and Precautions 
Patients should be advised to refrain from altering their normal exercise routine for the first 
28 days of treatment with AG-348 as symptoms of anemia could theoretically temporarily 
worsen before the eventual potential beneficial effect of AG-348 on the Hb level becomes 
evident. The elevated blood levels of 2,3-DPG commonly seen in patients with PK Deficiency 
may produce a right shift in the Hb-O2 dissociation curve. The effect of this is to enhance 
oxygen delivery at the tissue level, thus counteracting to some extent the physiologic 
consequences of the anemia. AG-348 was shown to produce rapid decreases (within the first 
12 hours following a single dose) in 2,3-DPG in two prior clinical trials with healthy adult male 
and female volunteers. In patients with PKD who have elevated 2,3-DPG levels, it is 
theoretically possible that decreases in 2,3-DPG that precede correction of the anemia could 
result in a temporary decrease of oxygen delivery at the tissue level resulting in clinical 
symptoms (e.g., increased fatigue). 
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10. 
STUDY ASSESSMENTS 
10.1. 
Schedule of Assessments 
The Schedules of Assessments for this study are provided in Appendix 15.1.  
After obtaining written informed consent, patients will undergo screening evaluations. The 
Screening visit is to be conducted within approximately 28 days prior to first dose of study 
treatment.  
During the Treatment period, patients will attend visits at baseline (Day 1), weekly through 
Week 3 (Days 8, 15, and 22), triweekly starting at Week 6 through Week 12 (Weeks 6, 9, and 
12) and monthly through Week 24 (Weeks 16, 20, and 24). Patients who safely tolerate and 
demonstrate one or more indicators of clinical activity of AG-348 through Week 24 may be 
eligible to immediately enter a separate extension study for continued treatment upon agreement 
of the treating Investigator and the Medical Monitor. For patients who finish treatment, Study 
Discharge will occur 4 weeks (Week 28 or earlier) following the last dose of study treatment at 
the final follow-up assessment. 
Whenever more than one assessment is scheduled for the same nominal time, the assessments 
should be performed in the order of least invasive to most invasive assessment (e.g., VS first, 
ECG, blood draw). The timing of these assessments should allow the PK blood draw to occur at 
the exact nominal time. The order of procedures may be revised with prior discussion between 
Sponsor and Site. 
Minor adjustments to the timing, number of planned safety monitoring procedures (e.g., VS, 
ECG, blood draw), and PK/PD assessments may be made during the course of the study based on 
collected data to ensure appropriate safety monitoring and will not require a protocol 
amendment. These minor changes will require prior approval from the Sponsor’s Medical 
Monitor (or Responsible Medical Officer) as well as appropriate documentation in the study 
records. The addition of new safety monitoring procedures or other assessments will require a 
protocol amendment. 
10.2. 
Informed Consent and Confirmation of Eligibility 
A complete description of the study is to be presented to each potential patient and a signed and 
dated informed consent is to be obtained before any study specific procedures are performed.  
Patient’s eligibility will be confirmed at Screening and within 24 hours prior to study treatment 
dose. If a patient is determined to be ineligible, the patient will be excluded from participation. 
10.3. 
Demographic Data, Medical and Medication History 
Patient demographic data, including gender, date of birth, age, race, and ethnicity, will be 
obtained at Screening. Collection of demographic data will be modified by country regulatory 
requirements, as appropriate.  
Medication history, including all relevant prior medical history and current medical conditions, 
will be obtained at the Screening assessment and on Day -1; all concomitant medications 
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administered and procedures conducted within 28 days prior to Day 1 should be reported in the 
source documentation and eCRF. Investigators will be asked to provide information on the 
patient’s history of any medical diagnoses (e.g., iron overload) and surgical procedures (e.g., 
splenectomy, cholecystectomy) pertaining to their diagnosis of PK deficiency and prior available 
complete blood counts (CBCs) over the preceding 6 months and transfusion history over the 
preceding 12 months prior to the date of signing informed consent.  
10.4. 
PKR Enzymatic Assay and PKR Genotyping 
Assessments for PKR enzymatic activity and PKR genotyping will be performed at Screening 
only for confirmation of study eligibility. PKR enzymatic assays will be conducted at 
 
. PKR genotyping will be conducted at 
 
 
10.5. 
Safety Assessments 
10.5.1. 
Physical Examination, Height, and Weight 
A complete physical examination (genital and rectal examinations will be performed at the 
discretion of the Investigator) will be obtained at Screening, Baseline, Week 12, and the Follow 
up Visit (Week 28). For patients rolling over into extension study, the last physical examination 
will occur at Week 24. Limited focused physical examinations will be performed at all other 
visits. Any findings will be recorded on the eCRF. Height will be collected at Screening only. 
10.5.2. 
Vital Signs 
Vital signs, including systolic and diastolic BP, heart rate, respiratory rate, and temperature, will 
be obtained according to the according to the Schedule of Assessments (Appendix 15.1). Vital 
sign measurements will be repeated if clinically significant or machine/equipment errors occur. 
Out of range BP or heart rate measurements will be repeated at the Investigator’s discretion. Any 
confirmed, clinically significant vital sign measurements will be recorded as AEs. 
10.5.3. 
Electrocardiogram  
A 12-lead ECG will be obtained according to the Schedule of Assessments (Appendix 15.1). The 
ECGs will be measured using an ECG machine that calculates the heart rate and measures PR, 
QRS, QT, QTcB (Bazett correction formula), and QTcF (Frederica’s correction). Only QTcF 
(not QTcB) will be used for determination of eligibility. 
The 12-lead ECGs should be obtained following 5 minutes of recumbency. The Screening ECG 
will be performed at least 7 days prior to Day 1 dosing. ECGs will be repeated if clinically 
significant abnormalities are observed, if artifacts are present, or if machine/equipment errors 
occur. Any confirmed, clinically significant ECG findings will be recorded as AEs. 
10.5.4. 
Safety Laboratory Assessments 
10.5.4.1. Hematology, Serum Chemistry, Coagulation Studies, and Urinalysis 
Laboratory values obtained prior to Screening and RBC antibodies obtained at Screening will be 
performed at a local laboratory. On-study clinical laboratory evaluations are to be performed by 
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a central laboratory. If Investigators believe that it is clinically indicated to obtain safety 
laboratory results from their own local laboratories on the day of the subject's visit, and before 
the results are returned from the designated central laboratory, they are free to exercise their 
discretion to do so. Investigators should be aware that since the Hb level is a secondary endpoint 
of the study, it behooves them to minimize blood volumes drawn, and samples for the central lab 
must still be collected, since it will serve as the official lab result for this study. 
Clinical laboratory evaluations are to be collected according to the Schedule of Assessments 
(Appendix 15.1). In addition, all clinically significant laboratory abnormalities noted on testing 
will be followed by repeat testing and further investigated according to the judgment of the 
Investigator. Please note that serum free and total testosterone will be collected at 2 time points 
during Screening at least 2 days apart in addition to Week 1/Day 1 (total of 3 time points prior to 
Day 1 dosing).  
The following safety laboratory parameters are to be determined: 
Hematology: 
HCT, Hb, RBC count, absolute reticulocyte count, percent 
reticulocyte count, MCV, mean corpuscular Hb (MCH), mean 
corpuscular Hb concentration (MCHC), red cell distribution width 
(RDW), white blood cell (WBC) count with manual differential, 
ANC, and absolute lymphocyte count (ALC), and platelet count. 
G6PD and RBC antibody screen will be performed at Screening only 
Other 
EPO, Hp, COHb 
Serum Chemistry: 
alkaline phosphatase (ALP), sodium, potassium, chloride, calcium, 
magnesium, phosphorus, carbon dioxide (CO2), albumin, total 
protein, glucose, blood urea nitrogen (BUN), creatinine, uric acid, 
LDH, ALT, AST, total bilirubin, and indirect bilirubin. 
Sex Hormones: 
testosterone (total and free), estrone, and estradiol. FSH will only be 
performed at Screening for female patients only for confirmation of 
post-menopausal status. 
Bone Turnover 
serum NTX and CTX. 
Lipids 
total cholesterol, HDL-C, triglycerides. 
Iron Panel 
iron (Fe), total iron-binding capacity (TIBC), percent saturation, 
ferritin. 
Coagulation Studies: 
fibrinogen, aPTT, INR. 
Urinalysis: 
color and appearance, pH, specific gravity, ketones, protein, glucose, 
bilirubin, nitrite, urobilinogen, pregnancy screen (dipstick), occult 
blood. Microscopic inspection of sediment will only be performed 
for cause or to investigate an abnormal dipstick finding per the 
Investigator’s discretion. 
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10.5.4.2. Screening Serology 
A blood sample for serology, including HBsAg, HCV Ab screen, and HIV1 and HIV2 Ab, is to 
be collected from all patients at Screening.  
10.5.5. 
Adverse Events 
Each patient will be carefully monitored for the development of any AEs throughout the study 
from signing of the informed consent through all scheduled study follow-up visits, or withdrawal 
of consent, whichever occurs first. In addition, SAEs that are assessed as possibly or probably 
related to study treatment that occur > 30 days post-treatment also are to be reported. 
AEs will be evaluated by the Investigator and recorded as described in the Schedule of 
Assessments. On dosing visits, all AEs (elicited and spontaneously reported) will be 
continuously evaluated by the Investigator and recorded. At any non-dosing day visit, AEs will 
be evaluated by the Investigator and recorded. Any AEs already documented at a previous 
assessment and designated as ongoing will be reviewed at subsequent visits or assessment time 
points as necessary. If these AEs have resolved, this will be documented. 
All AEs will be graded using the CTCAE grading system (Appendix 15.2 ). 
Complete details on AE monitoring are provided in Section 11. 
10.6. 
Pharmacokinetic Assessments 
10.6.1. 
Blood Sample Collection and Pharmacokinetic Measurements During Dose 
Escalation 
The first approximately 10 patients treated, contingent on clinical site feasibility, will undergo 
extensive PK sampling as detailed in Appendix 15.1, Table 5. The remainder of treated patients 
will undergo limited PK sampling as detailed in Appendix 15.1, Table 6. In-clinic visits on 
Day 8 and Day 22 may be performed by the patient’s primary care physician if necessary and 
must be approved by the Sponsor on a case by case basis; in these instances PK sampling would 
not be required. 
The collection times for post-dose PK samples will start from the time that dosing is completed. 
(For example, a PK draw at 30 minutes will be collected 30 minutes after the last capsule has 
been ingested.) The completion time of each dose will be recorded. 
Procedures for sample collection and processing will be provided in a separate study manual. 
The actual time point of each sample collection will be recorded. 
Samples for PK and PD assessments may be retained for up to 2 years from collection.  
10.7. 
Pharmacodynamic Assessments 
The first approximately 10 patients treated, contingent on clinical site feasibility, will undergo 
extensive PD sampling for 2,3-DPG and ATP as detailed in Appendix 15.1, Table 5. The 
remainder of treated patients will undergo limited PD for 2,3-DPG and ATP sampling as detailed 
in Appendix 15.3, Table 6. 
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The collection times for post-dose PD samples will start from the time that dosing is completed. 
(For example, a PD draw at 30 minutes will be collected 30 minutes after the last capsule has 
been ingested.) The completion time of each dose will be recorded. 
Procedures for sample collection and processing will be provided in a separate study manual. 
The actual time point of each sample collection will be recorded. 
Pharmacodynamic assessments will include 2,3-DPG, ATP,
 
In-clinic visits on Day 8 and Day 22 may be 
performed by the patient’s primary care physician if necessary and must be approved by the 
Sponsor on a case by case basis; in these instances PK sampling would not be required. 
Figure 4 provides a brief schematic outlining the PKR reaction and how each of these PD 
assessments fits into a complete mechanistic understanding of the action of AG-348. 
Figure 4: 
PKR Enzymatic Reaction 
 
The PKR enzyme catalyzes the PEP to pyruvate reaction, with concomitant formation of ATP. 
17. Binding of AG-348 to the PKR tetramer can be assessed through an ex-vivo biochemical 
assay of cell lysates from AG-348 treated patients. Because WBCs contain a high level of 
pyruvate kinase from a non-PKR pyruvate kinase isoform, WBCs are first removed by 
filtration before the purified red cells are frozen.
 
 
 
18. 
 
 
 
 
 
 
 
 
 
19. AG-348 target engagement 
 has been 
shown in preclinical models and healthy volunteer clinical studies to result in 
accumulation of ATP and depletion of the upstream metabolite 2,3-DPG. Therefore, 
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levels of these metabolites will be measured by mass spectrometry in frozen whole blood 
samples. 
The first approximately 10 patients treated, contingent on clinical site feasibility, will undergo 
extensive PD sampling as detailed in Appendix 15.1, Table 5. The remainder of treated patients 
will undergo limited PD sampling as detailed in Appendix 15.1, Table 6. The collection times for 
post-dose PD samples will start from the time that dosing is completed. (For example, a PD draw 
at 30 minutes will be collected 30 minutes after the last capsule has been ingested.) The 
completion time of each dose will be recorded. 
Blood samples will be stored at the site and regularly transported at -80°C ±10 C to the 
bioanalytical laboratory for analysis. Procedures for sample collection and processing will be 
provided in a separate study manual. 
10.8. 
Ordering of Blood Sample Collection 
When more than 1 blood sample is collected at the same nominal time, the samples will be 
collected in the following order: 
1. Safety laboratory assessments 
2. PK  
3. PD (2,3 DPG, ATP) 
 
 
 
10.9. 
Sample Processing, Storage, and Shipment 
Instructions for the processing, storage and shipment of all study samples for central analysis 
will be provided in a separate study manual. 
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11. 
ADVERSE EVENTS 
Monitoring of AEs will be conducted throughout the study. Adverse events and SAEs will be 
recorded in the source documentation and eCRF from time of the signing informed consent 
through the Follow-up Visits. All AEs should be monitored until they are resolved or are clearly 
determined to be due to a patient’s stable or chronic condition or intercurrent illness(es). 
All SAEs will be followed through 30 days after the last dose of study treatment or until the SAE 
has resolved. Any SAEs that are assessed as possibly or probably related to study treatment that 
occur > 30 days post-treatment also are to be reported. 
11.1. 
Definition of Adverse Events 
11.1.1. 
Adverse Event (AE) 
An AE is any untoward medical occurrence associated with the use of a drug in humans, whether 
or not considered drug related. An AE (also referred to as an adverse experience) can be any 
unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a drug, without any judgment about causality. An AE can 
arise from any use of the drug (e.g., off-label use, use in combination with another drug) and 
from any route of administration, formulation, or dose, including an overdose. 
11.1.2. 
Suspected Adverse Reaction 
A suspected adverse reaction is any AE for which there is a reasonable possibility that the drug 
caused the AE. For the purposes of expedited safety reporting, ‘reasonable possibility’ means 
that there is evidence to suggest a causal relationship between the drug and the AE.  
11.1.3. 
Unexpected Adverse Event 
An unexpected AE is one for which the nature or severity of the event is not consistent with the 
applicable product information, e.g., the Investigator’s Brochure. 
11.1.4. 
Serious Adverse Event 
An AE or suspected adverse reaction is considered serious (SAE) if, in the view of either the 
Investigator or Sponsor, it results in any of the following outcomes: 
• Death; 
• Life-threatening. Life-threatening means that the patient was at immediate risk of 
death from the reaction as it occurred, i.e., it does not include a reaction which 
hypothetically might have caused death had it occurred in a more severe form; 
• In-patient hospitalization or prolongation of existing hospitalization. Hospitalization 
admissions and/or surgical operations scheduled to occur during the study period, but 
planned prior to study entry are not considered AEs if the illness or disease existed 
before the patient was enrolled in the study, provided that it did not deteriorate in an 
unexpected manner during the study (e.g., surgery performed earlier than planned); 
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• A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions; 
• Congenital anomaly/birth defect; 
• Important medical event. An important medical event is an event that may not result 
in death, be life-threatening, or require hospitalization but may be considered an SAE 
when, based upon appropriate medical judgment, it may jeopardize the patient or 
patient and 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 convulsions that do not result in in-patient hospitalization, 
or the development of drug dependency or drug abuse. 
11.1.4.1. Potential Severe Drug-Induced Liver Injury 
The document entitled FDA Guidance for Industry, Drug-Induced Liver Injury: Premarketing 
Clinical Evaluation (FDA 2009) provides guidance on how the measurement of various 
laboratory parameters may be used to assess a given drug’s potential to cause severe liver injury 
(i.e., irreversible liver failure that is fatal or requires liver transplantation). Such cases are 
suggested by the following three components: 
1. The drug causes hepatocellular injury, generally shown by a higher incidence of 3-fold or 
greater elevations above the ULN of ALT or AST than the (non-hepatotoxic) control 
drug or placebo; 
2. Among trial patients showing such aminotransferase elevations, often with 
aminotransferases much greater than 3×ULN, one or more also show elevation of serum 
total bilirubin to > 2×ULN, without initial findings of cholestasis (elevated serum 
elevated serum ALP); 
3. No other reason can be found to explain the combination of increased aminotransferases 
and total bilirubin, such as viral hepatitis A, B, or C; pre-existing acute liver disease; or 
another drug capable of causing the observed injury.  
Clinical safety laboratory results compatible with the definition of drug-induced liver injury 
(DILI) stated above must be repeated for confirmation as soon as possible, and if confirmed, will 
be scored as an unacceptable AE and reported to FDA as a serious unexpected AE. 
11.2. 
Procedures for Reporting Adverse Events and Serious Adverse 
Events  
Each patient must be carefully monitored for the development of any AEs. This information 
should be obtained in the form of non-leading questions (e.g., “How are you feeling?”) and from 
signs and symptoms detected during each examination, observations of study personnel, and 
spontaneous reports from patients. 
All AEs (serious and non-serious) spontaneously reported by the patient and/or in response to an 
open question from study personnel or revealed by observation, physical examination, or other 
diagnostic procedures will be recorded in the source documentation and eCRF. Any clinically 
relevant deterioration in laboratory assessments or other clinical findings is considered an AE 
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and must be recorded in the appropriate source documentation and eCRF. Treatment-emergent 
abnormal clinical laboratory results should generally be reported as AEs if there are 
accompanying symptoms; if additional diagnostic evaluations or medical (including drug 
therapy) or surgical interventions are undertaken; if a change in study drug dosing or study drug 
discontinuation is required; or, if the laboratory result is considered clinically significant by the 
Investigator. When possible, signs and symptoms indicating a common underlying pathology 
should be noted as one comprehensive event. 
All SAEs that occur during the course of the study must be promptly reported by the Investigator 
to 
 Global Safety and Pharmacovigilance (see below). Deaths and AEs assessed 
as life-threatening are to be reported immediately and SAEs that meet other criteria are to be 
reported within 24 hours from the point in time when the Investigator becomes aware of the 
SAE. All SAEs must be reported whether or not they are considered causally related to AG-348. 
Serious adverse event forms will be completed and the information collected will include subject 
number, a narrative description of the event, and an assessment by the Investigator as to the 
severity of the event and relatedness to study drug. Follow-up information on the SAE may be 
requested by the Sponsor or Medical Monitor. 
Contact Information: 
 Safety Management 
SAE Hotline (United States): 
 
