Conditions: Sickle Cell Disease
Keywords: HbS polymerization pyruvate kinase 2,3- diphosphoglycerate ATP in red blood cells acute sickle pain
Allocation: NA | Intervention model: SEQUENTIAL
Enrollment: 17
Healthy volunteers: no

Brief summary:
Background:

Sickle Cell Disease (SCD) is an inherited blood disorder. People with SCD have abnormal hemoglobin in their red blood cells. Researchers are investigating the safety and efficacy of an investigational medicine called AG-348 (mitapivat sulfate) to determine if it will help people with SCD.

Objective:

To test the tolerability and safety of AG-348 in people with SCD.

Eligibility:

People ages 18 and older with SCD.

Design:

Participants will have 8 visits over approximately 14 weeks. At the first visit participants will be screened with a medical history, a physical exam, blood and urine testing, and an EKG. During the following 5 visits, participants will stay at the clinic for 1 night each. Participants will take study drug in increasing doses up to visit 6, after which the drug will be tapered off. All visits will include physical exam, blood, and urine tests. The last visit will occur 4 weeks after stopping the drug. Participants will provide DNA from the blood samples they provide. The DNA will be tested for an inherited gene that can cause differences in response to the study drug. Researchers may also test other genes to see if they can find any genes that interact with SCD.

Detailed description:
Sickle cell disease (SCD) is a multisystem disorder associated with episodes of acute clinical events and progressive organ damage. Episodic pain, triggered by micro-vasoocclusion induced by sickled red blood cells, is the most common acute complication and the leading cause of hospitalization. Management strategies for SCD have evolved very slowly, and treatment of acute pain is still limited to supportive therapy with opioid medication. Until recently in 2017, the only approved therapy for SCD was hydroxyurea (HU), indicated to reduce frequency of acute painful crises but not universally effective. In addition to HU, transfusions with normal red blood cells are widely used to treat severe sickle crises, but this strategy has limitations (not uniformly accessible, accompanied by risks of alloimmunization, hemolytic transfusion reactions and transfusional iron overload). The only curative treatment is bone marrow transplantation, but this option carries significant risks and is limited by the availability of histocompatible donors.

As the root cause of SCD is polymerization of deoxy-HbS, there is a strong rationale for exploring agents that could inhibit/ reduce the polymerization process itself. HbS polymerizes only when deoxygenated, its oxygenation is influenced by a few factors, one key factor being the 2,3- diphosphoglycerate (2,3-DPG) concentration in the red blood cell. 2,3-DPG decreases oxygen binding by preferentially binding to the low oxygen-affinity T conformation of HbS and also stabilizes the T form of hemoglobin S and HbS fiber. In addition, increased 2,3- DPG concentration decreases intracellular red cell pH further promoting HbS polymerization. 2,3-DPG is an intermediate substrate in the glycolytic pathway, the only source of ATP production in red blood cells. Pyruvate kinase (PK) is a key enzyme in the final step of glycolysis; PK converts phosphoenolpyruvate to pyruvate, creating 50% of the total red cell ATP that is essential for maintaining integrity of the red cell membrane. Reduced PK activity leads to accumulation of the upstream enzyme substrates, including 2,3- DPG, that favors polymerization of deoxy-HbS. In humans with SCD, and even in sickle carriers who are generally asymptomatic, reduced oxygen affinity will favor deoxygenation of HbS and its polymerization, and thus sickling. Indeed, the combination of PK deficiency and sickle cell trait causing an acute sickling syndrome has been previously reported in two cases.

Current approaches to reduce HbS polymerization include fetal hemoglobin induction via multiple strategies and drugs that targets HbS polymerization through increasing affinity of hemoglobin for oxygen (eg. Oxbryta (TM) / Voxelotor / GBT440).

Increasing red cell PK (PK-R) activity, leading to a decrease in intracellular 2,3-DPG concentration, presents a potentially attractive therapeutic target for thwarting HbS polymerization and acute sickle pain. AG-348 / mitapivat is a novel, orally bioavailable, small molecule allosteric activator of PK-R, that is currently in Phase II/III clinical trials in humans with PK deficiency (NCT02476916, NCT03548220 / AG348-C-006; NCT03559699 / AG348-C-007). Overview of the preclinical AG-348 data and other data support dose-dependent changes in blood glycolytic intermediates consistent with glycolytic pathway activation at all multiple ascending doses tested, supporting the potential role of AG-348 in the treatment of sickle cell disease. The overall objective of the present study is to determine the clinical safety and tolerability of AG-348 in subjects with SCD.

Arms:
arm 1: Intra-patient dose escalating study, starting with 5 mg twice a day, increasing to 20 mg twice a day, to maximum 50 mg or 100 mg twice a day. Dosing period is every 2 weeks at each dose level. Dose taper will start on Day 42 (50 mg) or Day 56 (100 mg) with dose reduced over 12 to 15 days.

Interventions: intervention 1: AG-348
Intervention descriptions: intervention 1: This is a nonrandomized, intra-patient dose escalating clinical study. AG-348 will be administered starting at 5 mg twice a day, increasing to 20 mg twice a day, to maximum 50 mg or 100 mg twice a day. Dosing period is every 2 weeks at each dose level. Dose taper will start on Day 42 (50 mg) or Day 56 (100 mg) with dose reduced over 12 to 15 days.

Protocol document:
Prot_SAP_001.pdf: 
February 7, 2021
1
CLINICAL RESEARCH PROJECT
Protocol # 19H0097
IND # 142982
IND Holder: NHLBI OCD
Date: April 1, 2021
Title: A Pilot Study to Evaluate the Safety, Tolerability, Pharmacokinetics, and 
Pharmacodynamics of escalating multiple oral doses of AG-348 in subjects with stable sickle 
cell disease
Abbreviated title: A Dose-Finding study of AG-348 in sickle cell disease
Other identifying words: HbS polymerization, pyruvate kinase, 2,3- diphosphoglycerate and 
ATP in red blood cells, acute sickle pain.
Principal and Accountable Investigator:
Swee Lay Thein, M.D., NHLBI (E) 
Bldg 10, CRC 6-6S241A
Subjects:
Number : N=15 (to enroll 20-25, over approximately 1 year, for 15 patients who have completed the full 
study of multiple doses)
Sex: Male & Female Age range: 18-70 years
Project involves ionizing radiation? No (medically and research indicated)
Off-site project?
Yes (for Pharmacokinetics and Pharmacodynamics assays 
of IND only)
Multi-institutional project?
No
DSMB Involved?
Yes
Tech Transfer?
CRADA


February 7, 2021
2
Table of Contents
Contents
CLINICAL RESEARCH PROJECT..............................................................................................................1
PRECIS ..........................................................................................................................................................5
1.0
OBJECTIVES Primary Objective: ..................................................................................................7
2.0 BACKGROUND.....................................................................................................................................7
2.1. Sickle cell disease....................................................................................................................................7
2.2
Epidemiology...................................................................................................................................7
2.3
Clinical Manifestations....................................................................................................................8
2.4
Economic Impact of SCD................................................................................................................9
2.5
Pathophysiology of Sickle Cell Disease..........................................................................................9
2.6
Available treatments for SCD........................................................................................................10
2.7
Current Experimental Nongenetic Drug Therapies .......................................................................11
2.8
Investigational Agent AG348 and pyruvate kinase deficiency .....................................................12
2.9
Clinical and Scientific Justification...............................................................................................13
2.9.1
Further Scientific Rationale...........................................................................................................14
3.0 STUDY DESIGN...................................................................................................................................19
4.0
SUBJECT RECRUITMENT AND REGISTRATION .................................................................21
5.0
ELIGIBILITY ASSESSMENT .....................................................................................................22
5.1
Inclusion Criteria ...........................................................................................................................22
5.2
Exclusion Criteria ..........................................................................................................................23
6.0
SAMPLE COLLECTION, STORAGE, AND TRACKING PLAN .............................................27
6.1
Clinical Evaluation of the Subject .................................................................................................27
6.4
Research Sample Collection..........................................................................................................33
6.4.1 Pharmacokinetics:..........................................................................................................................33
6.4.2 Pharmacodynamics:.......................................................................................................................34
7.0
DRUG ADMINISTRATION – AG-348 .......................................................................................36
7.1
Drug Accountability and Compliance: ..........................................................................................36
8.0
DATA AND BIOSPECIMEN MANAGEMENT PLAN..............................................................37
8.1
Data Collection:.............................................................................................................................37
8.2
End of Study Procedures: ..............................................................................................................37
8.3
Data Sharing: .................................................................................................................................37
8.4
Future use of biospecimens: ..........................................................................................................38