SAE Fax (United States): 
 
SAE Hotline (France): 
 
SAE Fax (France): 
  
SAE Hotline (Netherlands): 
 
SAE Fax (Netherlands): 
 
SAE Hotline (Italy): 
 
SAE Fax (Italy): 
 
SAE Hotline (United Kingdom): 
 
SAE Fax (United Kingdom): 
 
E-mail: 
 
If there are serious, unexpected adverse drug reactions associated with the use of AG-348, the 
Sponsor will notify the appropriate regulatory agency(ies) and all participating Investigators on 
an expedited basis. The local 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. 
All AEs, whether serious or not, will be described in the source documents in the database. All 
new events, as well as those that worsen in intensity or frequency relative to baseline, which 
occur after signing the informed consent through the final Follow-up Visit (Day 29 ± 3 days) 
must be recorded. Adverse events that are ongoing at the time of treatment discontinuation 
should be followed up to 30 days after the last dose of study treatment. All SAEs will be 
followed up to 30 days after the last dose of study treatment, or until the SAE has resolved.  
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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); 
• The date of onset of the event; 
• The date of resolution of the event; 
• Whether the event is serious or not; 
• Intensity of the event (see below for definitions); 
• Relationship of the event to study treatment (see below for definitions); 
• Action taken: none; change in the study drug administration (e.g., temporary 
interruption in dosing); drug treatment required; non-drug treatment required; 
hospitalization or prolongation of hospitalization required (complete SAE page); 
diagnostic procedure performed; patient discontinued from the study (complete End 
of Study visit); 
• Outcome: patient recovered without sequelae; patient recovered with sequelae; event 
ongoing; patient died (notify the Medical Monitor immediately, and complete the 
SAE form). 
Intensity of all AEs will be graded according to the NCI CTCAE Version 4.03 (Appendix 15.1). 
Relationship to study drug administration will be determined by the Investigator according to 
the following criteria: 
• Not Related:  Exposure to the study treatment did not occur, or the occurrence of the 
AE is not reasonably related in time, or the AE is considered unlikely to be related to 
the study treatment. 
• Possibly Related:  The study treatment and the AE were reasonably related in time, 
and the AE could be explained equally well by causes other than exposure to the 
study treatment. An attribution of possibly related means that there are facts in 
evidence to suggest a possible relationship. 
• Probably Related:  The study treatment and the AE were reasonably related in time, 
and the AE was more likely explained by exposure to the study treatment than by 
other causes, or the study treatment was the most likely cause of the AE. An 
attribution of probably related means that there are facts in evidence to suggest a 
probable relationship. 
For the purpose of safety analyses related to final database review, all AEs that are classified as 
possible or probable will be considered treatment-related AEs. 
11.3. 
Pregnancy Reporting 
Pregnancy is neither an AE nor an SAE, unless a complication relating to the pregnancy occurs 
(e.g., spontaneous abortion, which may qualify as an SAE). However, any pregnancy in a 
participating female patient or a female partner of a participating male patient that occurs during 
this study or within 30 days following the last dose of AG-348 must be reported to the Sponsor 
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or Medical Monitor within 30 days of being notified of the pregnancy. The Investigator must 
follow up and document the course and outcome of all pregnancies even if the patient was 
discontinued from the study or if the study has finished. The female patient or partner of a male 
patient should receive any necessary counseling regarding the risks of continuing the pregnancy 
and the possible effects on the fetus. Monitoring should continue until conclusion of the 
pregnancy. 
All outcomes of pregnancy must be reported by the Investigator to the Sponsor or Sponsor’s 
designee on a Pregnancy Outcome Report form within 30 days after he/she has gained 
knowledge of the delivery or elective abortion. 
Any SAE that occurs during pregnancy must be recorded on the SAE report form (e.g., maternal 
serious complications, spontaneous or therapeutic abortion, ectopic pregnancy, stillbirth, 
neonatal death, congenital anomaly, or birth defect) and reported within 24 hours in accordance 
with the procedure for reporting SAEs. 
All patients, male and female, must agree to use effective contraception during the entire study 
and for 30 days following the last dose of AG-348. 
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12. 
STATISTICAL METHODS 
The primary objective of this Phase 2 study is to evaluate the safety and tolerability of up to 
24 weeks of AG-348 in patients with PK Deficiency. Therefore, analyses will be primarily 
descriptive in nature; no formal hypothesis testing will be conducted. All analyses will be 
conducted separately within each dose arm, or pooled where appropriate. 
12.1. 
Sample Size Estimation 
Due to the rare disease setting, the minimal sample size in each dose arm may be determined by 
feasibility. In order to evaluate the primary objective of safety and tolerability of AG-348 in 
Arm 1 and Arm 2, up to a maximum of 25 patients may be randomized onto each arm. The 
actual number of patients enrolled into Arms 1 and 2 will depend on the safety reviews and 
decisions made by the DRT. In addition, up to 25 additional patients may be enrolled to evaluate 
an additional dose arm (Arm 3; see Section 7.1). As for Arms 1 and 2, the actual enrollment in a 
potential Arm 3 will depend on the safety reviews and decisions made by the DRT. Therefore, up 
to approximately 75 total patients may be enrolled in this study across 2 to 3 dose arms. 
Table 3 provides the probability within a dose arm of detecting 1 or more AEs with varying 
sample size and the true underlying AE rates. 
Table 3: 
Sample Size Estimation 
Sample Size 
True Underlying AE Rate 
15% 
10% 
5% 
5 
56% 
41% 
23% 
10 
80% 
65% 
40% 
15 
91% 
79% 
54% 
25 
98% 
93% 
72% 
 
12.2. 
Populations for Analysis 
The following patient populations (i.e., analysis sets) will be evaluated and used for presentation 
of the data: 
• Safety Analysis Set: All patients who are enrolled and receive any dose of study 
treatment. The Safety Analysis Set will be the primary set for the analysis of safety 
data. Patients will be classified according to treatment received, where treatment 
received is defined as the assigned treatment if it is received at least once, or as the 
first treatment received if assigned treatment is never received. Unless otherwise 
stated, the Safety Analysis Set will be the default analysis set for all data analyses. 
• Pharmacokinetic (PK) Analysis Set: All patients in the Safety Analysis Set with 
sufficient plasma sample data to assess PK parameters. Results of the potential PD 
activity of AG-348 will also be based on the PK analysis set.  
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• Efficacy Analysis Set: All patients who are enrolled and achieve at least 50% 
compliance with their assigned dose intensity for at least 4 weeks of continuous 
dosing. Additional efficacy analyses may be performed on subsets of patients with 
other degrees of compliance with their assigned dose intensity and/or duration of 
dosing. The Efficacy Analysis Set will be the primary set for the analysis of 
preliminary clinical activity data. Patients will be classified according to assigned 
treatment. 
 
 
 If such analyses are performed, they will be 
described in a separate PK Statistical Analysis Plan (SAP) and may be reported separately in a 
stand-alone report. 
12.3. 
Procedures for Handling Missing, Unused, and Spurious Data 
No imputation will be performed for missing data elements. When summarizing AE data, partial 
dates will be imputed as described in the SAP. Additional rules addressing the handling of 
missing data will be detailed in the SAP. 
12.4. 
Interim Analysis 
No formal statistical interim analysis will be conducted. Safety data will be reviewed on an 
ongoing basis by the DRT, who will meet to review safety, PK, PD, and preliminary clinical 
activity data at regular intervals (approximately every 6 weeks) throughout the duration of the 
study. The DRT’s decisions to suspend, terminate, or open a potential third dosing arm, or re-
assign patients’ dosing in a terminated arm to match the dose and schedule of another arm of the 
study will be based on the totality of the data including, safety, PK, PD, and preliminary clinical 
activity (e.g., changes in Hb levels). 
Additional interim reviews of data may be conducted to support decision making concerning the 
current clinical study, the sponsor’s development programs in general, or in case of any safety 
concerns. 
12.5. 
Statistical Methodology 
12.5.1. 
General Methods 
This study will be primarily descriptive in nature; therefore, there will be no formal hypothesis 
testing. Summaries will be produced for disposition, baseline disease characteristics and 
demographic data including genotype, safety measurements, PK, PD parameters and indicators 
of clinical activity. Data from each AG-348 dose group will be analyzed separately, and pooled 
across all dose groups where appropriate. All data will also be listed by individual patient. 
Categorical variables will be summarized by frequency distributions (number and percentages) 
and continuous variables will be summarized by descriptive statistics (mean, standard deviation 
[SD], median, minimum, and maximum). 
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12.5.2. 
Disposition 
A summary of the disposition of patients will be presented, including the number enrolled, the 
number treated, and the reasons for study discontinuation. Entry criteria and protocol deviations 
will be listed. 
12.5.3. 
Exposure and Safety Analyses 
Patients will receive multiple PO doses of AG-348 over a 24-week treatment period. The actual 
dose and duration in days of AG-348, as well as the dose intensity (computed as the ratio of 
actual dose received and actual duration) and the relative dose intensity (computed as the ratio of 
dose intensity and planned dose received/planned duration) will be listed and summarized using 
descriptive statistics by dose arm. 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) 
and the incidence of TEAEs (new or worsening from baseline) will be summarized by primary 
system organ class (SOC), preferred term (PT), CTCAE Version 4.03 severity, outcome, action 
taken with study drug, and relationship to the study drug by dose group. Separate summaries will 
be produced for all TEAEs, treatment-related AEs (those considered by the Investigator as at 
least possibly drug related), SAEs, discontinuations due to AEs, and AEs ≥ Grade 3 severity. 
Individual patient listings will be provided for deaths, SAEs, AEs leading to interruption and/or 
reduction of study drug dose, and AEs leading to discontinuation of treatment. 
Analyses of AEs will be performed for those events that are considered treatment-emergent, 
where treatment-emergent is defined as any AE with onset beginning at the day of first 
administration of study drug, throughout the treatment period until 30 days after the last dose of 
study drug, or any event that was present at baseline but worsened in intensity or was 
subsequently considered drug-related by the Investigator through the end of the study. 
Descriptive statistics will be provided for clinical laboratory values (e.g., hematology, serum 
chemistry, coagulation studies, urinalysis) and VS data, presented as both actual values and 
changes from baseline relative to each on-study evaluation. Shift analyses will be conducted for 
selected laboratory parameters based on the baseline CTCAE grade to maximum CTCAE grade. 
Where applicable CTCAE terms do not exist, a grading system based on the upper and/or lower 
limits of normal will be used to classify the results. 
No formal hypothesis-testing analysis of AE incidence rates will be performed. 
Electrocardiogram analyses will include individual patient listings and summaries of abnormal 
and clinically significant ECG results. Actual values and changes from baseline in PR, QRS, and 
heart-rate corrected QT interval (QTc) intervals will be summarized by visit and dose arm. Full 
details of the QTc analysis including correction methods used will be described in the SAP. 
Concomitant medications will be coded using the WHO Drug Dictionary. Concomitant 
medications prior to and after the start of the study drug will be listed by patient, and 
summarized by ATC term and dose arm. 
12.5.4. 
Pharmacokinetic Analyses 
Descriptive statistics will be used to summarize PK parameters for the parent compound AG-348 
and the metabolite AGI-8702 for each dose group, and where appropriate, for the entire 
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Protocol AG348-C-003, Version 1.0 
05 January 2015 
 
population. Pharmacokinetic parameters will be summarized using the following descriptive 
statistics: n, mean, SD, coefficient of variation %, median, minimum and maximum, geometric 
mean, and geometric coefficient of variation. PK analyses will be described in a separate PK 
SAP. 
12.5.5. 
Pharmacodynamic Analyses 
Descriptive statistics will be used to summarize PD parameters for 2,3-DPG and ATP for each 
dose group, and where appropriate for the entire population. Pharmacodynamic parameters will 
be summarized using the following descriptive statistics: n, mean, SD, coefficient of variation %, 
median, minimum, and maximum, geometric mean, and geometric coefficient of variation %. PD 
analyses will be described in a separate PD SAP. 
12.5.6. 
Aromatase Hormone Analysis  
The analyses of serum sex hormones will use appropriate graphic displays and statistical 
analyses to evaluate patient change from baseline for each parameter. These analyses will 
include summaries of actual values and change from baseline using appropriate descriptive 
statistics (mean, SD, median, min and max) as described above.  
 
These analyses will present information by each dose arm, and analyses of a pooled 
AG-348 cohort. Additional details regarding these analyses will be provided in the SAP. 
12.5.7. 
Clinical Activity 
Details on analyses to evaluate indicators of potential clinical activity of AG-348 in patients with 
PK Deficiency will be described in the SAP. These will include changes in Hb, HCT, 
reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, ferritin, and transferrin 
saturation (serum iron/iron binding capacity). Characterization of Hb response which will 
include, but is not limited to percent of patients with increase in Hb, time to Hb response, and 
duration of Hb response will be explored. 
12.6. 
Procedures for Reporting Deviations to Original Statistical Analysis 
Plan 
All deviations from the original SAP will be provided in the final clinical study report. 
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13. 
ADMINSTRATIVE REQUIREMENTS 
13.1. 
Good Clinical Practice 
The study will be conducted in accordance with the International Conference on Harmonization 
(ICH) for Good Clinical Practice (GCP) and the appropriate regulatory requirement(s). The 
Investigator will be thoroughly familiar with the appropriate use of the study drug as described in 
the protocol and Investigator’s Brochure. Essential clinical documents will be maintained to 
demonstrate the validity of the study and the integrity of the data collected. Master files should 
be established at the beginning of the study, maintained for the duration of the study, and 
retained according to the appropriate regulations. 
13.2. 
Ethical Considerations 
The study will be conducted in accordance with ethical principles founded in the Declaration of 
Helsinki (Appendix 15.4). 
The Investigator must obtain IRB approval for the investigation and must submit written 
documentation of the approval to the Sponsor before he or she can enroll any patient into the 
study. The IRB will review all appropriate study documentation in order to safeguard the rights, 
safety, and well-being of the patients. The study will only be conducted at sites where IRB 
approval has been obtained. The protocol, Investigator’s Brochure, informed consent, 
advertisements (if applicable), written information given to the patients, signed Dose Escalation 
Interim Safety Reports, annual progress reports, and any revisions to these documents will be 
provided to the IRB. The IRB is to be notified of any amendment to the protocol in accordance 
with local requirements. Progress reports and notifications of serious unexpected adverse drug 
reactions are to be provided to the IRB according to local regulations and guidelines. 
13.3. 
Patient Information and Informed Consent 
The Investigator or trained designee will ensure that the patient is given full and adequate oral 
and written information about the nature, purpose, possible risk, and benefit of the study. Patients 
must also be notified that they are free to discontinue from the study at any time. The patient 
should be given the opportunity to ask questions and allowed time to consider the information 
provided. 
After the study has been fully explained, written informed consent will be obtained from the 
patient prior to study participation.  
The patient’s signed and dated informed consent must be obtained before conducting any study-
related procedures. The Investigator must maintain the original, signed consent form. A copy of 
the signed form must be given to the patient. 
The method of obtaining and documenting the informed consent and the contents of the consent 
will comply with ICH-GCP and all applicable regulatory requirement(s). 
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13.4. 
Patient Confidentiality 
In order to maintain patient privacy, all source documents, study drug accountability records, 
study reports and communications will identify the patient by the assigned patient number. The 
Investigator will grant monitor(s) and auditor(s) from the Sponsor or its designee and regulatory 
authority(ies) access to the patient’s original medical records for verification of data gathered on 
the source documents and to audit the data collection process. The patient’s confidentiality will 
be maintained and will not be made publicly available to the extent permitted by the applicable 
laws and regulations. 
13.5. 
Protocol Compliance 
The Investigator will conduct the study in compliance with the protocol. Modifications to the 
protocol should not be made without agreement of both the Investigator and the Sponsor. 
Changes to the protocol will require written IRB approval/favorable opinion prior to 
implementation, except when the modification is needed to eliminate an immediate hazard(s) to 
patients. The IRB may provide, if applicable, where regulatory authority(ies) permit, expedited 
review and approval/favorable opinion for minor change(s) in ongoing studies that have the 
approval/favorable opinion of the IRB. The Sponsor or designee will submit all protocol 
modifications to the regulatory authority(ies) in accordance with the governing regulations.  
When immediate deviation from the protocol is required to eliminate an immediate hazard(s) to 
patients, the Investigator will contact the Sponsor’s Medical Monitor (or Responsible Medical 
Officer), if circumstances permit, to discuss the planned course of action. Any departures from 
the protocol must be fully documented in the source documents/database. 
13.6. 
Data Management 
A paper or eCRF will be completed for each patient or an electronic data capture system will be 
used. The electronic data capture system (EDC) (ClinBase™) is a software tool designed to 
ensure quality assurance and facilitate data capture during clinical trials. Through a system 
regulated workflow that includes barcode scanning and interfaces to medical equipment to avoid 
manual data entry, study operations performance is controlled and captured in real time. The 
system is fully Code of Federal Regulations (CFR) 21 Part 11 compliant. Source documentation 
supporting the data should indicate participation in the study and should document the dates and 
details of study procedures, AEs, and patient status. The Investigator, or trained designee should 
complete and the Investigator should verify the source documents as the information is collected. 
Any outstanding entries must be completed immediately after the final examination. An 
explanation should be given for all missing data. The Investigator will ensure the accuracy, 
completeness, and timeliness of the data reported to the Sponsor. Data collection processes and 
procedures will be reviewed and validated to ensure completeness, accuracy, reliability, and 
consistency. A complete audit trail will be maintained of all data changes. The Investigator or 
designee will cooperate with the Sponsor’s representative(s) for the periodic review of study 
documents to ensure the accuracy and completeness of the data capture system at each scheduled 
monitoring visit. Electronic consistency checks and manual review will be used to identify any 
errors or inconsistencies in the data. This information will be provided to the respective study 
sites by means of electronic or manual queries. The Investigator or designee will prepare and 
maintain adequate and accurate study documents (medical records, ECGs, AE, and concomitant 
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medication reporting, raw data collection forms, etc.) designed to record all observations and 
other pertinent data for each patient receiving study treatment. The Investigator will allow 
Sponsor representatives, contract designees, authorized regulatory authority inspectors, and the 
IRB to have direct access to all documents pertaining to the study. 
13.7. 
Source Document/Case Report Form Completion 
Source documents will be completed for each study patient. It is the Investigator’s responsibility 
to ensure the accuracy, completeness, and timeliness of the data reported in the patient’s source 
document. The source document should indicate the patient’s participation in the study and 
should document the dates and details of study procedures, AEs and patient status. 
The Investigator, or designated representative, should complete the source document as soon as 
possible after information is collected, preferably on the same day that a study patient is seen for 
an examination, treatment, or any other study procedure. Any outstanding entries must be 
completed immediately after the final examination. An explanation should be given for all 
missing data. 
The Investigator will sign and date each required assessment for all study patients. The 
Investigator will retain all completed source documents. 
13.8. 
Direct Access to Source Data 
The study will be monitored by the Sponsor or its designee. Monitoring will be done by personal 
visits from a representative of the Sponsor (site monitor) and will include on-site review of the 
source documents for completeness and clarity, cross-checking with source documents, and 
clarification of administrative matters will be performed. The review of medical records will be 
performed in a manner to ensure that patient confidentiality is maintained. 
The site monitor will ensure that the investigation is conducted according to protocol design and 
regulatory requirements by frequent communications (letter, telephone, e-mail, and fax). 
All unused study drug and other study materials are to be returned to the Sponsor or designee 
after the study has been completed and the database has been locked. 
Regulatory authorities, the IRB, and/or the Sponsor’s clinical quality assurance group or 
designee may request access to all source documents, database, and any other applicable study 
documentation for an on-site audit or inspection. Direct access to these documents must be 
guaranteed by the Investigator, who must provide support at all times for these activities. 
13.9. 
Record Retention 
The Investigator will maintain all study records according to ICH-GCP and applicable regulatory 
requirement(s). Records will be retained for at least 2 years after the last marketing application 
approval or 2 years after formal discontinuation of the clinical development of the investigational 
product or according to applicable regulatory requirement(s). If the Investigator withdraws from 
the responsibility of keeping the study records, custody must be transferred to a person willing to 
accept the responsibility. The Sponsor must be notified in writing if a custodial change occurs. 
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13.10. 
Liability and Insurance 
The Sponsor has subscribed to an insurance policy covering, in its terms and provisions, its legal 
liability for injuries caused to participating persons and arising out of this research performed 
strictly in accordance with the scientific protocol as well as with applicable law and professional 
standards.  
13.11. 
Publication of Study Findings and Use of Information 
All information regarding AG-348 supplied by the Sponsor or designee to the Investigator is 
privileged and confidential information. The Investigator agrees to use this information to 
accomplish the study and will not use it for other purposes without consent from the Sponsor. It 
is understood that there is an obligation to provide the Sponsor with complete data obtained 
during the study. The information obtained from the clinical study will be used towards the 
development of AG-348 and may be disclosed to regulatory authority(ies), other Investigators, 
corporate partners, or consultants as required. 
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14. 
LIST OF REFERENCES 
 
Beutler E, and Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene 
frequency in the general white population. Blood. 2000;95(11):3585-8. 
Christensen RD, Yaish HM, Johnson CB, Bianchi P, and Zanella A. Six children with pyruvate 
kinase deficiency from one small town: molecular characterization of the PK-LR gene. J Pediatr. 
2011;159(4):695-7. 
FDA. Guidance for Industry: Drug-Induced Liver Injury: Premarketing Clinical Evaluation. 
2009. 
Ghidini A, Sirtori M, Romero R, Yarkoni S, Solomon L, and Hobbins JC. Hepatosplenomegaly 
as the only prenatal finding in a fetus with pyruvate kinase deficiency anemia. Am J Perinatol. 
1991;8(1):44-6. 
Hennekam RC, Beemer FA, Cats BP, Jansen G, and Staal GE. Hydrops fetalis associated with 
red cell pyruvate kinase deficiency. Genet Couns. 1990;1(1):75-9. 
Rider NL, Strauss KA, Brown K, Finkenstedt A, Puffenberger EG, Hendrickson CL, et al. 
Erythrocyte pyruvate kinase deficiency in an old-order Amish cohort: longitudinal risk and 
disease management. Am J Hematol. 2011;86(10):827-34. 
Tanphaichitr VS, Suvatte V, Issaragrisil S, Mahasandana C, Veerakul G, Chongkolwatana V, et 
al. Successful bone marrow transplantation in a child with red blood cell pyruvate kinase 
deficiency. Bone Marrow Transplant. 2000;26(6):689-90. 
van Wijk R, and van Solinge WW. The energy-less red blood cell is lost: erythrocyte enzyme 
abnormalities of glycolysis. Blood. 2005;106(13):4034-42. 
Zanella A, Fermo E, Bianchi P, and Valentini G. Red cell pyruvate kinase deficiency: molecular 
and clinical aspects. Br J Haematol. 2005;130(1):11-25. 
 
 
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15. 
APPENDICES 
 
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Protocol AG348-C-003, Version 1.0 
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15.1. 
Schedules of Assessments 
Table 4: 
Schedule of Assessments:  
Timing 
Pre-
Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow Up 
Visit 
Screening 
Baseline 
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
1 
81 
15 
221 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Written Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
PK enzyme assay 
(confirmation of PK 
Deficiency) 
X 
 
 
 
 
 
 
 
 
 
 
 
PKR Genotype 
(for randomization) 
X 
 
 
 
 
 
 
 
 
 
 
 
UGT1A1 Genotype 
X 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical/Surgical History 
(General and PK 
Deficiency-specific)2 
X 
 
 
 
 
 
 
 
 
 
 
 
Medication History 
X 
 
 
 
 
 
 
 
 
 
 
 
Transfusion History 
X 
 
 
 
 
 
 
 
 
 
 
 
Confirmation of 
Vaccinations 
(Splenectomized Patients) 
X 
 
 
 
 
 
 
 
 
 
 
 
Physical Examination3/ 
Height3 and Weight 
X 
X 
 
 
X 
 
X 
X 
X 
X 
X 
X 
Performance Status 
X 
X 
 
 
X 
 
X 
X 
X 
X 
X 
X 
Vital signs  
(BP, HR, RR, T)4 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG5 
X 
X 
X 
 
X 
 
 
 
 
 
X 
X 
Laboratory Evaluations6: 
 
 
 
 
 
 
 
 
 
 
 
 
HBsAg, HCV Ab, HIV1 
and 2 Ab 
X 
 
 
 
 
 
 
 
 
 
 
 
RBC antibody Screen  
X 
 
 
 
 
 
 
 
 
 
 
 
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Timing 
Pre-
Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow Up 
Visit 
Screening 
Baseline 
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
1 
81 
15 
221 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Hematology (CBC)7 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin8 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
EPO levels9 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
G6PD screen 
X 
 
 
 
 
 
 
 
 
 
 
 
Serum Chemistry10 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Iron Panel11 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Carboxyhemoglobin 
(COHb) 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
 
Coagulation Studies12 
X 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Urinalysis13 
X 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Serum or Urine 
Pregnancy14 
X 
X 
 
 
 
 
 
 
 
 
 
 
Lipids15 
 
X 
 
 
 
X 
 
X 
 
 
X 
X 
Hormonal Testing16 
X 
X 
 
 
 
 
 
X 
 
 
X 
X 
Serum NTX and CTX 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Randomization17 
X 
 
 
 
 
 
 
 
 
 
 
 
Study Drug Administration 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Dispense Study Drug18 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
PK blood sampling19 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
PD Assessments19 
 
 
 
 
 
 
 
 
 
 
 
 
2,3 DPG/ATP 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
Adverse Events21 
 
Continuous 
X 
Transfusion Record 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant Medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
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Timing 
Pre-
Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow Up 
Visit 
Screening 
Baseline 
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-28 to -1 
1 
81 
15 
221 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Rollover to extension study 
 
 
 
 
 
 
 
 
 
 
X 
 
Abbreviations:  Ab = antibody; ATP = adenosine triphosphate; BP = blood pressure; CBC= complete blood count; COHb = carboxyhemoglobin; CTX = C-terminal 
telopeptide; D = day; DPG = diphosphoglycerate; ECG = electrocardiogram; EPO = erythropoietin; FSH = follicle stimulating hormone; G6PD = glucose-6-phosphate-
dehydrogenase; HBsAg = hepatitis B surface antigen; HCV Ab = hepatitis C virus antibody; HDL-C = high-density lipoprotein-C; HIV = human immunodeficiency virus; HR = 
heart rate; NTX = N-terminal telopeptide; PD = pharmacodynamic; PK = pharmacokinetic; PKD = pyruvate kinase deficiency; PKR = pyruvate kinase isoform R; RR = resting 
rate; W = week. 
Whenever more than one assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive assessment (e.g., 
VS, ECG, blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time. The order of procedures may be revised with prior 
discussion between Sponsor and Site. 
 
1 In-clinic visits on Day 8 and Day 22 may be performed by the patient’s primary care physician if necessary and must be approved by the Sponsor on a case by case basis; in 
these instances PK/PD sampling would not be required and dispensing of study medication would not be performed. 
2 Medical history, including all relevant prior medical history, current medical conditions, and hematology profile (CBCs) over prior 6 months, will be obtained at the Screening 
assessment.  
3 A complete physical examinations (genital and rectal examinations will be performed at the discretion of the Investigator) will be obtained at Screening, Baseline, Week 12, and 
the Follow up Visit (Week 28), or Week 24 for patients rolling over into extension study. Limited focused physical examinations will be performed at all other specified visits. 
Height to be collected at Screening only. 
4 Vital signs will include systolic and diastolic blood pressure, heart rate, respiratory rate, and temperature.  
5 12-lead ECGs are to be conducted after 5 minutes of recumbency. Screening ECG will be performed at least 7 days prior to Day 1 dosing. 
6 Laboratory evaluations (hematology, serum chemistry, coagulation studies, and urinalysis) are to be collected in the morning. These should be collected following an overnight 
fast on Baseline Day 1 Week 6 (Day 43), Week 12 (Day 85), Week 24 (Day 169), and Follow-up Week 28 (Day 197), when the lipid samples are also included. 
7 Complete blood count (CBC) to be performed at Screening and prior to dosing on Day 1 and will include HCT, Hb, red blood cell (RBC) count, absolute reticulocyte count, 
percent reticulocyte count, mean corpuscular volume (MCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW), 
white blood cell (WBC) count with manual differential, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and platelet count.  
8 Haptoglobin will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28. 
9 Erythropoietin (EPO) levels will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28. 
10 Alkaline phosphatase, sodium, potassium, chloride, calcium, magnesium, phosphorus, carbon dioxide (CO2), albumin, total protein, glucose, blood urea nitrogen (BUN), 
creatinine, uric acid, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin and indirect bilirubin. 
11 Iron (Fe), total iron-binding capacity (TIBC), percent saturation, and ferritin will be performed prior to dosing on Day 1, at the end of Week 12 and at the end of Week 24. 
12 Fibrinogen, activated partial thromboplastin time (aPTT), and international normalized ratio (INR) will be performed at Screening, prior to dosing on Day 1, at the end of Week 
3, the end of Week 12, the end of Week 24, and the end of Week 28. 
13 Color, appearance, pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite, urobilinogen, and occult blood. Microscopic inspection of sediment should only be performed 
for cause or to investigate an abnormal dipstick finding per the Investigator’s discretion. Urinalysis will be performed at Screening, prior to dosing on Day 1, at the end of Week 
3, the end of Week 12, the end of Week 24, and the end of Week 28. 
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14 Must be repeated at any point throughout the study period if pregnancy is clinically suspected. 
15 Total cholesterol, HDL-C, and triglyceride samples will be collected in the morning following an overnight fast. 
16 Estrone, estradiol, and testosterone (free and total). The first serum estrone and estradiol samples will be collected on Baseline/Day 1. Serum free and total testosterone will be 
collected at 2 time points during Screening at least 2 days apart in addition to Baseline/Day 1 (total of 3 time points pre dose on Day 1). Serum estrone, estradiol, and free and 
total testosterone will then follow the schedule indicated on Weeks 12, 24, and 28. FSH will only be performed at Screening for female patients only for confirmation of post-
menopausal status. 
17 Randomization will be performed following PKR genotyping and prior to dosing on Day 1. 
18 Study drug will be dispensed on a 28-day schedule, or on an alternate schedule (< 28 days) as needed to accommodate patient visit schedule and dose modifications. 
19 For the first 10 patients treated, extensive PK/PD sampling will be conducted on Days 1 and 15 (see Appendix 15.1, Table 5 for details), followed by limited PK/PD sampling 
from Week 3 to Week 24 (see Appendix 15.1, Table 6 for details). Limited PK/PD sampling will be conducted on the remainder of patients treated (see Appendix 15.1 , 
Table 6). See Section 10.6.1, Section 10.7, and Section 10.9 for details on blood sampling for PK and PD assessments, respectively, and guidelines on sample processing 
and storage.  
 
  
 All patients will be evaluated for AEs from the time they sign informed consent until they complete all scheduled study follow-up visits or withdraw consent, whichever occurs 
first.  
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Table 5: 
Schedule of Assessments: Extensive PK/PD Sampling 
Sample Timing/Interval 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Visit 
Baseline / D1 
W2 / D15 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
Study Day 
1/15 
22 
43 
64 
85 
113 
141 
169 
Visit Window 
± 2 D (D15) 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Timing 
Pre-
dose1 
30 
min2 
1 hr2 
2 hr2 
4 hr3 
8 hr3 
12 
hr3 
Pre-
dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
PK blood sample 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
2,3 DPG/ATP 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; D = day; DPG = diphosphoglycerate; PD = pharmacodynamic; PK = pharmacokinetic; PKR = pyruvate kinase isoform R; 
W = week. 
 
1 The acceptable time window will be within 60 minutes prior to study treatment dose administration for the pre-dose PK/PD sample. 
2 The acceptable time window will be within ± 5 minutes of the scheduled collection time for the 30 minute, 1 and 2 hour PK/PD samples. 
3 The acceptable time window will be within ± 30 minutes of the scheduled collection time for the 4, 8, and 12 hour PK/PD samples. 
4 To be collected on Day 1 only. 
 
  
6 If the 12 hour time point cannot be collected at site, an 8 hour time point may be collected instead. 
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Table 6: 
Schedule of Assessments: Limited PK/PD Sampling 
Sample Timing/Interval 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Visit 
Baseline / 
D1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
Study Day 
1 
15 
22 
43 
64 
85 
113 
141 
169 
Visit Window 
- 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Timing 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
Pre-dose1 
PK blood sample 
X 
X 
X 
X 
X 
X 
X 
X 
X 
2,3 DPG/ATP 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; D = day; DPG = diphosphoglycerate; PD = pharmacodynamic; PK = pharmacokinetic; 
 
W = week. 
 
1 The predose blood sample for plasma PK/PD analysis should be collected within 60 minutes prior to study treatment dose administration. 
 
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15.2. 
National Cancer Institute Common Terminology Criteria for 
Adverse Events 
 
The NCI CTCAE, Version 4.03, can be accessed using the following link:   
http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_8.5x11.pdf 
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15.3. 
Potential Drug Interactions 
Strong inhibitors of CYP3A4 (listed in Table 7) are not permitted for use with AG-348. Based on 
modeling of AG-348 metabolism and tolerability data from the Phase 1 MAD study, it is thought 
that moderate inhibitors of CYP3A4 do not pose a risk to patients dosed with AG-348. 
In-vivo data from the Phase 1 MAD show AG-348 induces CYP3A4 and induces its own 
metabolism, presumably via CYP3A4. The use of strong CYP3A4 inducers in combination with 
AG-348 is expected to reduce AG-348’s efficacy. Therefore, administration of strong CYP3A4 
inducers (listed in Table 8) with AG-348 is not permitted. Strong inhibitors of drug transport 
(listed in Table 9) are not permitted for use with AG-348. 
Digoxin is not permitted for use with AG-348. 
Induction of CYP3A4 by AG-348 is expected to reduce the efficacy of certain sensitive co-
medications. Co-administration of AG-348 with the drugs in Table 10 should be done with 
caution, as their efficacy may be reduced.  
Of note, in accordance with Inclusion Criteria 14, women in the trial utilizing oral contraception 
must utilize barrier methods while taking AG-348. 
The expected patient co-medications deferoxamine, deferasirox and oral penicillin are not 
expected to interact with AG-348. 
Table 7: 
Strong and Moderate CYP3A4 Inhibitors 
Strong CYP3A4 Inhibitors: Contraindicated 
Moderate CYP3A4 Inhibitors: No Action 
Indinavir 
Aprepitant 
Nelfinavir 
Erythromycin 
Ritonavir 
Fluconazole 
Clarithromycin 
Grapefruit juice1 
Itraconazole 
Verapamil 
Ketoconazole 
Diltiazem 
Nefazodone 
 
Saquinavir 
 
Suboxone 
 
Telithromycin 
 
Strong Inhibitor; > 5 fold increase in AUC 
Moderate Inhibitor; > 2 fold, < 5 fold increase in AUC 
1 Although classified as a moderate CYP3A4 inhibitor, grapefruit and grapefruit juice are prohibited. 
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Table 8: 
Strong CYP3A4 Inducers 
Strong CYP3A4 Inducers:  Contraindicated 
Efavirenz 
Phenytoin 
Nevirapine 
Pioglitazone 
Carbamazepine 
Rifabutin 
Glucocorticoids 
Rifampin 
Modafinil 
St. John's Wort 
Oxcarbazepine 
Troglitazone 
Phenobarbital 
 
 
Table 9: 
Strong P-glycoprotein Inhibitors 
Strong P-gp Inhibitors:  Contraindicated 
Amiodarone 
Felodipine 
Azithromycin 
Itraconazole 
Captopril 
Ketoconazole 
Carvedilol 
Lopinavir 
Clarithromycin 
Ritonavir 
Conivaptan 
Quercetin 
Cyclosporine 
Quinidine 
Diltiazem 
Ranolazine 
Dronedarone 
Ticagrelor 
Erythromycin 
Verapamil 
 
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Table 10:  
Sensitive CYP3A4 Substrates 
Sensitive CYP3A4 Substrates:  Substitute or Use with Caution 
Macrolide 
antibiotics: 
Prokinetic: 
Steroid 6beta-OH: 
 
 
Clarithromycin 
Cisapride 
Estradiol 
Finasteride 
Terfenadine 
Erythromycin  
 
hydrocortisone 
Gleevec 
Torisel 
Telithromycin 
Antihistamines: 
progesterone 
Haloperidol 
Trazodone 
Astemizole 
Testosterone 
Irinotecan 
Vemurafenib 
Anti-arrhythmics: 
Chlorpheniramine 
 