February 7, 2021
3
8.4
Loss or Destruction of Data:..........................................................................................................38
8.5
Publication Policy:.........................................................................................................................38
9.0
BIOSTATISTICAL CONSIDERATIONS ...................................................................................39
9.1
Primary Objective..........................................................................................................................39
9.2
Endpoints .......................................................................................................................................39
9.2.1 Primary Endpoint...........................................................................................................................39
9.2.2 Secondary Endpoints .....................................................................................................................39
9.2.3 Exploratory Endpoint.....................................................................................................................40
9.3
Sample Size ...................................................................................................................................40
9.4
Methods of statistical analysis:......................................................................................................40
9.5
Criteria for Study Termination ......................................................................................................41
9.6
Stopping Rules:..............................................................................................................................41
10.0
OFF-STUDY CRITERIA..............................................................................................................43
11.0
DATA SAFETY AND MONITORING PLAN ............................................................................44
11.1
Safety Monitoring..........................................................................................................................44
12.0
ADVERSE EVENT REPORTING ...............................................................................................45
12.1
Adverse Events ..............................................................................................................................45
12.1.2 Adverse event management:..........................................................................................................45
Exclusions to data reporting:........................................................................................................................46
12.1.3.
Dose modifications due to Adverse Events............................................................................46
12.1.4 Dose Taper Regimens....................................................................................................................47
12.1.5 Adverse Events of Special Interest................................................................................................49
12.2
Grading of Adverse Events............................................................................................................50
12.3
Attribution of Adverse Events.......................................................................................................51
13.0
NIH INTRAMURAL IRB AND NHLBI CD REPORTING ........................................................51
13.2
Reports to the IRB at the time of Continuing Review:..................................................................51
13.3
Reports to the CD: .........................................................................................................................52
14.0
IND SPONSOR REPORTING CRITERIA ..................................................................................52
14.1
Reporting Pregnancy......................................................................................................................52
15.0
HUMAN SUBJECT PROTECTION.............................................................................................53
15.1
Rationale for Subject Selection......................................................................................................53
15.2
Informed Consent Processes and Procedures ................................................................................53
15.3
Patient Advocate............................................................................................................................54
15.4
Privacy and Confidentiality ...........................................................................................................54


February 7, 2021
4
15.5
Risks and Discomforts...................................................................................................................54
15.5.2 Related to blood draws & IV.........................................................................................................57
15.5.3 Related to cardiac monitoring (during screening) .........................................................................57
15.6
Conflict of Interest.........................................................................................................................57
16.0 COMPENSATION & REIMBURSEMENT .......................................................................................57
Financial Compensation...............................................................................................................................57
17.0
PHARMACEUTICALS – AG-348 ...............................................................................................58
18.0 Reference..............................................................................................................................................62


February 7, 2021
5
PRECIS
Sickle cell disease (SCD) is a multisystem disorder associated with episodes of acute clinical 
events and progressive organ damage. Episodic pain, triggered by micro-vasoocclusion induced 
by ‘sickled’ red blood cells, is the most common acute complication and the leading cause of 
hospitalization. Management strategies for SCD have evolved very slowly, and treatment of 
acute pain is still limited to supportive therapy with opioid medication. Until recently in 2017, 
the only approved therapy for SCD was hydroxyurea (HU), indicated to reduce frequency of 
acute painful crises but not universally effective. In addition to HU, transfusions with normal red 
blood cells are widely used to treat severe sickle crises, but this strategy has limitations (not 
uniformly accessible, accompanied by risks of alloimmunization, hemolytic transfusion reactions 
and transfusional iron overload). The only curative treatment is bone marrow transplantation, but 
this option carries significant risks and is limited by the availability of histocompatible donors.
As the root cause of SCD is polymerization of deoxy-HbS, there is a strong rationale for 
exploring agents that could inhibit/reduce the polymerization process itself. HbS polymerizes 
only when deoxygenated, its oxygenation is influenced by a few factors, one key factor being the 
2,3- diphosphoglycerate (2,3-DPG) concentration in the red blood cell. Increased intracellular 
2,3-DPG decreases oxygen binding and stabilizes the deoxygenated form (T form) of 
hemoglobin. In addition, increased 2,3-DPG concentration decreases intracellular red cell pH 
further promoting HbS polymerization. 2,3-DPG is an intermediate substrate in the glycolytic 
pathway, the only source of ATP production in red blood cells. Pyruvate kinase (PK) is a key 
enzyme in the final step of glycolysis; PK converts phosphoenolpyruvate to pyruvate, creating 
50% of the total red cell ATP that is essential for maintaining integrity of the red cell membrane. 
Reduced PK activity leads to accumulation of the upstream enzyme substrates, including 2,3- 
DPG, that favours polymerization of deoxy-HbS. In humans with SCD, and even in sickle 
carriers who are generally asymptomatic, reduced oxygen affinity will favour deoxygenation of 
HbS and its polymerisation, and thus sickling. Indeed, the combination of PK deficiency and 
sickle cell trait causing an acute sickling syndrome has been previously reported in two cases.
Current approaches to reduce HbS polymerization include fetal haemoglobin induction via 
multiple strategies and drugs that targets HbS polymerization through increasing affinity of 
hemoglobin for oxygen (eg. Oxbryta™ / Voxelotor / GBT440). Increasing red cell PK (PK-R)


February 7, 2021
6
activity, leading to a decrease in intracellular 2,3-DPG concentration, presents a potentially 
attractive therapeutic target for thwarting HbS polymerization and acute sickle pain. AG-348 is a 
novel, orally bioavailable, small molecule allosteric activator of PK-R, that is currently in Phase 
II/III clinical trials in humans with PK deficiency (NCT02476916, NCT03548220 / AG348-C- 
006; NCT03559699 / AG348-C-007). Overview of the preclinical AG-348 data and other data 
support dose-dependent changes in blood glycolytic intermediates consistent with glycolytic 
pathway activation at all multiple ascending doses tested, supporting the potential role of AG- 
348 in the treatment of sickle cell disease. The overall objective of the present study is to 
determine the clinical safety and tolerability of AG-348 in subjects with SCD.


February 7, 2021
7
1.0 OBJECTIVES 
Primary Objective:
1.1
To assess the clinical safety and tolerability of multiple escalating doses of AG-348, 
an allosteric activator of the enzyme pyruvate kinase, in subjects with stable sickle 
cell disease (SCD).
Secondary Objectives:
1.2
To assess the pharmacokinetics of AG-348 in stable SCD subjects after multiple 
escalating oral doses.
1.3
To understand the mechanisms of action of AG-348 on the glycolytic pathway in 
sickle cell disease through laboratory studies. Specific pharmacodynamics 
objectives include: the levels of 2,3-DPG, PK-R and ATP, and oxygen dissociation 
sickling in red blood cells from stable SCD subjects treated ex-vivo as well as in- 
vivo.
1.4
To evaluate the relationship between AG-348 pharmacokinetics and safety 
parameters.
2.0 BACKGROUND
2.1. Sickle cell disease
Sickle cell disease (SCD) is an inherited hemoglobin disorder caused by the presence of 
hemoglobin S (HbS) which results from the substitution of valine for glutamic acid in position 6 
of the β chain of hemoglobin (βGlu6Val), a consequence of a specific single base substitution in 
the -globin gene (HBB c.20A>T; rs334). SCD is an autosomal recessive disorder, individuals 
with only one copy of the S allele are carriers of the condition (HbAS). This sickle cell ‘trait’ is a 
benign condition and generally does not cause any clinical disability. The genetic causes of SCD 
include homozygosity for the rs334 mutation (HbSS) (generally known as sickle cell anemia; 
SCA) and compound heterozygosity between rs334 and mutations that lead to other structural 
variants of β-globin (such as HbC), or to reduced levels of β-globin production (β-thalassemia). In 
patients of African ancestry, HbSS is the majority cause of SCD (65-70%), followed by HbSC 
(about 30%), with HbS/β-thalassemia being responsible for the majority of the rest.
2.2 Epidemiology
The HbS mutation is a prototype of a balanced polymorphism. Carriers for the βS gene (HbAS) are 
protected from falciparum malaria and have a survival advantage over normal (HbAA) individuals
1. This is, however, offset by the increased fatalities in HbSS individuals. Although the sickling
disorders occur predominantly in individuals of African descent, the disease is also prevalent 
throughout the Mediterranean, Middle East and parts of India, the Caribbean, and South and