LAAM 
Vincristine 
Quinidine→3-OH  
Terfenadine 
Miscellaneous: 
Lidocaine 
Zaleplon 
 
 
Alfentanil 
Methadone 
Ziprasidone 
Benzodiazepines: 
Calcium Channel 
Blockers: 
Aprepitant 
Nateglinide 
Zolpidem 
Alprazolam 
Amlodipine 
Aripiprazole 
Nevirapine 
 
Diazepam→3OH 
Diltiazem 
Boceprevir 
Ondansetron 
 
Midazolam 
Felodipine 
Buspirone 
Pimozide 
 
Triazolam 
Lercanidipine 
Carbamazepine 
Propranolol 
 
 
Nifedipine 
Cafergot 
Quetiapine 
 
Immune Modulators: 
Nisoldipine 
Caffeine→TMU 
Quinine 
 
Cyclosporine 
Nitrendipine 
Cilostazol 
Risperidone 
 
Tacrolimus (FK506) 
Verapamil 
Cocaine 
Romidepsin 
 
 
 
Codeine-N-
demethylation 
Salmeterol 
 
HIV Antivirals: 
HMG CoA 
Reductase 
Inhibitors: 
Dapsone 
Sildenafil 
 
Indinavir 
Atorvastatin 
Dexamethasone 
Sirolimus 
 
Nelfinavir 
Cerivastatin  
Dextromethorphan 
Sorafenib 
 
Ritonavir 
Lovastatin 
Docetaxel 
Sunitinib 
 
Saquinavir 
Simvastatin 
Domperidone 
Tamoxifen 
 
 
 
Eplerenone 
Taxol 
 
 
 
Fentanyl 
Telaprevir 
 
 
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15.4. 
Declaration of Helsinki 
WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI: 
Recommendations Guiding Medical Doctors in Biomedical Research Involving Human Subjects 
Adopted by the 18th World Medical Association (WMA) General Assembly, Helsinki, Finland, 
June 1964 and amended by the 29th WMA General Assembly, Tokyo, Japan, October 1975, 35th 
WMA General Assembly, Venice, Italy, October 1983, and the 41st WMA General Assembly, 
Hong Kong, September 1989, the 48th WMA General Assembly, Somerset West, Republic of 
South Africa, October 1996; 52nd WMA General Assembly, Edinburgh, Scotland, October 2000; 
53rd WMA General Assembly, Washington 2002 (Note of Clarification on paragraph 29 added); 
55th WMA General Assembly, Tokyo 2004 (Note of Clarification on Paragraph 30 added); and 
59th WMA General Assembly, Seoul, October 2008. 
A. 
INTRODUCTION  
1. The World Medical Association (WMA) has developed the Declaration of Helsinki as a 
statement of ethical principles for medical research involving human subjects, including 
research on identifiable human material and data.  
The Declaration is intended to be read as a whole and each of its constituent paragraphs 
should not be applied without consideration of all other relevant paragraphs.  
2. Although the Declaration is addressed primarily to physicians, the WMA encourages 
other participants in medical research involving human subjects to adopt these principles.  
3. It is the duty of the physician to promote and safeguard the health of subjects, including 
those who are involved in medical research. The physician's knowledge and conscience 
are dedicated to the fulfilment of this duty.  
4. The Declaration of Geneva of the WMA binds the physician with the words, "The health 
of my subject will be my first consideration," and the International Code of Medical 
Ethics declares that, "A physician shall act in the subject's best interest when providing 
medical care."  
5. Medical progress is based on research that ultimately must include studies involving 
human subjects. Populations that are underrepresented in medical research should be 
provided appropriate access to participation in research.  
6. In medical research involving human subjects, the well-being of the individual research 
subject must take precedence over all other interests.  
7. The primary purpose of medical research involving human subjects is to understand the 
causes, development and effects of diseases and improve preventive, diagnostic and 
therapeutic interventions (methods, procedures and treatments). Even the best current 
interventions must be evaluated continually through research for their safety, 
effectiveness, efficiency, accessibility and quality.  
8. In medical practice and in medical research, most interventions involve risks and 
burdens.  
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9. Medical research is subject to ethical standards that promote respect for all human 
subjects and protect their health and rights. Some research populations are particularly 
vulnerable and need special protection. These include those who cannot give or refuse 
consent for themselves and those who may be vulnerable to coercion or undue influence.  
10. Physicians should consider the ethical, legal and regulatory norms and standards for 
research involving human subjects in their own countries as well as applicable 
international norms and standards. No national or international ethical, legal or regulatory 
requirement should reduce or eliminate any of the protections for research subjects set 
forth in this Declaration.  
B. 
BASIC PRINCIPLES FOR ALL MEDICAL RESEARCH  
11. It is the duty of physicians who participate in medical research to protect the life, health, 
dignity, integrity, right to self-determination, privacy, and confidentiality of personal 
information of research subjects.  
12. Medical research involving human subjects must conform to generally accepted scientific 
principles, be based on a thorough knowledge of the scientific literature, other relevant 
sources of information, and adequate laboratory and, as appropriate, animal 
experimentation. The welfare of animals used for research must be respected.  
13. Appropriate caution must be exercised in the conduct of medical research that may harm 
the environment.  
14. The design and performance of each research study involving human subjects must be 
clearly described in a research protocol. The protocol should contain a statement of the 
ethical considerations involved and should indicate how the principles in this Declaration 
have been addressed. The protocol should include information regarding funding, 
Sponsors, institutional affiliations, other potential conflicts of interest, incentives for 
subjects and provisions for treating and/or compensating subjects who are harmed as a 
consequence of participation in the research study. The protocol should describe 
arrangements for post-study access by study subjects to interventions identified as 
beneficial in the study or access to other appropriate care or benefits.  
15. The research protocol must be submitted for consideration, comment, guidance and 
approval to a research ethics committee before the study begins. This committee must be 
independent of the researcher, the Sponsor and any other undue influence. It must take 
into consideration the laws and regulations of the country or countries in which the 
research is to be performed as well as applicable international norms and standards but 
these must not be allowed to reduce or eliminate any of the protections for research 
subjects set forth in this Declaration. The committee must have the right to monitor 
ongoing studies. The researcher must provide monitoring information to the committee, 
especially information about any SAEs. No change to the protocol may be made without 
consideration and approval by the committee.  
16. Medical research involving human subjects must be conducted only by individuals with 
the appropriate scientific training and qualifications. Research on subjects or healthy 
volunteers requires the supervision of a competent and appropriately qualified physician 
or other health care professional. The responsibility for the protection of research subjects 
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05 January 2015 
 
must always rest with the physician or other health care professional and never the 
research subjects, even though they have given consent.  
17. Medical research involving a disadvantaged or vulnerable population or community is 
only justified if the research is responsive to the health needs and priorities of this 
population or community and if there is a reasonable likelihood that this population or 
community stands to benefit from the results of the research.  
18. Every medical research study involving human subjects must be preceded by careful 
assessment of predictable risks and burdens to the individuals and communities involved 
in the research in comparison with foreseeable benefits to them and to other individuals 
or communities affected by the condition under investigation.  
19. Every clinical trial must be registered in a publicly accessible database before recruitment 
of the first subject.  
20. Physicians may not participate in a research study involving human subjects unless they 
are confident that the risks involved have been adequately assessed and can be 
satisfactorily managed. Physicians must immediately stop a study when the risks are 
found to outweigh the potential benefits or when there is conclusive proof of positive and 
beneficial results.  
21. Medical research involving human subjects may only be conducted if the importance of 
the objective outweighs the inherent risks and burdens to the research subjects.  
22. Participation by competent individuals as subjects in medical research must be voluntary. 
Although it may be appropriate to consult family members or community leaders, no 
competent individual may be enrolled in a research study unless he or she freely agrees.  
23. Every precaution must be taken to protect the privacy of research subjects and the 
confidentiality of their personal information and to minimize the impact of the study on 
their physical, mental and social integrity.  
24. In medical research involving competent human subjects, each potential subject must be 
adequately informed of the aims, methods, sources of funding, any possible conflicts of 
interest, institutional affiliations of the researcher, the anticipated benefits and potential 
risks of the study and the discomfort it may entail, and any other relevant aspects of the 
study. The potential subject must be informed of the right to refuse to participate in the 
study or to withdraw consent to participate at any time without reprisal. Special attention 
should be given to the specific information needs of individual potential subjects as well 
as to the methods used to deliver the information. After ensuring that the potential subject 
has understood the information, the physician or another appropriately qualified 
individual must then seek the potential subject's freely-given informed consent, 
preferably in writing. If the consent cannot be expressed in writing, the non-written 
consent must be formally documented and witnessed.  
25. For medical research using identifiable human material or data, physicians must normally 
seek consent for the collection, analysis, storage and/or reuse. There may be situations 
where consent would be impossible or impractical to obtain for such research or would 
pose a threat to the validity of the research. In such situations the research may be done 
only after consideration and approval of a research ethics committee.  
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26. When seeking informed consent for participation in a research study the physician should 
be particularly cautious if the potential subject is in a dependent relationship with the 
physician or may consent under duress. In such situations the informed consent should be 
sought by an appropriately qualified individual who is completely independent of this 
relationship.  
27. For a potential research subject who is incompetent, the physician must seek informed 
consent from the legally authorized representative. These individuals must not be 
included in a research study that has no likelihood of benefit for them unless it is intended 
to promote the health of the population represented by the potential subject, the research 
cannot instead be performed with competent persons, and the research entails only 
minimal risk and minimal burden.  
28. When a potential research subject who is deemed incompetent is able to give assent to 
decisions about participation in research, the physician must seek that assent in addition 
to the consent of the legally authorized representative. The potential subject's dissent 
should be respected.  
29. Research involving subjects who are physically or mentally incapable of giving consent, 
for example, unconscious subjects, may be done only if the physical or mental condition 
that prevents giving informed consent is a necessary characteristic of the research 
population. In such circumstances the physician should seek informed consent from the 
legally authorized representative. If no such representative is available and if the research 
cannot be delayed, the study may proceed without informed consent provided that the 
specific reasons for involving subjects with a condition that renders them unable to give 
informed consent have been stated in the research protocol and the study has been 
approved by a research ethics committee. Consent to remain in the research should be 
obtained as soon as possible from the subject or a legally authorized representative.  
30. Authors, editors and publishers all have ethical obligations with regard to the publication 
of the results of research. Authors have a duty to make publicly available the results of 
their research on human subjects and are accountable for the completeness and accuracy 
of their reports. They should adhere to accepted guidelines for ethical reporting. Negative 
and inconclusive as well as positive results should be published or otherwise made 
publicly available. Sources of funding, institutional affiliations and conflicts of interest 
should be declared in the publication. Reports of research not in accordance with the 
principles of this Declaration should not be accepted for publication.  
C 
ADDITIONAL PRINCIPLES FOR MEDICAL RESEARCH 
COMBINED WITH MEDICAL CARE  
31. The physician may combine medical research with medical care only to the extent that 
the research is justified by its potential preventive, diagnostic or therapeutic value and if 
the physician has good reason to believe that participation in the research study will not 
adversely affect the health of the subjects who serve as research subjects.  
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32. The benefits, risks, burdens and effectiveness of a new intervention must be tested 
against those of the best current proven intervention, except in the following 
circumstances:  
• The use of placebo, or no treatment, is acceptable in studies where no current proven 
intervention exists; or  
• Where for compelling and scientifically sound methodological reasons the use of 
placebo is necessary to determine the efficacy or safety of an intervention and the 
subjects who receive placebo or no treatment will not be subject to any risk of serious 
or irreversible harm. Extreme care must be taken to avoid abuse of this option.  
33. At the conclusion of the study, subjects entered into the study are entitled to be informed 
about the outcome of the study and to share any benefits that result from it, for example, 
access to interventions identified as beneficial in the study or to other appropriate care or 
benefits.  
34. The physician must fully inform the subject which aspects of the care are related to the 
research. The refusal of a subject to participate in a study or the subject's decision to 
withdraw from the study must never interfere with the subject-physician relationship.  
35. In the treatment of a subject, where proven interventions do not exist or have been 
ineffective, the physician, after seeking expert advice, with informed consent from the 
subject or a legally authorized representative, may use an unproven intervention if in the 
physician's judgement it offers hope of saving life, re-establishing health or alleviating 
suffering. Where possible, this intervention should be made the object of research, 
designed to evaluate its safety and efficacy. In all cases, new information should be 
recorded and, where appropriate, made publicly available. 
 
 
89 
 

SAP_001.pdf: 
Official Title: 
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, 
Pharmacokinetic and Pharmacodynamic Study of AG-348 in Adult Patients 
with Pyruvate Kinase Deficiency 
NCT Number: 
NCT02476916 
Document Date: 
SAP Version 1: 15 June 2017 


 
 
 
 
 
 
 
STATISTICAL ANALYSIS PLAN (METHODS)   
PROTOCOL AG348-C-003 CORE PERIOD 
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, 
Efficacy, Pharmacokinetic and Pharmacodynamic Study of 
AG-348 in Adult Patients with Pyruvate Kinase Deficiency 
 
 
 
 
 
 
Sponsor: 
Agios Pharmaceuticals, Inc. 
88 Sidney Street 
Cambridge, MA  02139-4169 
Tel: (617) 649-8600 
Fax: (617) 649-8618 
 
 
 
 
Analysis Plan Date: 
15 June 2017 
Analysis Plan Version: 
Version 1.0 
 
Confidentiality Statement 
The information contained herein is confidential and the proprietary property  
of Agios Pharmaceuticals, Inc. and any unauthorized use or disclosure  
of such information without the prior written authorization of  
Agios Pharmaceuticals, Inc. is expressly prohibited. 


Core Period Statistical Analysis Plan (Methods) 
 
 
 
 
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APPROVAL SIGNATURE PAGE 
Protocol Title: 
A Phase 2, Open Label, Randomized, Dose Ranging, Safety, 
Efficacy, Pharmacokinetic and Pharmacodynamic Study of AG-
348 in Adult Subjects with Pyruvate Kinase Deficiency 
 
Protocol Number: 
AG348-C-003 
 
 
 
 
 
 
Author Signatory: 
, PhD 
Study Biostatistician 
Agios Pharmaceuticals, Inc. 
 
Signature:______________________________________ 
 
Date:__________________________________________ 
 
Approver Signatory: 
, MD, PhD 
 
 
Agios Pharmaceuticals, Inc. 
 
Signature:______________________________________ 
 
Date:__________________________________________ 
 
, MD 
 
On Behalf of Agios 
Pharmaceuticals, Inc. 
 
Signature:______________________________________ 
 
Date:__________________________________________ 
 
, PhD 
 
Agios Pharmaceuticals, Inc. 
 
Signature:______________________________________ 
 
Date:__________________________________________ 


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1 
TABLE OF CONTENTS 
1 
TABLE OF CONTENTS ................................................................................................ 3 
List of Tables........................................................................................................................ 5 
2 
LIST OF ABBREVIATIONS ......................................................................................... 6 
3 
SUMMARY OF MODIFICATIONS ............................................................................. 8 
3.1 
Modifications From the Approved Clinical Study Protocol ....................................... 8 
3.2 
Modifications From the Approved Statistical Analysis Plan ..................................... 9 
4 
INTRODUCTION ......................................................................................................... 10 
5 
STUDY OBJECTIVES (Core Period Only) ................................................................ 10 
5.1 
Primary Objective ..................................................................................................... 10 
5.2 
Secondary Objectives ............................................................................................... 10 
 
 11 
6 
STUDY ENDPOINTS ................................................................................................... 11 
6.1 
Safety Endpoints ....................................................................................................... 11 
6.2 
Clinical Endpoints .................................................................................................... 11 
6.2.1 
Additional Endpoints ........................................................................................ 11 
7 
STUDY DESIGN ........................................................................................................... 12 
7.1 
Overall Design .......................................................................................................... 12 
7.2 
Sample Size and Power ............................................................................................ 13 
7.3 
Randomization .......................................................................................................... 14 
7.4 
Blinding and Unblinding .......................................................................................... 14 
8 
ANALYSIS SETS .......................................................................................................... 14 
8.1 
Safety Analysis Set ................................................................................................... 14 
8.2 
Efficacy Analysis Set ............................................................................................... 16 
9 
Analysis Periods ............................................................................................................. 16 
10 STATISTICAL ANALYSIS ......................................................................................... 17 
10.1 General Considerations ............................................................................................ 17 
10.2 Background Characteristics ...................................................................................... 18 
10.2.1 
Subject Disposition ........................................................................................... 18 
10.2.2 
Demographics and Baseline Characteristics ..................................................... 19 
10.2.3 
Prior and Concomitant Medications ................................................................. 20 
10.2.4 
Study Drug Exposure........................................................................................ 21 
10.2.5 
Study Drug Compliance ................................................................................... 21 
10.2.6 
Actual Dose Intensity and Relative Dose Intensity .......................................... 21 
10.2.7 
Study Drug Modification .................................................................................. 22 
10.2.8 
Protocol Deviations .......................................................................................... 22 
10.3 Efficacy Analysis ...................................................................................................... 22 
10.3.1 
Analyses of Hemoglobin (Hb) .......................................................................... 22 
10.3.2 
Other Preliminary Clinical Activity Indicators ................................................ 23 
10.4 Safety Analysis ......................................................................................................... 23 
10.4.1 
Adverse Events ................................................................................................. 24 
10.4.2 
Clinical Laboratory ........................................................................................... 26 
10.4.3 
Standard Digital ECG ....................................................................................... 27 


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10.4.4 
Vital Signs ........................................................................................................ 27 
10.4.5 
Physical Examination ....................................................................................... 27 
10.4.6 
Assessment of Aromatase Inhibitor Activity of AG348 .................................. 27 
10.4.7 
Menstrual Cycle Diary ...................................................................................... 28 
11 INTERIM AND DMC ANALYSES ............................................................................. 28 
11.1 Interim analysis/DRTs .............................................................................................. 28 
11.2 DMC Analysis .......................................................................................................... 28 
12 APPENDICES ................................................................................................................ 29 
Appendix A 
Schedule of Assessments ........................................................................... 30 
Appendix B 
Preferred Reporting Units .......................................................................... 39 
Appendix C 
Imputation Rules for Missing Prior/Concomitant Medication Dates ........ 40 
Appendix D 
Handling of Missing Dates in Adverse Events .......................................... 41 
Appendix E 
PKR genotype information and the corresponding mutation description and 
classification ............................................................................................................. 43 
Appendix F 
MedDRA Terms List for AEs of Endocrinological Interest (Menopausal 
Symptoms) ................................................................................................................ 45 
  
 
 