February 7, 2021
8
Central America, where falciparum malaria was endemic. Within these regions the HbS gene 
frequency ranges from 10–30%. Due to the recent global population migration, however, SCD has 
become an important part of clinical practice in many regions, that are well beyond its early 
geographic regions, including the Caribbean islands, Brazil, the United States, the United 
Kingdom, France and many parts of other European countries 2.
Around the world, it is estimated that more than 300,000 children are born with sickle cell anemia 
each year 3. In the US, SCD affects an estimated 100,000 Americans (vast majority African- 
Americans and the rest of Hispanic descent), and about one in 365 African-American newborns 4.
2.3 Clinical Manifestations
Individuals with SCD have chronic hemolytic anemia interrupted by acute and recurrent clinical 
events, that vary greatly in frequency and severity, the most common being acute pain, frequently 
referred to as acute vaso-occlusive crises (VOC).
Symptoms do not develop in babies until after 4–6 months of age when adult type hemoglobin 
becomes the predominant hemoglobin. Until then, neonates are protected from the effects of SCD 
due to the presence of residual fetal hemoglobin (HbF). Hand-foot syndrome, or dactylitis, is often 
one of the first indications of SCD, presenting as painful swelling of the bones of the hand or foot
5. At this stage, the infant is usually anemic with mild jaundice and has a palpably enlarged spleen.
Splenomegaly usually resolves due to repeated infarctions of the spleen and it is unusual to be able 
to feel the spleen after the first decade of life, although functional hyposplenism may occur in as 
many as 90% of children with SCA between the ages of 6 months and 3 years. Typically, these 
children have a hemoglobin level that varies between 6 and 8 gm/dL and a reticulocyte count of 
10 to 20%.
The clinical course of SCD is notoriously variable. Progressive damage to a range of organs is a 
constant feature of the disease. The chronic hemolysis of SCD is punctuated by acute exacerbations 
of the illness, the most common manifestations are the vaso-occlusive crises characterized by acute 
painful episodes due to blockage of small vessels triggered by the sickled erythrocytes and tissue 
infarction. Microvascular occlusion occurs in different organs including: spleen, as acute splenic 
sequestration  commonly in the first two years of life; lungs as “acute chest syndrome” (ACS). 
ACS presents with a clinical picture (including cough, shortness of breath and signs of 
consolidation and hypoxemia) that is difficult to distinguish from acute pneumonia. Vaso- 
occlusion affecting the CNS manifests either as fits, transient neurological symptoms resembling 
ischemic attacks, or a fully developed stroke. Vaso-occlusion of the outflow vessels from the 
corpora cavernosa by sickled erythrocytes causes priapism, an unwanted and painful erection of 
the penis that lasts for >2 hours.
Repeated vaso-occlusive events and the chronic uncompensated hemolytic anemia ultimately 
result in end organ damage and almost any organ can be affected. Virtually every patient with SCD 
has some form of renal impairment, with progressive inability to concentrate urine, polyuria, 
nocturia and enuresis, which is common in children. Eventually the glomerular damage causes


February 7, 2021
9
chronic renal failure, particularly in patients over 40 years of age. Other common clinical 
conditions include gallstones, recurrent chronic leg ulceration (more common in those patients 
with severe anemia) and proliferative retinopathy leading to progressive visual loss, which is more 
common in patients with HbSC disease. The vertebral bodies, femoral and humeral heads are 
particularly prone to infarction and avascular necrosis of the femoral head may lead to total 
disability, frequently requiring a total hip prosthesis.
Acute anemia, usually defined as a decline in hemoglobin of >2g/dL from steady state levels 6, is
a common feature of painful crises but can also result from sequestration of erythrocytes in deep 
vascular beds, most commonly within the spleen (acute splenic sequestration) or liver (acute 
hepatic sequestration), temporary marrow aplasia secondary to Parvovirus B19 infection 7, or from 
increased hemolysis secondary to serious acute infections or delayed transfusion reactions. 
Infection is the major cause of death in children, with hyposplenism increasing the susceptibility 
to encapsulated bacteria, in particular Steptococcus pneumonia. Causes of death in adults are more 
variable and include infection, acute chest syndrome, liver failure, stroke and heart failure.
2.4 Economic Impact of SCD
Less than 50 years ago in 1960, SCD was regarded as a “disease of childhood” 8 and few 
children survived beyond their teens, while 25 years later, the Cooperative Study of Sickle Cell 
Disease (CSSCD) reported a median age at death of 42 years for males and 48 years for females 
with HbSS and that 85% HbSS patients will survive to adulthood 9. More recent studies 
confirmed that the majority of newborns (94 to 99%) in well-resourced countries will now 
survive to adulthood 10-12 but early mortality remains. Survival of patients with SCD in well- 
resourced and medium-resourced settings, has improved greatly in the last 60 years, and 
continues to improve 13-15. Nonetheless, the life expectancy of patients with SCD is still 
shortened by 20 to 30 years compared to the general population 4,16,17. Patients with SCD also 
have a poor quality of life beset with varying degrees of multi-system organ damage and chronic 
pain, further impacted by intermittent unpredictable episodes of acute pain. In a two-year study 
of 1,189 SCD patients in Illinois, 8,403 hospital admissions were reported. From these 
hospitalizations, over 97% of admitting diagnoses were for acute pain crises, of which 86% 
required emergency services as the primary source of admission 18. A study of 4,294 patients 
who were enrolled in Florida Medicaid program demonstrated that a lifetime cost of care in 
average was $460,151 per patient with SCD 19. The majority (80.5%) of SCD-related costs were 
associated with inpatient hospital care. Few options are available for treating acute pain crises, 
which are largely managed symptomatically with fluids and analgesics.
2.5 Pathophysiology of Sickle Cell Disease
The root cause of SCD is polymerization of deoxy-HbS. Low oxygen conditions predispose deoxy- 
HbS to polymerize which causes the red blood cells to become distorted and bizarrely shaped,


February 7, 2021
10
some adopting a “sickled” shape. Sickled red blood cells are rigid and adhesive, and together with 
white blood cells and platelets, cause blockages in the fine blood vessels (microvascular occlusion) 
leading to ischemia and depletion of oxygen to tissues. The body experiences this as an intense 
pain episode (known as acute painful crisis), acute organ damage, stroke or even acute multi-organ 
failure, depending upon the site and severity of the vascular blockage. Sickled RBCs have a 
shortened lifespan of 16 days compared to the average of 120 for normal RBCs, leaving patients 
with SCD in a constant state of uncompensated hemolytic anemia. The chronic anemia and 
recurrent vaso-occlusive events result in multiple organ co-morbidities and reduced life 
expectancy.
2.6 Available treatments for SCD
Management strategies for SCD have evolved very slowly, and treatment of SCD remains a serious 
unmet medical need. Until July 2017, when Endari (L-glutamine oral powder) was licensed by the 
US Food and Drug Administration (FDA), hydroxyurea (HU) was the only disease-modifying drug 
ever to be licensed for the treatment of SCA by the FDA. HU is the first of two FDA-approved anti-
sickling agents. HU is indicated to reduce the frequency of painful crises but its use is limited by 
its side-effect profile, including neutropenia and thrombocytopenia 20,21. The clinical effectiveness 
of HU is largely due to induction of HbF, but it is not universally effective. HbF is not evenly 
distributed unlike the rare natural genetic variants (Hereditary Persistence of Fetal Hemoglobin, 
HPGH) in which the HbF increase is evenly distributed among the red blood cells . Voxelotor is 
the second anti-sickling agent that was approved by the FDA in November 2019 for the treatment 
of SCD in adults and pediatric patients 12 years of age and older. Voxelotor inhibits sickling by 
preferentially binding to the high-oxygen affinity, non-polymerizing R conformation of 
hemoglobin, thereby reducing the concentration of the polymerizing T conformation 23. Other 
treatment options include transfusions with normal blood to alleviate symptomatic anemia, treat 
severe sickle crises (eg, acute chest syndrome), and treat cerebrovascular stroke 24. Blood 
transfusions, however, have substantial limitations: the treatments are expensive, not uniformly 
accessible, and are accompanied by risks including alloimmunization, hemolysis, and transfusional 
iron overload. Another option is to reduce/prevent frequency of VOC in those patients with 
recurrent acute pain, an indication for Adakveo® (crizanlizumab / anti-P selectin) that was 
approved by FDA in November 2019 25. The only curative treatment is bone marrow 
transplantation from a histocompatible donor 26,27, but bone marrow transplantation carries 
significant risks and is limited by the availability of histocompatible donors 28.
As curative options are limited to a small subgroup, and current treatment options are not 
universally effective, there is a need for novel therapies that target the other mechanisms of sickling
29. HU has multiple mechanisms of action that affect various points on the pathophysiological
pathway of SCD but the main mechanism of action of HU is the therapeutic induction of fetal 
hemoglobin (HbF) which has an inhibitory effect on HbS polymerization. However, the inter- 
individual response to HbF induction by HU is highly variable. HU-induced HbF increase is