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List of Tables 
Table 3-1 Modifications from the approved clinical protocol .................................................. 8 
Table 7-1 Sample Size Estimation .......................................................................................... 14 
Table 8-1 Initial Treatment Group Assignments .................................................................... 14 
Table 8-2 Actual Dose Group Assignment ............................................................................. 15 
Table 8-3 Randomized Treatment Group Assignment ........................................................... 16 
Table 12-1 Clinical Laboratory Preferred Reporting Units .................................................... 39 
Table 12-2 Prior, Concomitant, and Post Categorization of a Medication ............................. 40 


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2 
LIST OF ABBREVIATIONS 
Abbreviation 
Term 
2,3-DPG 
2,3-diphosphoglycerate 
AE 
Adverse event 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Aminotransferase 
AST 
Aspartate Aminotransferase 
ATC 
Anatomic Therapeutic Class 
ATP 
Adenosine triphosphate 
BID 
Twice daily 
BMI 
Body mass index 
CI 
Confidence Interval 
COHb 
Carboxyhemoglobin 
CPAP 
clinical pharmacology analysis plan 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTX 
Serum C-terminal telopeptide 
CTWG 
Clinical Trial Working Group 
DBP 
Diastolic Blood Pressure 
DRT 
Data Review Team 
DXA 
Dual-energy x-ray absorptiometry 
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EPO 
Erythropoietin 
Hb 
Hemoglobin 
HCT 
Hematocrit 
HDL-C 
High-density lipoprotein-cholesterol 
Hp 
Haptoglobin 
HLT 
MedDRA High Level Term 
HR 
Hemoglobin Response 
LDH 
Lactate dehydrogenase 
MedDRA 
Medical Dictionary for Regulatory Activities 
max 
Maximum value 


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Abbreviation 
Term 
min 
Minimum value 
NTX 
Serum N-terminal telopeptide 
PD 
Pharmacodynamic 
PO 
Administered orally 
PK 
Pharmacokinetic 
PKD 
Pyruvate kinase deficiency 
PKR 
Pyruvate kinase isoform R 
PR 
The portion of the ECG wave from the beginning of the P wave to 
the beginning of the QRS complex 
PT 
Preferred Term 
q12h 
Every 12 hours 
QD 
Once-daily 
QOD 
Every Other Day 
QTc 
Heart-rate corrected QT interval 
QTcB 
Corrected QT interval - Bazett correction formula 
QTcF 
Corrected QT interval - Fridericia’s method 
RBC 
Red blood cell (count) 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SBP 
Systolic Blood Pressure 
SD 
Standard deviation 
SOC 
System organ class 
TEAE 
Treatment-emergent adverse event 
TIBC 
Total iron binding capacity 
VS 
Vital signs 
WHO 
World Health Organization 
WHODDE 
World Health Organization Drug Dictionary Enhanced 


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3 
SUMMARY OF MODIFICATIONS 
3.1 
Modifications From the Approved Clinical Study Protocol 
Table 3-1 Modifications from the approved clinical protocol 
Protocol 
Section 
Protocol Language 
SAP Changes 
Rationale 
Section 12.2 
Efficacy Analysis Set 
defined as all subjects 
who are enrolled and 
achieve at least 50% 
compliance with their 
assigned dose intensity 
for at least 4 weeks of 
continuous dosing, is 
specified to be the 
primary set for the 
analysis of preliminary 
clinical activity data. 
Efficacy Analysis 
Set will include all 
subjects who 
received any study 
treatment for at least 
3 weeks (Section 
8.2). 
A considerable number of subjects 
changed their actual dose from the 
randomized dose, usually within the 
first few weeks of dosing due to 
excessive Hb increase (a measure of 
efficacy). Therefore compliance as 
originally defined may not be 
relevant to efficacy measurement. In 
addition, the effect of PKR 
activators can usually be detected 
within a few weeks. The 3 weeks 
minimum treatment duration is 
considered sufficient to reflect the 
treatment effect. The Efficacy 
Analysis Set definition is updated to 
better reflect the effect of the study 
drug treatment on Hemoglobin 
response and other related clinical 
endpoints as detailed in Section 
10.3. 
Section 5.2 
Section 6.2 
ETCO (Core Period 
only) is specified as 
part of the 3rd bullet 
under the Second 
Objectives and 
subsequently 
considered one of the 
clinical activity 
endpoint 
ETCO is removed 
from the 
objectives/endpoints 
ETCO is not measured in the study 
as determined by the Clinical Trial 
Working Group (CTWG) in May 
2016. 
Section 
12.5.3 
The actual dose and 
duration in days of AG-
348, as well as the dose 
intensity (computed as 
Actual Dose 
intensity is defined 
as the ratio of actual 
dose and actual 
The protocol definition for ‘dose 
intensity’ is the Actual dose 
intensity. To avoid future confusion 
it with planned dose intensity, the 


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the ratio of actual dose 
received and actual 
duration) and the 
relative dose intensity 
(computed as the ratio 
of dose intensity and 
planned dose 
received/planned 
duration) will be listed 
and summarized using 
descriptive statistics by 
dose arm. 
duration of study 
drug exposure. 
Relative dose 
intensity is defined 
as the ratio of actual 
dose intensity and 
planned dose 
intensity, 
terms are updated.   
3.2 
Modifications From the Approved Statistical Analysis Plan 
This is the first version of the final Statistical Analysis Plan for Core Period. 


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4 
INTRODUCTION 
This SAP describes the planned final analyses for the Study AG348-C-003 Core Period data 
and is based on the  
• approved clinical study protocol (Version 5.0, dated 30 March 2016), 
• approved electronic case report form (eCRF) (Version 4.0, dated 28 June, 2016),  
• eCRF Completion Guidelines (Version 8.0, dated 11 August 2016). 
Study AG348-C-003 is a Phase 2, open label, randomized, dose ranging study in subjects 
aged 18 years or older with pyruvate kinase deficiency. This study is designed to evaluate the 
safety, efficacy, pharmacokinetics (PK) and pharmacodynamics (PD) of AG-348. 
Study AG-348-C-003 is divided into a Core Period and an Extension Period as detailed in the 
Protocol and summarized in Section 7.1 of this document. This SAP (Methods) documents 
the planned final statistical analyses of safety and efficacy analysis in the Core Period 
introduced in the study protocol, and describes the corresponding data presentations. It also 
documents additional safety and efficacy analyses not specified in the protocol, which will 
provide supportive information to the scientific understanding of the drug entity in the Core 
Period. Data from the Extension Period that becomes available at the Core Period database 
lock will also be included and summarized as appropriate.  
The study will also evaluate the pharmacokinetic (PK) and pharmacodynamic 
(PD) characteristics of AG-348 in this patient population. PK and PD analyses will be 
documented separately in the clinical pharmacology analysis plan (CPAP) for the study. 
The Agios Biostatistics and Statistical Programming (or delegates) will perform the statistical 
analysis of the efficacy and safety data; SAS (Version 9.2 or higher) will be used to generate 
all statistical outputs (tables, figures, listings, and datasets). The SAP will be finalized and 
approved prior to clinical data lock for the Core Period.  
5 
STUDY OBJECTIVES (CORE PERIOD ONLY) 
5.1 
Primary Objective 
To evaluate the safety and tolerability of up to 24 weeks of AG-348 administration in 
subjects with PKD. 
5.2 
Secondary Objectives 
• Evaluate the PK of AG-348 and the metabolite AGI-8702 (to be analyzed per CPAP). 
• Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348 (to be 
analyzed per CPAP). 
• Evaluate indicators of clinical activity of AG-348 in subjects with PKD, including 
changes in Hemoglobin (Hb), hematocrit (HCT), reticulocyte count, haptoglobin (Hp), 
carboxyhemoglobin (COHb), lactate dehydrogenase (LDH), total and indirect bilirubin, 


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erythropoietin (EPO), hepcidin, ferritin, and transferrin saturation (serum iron/iron 
binding capacity). 
6 
STUDY ENDPOINTS 
6.1 Safety Endpoints 
• Monitoring of AEs, including determination of SAEs and AEs leading to discontinuation; 
safety laboratory parameters (hematology, chemistry, urinalysis, coagulation); physical 
examination findings; vital signs (VS); 12 lead electrocardiograms (ECGs); and DXA 
scans. Adverse events will be graded using CTCAE, Version 4.03. Serum sex hormone 
levels (testosterone [total and free], estrone, and estradiol), bone turnover markers (serum 
osteocalcin-N-mid and serum C-terminal telopeptide [CTX], N-terminal telopeptide 
[NTX]), 25-hydroxy vitamin D2 and D3, total cholesterol, high-density lipoprotein-
cholesterol (HDL-C), and triglycerides will be monitored for evidence of potential 
inhibition of aromatase by AG-348. Menstruating female subjects will also keep a paper-
based menstrual cycle diary throughout the Core and Extension Periods. 
6.2 Clinical Endpoints 
• Monitoring of potential indicators of clinical activity will include evaluating changes in 
Hb, HCT, reticulocyte count, Hp, COHb, LDH, total and indirect bilirubin, EPO, 
hepcidin, ferritin, and transferrin saturation. 
6.2.1 Additional Endpoints 
Note that the endpoints included in this section are not specified as endpoints in protocol. 
They are analyzed to provide additional clinical information related to Hb:  
• Hb response (HR) rate in the Core Period (detailed in Section 10.3.1) 
• Time to the first Hb change >=1.5 g/dL among subjects with HR in the Core Period. 
• Maximum Hb change from baseline in the Core Period 
• Average Hb change from baseline in the Core Period 


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7 
STUDY DESIGN  
7.1 
Overall Design  
Study AG348-C-003 is a Phase 2, first-in-patient, open label, two arm, multicenter, 
randomized, dose-ranging study in adult subjects with Pyruvate Kinase Deficiency (PKD); 
the study is divided into a Core Period and an Extension Period. During the Core Period, 
subjects receive AG-348 for up to 24 weeks; subjects who are eligible can enter the 
Extension Period to receive AG-348 for up to 2 years following the end of the Core Period. 
Subjects with PKD confirmed by red blood cell (RBC) pyruvate kinase enzymatic assay 
performed at Screening will be eligible to participate in this study. At Week 25 (Week 24 
visit scheduled at Day 169), subjects who safely tolerate AG-348 and demonstrate clinical 
activity of AG-348 may be eligible to immediately roll over to the Extension Period for 
continued treatment. Subjects who complete treatment at the end of the Core Period (24 
weeks) will undergo follow-up assessments 4 weeks after the last dose of study drug. If a 
subject discontinues at any other time (including early discontinuation or discontinuation 
during the Core or Extension Period), the follow-up assessments will be conducted 4 weeks 
after discontinuation. Subjects with toxicity suspected to be related to study drug will 
continue follow-up until the adverse event (AE) resolves, is declared chronic by the 
Investigator, or the subject is lost to follow-up.  
For the Core Period, approximately 25 subjects will be initially randomized on an open-label 
1:1 basis to each of two twice-daily (BID) doses of AG-348 (approximately 50 subjects total, 
see Figure 7-1). The dose of Arm 1 is 300 mg of AG-348 administered orally (PO) every 
12 hours (q12h, BID). The dose of Arm 2 is 50 mg of AG-348 administered PO q12h (BID). 
Randomization will be stratified by PKR mutation in order to maintain balance as much as 
possible across the dose arms for the specific mutations expected to be most frequently 
enrolled. The PKR mutation stratification factor will consist of 4 levels (R510Q, R486W, and 
R479H) and all other mutations (“other”). Mutation status is defined by the presence of at 
least one of the indicated mutations; subjects with more than one stratified mutation will be 
assigned based on Sponsor’s discretion. 
Per study protocol, during the conduct of the study, a Data Review Team (DRT) has been 
established to review study data on a regular basis and adapt the study design, dose and 
schedule of AG-348. Based on the DRT’s recurring reviews, the DRT may add 1 new dose 
arm (Arm 3) to enroll up to 25 subjects at a dose to be determined; the dose for Arm 3 may 
be lower or higher than Arm 1 and Arm 2 doses, but will not exceed 360 mg q12h, as this 
was the highest dose that demonstrated acceptable safety and tolerance in the 14-day multiple 
BID dosing study in healthy volunteers.  
As detailed in Section 11.1, 12 DRT meetings were held during the conduct of the study 
before the Core Period database lock. The study remains as a 2-arm study because the DRT 
hasn’t exercised the option to add a 3rd arm. However, multiple patients have had their 
randomized dose changed, as allowed by the protocol. In some, the dose was decreased for 
adverse events or when the Hb exceeded the midpoint of the normal range. In some subjects 
initially randomized to the 50 mg BID arm, the dose was increased to 300 mg BID after at 
least 12 weeks of treatment if there was no Hb improvement noted with the initial lower 


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dose. As a result, subjects in the trial are being treated across a range of doses in the Core 
Period, rather than falling exclusively into the two originally assigned dose groups of 50 mg 
BID and 300 mg BID (Initial Dose Groups).  Additional analyses were planned to fully 
evaluate the effect of treatment on both efficacy and safety as detailed in Section 10.  
Figure 7-1 Study Schema for Core Period 
 
Abbreviations:  BID (q12h) = twice-daily (every 12 hours); DRT = data review team; PKR = pyruvate kinase 
red blood cell isoform; TBD = to be determined; w = weeks.  
7.2 
Sample Size and Power 
Due to the rare disease setting, the minimal sample size in each dose arm may be determined 
by feasibility. In order to evaluate the primary objective of safety and tolerability of AG-348 
in Arm 1 and Arm 2, up to a maximum of 25 patients may be randomized onto each arm. The 
actual number of patients enrolled into Arms 1 and 2 will depend on the safety reviews and 
decisions made by the DRT. In addition, up to 25 additional patients may be enrolled to 
evaluate an additional dose arm (Arm 3; see Section 7.1). As for Arms 1 and 2, the actual 
enrollment in a potential Arm 3 will depend on the safety reviews and decisions made by the 
DRT. Therefore, up to approximately 75 total patients may be enrolled in this study across 2 
to 3 dose arms. 
Table 4 provides the probability within a dose arm of detecting 1 or more AEs with varying 
sample size and the true underlying AE rates. 


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Table 7-1 Sample Size Estimation 
Sample Size 
True Underlying AE Rate 
15% 
10% 
5% 
5 
56% 
41% 
23% 
10 
80% 
65% 
40% 
15 
91% 
79% 
54% 
25 
98% 
93% 
72% 
 
7.3 
Randomization 
According to the initial study design, approximately 50 subjects (25 /treatment arm), who 
meet the eligibility criteria, will be randomized (1:1) to 1 of the 2 treatment arms, stratified 
by PKR mutation in order to maintain balance as much as possible across the dose arms for 
the specific mutations expected to be most frequently enrolled. The PKR mutation 
stratification factor will consist of 4 levels (R510Q, R486W, and R479H) and all other 
mutations (“other”). Mutation status is defined by the presence of at least one of the indicated 
mutations; subjects with more than one stratified mutation will be assigned based on 
Sponsor’s discretion. 
Based on the DRT’s recurring reviews, the DRT may add 1 new dose arm (Arm 3) to enroll 
up to 25 subjects at a dose to be determined; the dose for Arm 3 may be lower or higher than 
Arm 1 and Arm 2 doses, but will not exceed 360 mg q12h. 
7.4 
Blinding and Unblinding 
Not applicable. This is an open-label study. 
8 
ANALYSIS SETS 
The following analysis sets will be defined: Safety Analysis Set and Efficacy Analysis Set 
All Subjects will be referenced to include all subjects who were randomized or dosed. 
Listings will be referenced using All Subjects, unless otherwise specified. 
8.1 
Safety Analysis Set 
Safety Analysis Set will include all subjects who received at least one dose of study drug. 
The Safety Analysis Set will be used for safety analysis, unless specified otherwise. The 
primary safety analyses will be by Initial Treatment Group as in Table 8-1. 
Table 8-1 Initial Treatment Group Assignments 


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Initial Treatment 
Group 
Assignment 
300 mg BID 
Subjects who were randomized to 300 mg BID and received the 
dose as least once; and subjects randomized to 50 mg BID but who 
never received the dose and first received 300 mg BID dose 
50 mg BID 
Subjects randomized to 50 mg BID and received the dose as least 
once; and subjects randomized to 300 mg BID, never received the 
dose and first received 50 mg BID dose  
The study allowed for dose modifications as described in Section 7.1, which leads to a range 
of doses that subjects received. To better reflect the analysis with appropriate treatment 
assignment, additional analyses will be conducted by Actual Dose Group, defined as the 
treatment (total daily dose) the subject received for the longest duration in the Core Period, as 
shown in Table 8-2.  
Table 8-2 Actual Dose Group Assignment 
Actual Dose Group* 
Assignment 
<25 mg BID 
Subjects who received less than 50 mg daily dose, including 5 mg 
QD, 25 mg QD or 25 mg QOD for the longest duration during the 
Core Period 
25 mg BID 
Subjects who received 25 mg BID for the longest duration during 
the Core Period 
50 mg BID 
Subjects who received 50 mg BID for the longest duration during 
the Core Period 
100 mg BID 
Subjects who received 100 mg BID for the longest duration during 
the Core Period 
200 mg BID 
Subjects who received 200 mg BID for the longest duration during 
the Core Period 
300 mg BID 
Subjects who received 300 mg BID for the longest duration during 
the Core Period 
* Additional treatment arms may appear as subjects change their treatment during the Core 
Period, and will be allocated accordingly. 