February 7, 2021
11
unevenly distributed among the RBCs (F-cells), resulting in limited protection from sickling unlike 
the pancellular HbF increase seen in patients with sickle hereditary persistence of fetal hemoglobin 
(S/HPFH) 30. Few other therapies developed for the treatment of SCD seek to target the underlying 
mechanism of the disease, i.e., preventing the polymerization of HbS and sickling of red blood 
cells (RBCs) that cause the clinical complications of SCD (Bunn 1997).
2.7 Current Experimental Nongenetic Drug Therapies
The last 2 decades have witnessed a surge in the development of drugs that target HbS 
polymerization 23,29, and those that prevent or reverse vaso-occlusion 25,31. The best established 
strategy for mitigating HbS polymerization is the induction of fetal hemoglobin (HbF) synthesis, 
and both gene therapy and pharmacological approaches are actively being investigated 32,33. 
Induction of HbF is a major component of the efficacy of HU therapy but the variability in HbF 
increase and its uneven distribution among the RBCs results in limited protection from sickling 22. 
In recent years, advances in unravelling the molecular mechanisms controlling globin gene 
expression has led to alternative approaches to HbF induction that fall into 2 groups - those that 
affect chromatin regulators (such decitabine on DNA methylation and histone deacetylase 
(HDAC) inhibitors) and, the others that affect DNA-binding transcription factors 34-36. Another 
HbF-inducing agent under investigation is Metformin, a prescription drug currently in use for type 
II diabetes mellitus (NCT02981329).
A second ant-sickling agent that was approved by the FDA in November 2019 for the treatment of 
SCD is voxelotor. Voxelotor is an allosteric hemoglobin modifier that binds to the N-terminal 
valine residue of the -globin chain of HbS and increases its oxygen affinity. To date, there is no 
evidence that it reduces frequency of pain crises, organ damage and survival, although the increase 
in hemoglobin is dose-dependent and accompanied by decreased markers of hemolysis, suggestive 
of reduced sickling 23. There is also some concern that Hb molecules with the drug bound are in a 
conformation that delivers very little oxygen, especially detrimental in a disease characterized by 
decreased oxygen delivery 37. Hopefully, these concerns are addressed in current multicenter phase 
3 clinical studies in both adults (http://www.clinicaltrials.gov/, NCT03036813) and children 
(http://www.clinicaltrials.gov/, NCT02850406).
Most of the nongenetic approaches currently being assessed in clinical trials are aimed at using 
drugs to ameliorate the downstream sequelae of sickle cell hemoglobin (HbS) polymerization, such 
as adhesion of red cells to vascular endothelium, leukocytes, and platelets, as well as inflammation, 
coagulation, and nitric oxide scavenging 31. From the perspective of preventing acute painful 
crises, crizanlizumab, a humanized antibody to P selectin holds much promise. In a double-blind, 
randomized, placebo-controlled phase II trial, adult participants (on or off HU), Crizanlizumab 
showed a significant reduction in sickle related pain crises and low incidence of adverse events 38. 
Crizanlizumab was approved by the FDA in November 2019for reducing VOCs in SCD. A phase 
2 trial has shown efficacy of GMI-1070 (rivipansel), a pan-selectin inhibitor, that seems to shorten 
the duration of acute sickle pain crises accompanied by a marked reduction in


February 7, 2021
12
opioid
use
39.
Rivipansel
is
currently
undergoing
phase
3
investigation 
(http://www.clinicaltrials.gov/, #NCT01119833).
2.8 Investigational Agent AG348 and pyruvate kinase deficiency
Investigational drug AG-348 (formerly known as AGI-1480 and AGX-0841) is a potent, broad- 
spectrum activator of alleles of the red blood cell (RBC)-specific form of pyruvate kinase (PKR). 
PKR is 1 of 4 pyruvate kinase isoenzymes expressed in human tissues from 2 distinct genes. PKR 
and liver-type pyruvate kinase (PKL) are generated from the PKLR gene by 2 separate tissue- 
specific promoters (https://www.ncbi.nlm.nih.gov/gene/5315), while PKM1 and PKM2 are from 
the 
PKM 
gene 
via 
differential 
splicing 
of 
the 
ribonucleic 
acid 
(https://www.ncbi.nlm.nih.gov/gene/5315). PKLR is located on chromosome 1q21. The difference 
between the red cell (PKR) and liver (PKL) isoforms is the 32 amino acids at the N- terminus 
generating a different promoter; red cell and liver PK are identical except for exon 1 40. AG-348 is 
an allosteric activator of PKR, PKL, and PKM2, with similar potency against each.
Pyruvate Kinase deficiency
Hereditary RBC enzymopathies are genetic disorders arising from mutations in genes encoding 
red cell enzymes 41,42. These red cell enzymopathies cause a specific type of anemia termed 
hereditary/congenital nonspherocytic hemolytic anemia (HNSHA) 42. Glycolysis is the only source 
of ATP production in red blood cells 43 (Fig. 1). Enzyme deficiencies in the glycolytic pathway 
(including pyruvate kinase) leads to the accumulation of upstream enzyme substrates, including 
2,3-DPG (Fig. 1). PK converts phosphoenolpyruvate (PEP) to pyruvate, creating 50% of the total 
red cell ATP. Red cell longevity is dependent on ATP produced during glycolysis. PK deficiency 
leads to less intracellular ATP, and thereby shortening the red cell lifespan 44.
Mutations in PKLR gene cause PK deficiency in RBCs and hemolysis. PK deficiency (PKD) is 
rare (estimated at 1:20,000 in the general white population 45 with various prevalence estimates of 
0.24%, 1.1%, <0.1%, 1.9%, and 3.2 % reported in populations of Spain, Turkey, Asia, Hong Kong 
(Chinese), southern Iran, and Saudi Arabia, respectively 46-50. Over 250 PKLR mutations have been 
described, causing a range of quantitative deficiencies and dysfunctional PK enzymes 51. The 
RBCs from patients with PK deficiency are characterized by changes in metabolism associated 
with defective glycolysis, including a deficiency in adenosine triphosphate (ATP) levels. Levels 
of 2,3-diphosphoglycerate (2,3-DPG), phosphoenolpyruvate (PEP), and other glycolytic 
intermediates upstream of the PKR reaction are reported to be elevated in patients with PK 
deficiency 42, reflecting the constriction of glycolysis at the PKR step. The range of PKLR 
mutations lead to a range of functional PK deficiencies, and diverse clinical manifestations of 
PKD, ranging from mild or fully compensated forms to life-threatening neonatal anemia, all with 
the usual hallmark of chronic hemolysis 44,52,53.
Drug intervention with AG-348 has been previously shown to restore glycolytic pathway activity 
and normalize RBC metabolism in PK deficient patients with minimal accompanying side effects. 
Two completed clinical Phase 1 studies (randomized, placebo-controlled, double-blind) assessed


February 7, 2021
13
the safety, tolerability and pharmacokinetics/pharmacodynamics of AG-348 in single ascending 
dose (SAD; http//:www.Clinical Trials.gov: NCT02108106), and multiple ascending dose (MAD; 
http//:www.Clinical Trials.gov: NCT02149966) and the results were recently published 54. Dose- 
dependent changes in blood glycolytic intermediates consistent with glycolytic pathway activation 
were observed at all multiple ascending doses, supporting the potential role of AG348 in the 
treatment of sickle cell disease. The most common treatment-related AEs in AG-348-treated 
subjects were headache and nausea.
AG-348 is currently in Phase 2/3 clinical trials in subjects with PK deficiency.
Figure 1. The Emden-Meyerhof pathway (Glycolysis). G6P, glucose-6-phosphate; F6P, fructose- 
6-phophate; F1,6P, fructose 1,6-phosphate; DHAP, dihydroxyacetone phosphate; G3P, glucose- 
3-phosphate; 1,3-DPG, 1,3-diphosphoglycerate; 2,3-DPG, 2,3-diphosphoglycerate; 3-PG, 3- 
phosphoglycerate; 2-PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate (picture courtesy:52).
2.9 Clinical and Scientific Justification
Attempts to target downstream sequelae of the sickling process have not proved to be effective in


February 7, 2021
14
the reducing vaso-occlusive crisis frequency 55-63. More effort should be directed toward preventing 
or mitigating the HbS polymerization process itself, the root cause of sickle cell pathology.
Reduce concentration of 2,3-diphosphoglycerate
Polymerization of HbS and its oxygenation are influenced by a few factors, one key factor is 2,3- 
diphosphoglycerate (2,3-DPG) concentration in RBCs 29,64. 2,3-DPG is an allosteric effector on 
hemoglobin oxygen binding. It has a greater binding affinity to deoxygenated hemoglobin (the T 
state) than to oxygenated hemoglobin (the R state). 2,3-DPG thus stabilizes the quaternary T form 
of deoxy-hemoglobin and decreases oxygen binding. Thus, lowering 2,3-DPG intracellular 
concentration would increase the fraction of HbS in the non-polymerizing R quaternary structure
65.  In  addition,  decreasing  2,3-DPG  concentration  increases  HbS  solubility  because  of  an
accompanying increase in intracellular pH. Studies have shown that 2,3-DPG levels are 
significantly elevated in sickle cell patients and that 2,3-DPG promotes HbS polymerization 66,67. 
Further, adding 2,3-DPG lowers oxygen affinity of HbS 66. Increased 2,3-DPG concentration and 
decreased intracellular red cell pH have both been shown to boost deoxy-HbS polymerization 68,69. 
While increased 2,3-DPG concentration and reduction of hemoglobin oxygen affinity is beneficial 
in anemia caused by PK deficiency 70,71, increased 2,3-DPG levels and decreased intracellular red 
cell pH are detrimental in the presence of HbS, as they boost deoxy-HbS polymerisation. In SCD, 
and even in sickle trait (where intracellular HbS concentration is 35-40%), the reduced oxygen 
affinity will favor deoxy-HbS polymerisation - and thus sickling 72.
2.9.1 Further Scientific Rationale
Further scientific rationale for proposing the study stems from the PI’s two clinical 
observations:
1. Two case reports (including one from the PI’s group) exist of sickle carriers, typically a totally 
benign condition, but with typical sickle cell phenotype 73,74. Each of the proband had co-inherited 
mutations in the PKLR gene in addition to being a carrier for sickle cell. One report supported the 
role of 2,3-DPG in in-vitro functional assays. Unfortunately, in the second case reported by the PI, 
we could not obtain fresh blood for oxygen dissociation and other studies as patient had left the 
country.
2. Genetic association studies led by the PI in a sickle cohort in King’s College London 
(where PI was located prior to relocation to the NHLBI) showed significant association 
between 4 genetic variants in the PKLR gene and hospitalization rate, a marker of sickle cell 
vaso-occlusive pain crises (Kate Gardner, PhD thesis King’s College London; manuscript in 
preparation). The 4 PKLR variants are fairly common among individuals of African descent. 
We confirmed an allele frequency of 14% of these PKLR intronic variants in the NHLBI 
sickle cell cohort, and we have preliminary evidence from work here at the NHLBI that they 
are associated with reduced PK-R messenger activity. We are currently investigating the 
impact of these PKLR variants on PK, 2,3-DPG and ATP levels.
Because 2,3-DPG plays such a critical role in potentiating HbS polymerization, there is a


February 7, 2021
15
compelling rationale for the development of drugs that target the enzymatic pathway responsible 
for its remarkably high (5 mM) concentration in red blood cells. Apart from reducing 2,3-DPG 
levels, activation of PKR will also concomitantly generate more ATP, a key molecule for 
maintaining RBC membrane integrity.
Prompted by these clinical observations, we have performed studies on ex-vivo incubation 
of sickle RBCs with AG-348, and the preliminary data show clear left shifts in the oxygen 
dissociation curve for red cells from two sickle trait patients (so there is no interference 
resulting from fiber formation) and inhibition of sickling for cells from a patient with 
homozygous SS disease at concentrations of 10 micromolar (see figure 2). While 
encouraging, we would caution how we interpret the result from ex-vivo experimentation 
with respect to implications for dosing in humans, especially how it relates to the effect on 
downstream PD markers.


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Fig. 2a. Oxygen dissociation curves in 20 mM phosphate-buffered saline, pH 7.4, 37 C for 
two sickle trait patients. Cells are incubated for 4 or 24 hrs in 0, 10, 30, and 100 M AG- 
348.
Fig. 2b. Fraction sickled vs time following deoxygenation with 95% nitrogen, 5% oxygen for 
homozygous SS patient after 2 hr or 19 hr incubation with 0 (control), 10, 30, and 100 M AG- 
348 in 20 mM phosphate-buffered saline, pH 7.4, 37 C. Images of cells in wells of 384 well 
plate collected every 15 minutes in Biotek Lionheart microscope system.
We have also started recruiting for a prospective protocol in October 2018 on “Genotype - 
Phenotype Correlation of PKLR Variants with Pyruvate Kinase, 2,3-diphosphoglycerate 
and Adenosine Triphosphate Activities in Red Blood Cells of Patients with Sickle Cell


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Disease”, and as of December 2019, more than 300 subjects with AA, AS and SS have been 
enrolled and sampled. This study will inform any potential variability observed in the response 
of SCD patients to AG-348.
Justification for AG348
AG-348 is a novel, first-in-class, orally bioavailable, small molecule allosteric activator of the 
PKR, PKL, and PKM2 isoenzymes, with similar activity for each. AG-348 acts by directly binding 
to the PKR tetramer and allosterically enhancing its affinity for PEP. Deficiency in PK activity 
leads to defective glycolysis, including a buildup of PEP and the intermediate 2,3-DPG, and 
lowered levels of ATP, changes that impair metabolism of RBCs and their progenitors. The effect 
of AG-348 on PKR activity and a number of downstream pathway markers was evaluated in both 
human and murine RBCs and whole blood. In blood samples from PKR deficient patients, ex vivo 
treatment with AG-348 increased PKR activity (by 1·3- to 3·4-fold) and induced metabolic 
changes, including increased levels of ATP (by 1·3- to 2·4-fold), consistent with increased 
glycolytic pathway activity 75. Mice with wild-type PK-R treated with AG-348 had increased PKR 
activity levels, as well as increased ATP and decreased 2,3-DPG levels consistent with in vivo 
activation of PK-R 75. Moreover, in the Clinical Phase 1 study of healthy adult subjects, the 
concentration of 2,3-DPG decreased after a single dose of AG-348 in a dose-dependent manner to 
a minimum at 24 hours post dose and then returned to baseline values by 72 to 120 hours post 
dose. AG-348 was well tolerated at doses ranging from 15 to 360 mg administered q12h over 14 
days, and pharmacodynamic changes in blood consistent with increased activity of the glycolytic 
pathway were observed at these levels. Collectively, these observations lead to the hypothesis that 
activation of PKR through AG-348 may lead to decreased 2,3-DPG that could have anti-HbS 
polymerizing effect. The concomitantly increased ATP generation may also help improve red cell 
survival. Of particular relevance to SCD, we noted that AG-348 showed potent activation (defined 
as >2-fold activation) not only of mutant isoforms of PKR, but also wild type PKR. We therefore 
propose this dose-escalating study of AG348 in patients with SCD in a clinical trial designed to 
evaluate its safety (maximum tolerated dose) and tolerability.
In the SAD and MAD studies, the concentration of 2,3-DPG decreased in a dose-dependent
manner and returned to levels close to baseline by 72 hours following the final dose of AG-
348. (see figure 3 below)