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8.2 
Efficacy Analysis Set 
Efficacy Analysis Set will include all subjects who enrolled and received any study 
treatment for at least 3 weeks. The Efficacy Analysis Set will be the set used for efficacy 
analysis, unless specified otherwise. The treatment assignment for Efficacy Analysis Set will 
primarily be by Randomized Treatment Group, as shown in Table 8-3. 
Table 8-3 Randomized Treatment Group Assignment 
Randomized Treatment Group 
Assignment 
300 mg BID 
Subjects randomized to 300 mg BID  
50 mg BID 
Subjects randomized to 50 mg BID  
Additional analysis will also be conducted based by Actual Dose Group as shown in Table 
8-2. 
9 
ANALYSIS PERIODS 
Three dosing periods, including the Core Dosing Period, Extension Dosing Period, and 
Cumulative Dosing Period will be defined. For each dosing period, the safety analysis will be 
based on their treatment-emergent period as defined below; and the efficacy analysis will be 
based on the corresponding efficacy window.  
The Core Dosing Period is from the first dose date to the last dose date in the Core Period; 
for subjects who continued treatment in Extension Period, the last dose date in the Core 
Period will be imputed using the actual Week 24 visit date -1. 
• The Safety Analysis will be based on treatment-emergent period in the Core Dosing 
Period, defined as the period from the date of the first dose in the Core Period to 1) prior 
to the first dose in the Extension Period for subjects who continued being dosed in the 
Extension Period; or to 2) the safety follow-up (if available), 30 days post last dose in the 
Core Dosing Period, whichever is earlier for the subjects who did not enter Extension 
Period.  
• The Efficacy Analysis will be based on the efficacy window which is from the date of the 
first dose to 1) prior to first dose in the Extension Period for subjects who continued 
being dosed in the Extension Period; or to 2) 1 day after the end of core dosing period (to 
account for the assessment collected the next day after the last dose) for subjects who did 
not dose in Extension Period. 
The Extension Dosing Period is from actual Week 24 visit date to the last dose date in the 
Extension Period for subjects who continued treatment in Extension Period. 
• The Safety Analysis will be based on the Treatment-emergent period in Extension Dosing 
Period, which is from the date of the first dose in the Extension Period to 1) the safety 
follow-up (if available) or 30 days post last dose whichever is earlier for the subjects who 


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completed or discontinued treatment in Extension Period; or 2) to the date of Core Period 
database lock for subjects who are still on treatment in the Extension Period.  
• The Efficacy Analysis will be based on the efficacy window which is from the first dose 
in the Extension to 1 day after the last dose in the Extension Dosing Period.  
The Cumulative Dosing Period is from the date of the first dose (in the Core Period) to the 
last dose date (in the Core period for subjects who did not dose in Extension Period and in 
the Extension Period for subjects who dosed in Extension Period). For subjects who are still 
on treatment in the extension period, the date of Core Period database lock will be used as the 
subject’s last dose date.  
• The Safety Analysis will be based on the Treatment-emergent period in Cumulative 
Period, defined as from the first dose date to the safety follow-up visit (if available) or 30 
days post last dose date whichever is earlier.  
• The Efficacy Analysis will be based on the efficacy window which is from the first dose 
date to 1 day (inclusive) after last dose in the Cumulative Dosing Period.  
10 
STATISTICAL ANALYSIS 
10.1 
General Considerations 
The Schedule of Assessments is provided in Appendix A.  
Continuous variables will be summarized using the following descriptive summary 
statistics: the number of subjects (n), mean, standard deviation (SD), median, minimum value 
(min), and maximum value (max).  
The precision of the measurement in raw data for other continuous variables will be used to 
determine the number of decimal places to present in tables, figures, and derived listings. 
Minimum and maximum values will be reported with the same precision as the units of 
measure. The mean and median will be reported to 1 greater decimal place. SD will be 
reported to 2 greater decimal place. 
Categorical variables will be summarized using counts and percentages. Percentages will be 
presented to 1 decimal place.  
Baseline Value: Unless otherwise specified, the baseline value will be defined as the most 
recent non-missing measurement (scheduled or unscheduled) collected before the initial 
administration of study drug. Unscheduled visit measurements will be considered for 
derivation of baseline.  
• The baseline of Hb will be calculated as the arithmetic average of all available central 
lab results within 6 weeks (42 days) prior to the first dose, excluding Hb within 2 
months (61days) of a transfusion. 
Change (Absolute Change) from baseline: will be calculated as Postbaseline value - 
Baseline value.  
Relative change from baseline: will be calculated and expressed in percentages as 
100×(Postbaseline value - Baseline value)/Baseline value. 


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Analysis visits: Nominal visits will be used in the by-visit summaries. If there are multiple 
assessments collected with the same nominal visit, the assessment closest to the targeted visit 
date will be used. The actual study day will be included in listings.  
Unscheduled Visits: Unscheduled visit measurements will be included in listings and for the 
analysis of max/min values and max/min changes from baseline values. 
Central and Local Lab: Only central lab data will be used for by-visit summary including 
the actual values and their changes from baseline. Local lab data, in addition to central lab 
data, will be included in shift tables and the summary tables of categorized worst value or 
change for safety analysis. Both central and local labs will be included in the listings. 
Repeated Measurements: For repeated measurements, i.e., measurements with exactly the 
same collection date and time (if time is available), the average of the repeated measurements 
will be used to reflect the value at the corresponding measurement date and time. Actual 
values will be presented in the data listings. 
Incomplete/Missing data will not be imputed, unless otherwise specified; i.e., all missing 
values will remain as missing in all statistical analyses and listings, unless otherwise 
specified.  
Outliers: No formal statistical analyses will be performed to detect and/or remedy the 
presence of statistical outliers. 
10.2 
Background Characteristics  
10.2.1 Subject Disposition 
The number and percentage of subjects in the following analyses sets will be summarized:  
• Randomized (number only) 
• Treated (Number, with percentage calculated based on the number of subjects 
randomized) 
• Efficacy Analysis Set 
• Safety Analysis Set 
The percentage will be calculated based on the number of treated subjects.  
The number and percentage of subjects in each disposition category below will be 
summarized with the number in the Safety Analysis Set as the denominator: 
• Completed Core Period treatment 
o Extension enrollment status (Yes vs. No) 
• Prematurely discontinued treatment in Core Period 
o Reasons for discontinuations 


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Among subjects who enrolled into Extension, the number and percentage of subjects in the 
following category will be provided, with the number of Safety Analysis Set as the 
denominator: 
• On Treatment (at the time of Core Period Database Lock) 
• Completed Extension Period treatment 
• Prematurely discontinued treatment in Extension Period 
o Reasons for discontinuations 
Of the subjects who completed or discontinued study, the reason they are off study. 
The disposition table will be provided by Initial Treatment Group and repeated by the Actual 
Dose Group. 
A listing will be provided including 1) subjects who discontinued treatment in the Core 
Period; 2) subjects who completed treatment in the Core Period but did not roll into the 
Extension Period; 3) and subjects who discontinued treatment in the Extension Period.  
10.2.2 Demographics and Baseline Characteristics 
Demographics and baseline characteristics will be summarized based on the Safety Analysis 
Set by the Initial Treatment Group, and repeated by the Actual Dose Group:  
• Age at informed consent (in years) 
• Sex (female and male) 
o Female subjects will further be summarized by their child bearing potential status 
(Yes, No, and missing), with the percentage calculated using number of female 
subjects; 
o Female subjects with child bearing potential will be summarized by their usage of 
adequate birth control (Yes, No, and missing), with the percentage calculated 
using number of female subjects with  child bearing potentail;  
• Race (White, Black or African American, Asian, American Indian or Alaska Native, 
Native Hawaiian or Other Pacific Islander, Not Reported, Other [including subjects 
reported with one race])  
• Ethnicity (Hispanic or Latino, not Hispanic or Latino, and Not Reported) 
For subjects who reported multiple races, they will be considered as ‘Other’. 
• Baseline characteristics include 
− Weight, height and body mass index (BMI) 
− Hemoglobin level 
− DXA Scan by location (Hip, Spine, and Femoral Neck): Bone mineral density and 
their corresponding T-scores and Z-scores 


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• PKR genotype related 
− PKR stratification factors (R510Q, R486W, R479H, and Others),  
− PKR mutations for each allele 
− PKR mutation groups (missense/missense; missense/non-missense; non-
missense/non-missense) 
• Disease characteristics include 
− Age at PKD diagnosis  
− Splenectomy history (Yes or No): Of those with splenectomy, their age at 
splenectomy, type (total or partial) and effect on transfusion burden (reduced or 
not) and baseline hemoglobin (increased or not),  
− Cholecystectomy history (Yes vs. No), age at cholecystectomy among those with 
splenectomy, 
− Chelation history (Yes or No), 
− Phlebotomy during the 12 months prior to enrollment 
Demographics, baseline characteristics and disease characteristics will be included in subject 
level listings. 
Targeted medical history by reported term and Medical history by System organ class (SOC) 
and Preferred Term (PT) summary will be presented for the Safety Analysis Set. 
10.2.3 Prior and Concomitant Medications 
Medications used in this study will be coded by using the World Health Organization Drug 
Dictionary Enhanced (WHODDE) and categorized as the following: 
• Prior medication: any medication that started before the first dose date of study drug of 
the Core Dosing Period, regardless of when the medication ended. 
• Concomitant medication: medication continued or newly received on or after the first 
dose date of study drug through the end of treatment-emergent period. 
Concomitant medication will be summarized for the Core Dosing Period and repeated for 
the Cumulative Dosing Period. 
• Post-treatment medication: medication continued or newly received after the treatment-
emergent period. 
A given medication can be classified as a prior medication, a concomitant medication, or a 
post-treatment medication; both prior and concomitant; both concomitant and post-treatment; 
or prior, concomitant, and post-treatment. If a medication has a missing or partially missing 
start/end date or time and it cannot be determined whether it was taken before initial dosing, 
concomitantly, or more than 30 days after the last dose of study drug, it will be considered as 
prior, concomitant, and post-treatment. 


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Prior medications, concomitant medications for the Core Dosing Period and concomitant 
medications for the Cumulative Dosing Period will be summarized descriptively by anatomic 
class (ATC) level 1, ATC level 2, and preferred name based on the Safety Analysis Set. Post-
treatment medication will only be included in the subject level listings.  
10.2.4 Study Drug Exposure 
Exposure will be summarized for Core Dosing Period and the Cumulative Dosing Period 
based on the Safety Analysis Set by Initial Treatment Group.  
Duration of study drug exposure is defined as: last dose date – first dose date + 1 day, 
regardless of any interruptions in dosing. Duration of study drug exposure will be 
summarized descriptively (n, mean, SD, median, min, and max) and in categories (>0 to ≤2 
weeks, >2 to ≤4 weeks, >4 to ≤8 weeks, >8 to ≤16 weeks, >16 to ≤24 weeks, >24 to ≤52 
weeks, >52 to ≤108 weeks and >108 weeks).  
10.2.5 Study Drug Compliance 
Compliance will be summarized for both the Core Dosing Period and the Cumulative Dosing 
Period based on the Safety Analysis Set by Initial Treatment Group. Given the lag in data 
entry of study drug compliance in the Extension period at the time of Core Period database 
lock, compliance during the Cumulative Dosing Period is only based on data reported and 
needs to be interpreted with caution.  
Actual dose is the total amount of study drug taken (mg), calculated as the sum of the amount 
of drug taken for all dosing regimens while the subject is in the corresponding dosing period. 
The doses for each dosing regimen is the duration of the dosing regimen times the dose times 
the frequency of the dose. Note that by this derivation, the missing exposure records during 
the core period is considered as no study drug taken during the corresponding interval. 
Planned dose is the planned total amount of study drug (mg), calculated as the sum of the 
planned drug amount expected to be taken for each interval in the corresponding dosing 
period. The planned drug amount expected to be taken for each interval is calculated as the 
duration of treatment times the dose times the dosing frequency for the exposure reported as 
‘Per Protocol’, ‘Dose Increased’, ‘Dose Reduced’ or ‘Dose Held’. For the records reported as 
‘Patient Missed Dose(s)’ or ‘Incorrect Dose’ or missing reason for dose change, the dose 
level and frequency from the last available compliance records prior will be used.  
Compliance will calculated as the ratio of actual dose and the planned dose. 
Actual dose, planned dose, and compliance will be summarized descriptively (n, mean, SD, 
median, min, and max). The number and percentage of subjects with compliance in different 
categories (i.e., <80%, ≥80% - <90%, ≥90% - <95%, vs ≥95%) will be summarized. 
10.2.6 Actual Dose Intensity and Relative Dose Intensity 
Actual Dose intensity is defined as the ratio of actual dose and actual duration of study drug 
exposure. Relative dose intensity is defined as the ratio of actual dose intensity and planned 
dose intensity, i.e., daily dose for randomized treatment (100 mg for those randomized to 50 


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mg BID group and 600 mg for those randomized to 300 mg BID group). Both dose intensity 
and relative dose intensity will be summarized for the Core Dosing Period and the 
Cumulative Dosing Period based on the Safety Analysis Set by Initial Treatment Group. 
10.2.7 Study Drug Modification 
Number and percentage of subjects with dose held or reduced, Number and percentage of 
subjects with dose increased will be summarized respectively based on the Safety Analysis 
Set by the Initial Treatment Group and repeated by the Actual Dose Group. The subjects with 
dose held or reduced will further be summarized by reason (Adverse event, increased 
hemoglobin, and others). 
10.2.8 Protocol Deviations 
All protocol deviations/violations as reported by the site monitor will be provided as a 
subject data listing. 
10.3 
Efficacy Analysis 
Efficacy summary tables listed below will be summarized based on the Efficacy Analysis Set 
overall, and by Randomized Treatment Group. The summary will also be repeated by the 
Actual Dose Group, unless specified otherwise.  
10.3.1 Analyses of Hemoglobin (Hb) 
Hb values and their changes from baseline will be summarized at scheduled visits (including 
visits from the Core Period and the visits from the Extension Period).  
Unless specified otherwise, only central lab results will be considered in the Hb related 
analyses. Hb results obtained within 2 months (61 days) after a red blood cell transfusion will 
be excluded. 
Hemoglobin response  
A hemoglobin response (HR) is defined as having post-baseline changes in Hb ≥1.5 g/dL at 
>50% assessments in the Core Dosing Period Efficacy Window. The Number and percentage 
of subjects with a HR will be summarized, along with the 95% CI based on exact binomial 
distribution.  
Mean (95% Confidence Interval [CI]) of Hb values at scheduled visits will be plotted by HR 
status (as defined below). Spaghetti plot for Hb assessments (excluding the Hb within 2 
months of transfusion) and spaghetti plot of Hb change from baseline will be provided by HR 
status. In spaghetti plots, both central and local lab will be included. In cases where there are 
both central and local labs on the same date, the central lab result will be used. 
The time to the first occurrence of Hb change from baseline >1 g/dL, ≥1.5 g/dL and ≥2 g/dL 
for subjects with a HR will be summarized using summary statistics.  
Maximum Hb change  
The Maximum Hb change from baseline will be summarized using descriptive statistics. In 
addition, categorical analysis (number and percentage of subjects) of maximum Hb change 


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(g/dL) from baseline (<1, 1 - <1.5, 1.5 - < 2, ≥2) will be summarized. A waterfall plot of 
maximum Hb change from baseline will be provided. These analyses will be repeated by HR 
status (Yes vs. No). 
The maximum Hb change from baseline considering only assessments collected while the 
subject was on the treatment same as the Actual Dose Group for at least 2 weeks will also be 
summarized for Hemoglobin responders.  
Average Hb change  
Average Hb change from baseline, defined as the arithmetic average of all Hb changes from 
baseline at all scheduled post-baseline visits based on central lab, excluding those obtained 
within 2 months (61 days) after a red blood cell transfusion, will be summarized using 
descriptive statistics. In addition, categorical analysis (number and percentage of subjects) of 
average Hb change (g/dL) from baseline (<1, 1 - <1.5, 1.5 - < 2, ≥2) will be summarized. 
Waterfall plot of average Hb change from baseline will be provided. These analyses will be 
repeated by HR status (Yes vs. No). 
The analyses for maximum Hb change and average Hb change will be repeated based on 
Efficacy Analysis Set by Actual Dose Group.  
The Hb by-visit summary, analysis of maximum Hb change and average Hb change, 
Hemoglobin response (Yes. vs. No) will be repeated by PKR genotype (Missense/Missense, 
Missense/Non-Missense, Non-Missense/Non-Missense) based on Efficacy Analysis Set by 
Actual Dose Group. 
All available Hb values, including central and local labs, for all subjects will be listed. 
10.3.2 Other Preliminary Clinical Activity Indicators 
Absolute values of HCT, percentage and absolute reticulocyte count, Hp, COHb, LDH, total 
and indirect bilirubin, EPO, and their changes from baseline will be summarized at each 
scheduled visit by HR status using summary statistics.  Mean (±SD) by visit will be plotted 
by HR status.  
For the summary of iron related parameters, including hepcidin, ferritin, iron, TIBC, and 
transferrin saturation, the summary will be separated for subjects reported on chelation over 
the efficacy analysis window vs. those reported no chelation. For subjects reported a change 
in chelation status during the efficacy analysis window, their related assessments will not be 
included in the summary table. Note that there is a testing method/kit change in hepcidin in 
the middle of the trial and the related results need to be interpreted with caution. 
These assessments will also be listed. 
10.4 Safety Analysis 
The overall safety profile of study drug will be assessed in terms of the following safety and 
tolerability endpoints:   
• Adverse events 


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• Clinical laboratory values (hematology, serum chemistry, coagulation studies, and 
urinalysis) 
• Assessment of Aromatase Inhibitor Activity: 
o DXA scans 
o Serum sex hormones  (testosterone [total and free], estrone, and estradiol) 
o Bone turnover markers (serum osteocalcin-N-mid and serum C-terminal 
telopeptide [CTX], N-terminal telopeptide [NTX]), 25-hydroxy vitamin D2 and 
D3, total cholesterol, high-density lipoprotein-cholesterol (HDL-C) 
• Standard digital electrocardiograms (ECGs) 
• Vital signs  
• Physical exams 
Safety endpoints will be analyzed based on the Safety Analysis Set by Initial Treatment 
Group and repeated by the Actual Dose Group, unless specified otherwise. The listings will 
be provided for the Cumulative Dosing Period. Only descriptive analysis of safety will be 
performed (i.e., no statistical testing will be performed). 
10.4.1 Adverse Events  
For analysis purpose, AEs will be categorized as pretreatment AEs, treatment-emergent 
adverse events (TEAEs), or post-treatment AEs: 
• Pretreatment AE: any AE that started in the pre-treatment period. 
• TEAE: any AE that increased in severity or that was newly developed in the treatment-
emergent period. 
• Post-treatment AE: any AE that increased in severity or that was newly developed after 
the end of treatment-emergent period. 
For AEs with missing or partial start dates, if there is no clear evidence that the AEs started 
before or after study treatment, then the AEs will be classified as TEAEs. 
Treatment-emergent AEs (TEAEs) for the Core Period and the Cumulative Period will be 
summarized, separately, based on Safety Analysis Set by Initial Treatment Group. Additional 
analyses based on the Safety Analysis Set by Actual Dose Group will also be provided.  
An overview of the TEAE profile will be provided, including number and percent of subjects 
for the following categories:  
• All TEAEs  
• TEAEs by relationship 
• TEAEs by maximum severity 
• TEAEs leading to treatment modification 
• TEAEs leading to treatment interruption 


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• TEAEs leading to treatment discontinuation 
• Serious TEAEs 
• Related serious TEAEs 
• TEAE leading to death  
Adverse events summary tables will be presented for TEAEs only and will include the 
following:   
• All TEAEs  
• Grade 3/4 TEAEs 
• Related TEAEs (including Possibly Related and Probably Related) 
• TEAEs by relationship 
• TEAEs leading to treatment modification 
• TEAEs leading to treatment interruption 
• TEAEs leading to treatment discontinuation  
• Serious TEAEs 
• Related serious TEAEs 
• TEAEs leading to death 
• TEAEs by treatment interval for the Core Dosing Period (>0 to ≤1 Week: [Day1, Day8], 
>1 to ≤2 Weeks: [Day9, Day15], >2 to ≤8 Weeks: [Day16, Day57], >0 to ≤8 Weeks: 
[Day1, Day57], >8 to ≤16 Weeks: [Day58, Day113], >16 to ≤24 Weeks: [Day114, 
Day169], >24 Weeks: [Day170, end of TEAE period]) 
Summaries will be presented by Medical Dictionary for Regulatory Activities (MedDRA) 
system organ class (SOC) and preferred term (PT) using frequency counts and percentages 
(i.e., number and percentage of subjects with an event). When summarizing the number and 
percentages of subjects, subjects with multiple occurrences of the same adverse event or a 
continuing adverse event will be counted once, and only the maximum severity level will be 
presented in the severity summaries, and the worst/highest relationship level in the 
relationship summaries.   
In addition to the summary of all TEAEs by SOC and PT, TEAEs in the Core Period by PT 
will also be provided based on the Safety Analysis Set by Initial Treatment Group. 
Additional Events of interest:  
AEs of endocrinological interest, defined by the AEs whose High level terms (HLTs) or PTs 
as listed in Appendix F will be summarized by total number of subjects with menopause 
symptoms, as well as by corresponding SOCs and PTs using frequency counts and 
percentages. 