February 7, 2021
18
Figure 3, panel D in: Yang H et al. Clinical Pharmacology in Drug Development 2018, 00(0) 1– 
14.
Patients with SCD have a WT PKR enzyme as found in healthy individuals. Therefore, 
pharmacokinetic/PD results from the MAD study of AG-348 conducted in healthy adult subjects 
(NCT02149966, referenced in the protocol on page 13) have been used to select doses for the 
study planned to be conducted in patients with SCD. Briefly, a simple Emax (maximum effect) 
model was used to describe the pharmacokinetic/PD correlation between plasma AG-348 AUC0- 
12 and blood AUC_Net_B0-12 for 2,3-DPG using results from the MAD study. The estimated 
plasma AG-348 AUC0-τ that resulted in 50% maximum inhibitory effect (IAUC50) on 2,3-DPG 
over the dosing interval at steady-state was estimated to be 750 ng•h/mL. This corresponded to 
the AG-348 plasma total exposure (AUC0-τ) achieved following multiple doses of AG-348 
below or near 15 mg q12h in the MAD study. Based on the mean observed values of 2,3-DPG 
blood AUC_Net_B0-12 and the predicted value of the maximum inhibitory effect of AG-348 on 
2,3-DPG blood AUC_Net_B0-12 of 3,381 μg•h/mL, approximately 74% of the maximum 
inhibitory effect was achieved at a dose of 60 mg q12h in the MAD study and over 99% of the 
maximum inhibitory effect was achieved at a dose of 360 mg q12h in the MAD study.
In a phase 2 study (DRIVE PK), subjects with PKD were randomized to receive either 50 or 300 
mg of AG-348 twice daily. Preliminary results of the DRIVE-PK study showed that several


February 7, 2021
19
subjects had to have their randomized dose level reduced because of excess increases in Hb or 
the occurrence of adverse events (AEs) such as insomnia, headache, or nausea) 54. Dose 
reductions led to the resolutions of most of these AEs and the maintenance of satisfactory 
efficacy where applicable. Therefore, in the ongoing phase 3 studies being conducted in subjects 
with PKD, individual dose optimization is incorporated to allow each subject to gradually 
increase his/her dose of AG-348 in order to identify a dose that confers maximum benefit with 
minimum risk to that subject. In the phase 3 PKD studies, all subjects will start at a low dose 
level (5 mg BID) with 2 sequential steps for dose level increases (ie, from 5 to 20 mg BID and 
from 20 to 50 mg BID), depending on safety and tolerability. In a phase 2 Agios sponsored study 
being conducted in patients with thalassemia, doses of 50 mg and 100 mg BID are being 
evaluated. Given there is no prior experience of administering AG-348 in patients with SCD, the 
starting dose in this study was selected to be 5 mg BID (similar to the starting dose in phase 3 
studies being conducted in patients with PKD), with the potential for intra-patient dose 
escalations to 20, 50, and 100 mg BID. In the AG-348-C-002 study, at 120 mg BID, there’s
~89% inhibitory effect on 2,3-DPG compared to 700 mg BID; ~85% inhibitory effect on 2.3-
DPG compared to 360 mg BID. Maximal Inhibition of 2,3-DPG was observed at about 360 mg 
BID. Studies of AG-348 at doses of 300mg (DRIVE-PK for PKD) suggest that 100 mg BID 
would have a comparable safety and tolerability profile to doses in the range of 5-50mg BID. 
The higher doses of 50 and 100 mg BID of AG-348 in the proposed study in adult subjects with 
SCD are expected to lower 2,3 DPG sufficiently to be able to study its effects on sickling, while 
concomitantly generating more ATP, a key molecule for maintaining RBC membrane integrity.
3.0 STUDY DESIGN
This is a non-randomized, dose-escalating clinical study designed to assess the safety and 
tolerability of escalating doses of AG348 in subjects with sickle cell disease (SCD).
Fifteen subjects with SCD (HbSS) will complete multiple dose levels of AG-348. Up to ten 
subjects will complete 3 dose levels, starting with 5 mg of AG-348 BID by mouth, escalating to 
20 mg BID, and then 50 mg BID, with a dosing period of 2 weeks at each dose level. Starting with 
amendment #5, at least 5 subjects will complete 4 dose levels, escalating from 50mg BID to
100 mg BID for 2 additional weeks to allow for assessment of the safety, tolerability, 
pharmacokinetics (PK), and pharmacodynamics (PD) of the 100 mg BID dose in subjects with 
SCD. If fewer than 10 subjects have successfully completed the 3 dose levels at the time of 
approval of amendment #5, then all remaining subjects will be enrolled onto the 4-dose level 
escalation scheme detailed in the amended protocol. For any subjects undergoing dose escalation 
at the time of approval of amendment #5 (i.e. prior to initiating the drug taper), the study team will 
discuss the higher 100 mg BID dose level with the subject, and if interested, the subject will be 
reconsented to complete the 4 dose levels. If 10 subjects who are on track to complete the 3 dose 
levels are enrolled prior to the approval of amendment #5, then enrollment will be held until the 
amendment is approved to allow for at least 5 subjects to complete all 4 dose levels.


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For subjects escalating to a maximum dose of 50 mg BID, subjects will be assessed for safety and 
tolerability every 2 weeks; in the absence of serious adverse effects, subjects will receive the 
escalating dose from 5 mg BID to 20 mg BID to 50 mg BID. Dose taper will start on Day 42 from 
their last dosing regimen of 50 mg twice daily using a rapid taper schedule over 12 days (Table 
7A). For subjects escalating to a maximum dose of 100 mg BID, subjects will also be assessed for 
safety and tolerability every 2 weeks; in the absence of serious adverse effects, subjects will receive 
the escalating dose from 5 mg BID to 20 mg BID to 50 mg BID to 100 mg BID. Dose taper will 
start on Day 56 with subjects reducing their last dosing regimen of 100 mg twice daily to stopping 
all together using a rapid taper schedule over 15 days (Table 7B). 
Subjects will be instructed to closely monitor for any signs or symptoms differing from baseline 
and to contact the study team immediately if they experience any change in their symptoms or 
overall health. If there is no change in their health, subjects will not need to come to the NIH for a 
provider visit or lab work. However, if a subject reports any symptoms concerning acute pain, the 
subject may be transitioned to a gradual dose taper per Investigator’s discretion and potentially be 
asked to come to the NIH for a clinical evaluation with a provider and lab work prior to the end of 
the taper. The rapid and gradual dose taper are described in section 12.1.4.
At any time, the investigator can suspend or alter dosing for reasons related to safety or tolerability. 
All patients may be admitted overnight for up to 2 days for observation and for completion of all 
blood collection time points when they commence the study, for each of the escalating dose of 20 
mg BID, 50 mg BID, and 100 mg BID, and at the beginning of dose taper.
Subjects should be advised not to discontinue dosing without first speaking with the treating 
Investigator except in case of medical emergency; abrupt discontinuation of AG-348 may result in 
withdrawal hemolysis. If a subject needs to discontinue study drug at any time during the study, 
guidance is provided in Section 10.0 Off Study Criteria.
All study visits will be conducted at the NIH Clinical Center. Study visits conducted electronically 
will not be routinely used and will only be conducted under extreme circumstances for existing 
subjects. In these scenarios, only approved electronic technology and telehealth platforms will be 
used to communicate with the subject and will allow study providers to obtain a medical history, 
perform a limited physical exam, and assess for adverse events. The initial consent and screening 
procedures for new subjects should take place at the NIH. All telehealth visits will be documented 
in the source documents similar to a routine in-person study visit. 
Even in the situation in which telehealth visits are used, subjects should have their laboratory 
studies drawn at the NIH if at all possible. If subjects are unable to present themselves to the NIH, 
the study team will directly order or will work with the subject’s local provider to order necessary 
clinical laboratory tests and cover the cost of all study-related laboratory testing. If laboratory tests 
are obtained outside of the NIH, then only clinical laboratory tests for safety will be ordered, and 
research labs will be omitted.


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  Figure 4. Dosing schemes.
Abbreviations: BID = twice daily; n/a = not applicable; QD = once daily; AM = daily in the 
morning; PM = daily at night.
A) Dosing scheme for subjects escalating to maximum study drug dose of 50 mg BID. Blood 
samples drawn for measurement of PK-R, 2,3-DPG and ATP levels, and Oxygen Sickling Assay 
at Screening, Day 1, Day 14 ( 3 days), Day 28 ( 3 days), Day 42 ( 3 days), Stop date (Day 54
 3 days) and 4 weeks ( 3 days) post-last dose, in addition to full blood counts, reticulocyte count, 
lactate dehydrogenase, renal and liver profile.
B) Dosing scheme for subjects escalating to maximum study drug dose of 100 mg BID. Blood 
samples drawn for measurement of PK-R, 2,3-DPG and ATP levels, and Oxygen Sickling Assay 
at Screening, Day 1, Day 14 ( 3 days), Day 28 ( 3 days), Day 42 ( 3 days), Day 56 ( 3 days), 
Stop date (Day 71  3 days) and 4 weeks ( 3 days) post-last dose, in addition to full blood counts, 
reticulocyte count, lactate dehydrogenase, renal and liver profile.
4.0 SUBJECT RECRUITMENT AND REGISTRATION
Subjects with steady state sickle cell disease will be recruited from the sickle cell clinic at the NIH CC. We 
will also recruit subjects through NIH using traditional recruitment methods such as referrals from other 
protocols, outside physician referral and self-referral.


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The study may also use the following strategies of recruitment including:

ClinicalTrials.gov website

Clinical Center Research Studies (“Search the Studies”) website

National Heart, Lung and Blood Institute (NHLBI) patient recruitment website

Disease advocacy organizations such as the Sickle Cell Disease Association of America and Sickle 
Cell Anemia Research Fund website

Twitter messages and chats with study investigators

Facebook Posts

Google AdWords

Use of Clinical Center Office of Patient Recruitment services including creation and 
distribution of study flyers and information through pre-existing recruitment avenues such 
as the NIH recruitment listserv.
The DIR Patient Recruitment Office (PRO) will work with study investigators to ensure accrual 
goals are being met. All recruitment materials and tools will use IRB-approved language and 
information to include standard recruitment contacts.
5.0 ELIGIBILITY ASSESSMENT
All patients with sickle cell anemia (HbSS) will be considered for enrollment.
5.1
Inclusion Criteria
For enrollment, subjects must meet all of the following criteria during the screening period:
5.1.1
Have provided signed written informed consent prior to performing any study 
procedure, including screening procedures.
5.1.2
Age between 18-70 years
5.1.3
Unequivocal diagnosis of HbSS confirmed by hemoglobin electrophoresis 
performed on patients at least 90 days after a blood transfusion if previously 
transfused, or DNA genotyping 
5.1.4
No transfusion in the 90 days prior to the first dose of study drug, or absence of HbA on 
hemoglobin analysis (by high-performance liquid chromatography; HPLC)
5.1.5
Have adequate organ function, as defined by:
a.
Serum aspartate aminotransferase (AST) ≤2.5 × Upper Limit of Normal 
(ULN) (unless the increased AST is assessed by the Investigator as due to 
hemolysis and/or hepatic iron deposition) and alanine aminotransferase (ALT)
≤2.5 × ULN (unless the increased ALT is assessed by the Investigator as due 
to hepatic iron deposition).


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b. Serum creatinine ≤1.25 × ULN. If serum creatinine is >1.25 × ULN, then 
glomerular filtration rate (based on creatinine) must be ≥60 mL/min.
c.
Absolute neutrophil count ≥1.0 × 109/L.
d. Hemoglobin ≥ 7 g/dL
e.
Platelet count ≥100 × 109/L.
f.
Activated partial thromboplastin time and international normalized ratio ≤1.5
× ULN, unless the subject is receiving therapeutic anticoagulants.
5.1.6
For women of reproductive potential, have a negative serum or urine pregnancy 
test during the screening period. Women of reproductive potential are defined as 
sexually mature women who have not undergone a hysterectomy, bilateral 
oophorectomy, or tubal occlusion; or who have not been naturally 
postmenopausal (i.e., who have not menstruated at all for at least the preceding 12 
months prior to signing informed consent and have an elevated follicle- 
stimulating hormone level indicative of menopause during the screening period).
5.1.7
For women of reproductive potential as well as men and their partners who are 
women of reproductive potential, be abstinent as part of their usual lifestyle, or 
agree to use 2 effective forms of contraception from the time of giving informed 
consent, during the study, and for 28 days for women and 90 days for men 
following the last dose of study treatment. An effective form of contraception is 
defined as hormonal oral contraceptives, injectables, patches, and barrier 
methods.
5.1.8
Be willing to comply with all study procedures for the duration of the study.
5.2
Exclusion Criteria
Subjects who meet any of the following criteria during screening will not receive AG348 
and will not be counted toward the final enrollment count for statistical purposes:
5.2.1
Documented pyruvate kinase deficiency
5.2.2
Have a significant medical condition that confers an unacceptable risk to 
participating in the study, and/or that could confound the interpretation of the 
study data. Such significant medical conditions include, but are not limited to the 
following:
a.
Poorly controlled hypertension (defined as systolic blood pressure [BP]
>150 mmHg or diastolic BP >90 mmHg) refractory to medical 
management.
b. History of recent (within 6 months prior to signing informed consent) 
congestive heart failure; myocardial infarction or unstable angina pectoris; 
hemorrhagic, embolic, or thrombotic stroke; deep venous thrombosis; or 
pulmonary or arterial embolism.


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c.
Cardiac dysrhythmias judged as clinically significant by the Investigator.
d. Heart-rate corrected QT interval-Fredericia's method (QTcF) >480 
msec with the exception of subjects with right or left bundle branch 
block.


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e.
Clinically symptomatic cholelithiasis or cholecystitis. Prior 
cholecystectomy is not exclusionary. Subjects with symptomatic 
cholelithiasis or cholecystitis may be rescreened once the disorder has 
been treated and clinical symptoms have resolved.
f.
History of drug-induced cholestatic hepatitis.
g. Iron overload sufficiently severe to result in a clinical diagnosis by the 
Investigator of cardiac (e.g., clinically significant impaired left ventricular 
ejection fraction), hepatic (e.g., fibrosis, cirrhosis), or pancreatic (e.g., 
diabetes) dysfunction.
h. Have a diagnosis of any other congenital or acquired blood disorder, or 
any other hemolytic process as defined by a positive direct antiglobulin 
test (DAT), except mild allo-immunization as a consequence of 
transfusion therapy.
i.
Positive test for hepatitis B surface antigen or hepatitis C virus (HCV) 
antibody (Ab) with signs of active hepatitis B or C virus infection. If the 
subject is positive for HCV Ab, a reverse transcriptase-polymerase 
chain reaction test will be conducted. Subjects with hepatitis C may be 
rescreened after receiving appropriate hepatitis C treatment.
j.
Positive test for human immunodeficiency virus 1 or 2 Ab.
k. Active infection requiring any use of systemic antimicrobial agents 
(parenteral or oral) or Grade ≥3 in severity (per National Cancer Institute 
Common Terminology Criteria for Adverse Events) within 2 months prior 
to signing informed consent.
l.
Diabetes mellitus judged to be under poor control by the Investigator or 
requiring >3 antidiabetic agents, including insulin (all insulins are 
considered 1 agent); use of insulin per se is not exclusionary.
m. History of any primary malignancy, with the exception of: curatively 
treated nonmelanomatous skin cancer; curatively treated cervical or breast 
carcinoma in situ; or other primary tumor treated with curative intent, no 
known active disease present, and no treatment administered during the 
last 3 years.


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26
n. Unstable extramedullary hematopoiesis that could pose a risk of imminent 
neurologic compromise.
o. Current or recent history of psychiatric disorder that, in the opinion of the 
Investigator or Medical Monitor, could compromise the ability of the 
subject to cooperate with study visits and procedures.
p. Are currently enrolled in another therapeutic clinical trial involving 
ongoing therapy with any investigational or marketed product or placebo. 
Sickle cell anemia subjects on hydroxyurea or L-glutamine will also be 
considered, provided that they have been on an unchanged dose of 
hydroxyurea or L-Glutamine for three months prior to enrollment.
q. Have exposure to any investigational drug, device, or invasive procedure 
within 3 months prior to the first dose of study treatment. All non-
investigational invasive procedures within 3 months of starting study 
treatment may be considered as a potential exclusion criteria per the PI’s 
discretion.
r.
Have had any prior treatment with a pyruvate kinase activator.
s.
Have received crizanlizumab or voxelotor in the 12 weeks prior to signing consent.
t.
Have a prior bone marrow or stem cell transplant.
u. Are currently pregnant or breastfeeding.
v. Are currently receiving medications that are strong inhibitors of cytochrome P450 
(CYP)3A4 or strong inducers of CYP3A4 that have not been stopped for a duration 
of at least 5 days or a timeframe equivalent to 5 half-lives (whichever is longer) 
prior to the first dose of AG-348.
w. Are currently receiving hematopoietic stimulating agents (e.g., 
erythropoietins, granulocyte colony stimulating factors, thrombopoietins) 
that have not been stopped for a duration of at least 28 days prior to the 
first dose of study treatment.
x. Have a history of allergy to sulfonamides if characterized by acute 
hemolytic anemia, drug-induced liver injury, anaphylaxis, rash of 
erythema multiforme type or Stevens-Johnson syndrome, cholestatic 
hepatitis, or other serious clinical manifestations.