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A listing of AEs will be provided. In addition, listings containing individual subject adverse 
event data for TEAEs leading to treatment interruption/interruption/discontinuation, SAEs 
and all deaths will be provided separately. In the listing of SAEs and death, a flag indicating 
the TEAE status will be included. 
10.4.2 Clinical Laboratory 
For the laboratory measurements, the raw values and change from baseline values of the 
continuous hematology, chemistry, special chemistry, coagulation and urinalysis results will 
be summarized at each scheduled time point. For parameters summarized in Table 12-1, the 
original reporting units will be used. All other parameters will be summarized in SI units.  
Listings containing individual subject hematology, chemistry, and coagulation values outside 
the reference ranges will be provided. This listing will include data from scheduled and 
unscheduled time points. 
Analysis on liver function test (LFT) 
• For transaminase (AST and ALT),  
1) Box plot of LFT value/ULN will be plotted against visit. 
2) Shift of LFTs by CTCAE grade from baseline to the worst grade during the 
treatment-emergent period will also be summarized for AST and ALT separately. 
In addition, subjects with AST shift or ALT shift will be summarized. In this AST 
or ALT pooled summary, the shift of the parameter with the worst grade in the 
treatment-emergent period is considered; when AST and ALT have the same 
worst grade in the treatment-emergent period, the shift of the parameter with the 
lowest baseline grade is considered. 
3) A listing of subjects with elevated AST and ALT results during the treatment-
emergent period will be presented. The listing will include both AST and ALT at 
all visits. 
• For all LFT results (alanine aminotransferase [ALT], aspartate aminotransferase [AST], 
and serum alkaline phosphatase [ALP]), a spaghetti plot (including all time points) will 
be provided under the unit of ULN:  
1) The plot for ALT will include subjects with at least one ALT>3×ULN during the 
treatment-emergent period.  
2) The plot for AST will include subjects with at least one AST>3×ULN during the 
treatment-emergent period,  
3) The plot for ALP will include subjects with at least one ALP>1.5×ULN during 
the treatment-emergent period, and  
Analysis on triglycerides and cholesterol 
Shift of triglycerides and cholesterol by CTCAE grade from baseline to the worst grade 
during the treatment-emergent period will be summarized.  Mean values (±SD) against visit 
will be provided for triglycerides, cholesterol and HDL-C. A listing of subjects with elevated 


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triglycerides during the treatment-emergent period will be presented. The listing will include 
triglycerides, cholesterol and HDL-C at all visits. 
10.4.3 Standard Digital ECG 
For the ECG measurements during the treatment-emergent period, a summary of raw values 
and change from baseline values will be provided at each scheduled time point for the 
following standard digital ECG measurements: PR, QT, and QT corrected for HR (QTc) 
intervals (Fridericia's correction [QTcF = QT/RR1/3], QRS duration, and HR. 
In addition, number and percentage of subjects with QTcF in categories of ≤450, >450-≤480, 
>480-≤500 and >500 and the changes in QTcF from baseline in categories of ≤0, >0-≤30, 
>30-≤60, and >60 will be provided.  
10.4.4 Vital Signs 
For the vital signs measurements, the raw values and change from baseline values will be 
summarized at each scheduled time point: weight, BMI (calculated as weight [kg]/height 
[m]2), with height using the last available height), systolic and diastolic blood pressure (mm 
Hg), body temperature (oC), HR (beats per minute), and respiratory rate (breaths per minute). 
To further evaluate the impact of triglycerides on weight, weight and their change from 
baseline will be summarized by visit separating by subjects who had at least 1 grade shift 
versus those remain at the same grade in triglycerides. This subgroup analysis will be done 
only by Actual Dose Group based on the Safety Analysis Set. 
10.4.5 Physical Examination 
PE findings will be presented as a data listing only. 
10.4.6 Assessment of Aromatase Inhibitor Activity of AG348 
• Sex Hormones: testosterone (total and free), estrone, and estradiol.  
For sex hormones, the by-visit summary will be provided by sex based on the Safety 
Analysis Set. A spaghetti plot will also be provided by Actual Dose Group and by sex. 
The spaghetti plot will be repeated to only include assessments within the efficacy 
analysis window of the Cumulative Dosing Period to present the subjects’ on-treatment 
hormone profile. Note that there is a testing method/kit change in free testosterone in the 
middle of the trial and the related results need to be interpreted with caution.  
• Bone Turnover: serum osteocalcin-N-mid, CTX, and NTX: The by-visit summary will be 
provided by sex. 
• Lipids: total cholesterol, HDL-C, triglycerides: The by-visit summary will be provided by 
sex. 
• DXA: bone mineral density at each visit (Baseline, week 24 in core period; month 18, 
month 30 in extension period) will be summarized; The summary will be provided by 
location (hip vs. spine vs. femoral neck) and by sex. 


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o For bone mineral density, in addition to the actual value and their change from 
baseline, the percent changes from baseline will also be summarized.  
o For the t-scores and z-scores, only the actual value and their change from baseline 
will be summarized.  Additional categorical (<-2.5, -2.5-<-1, -1-<0, >=0) 
summary will be provided for both t-score and z-score. 
All Sex Hormone, Bone Turnover, lipids, and DXA results will be listed. In the listing for 
Sex Hormone, the number of days of last menstrual cycle preceding the assessment 
(calculated as the assessment date – last menstrual cycle start date + 1) will be provided for 
female subjects. 
10.4.7 Menstrual Cycle Diary 
Menstrual cycle diary will be summarized by regular contraceptive status (oral 
contraceptives or depot injection) for subjects with child bearing potential. Total number of 
menstrual cycle will be summarized. Number and percentage of menstrual cycles that are 
heavier, lighter, longer, shorter, sooner and later than usual will be summarized, where the 
percentage will be calculated based on the total number of reported menstrual cycles.  
Of the women who reported at least one menstrual cycle difference from usual, number and 
percentage of subjects with at least one menstrual that is heavier, lighter, longer, shorter, 
sooner and later than usual will be provided. The summary will be separated for female 
subjects with child bearing potential who ever reported using regular contraceptives (oral 
contraceptives or depot injection) versus those who never reported using regular 
contraceptives.  
A listing of menstrual diary will be provided. 
11 
INTERIM AND DMC ANALYSES 
11.1 
Interim analysis/DRTs 
There were 12 DRT reviews of the ongoing data. Based on the on-going DRT review, the 
study retained the 2 planned arms (300 mg BID and 50 mg BID as initially designed.   
DRT13 will be held based on the final analysis described above.  
11.2 
DMC Analysis 
Not DMC is conducted for this study. 
 


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12 
APPENDICES 


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Appendix A Schedule of Assessments 
Note: All the sections referenced in the footnotes refer to sections in the protocol rather than the current SAP. 
Table 4-1: 
Schedule of Assessments: Core Period 
Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up1 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-42 to -1 
-40 to -12 
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Written Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
PK enzyme assay 
(confirmation of PK 
deficiency)4 
X 
 
 
 
 
 
 
 
 
 
 
 
 
PKR Genotype 
(for randomization) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
UGT1A1 Genotype 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medical/Surgical 
History (General and 
PK deficiency-specific)
5 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medication History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Transfusion History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Confirmation of 
Vaccinations 
(Splenectomized 
Patients) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Examination6/ 
Height6 and Weight 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
X 
X 


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Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up1 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-42 to -1 
-40 to -12 
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Performance Status 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
X 
X 
Vital signs  
(BP, HR, RR, T)7  
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG8 
X 
 
X 
X 
 
X 
 
 
 
 
 
X 
X 
DXA Scan9 
X 
 
 
 
 
 
 
 
 
 
 
X10 
 
Laboratory Evaluations
11 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBsAg, HCV Ab, 
HIV1 and 2 Ab 
X 
 
 
 
 
 
 
 
 
 
 
 
 
RBC antibody 
Screen  
X 
 
 
 
 
 
 
 
 
 
 
 
 
Hematology (CBC)12 
X 
X13 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin14 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
EPO levels15 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
G6PD screen 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Hepcidin 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
X 
Serum Chemistry16 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Iron Panel17 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Carboxyhemoglobin 
(COHb)18 
 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
 
ETCO19 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 


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Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up1 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-42 to -1 
-40 to -12 
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Coagulation Studies
20 
X 
 
X 
 
X 
 
 
 
X 
 
 
X 
X 
Urinalysis21 
X 
 
X 
 
X 
 
 
 
X 
 
 
X 
X 
Serum or Urine 
Pregnancy22 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
Lipids23 
 
 
X 
 
 
 
X 
 
X 
 
 
X 
X 
Hormonal Testing24 
X 
X25 
X 
 
 
 
 
 
X 
 
 
X 
X 
Serum 
osteocalcin-N-mid 
and CTX26 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
25-hydroxy Vitamin 
D2 and D3 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
Randomization27 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Study Drug 
Administration 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X28 
 
Dispense Study Drug29 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
PK blood sampling30 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
PD Assessments30 
 
 
 
 
 
 
 
 
 
 
 
 
 
2,3-DPG/ATP 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 


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Timing 
Pre-Treatment 
Month 1 
Months 2 and 3 
Months 4, 5 and 6 
Follow 
Up1 
Visit 
Screening 
Baseline /  
D1 
W1 
W2 
W3 
W6 
W9 
W12 
W16 
W20 
W24 
W28 
Study Day 
-42 to -1 
-40 to -12 
1 
83 
15 
223 
43 
64 
85 
113 
141 
169 
197 
Visit Window 
 
 
 
± 2 D 
± 2 D 
± 2 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
± 7 D 
Dispense/Collect 
Menstrual Cycle Diary
32 
 
 
X 
 
 
 
X 
 
X 
X 
X 
X 
X 
Adverse Events33 
 
 
Continuous 
X 
Transfusion Record34 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
Medications/Procedures 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Rollover to Extension 
Period35 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Abbreviations:  Ab = antibody; ATP = adenosine triphosphate; BP = blood pressure; CBC= complete blood count; COHb = carboxyhemoglobin; 
CTX = C-terminal telopeptide; D = day; DPG = diphosphoglycerate; DXA = Dual-energy x-ray absorptiometry; ECG = electrocardiogram; 
EPO = erythropoietin; ETCO = end tidal carbon monoxide; FSH = follicle stimulating hormone; G6PD = glucose-6-phosphate-dehydrogenase; HBsAg = 
hepatitis B surface antigen; HCV Ab = hepatitis C virus antibody; HDL-C = high-density lipoprotein-cholesterol; HIV = human immunodeficiency virus; HR 
= heart rate; PD = pharmacodynamic; PK = pharmacokinetic; PK deficiency = pyruvate kinase deficiency; PKR = pyruvate kinase isoform R; RR = resting 
rate; W = week. 
Whenever more than one assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive 
assessment (e.g., VS, ECG, blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time. The order of 
procedures may be revised with prior discussion between Sponsor and site. 
 
1 The Week 28 Follow-up Visit will only be conducted for patients who do not enter the Extension Period. 
2 To be performed at least 2 days after the first Screening Visit. 
3 In-clinic visits on Day 8 and Day 22 may be performed by the patient’s primary care physician if necessary and must be approved by the Sponsor on a case by 
case basis; in these instances PK/PD sampling would not be required and dispensing of study medication would not be performed. For the Day 8 visit 
performed by the patient’s primary care physician, the primary care medical office will collect a blood sample for hematology using the blood sample 
collection and shipping supplies from the kit prepared by the designated central laboratory. The kit will be sent to the primary care physician’s office. No 
other testing or procedures will be asked of the primary care physician on Day 8 [VS and serum chemistry will not be required]. The 12-lead ECG scheduled 
for Day 8 will instead be performed at the main study center on Day 15. For the Day 22 visit performed by the patient’s primary care physician, the primary 
care medical office will collect a blood sample for hematology using the blood sample collection and shipping supplies from the kit prepared by the 
designated central laboratory. The kit will be sent to the primary care physician’s office. No other testing or procedures will be asked of the primary care 


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physician on Day 22. [VS, serum chemistry, coagulation, haptoglobin, EPO level, carboxyhemoglobin, and PK/PD samples will not be required.] The 12-lead 
electrocardiogram scheduled for Day 22 will instead be performed at the main study center on Day 43 (Week 6). For patients having their Day 8 and/or 
Day 22 visits performed by the primary care physician, the Principal Investigator will him or herself, or have a qualified research nurse or other designated 
site staff member, make telephone contact with the patient to inquire about any adverse events. These must be recorded as if the patient appeared in the main 
study center. The Principal Investigator will exercise prudent clinical judgment in determining any clinical course of action to take based on any adverse 
events discovered. These telephone contacts on Day 8 and Day 22 must be explained to the patient in advance and scheduled in advance to maximize the 
likelihood of successfully making contact. 
4 May be performed either by a designated central laboratory or any participating investigative site’s local hematology laboratory. 
5 Medical history, including all relevant prior medical history, current medical conditions, and hematology profile (CBCs) over prior 6 months, will be obtained 
at the Screening assessment. 
6 A complete physical examination (including neurological examination; genital and rectal examinations will be performed at the discretion of the Investigator) 
will be obtained at Screening, Baseline, Week 12, and the Follow up Visit (Week 28), or Week 24 for patients rolling over to the Extension Period. The 
neurological examination must include an assessment of general orientation and mental status including level of alertness (assess as normal or abnormal and 
specify any abnormality). Limited focused physical examinations will be performed at all other specified visits. Height to be collected at Screening only. 
7 Vital signs will include systolic and diastolic blood pressure, heart rate, respiratory rate, and temperature.  
8 12-lead ECGs are to be conducted after 5 minutes of recumbency.  
9 If a DXA scan of the hip and spine with T and Z scores has been performed within 3 months preceding the first day of dosing, this may be used to meet this 
requirement.  
10 Week 24 DXA scan may be performed anytime between Weeks 24 and 28 and must be performed at the same imaging center on the same DXA machine as 
the original Screening DXA scan. 
11 Laboratory evaluations (hematology, serum chemistry, coagulation studies, and urinalysis) are to be collected in the morning. These should be collected 
following an overnight fast on Baseline Day 1 Week 6 (Day 43), Week 12 (Day 85), Week 24 (Day 169), and Follow-up Week 28 (Day 197), when the lipid 
samples are also included. 
12 Three Screening/Baseline samples will be collected for complete blood count (CBC). Samples will be collected in the AM on 3 different days; the samples 
collected on Baseline/Day 1 may comprise one of these; samples may be taken at the same time as Screening/Baseline hormone assessments (any 2 time 
points during Screening at least 2 days apart). CBC will include HCT, Hb, red blood cell (RBC) count, absolute reticulocyte count, percent reticulocyte count, 
mean corpuscular volume (MCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW), white 
blood cell (WBC) count with automated (or manual, as indicated) differential, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and 
platelet count. In the event that the designated central laboratory for the study is unable to provide a valid result for any specific component of the defined 
CBC for a specific patient, the site may be asked to have the test performed at their local laboratory. The result of the local CBC will be entered into the study 
database along with the local normal reference range. 
13 The second Screening hematology (CBC) should be drawn in the morning (does not have to be fasting), and may be drawn at the same time the patient returns 
for the second estradiol and free and total testosterone sample. 
14 Haptoglobin will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 28. 
15 Erythropoietin (EPO) levels will be performed prior to dosing on Day 1, at the end of Week 3, the end of Week 12, the end of Week 24, and the end of Week 
28. 
16 Alkaline phosphatase, sodium, potassium, chloride, calcium, magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, albumin, total protein, glucose, 
blood urea nitrogen (BUN), creatinine, uric acid, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total 
bilirubin and indirect bilirubin, (and estimated creatinine clearance or glomerular filtration rate for screening only, as appropriate). 


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17 Iron, total iron-binding capacity (TIBC), transferrin saturation, and ferritin will be performed prior to dosing on Day 1, at the end of Week 12 and at the end 
of Week 24. 
18 To be collected before the AG-348 morning dose is administered. 
19 End tidal carbon monoxide (ETCO) assessment by CoSense® End Tidal Carbon Monoxide Monitor to be performed only at a subset of investigative sites that 
elect to participate in this assessment and only during the Core Period. The screening ETCO may be performed at either Screening 1 or Screening 2. ETCO 
measurement should performed before administration of AG-348 on Baseline/Day 1 and on W2, W3, W6, W9 W12, W16, W20, W24, and W28 (W28 only if 
not going into Extension Period). 
20 Fibrinogen, activated partial thromboplastin time (aPTT), and international normalized ratio (INR) will be performed at Screening, prior to dosing on Day 1, 
at the end of Week 2, the end of Week 12, the end of Week 24, and the end of Week 28. 
21 Color, appearance, pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite, urobilinogen, and occult blood. Microscopic inspection of sediment 
should only be performed for cause or to investigate an abnormal dipstick finding per the Investigator’s discretion. Urinalysis will be performed at Screening, 
prior to dosing on Day 1, at the end of Week 2, the end of Week 12, the end of Week 24, and the end of Week 28. 
22 Must be repeated at any point throughout the study period if pregnancy is clinically suspected. 
23 Total cholesterol, HDL-C, and triglyceride samples will be collected in the morning following an overnight fast. 
24 Three Screening/Baseline samples will be collected for estradiol and testosterone (total and free). Samples will be collected in the AM on 3 different days; the 
samples collected on Baseline/Day 1 may comprise one of these; the Screening samples may be collected at any 2 time points during Screening at least 2 days 
apart. Serum estrone, estradiol, and free and total testosterone will then follow the schedule indicated on Day 1 and Weeks 12, 24, and 28. FSH will only be 
performed at Screening for female patients only for confirmation of post-menopausal status. 
25 The second Screening hormone testing will consist of estradiol and testosterone (free and total) only. Samples should be drawn in the AM (does not need to 
be fasting), and may be drawn at the same time the patient returns for the second CBC sample. 
26 Serum osteocalcin-N-mid and CTX will be drawn in the AM each time, approximately between 8-10 AM, and after an overnight fast of 10-12 hours. 
27 Randomization will be performed following PKR genotyping and prior to and as close as feasible to dosing on Day 1. 
28 Study drug administration is not required on W24/D169 for patients not continuing into the Extension Period. 
29 Study drug will be dispensed on a 28-day schedule, or on an alternate schedule (< 28 days) as needed to accommodate patient visit schedule and dose 
modifications. The amount of study drug dispensed should be sufficient to provide an adequate reserve supply of AG-348 to ensure uninterrupted dosing in 
the event of an unexpected delay for the next scheduled study visit (7 extra days of dosing supply is recommended during the Core Period). 
30 For the first 10 patients treated, extensive PK/PD sampling will be conducted on Days 1 and 15 (see protocol Appendix 15.1, Table 7 for details), followed by 
limited PK/PD sampling from Week 3 to Week 24 (see protocol Appendix 15.1, Table 8 for details). Limited PK/PD sampling will be conducted on the 
remainder of patients treated (see protocol Appendix 15.1, Table 8). See protocol Section 10.6, Section 10.7, and Section 10.9 for details on blood sampling 
for PK and PD assessments, respectively, and guidelines on sample processing and storage. 
 
 
32 Menstruating female patients will record their menstrual cycles (start, stop, characteristics) monthly. Paper-based menstrual cycle diaries will be dispensed at 
study visits approximately every month. The previous month’s diary will be collected at these visits as well. 
33 All randomized patients will be evaluated for AEs from the time they sign informed consent until they complete all scheduled study follow-up visits or 
withdraw consent, whichever occurs first.  
34 All transfusions must be recorded in the eCRF 
35 Patient must have completed 24 weeks of treatment and tolerated AG-348 (may have had dose modifications). Investigator and Medical Monitor or 
Responsible Medical Officer must agree with the patient continuing on treatment and patient must sign a separate ICF for the Extension Period. 


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Table 4-2: 
Schedule of Assessments: Extension Period 
Visit 
Month 9 
Month 12 
Month 15 
Month 18 
Month 21 
Month 24 
Month 27 
Month 30 
Follow-up 
Approximate Study Day 
259 
349 
439 
529 
619 
709 
799 
889 
919 
Visit Window 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
Physical Examination/Weight1 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Performance Status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs  
(BP, HR, RR, T)2 
X 
X 
X 
X 
X 
X 
X 
X 
X 
12-lead ECG3 
 
X 
 
X 
 
X 
 
X 
X 
DXA Scan 
 
 
 
X 
 
 
 
X 
 
Laboratory Evaluations4 
 
 
 
 
 
 
 
 
 
Hematology (CBC)5 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Haptoglobin 
 
X 
 
X 
 
X 
 
X 
X 
EPO levels6 
 
X 
 
X 
 
X 
 
X 
X 
Hepcidin 
 
X 
 
X 
 
X 
 
X 
 
Serum Chemistry7 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Iron Panel8 
 
X 
 
X 
 
X 
 
X 
 
Carboxyhemoglobin (COHb)
9 
 
X 
 
X 
 
X 
 
X 
 
Coagulation Studies10 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urinalysis11 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum or Urine Pregnancy12 
 
 
 
 
 
 
 
 
 
Lipids13 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Hormonal Testing14 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum osteocalcin-N-mid and 
CTX15 
 
 
 
X 
 
 
 
X 
 


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Visit 
Month 9 
Month 12 
Month 15 
Month 18 
Month 21 
Month 24 
Month 27 
Month 30 
Follow-up 
Approximate Study Day 
259 
349 
439 
529 
619 
709 
799 
889 
919 
Visit Window 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
± 2 W 
Study Drug Administration 
X 
X 
X 
X 
X 
X 
X 
X 
 
Dispense Study Drug16 
X 
X 
X 
X 
X 
X 
X 
 
 
PK blood sampling17 
X 
X 
X 
X 
X 
X 
X 
X 
 
PD Assessments18 
(2,3-DPG/ATP, 
) 
X 
X 
X 
X 
X 
X 
X 
X 
 
Dispense/Collect Menstrual 
Cycle Diary19 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Events20 
Continuous 
X 
Transfusion Record21 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
Medications/Procedures 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Abbreviations:  ATP = adenosine triphosphate; BP = blood pressure; CBC= complete blood count; COHb = carboxyhemoglobin; CTX = C-terminal telopeptide; 
DPG = diphosphoglycerate; DXA = Dual-energy x-ray absorptiometry; ECG = electrocardiogram; EPO = erythropoietin; HDL-C = high-density lipoprotein-
cholesterol; HIV = human immunodeficiency virus; HR = heart rate; PD = pharmacodynamic; PK = pharmacokinetic; PK deficiency = pyruvate kinase 
deficiency; PKR = pyruvate kinase isoform R; RR = resting rate; W = week. 
Whenever more than one assessment is scheduled for the same nominal time, the assessments should be performed in the order of least invasive to most invasive 
assessment (e.g., VS, ECG, blood draw). The timing of these assessments should allow the blood draw to occur at the exact nominal time. The order of 
procedures may be revised with prior discussion between Sponsor and site. 
 
1 A focused physical examination (including neurological examination; genital and rectal examinations will be performed at the discretion of the Investigator) 
will be obtained every 3 months; a complete physical examination will be performed at the Month 30 visit. The neurological examination must include an 
assessment of general orientation and mental status including level of alertness (assess as normal or abnormal and specify any abnormality). 
2 Vital signs will include systolic and diastolic blood pressure, heart rate, respiratory rate, and temperature 
3 12-lead ECGs are to be conducted after 5 minutes of recumbency.  
4 Laboratory evaluations (hematology, serum chemistry, coagulation studies, and urinalysis) are to be collected in the morning. These should be collected 
following an overnight fast. 
5 CBC will include HCT, Hb, red blood cell (RBC) count, absolute reticulocyte count, percent reticulocyte count, mean corpuscular volume (MCV), mean 
corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW), white blood cell (WBC) count with automated (or 
manual, as indicated) differential, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and platelet count. In the event that the designated 
central laboratory for the study is unable to provide a valid result for any specific component of the defined CBC for a specific patient, the site may be asked 


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to have the test performed at their local laboratory. The result of the local CBC will be entered into the study database along with the local normal reference 
range. 
6 Erythropoietin (EPO) levels will be performed prior to dosing at Month 12, Month 18, Month 24, and Month 30. 
7 Alkaline phosphatase, sodium, potassium, chloride, calcium, magnesium, phosphorus, carbon dioxide (CO2) or bicarbonate, albumin, total protein, glucose, 
blood urea nitrogen (BUN), creatinine, uric acid, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total 
bilirubin and indirect bilirubin. 
8 Iron, total iron-binding capacity (TIBC), transferrin saturation, and ferritin will be performed prior to dosing at Month 12, Month 18, Month 24, and 
Month 30. 
9 To be collected before the AG-348 morning dose is administered. 
10 Fibrinogen, activated partial thromboplastin time (aPTT), and international normalized ratio (INR) will be performed at each study visit. 
11 Color, appearance, pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite, urobilinogen, and occult blood. Microscopic inspection of sediment 
should only be performed for cause or to investigate an abnormal dipstick finding per the Investigator’s discretion. Urinalysis will be performed prior to 
dosing at each study visit. 
12 Must be obtained at any point throughout the study period if pregnancy is clinically suspected. Effective contraception must be continued throughout the 
Extension Period in all patients and any pregnancies must be reported (see protocol Section 11.3). 
13 Total cholesterol, HDL-C, and triglyceride samples will be collected in the morning following an overnight fast. 
14 Serum estrone, estradiol, and free and total testosterone.  
15 Serum osteocalcin-N-mid and CTX will be drawn in the AM each time, approximately between 8-10 AM, and after an overnight fast of 10-12 hours. 
16 Study drug will be dispensed on a 3-month schedule, or on an alternate schedule (< 3 months) as needed to accommodate patient visit schedule and dose 
modifications. The amount of study drug dispensed should be sufficient to provide an adequate reserve supply of AG-348 to ensure uninterrupted dosing in 
the event of an unexpected delay for the next scheduled study visit (14 extra days of dosing supply is recommended during the Extension Period). 
17 Predose; PK sampling will only include AG-348 and AGI-8702 concentrations. 
18 Predose. 
19 Menstruating female patients will record their menstrual cycles (start, stop, characteristics) monthly. Paper-based menstrual cycle diaries will be dispensed 
and collected at each study visit. 
20 All randomized patients will be evaluated for AEs from the time they sign informed consent until they complete all scheduled study follow-up visits or 
withdraw consent, whichever occurs first.  
21 All transfusions must be recorded in the eCRF. 
 


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Appendix B Preferred Reporting Units  
Table 12-1 Clinical Laboratory Preferred Reporting Units 
Clinical Laboratory 
Standard Unit 
Original/Preferred Unit 
Haemoglobin 
g/L 
g/dL 
Bilirubin Indirect 
umol/L 
mg/dL 
Bilirubin total 
umol/L 
mg/dL 
Ferritin 
pmol/L 
ng/mL 
Iron 
umol/L 
ug/dL 
Free Testosterone 
pmol/L 
pg/mL 
Total Testosterone 
nmol/L 
pg/mL 
Triglycerides 
mmol/L 
mg/dL 
Cholesterol 
mmol/L 
mg/dL 
HDL-C 
mmol/L 
mg/dL 
 
 


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Appendix C Imputation Rules for Missing Prior/Concomitant Medication Dates 
Imputation rules for missing or partial medication start/stop dates are defined below: 
1. Missing or partial medication start date: 
a. If only DAY is missing, use the first day of the month.  
b. If DAY and Month are both missing, use the first day of the year.  
c. If DAY, Month and Year are all missing, use a date before the first dose date. 
2. Missing or partial medication stop date: 
a. If only DAY is missing, use the last day of the month. 
b. If DAY and Month are both missing, use the last day of the year. 
c. If DAY, Month and year are all missing, assign ‘continuing’ status to stop 
date. 
In summary, the prior, concomitant or post categorization of a medication is described below. 
Table 12-2 Prior, Concomitant, and Post Categorization of a Medication 
Medication Start Date 
Medication Stop Date 
< First Dose Date of 
Study Drug 
≥ First Dose Date 
and ≤ End Date of 
Treatment-Emergent 
Period 
> End Date of 
Treatment-Emergent 
Period 
< First dose date of study drug 
P 
PC 
PCA 
≥ First dose date and  
≤ End date of Treatment-
Emergent period 
- 
C 
CA 
> End date of Treatment-
Emergent period 
- 
- 
A 
A: Post; C: Concomitant; P: Prior 
 
 
 


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Appendix D Handling of Missing Dates in Adverse Events 
For AEs with missing or partial start dates, if there is no clear evidence that the AEs started 
before the corresponding study period as appropriate, then the AEs will be classified as 
TEAEs. 
As an intermediate step for programming purpose, imputation rules for missing or partially 
missing AE start/end dates are defined below. 
If Year of AE start date is missing: 
If year of the AE start date is missing, then compare the full (or partial) AE end date to the 
first dose date. If the AE end date is before the first dose date then the AE should be 
considered as a pretreatment AE. Otherwise, the AE will be considered as TEAE. 
If Year of AE start date is not missing: 
If both day and month of the AE start date are missing: 
• If year of the AE start date = the year of the first dose date, impute the missing day and 
month of the AE start date using the day and month of the first dose date. If this leads to a 
date after the AE end date, then impute the missing day and month of the AE start date 
using the day and month of the AE end date. Note: 
o If the AE ended before first dose date, then the AE will be classified as a pre-
treatment AE. If the AE ended after the first dose date, then there is not enough 
evidence to determine whether the AE is a TEAE or not, and to be conservative, 
the AE will be classified as a TEAE. 
• If year of the AE start date ≠ the year of the first dose date, impute the missing day and 
month of the AE start date using the 1 and Jan. 
o If the year is before the year of the first dose date, then by imputing the missing 
day and month of the AE start date January 1, TEAE status will be pre-treatment.  
o If the year is after the year of the first dose date, then by imputing the missing day 
and month of the AE start date January 1, TEAE status will be TEAE if AE start 
date is on or before the last dose date +28 days; and will be post-treatment AE 
start date is after the last dose date+28 days. 
If only day of the AE start date is missing: 
• If the year of the AE start date = the year of the first dose date = the year of the AE end 
date, and the month of the AE start date = the month of the first dose date = the month of 
the AE end date, then impute the missing day of AE start date using the smaller non-
missing value of (day of the first dose, day of the AE end date). 
• If (the year of the AE start date = the year of the first dose date), and (the month of the 
AE start date = the month of the first dose date), and (the year of the AE start date < the 
year of the AE end date, or the month of the AE start date < the month of the AE end 
date), then impute the missing day of AE start date using the day of the first dose. 


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o If (the year of the AE start date = the year of the first dose date), and (the month of 
the AE start date = the month of the first dose date), and the AE is ongoing, then 
impute the missing day of AE start date using the day of the first dose. 
o Otherwise, impute the missing day of the AE start date as 1. 
Missing or partially missing AE stop date will not be imputed. 
 
 


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Appendix E PKR genotype information and the corresponding mutation 
description and classification 
Due to the evolving understanding on the PKR genotype, the mutations are mostly entered 
into eCRF as free text. These mutations have been reviewed by the team and the following 
mapping of each PKR genotype is mapped to the corresponding mutation description and 
classifications.  
Table 1 PKR genotype information and corresponding mutation description and classification 
Mutation 
Mutation description 
Mutation classification 
1003 G>A 
V335M 
Missense 
1008 A>AA 
insertion (frameshift) 
non-missense 
1010 G>A 
R337Q 
Missense 
1022 G>C 
G341A 
Missense 
1072 G>A 
G358R 
Missense 
1091 G>A 
G364D 
Missense 
1151 C>T 
T384M 
Missense 
1153 A>T 
R385W 
Missense 
1178 A>G 
N393S 
Missense 
1178 A>G, exon4IVS-IV+10G>T 
N393S/splicing 
Non-missense 
1179 T>A 
N393K 
Missense 
1223 C>T 
T408I 
Missense 
1228 A>G 
K410E 
Missense 
1318 G>T 
truncation 
Non-missense 
1373 G>A 
G458D 
Missense 
142_159del 
in-frame deletion 
Non-missense 
1435 C>T 
R479C 
Missense 
1436 G>A 
R479H 
Missense 
1442 C>T 
A481V 
Missense 
1456 C>T 
R486W 
Missense 
1463 G>A 
R488Q 
Missense 
1483 G>A 
A495T 
Missense 
1484 C>T 
A495V 
Missense 
1487 T>G 
V496G 
Missense 
1493 G>A 
R498H 
Missense 
1528 C>T 
truncation (R510stop) 
Non-missense 
1529 G>A 
R510Q 
Missense 
1574 G>A 
truncation 
Non-missense 
1574 G>GG  
(1612 G>GG for site 
)* 
insertion (frameshift) 
Non-missense 
1594 C>T  
(1633 CGG>TGG for site 
)* 
R532W 
Missense 
284-2A>C 
splicing 
Non-missense 


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307delC 
in-frame deletion 
Non-missense 
376-2A>C 
splicing 
Non-missense 
389 C>A 
S130Y 
Missense 
391_393delATC 
in-frame deletion 
Non-missense 
401 T>A 
V134D 
Missense 
494 G>T 
G165V 
Missense 
507+1G>A 
splicing 
Non-missense 
664 G>A 
G222R 
Missense 
695-3C>G 
splicing 
Non-missense 
-70 A>C 
splicing 
Non-missense 
721 G>T  
(760 GAG>TAG or 760 G>T for site 
)* 
truncation 
Non-missense 
721 G>T, 826delG 
truncation/frameshift 
Non-missense 
92 C>T 
(131 GCT>GTT or 131 C>T for site 
)* 
A31V 
Missense 
953_955delAAG 
in-frame deletion 
Non-missense 
exon10del 
deletion 
Non-missense 
exons3-9del 
deletion 
Non-missense 
*Site 
 used a different nomenclature to label the mutations (as indicated and reviewed by 
Translational Science)  
 
 
 
 


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Appendix F MedDRA Terms List for AEs of Endocrinological Interest 
(Menopausal Symptoms) 
Based on AG-348-C-003 List of Terms for analysis of Menopausal Symptoms Version 1.0 
(MedDRA Version 20.0) Dated 28Apr2017. 
 
HLT 
PTs 
Code 
Menstruation and 
uterine bleeding NEC 
Abnormal withdrawal bleeding 
10027335 
Anovulatory cycle 
Bleeding anovulatory 
Delayed menarche 
Dysfunctional uterine bleeding 
Dysmenorrhoea 
Menstrual discomfort 
Menstrual disorder 
Menstruation irregular 
Metrorrhagia 
Premature menarche 
Premenstrual cramps 
Premenstrual dysphoric disorder 
Premenstrual headache 
Premenstrual pain 
Premenstrual syndrome 
Retrograde menstruation 
Withdrawal bleed 
Menstruation with 
decreased bleeding 
Amenorrhoea 
10027341 
Hypomenorrhoea 
Menstruation delayed 
Oligomenorrhoea 
Pituitary amenorrhoea 
Menstruation with 
increased bleeding 
Menometrorrhagia 
10027342 
Menorrhagia 
Polymenorrhagia 
Polymenorrhoea 
Emotional and mood 
disturbances NEC 
Mood swings 
10027951 
Other PTs 
Hot flush 
10060800 


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HLTs are too broad 
Menopausal symptoms 
10027304 
Premature menopause 
10036601 
Acne 
10000496 
Flushing 
10016825 
Erectile dysfunction 
10061461 
Arthralgia  
10003239 
Arthritis 
10003246 
Alopecia 
10001760 
Vulvovaginal dryness 
10047791 
Vulvovaginal discomfort 
10047786 
Dysphoria 
10013954 
Night sweats 
10013954 
Mood altered 
10027940 
Premenstrual dysphoric disorder 
10051537 
Premenstrual syndrome 
10036618