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y. Have a history of allergy to AG-348 or its excipients (microcrystalline 
cellulose, croscarmellose sodium, sodium stearyl fumarate, and mannitol).
6.0 SAMPLE COLLECTION, STORAGE, AND TRACKING PLAN
Collection Practices:
6.1
Clinical Evaluation of the Subject
Pre-Study Evaluation- Screening:
Testing for screening evaluation will be conducted under another NHLBI screening protocol. 
Alternatively, if a test result is available from any other NHLBI protocol within 90 days of 
enrollment, that test result can also be used for screening purposes
a.
Informed Consent
b.
Inclusion/Exclusion Criteria evaluation
c.
Prior Concomitant medications
d.
History and physical exam including prior transfusion history and 
review of sleep history.
e.
Vital sign measurements including weight
f.
Oximetry
g.
Electrocardiogram (EKG)
h.
CBC with differential
i.
Cooximeter (venous) if hemoglobin value is not able to be obtained with a 
CBC
j.
Reticulocyte count
k.
Acute care panel (Sodium (NA), Potassium (K), Chloride (CL) Total 
CO2 (Bicarbonate), Creatinine, Glucose, Urea nitrogen, , Anion Gap), 
Mineral panel (Albumin, Calcium, Magnesium (Mg), Phosphorus), 
Hepatic panel (Alkaline Phosphatase, ALT/GPT, AST/GOT, Total 
Bilirubin, Direct Bilirubin)
l.
Iron Panel and ferritin
m. Coagulation screen (PT/INR, PTT, fibrinogen)
n.
DAT (Direct Antiglobulin Screen)
o.
Pregnancy test (serum or urine) for female subjects
p.
Total protein, creatinine kinase, uric acid, lactate dehydrogenase
q.
eGFR
r.
Urinalysis
s.
Hemoglobin electrophoresis
t.
Serum HBsAg, anti-HBc, anti-HBs, anti-HCV
u.
anti-HIV
v.
Calculated creatinine clearance


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Baseline values:
The most recent results of laboratory/procedure testing performed within 90 days of 
administration of the first dose of study drug will be considered subject’s baseline laboratory 
values.
Table 1A: Schedule of Events for Subjects Escalating to Maximum AG-348 Dose of 50 mg 
BID
VISIT 1
VISIT 2
VISIT 3
VISIT 4
VISIT 5
VISIT 6
VISIT 7
Day 1± 3 days 
(inpatient)
Day 14± 3
days 
(inpatient)
Day 28± 3
days 
(inpatient)
Day 42± 3
days 
(inpatient)
Day 54±
3 days
Follow- 
up± 3 days
Procedure
Consent
5mg BID
20mg BID
50mg BID
DoseTaper
Stop
4 week 
follow-up
Informed consent
X
Inclusion/exclusion criteria
X
History and Physical with
Transfusion history and 
sleep history
X
X
X
X
X
X
X
Vital sign measurements 
including weight
X
X
X
X
X
X
Oximetry
X
X
X
X
X
X
Electrocardiogram (EKG)
X
CBC with diff
X
X
X
X
X
X
*Cooximeter panel, venous
*X
*X
*X
*X
*X
*X 
Reticulocyte count
X
X
X
X
X
X
Acute Care, Mineral, 
Hepatic panels,
X
X
X
X
X
X
Fasting lipid panel
X
X
Coagulation screen 
(PT/INR, PTT, fibrinogen)
X
Urine or serum pregnancy 
test (if applicable)
X
X
X
X
X
X
Total Protein, Creatinine 
Kinase, Uric Acid, Lactate
X
X
X
X
X
X
eGFR
X
X
X
X
X
X
Urinalysis
X
Hemoglobin electrophoresis
X
X
X
X
X
X
Testosterone, Total and free
X
X
Estradiol, Serum
X
X
Estrone, serum
X
X
Research labs & 
pharmacokinetics of AG-
348


February 7, 2021
29
Pharmacokinetic blood
samples
X
X
X
X
X
X
X
Determination of PK-R
protein
X
X
X
X
X
X
X
ATP/2,3-DPG concentration
samples
X
X
X
X
X
X
X
Shift in p50 of hemoglobin 
from venous blood specimen
X
X
X
X
X
X
X
Percentage of sickled RBCs
X
X
X
X
X
X
X
Drug administration
X
X
X
X
Drug accountability and pill
count
X
X
X
X
Study diary review
X
X
X
X
X
Adverse event monitoring
X
X
X
X
X
X
X
      *If clinical hemoglobin value cannot be obtained with a CBC, a hemoglobin value by  venous cooximeter panel will be used instead 
for clinical monitoring. 
Table 1B: Schedule of Events for Subjects Escalating to Maximum AG-348 Dose of 100 mg 
BID
VISIT 1
VISIT 2
VISIT 3
VISIT 4
VISIT 5
VISIT 6
VISIT 7
VISIT 8
Day 1
± 3 days 
(inpatient)
Day 14
± 3 days 
(inpatient)
Day 28
± 3 days 
(inpatient)
Day 42
± 3 days 
(inpatient)
Day 56
± 3 days 
(inpatient)
Day 71
± 3 days
Follow-up
± 3 days
Procedure
Consent
5mg BID
20mg BID
50mg BID
100mg BID
DoseTaper
Stop
4 week 
follow-up
Informed consent
X
Inclusion/exclusion criteria
X
History and Physical with 
Transfusion history and sleep
history
X
X
X
X
X
X
X
X
Vital sign measurements 
including weight
X
X
X
X
X
X
X
Oximetry
X
X
X
X
X
X
X
Electrocardiogram (EKG)
X
CBC with diff
X
X
X
X
X
X
X
*Cooximeter panel, venous
*X
*X
*X
*X
*X
*X 
*X 
Reticulocyte count
X
X
X
X
X
X
X
Acute Care, Mineral, Hepatic 
panels,
X
X
X
X
X
X
X
Fasting lipid panel
X
X
Coagulation screen (PT/INR, 
PTT, fibrinogen)
X
Urine or serum pregnancy test 
(if applicable)
X
X
X
X
X
X
X
Total Protein, Creatinine 
Kinase, Uric Acid, Lactate
X
X
X
X
X
X
X
eGFR
X
X
X
X
X
X
X
Urinalysis
X
Hemoglobin electrophoresis
X
X
X
X
X
X
X
Testosterone, Total and free
X
X


February 7, 2021
30
Estradiol, Serum
X
X
Estrone, serum
X
X
Research labs & 
pharmacokinetics of AG-348
Pharmacokinetic blood
samples
X
X
X
X
X
X
X
X
Determination of PK-R protein
X
X
X
X
X
X
X
X
ATP/2,3-DPG concentration
samples
X
X
X
X
X
X
X
X
Shift in p50 of hemoglobin 
from venous blood specimen
X
X
X
X
X
X
X
X
Percentage of sickled RBCs
X
X
X
X
X
X
X
X
Drug administration
X
X
X
X
X
Drug accountability and pill
count
X
X
X
X
X
Study diary review
X
X
X
X
X
X
Adverse event monitoring
X
X
X
X
X
X
X
X
   *If clinical hemoglobin value cannot be obtained with a CBC, a hemoglobin value by cooximetry panel will be used instead for clinical 
monitoring. 
6.2
Monitoring During AG-348 Administration for Safety and Tolerability 
Time Points: See Schedule of Events Tables 1A and 1B.
a. History and physical exam, including sleep history.
b. CBC with differential
c. Cooximeter panel (venous) labs if hemoglobin value is not able to be obtained with a CBC
d. Acute care panel (Sodium (NA), Potassium (K), Chloride (CL) Total CO2 
(Bicarbonate), Creatinine, Glucose, Urea nitrogen, eGFR, Anion Gap), Mineral 
panel (Albumin, Calcium, Magnesium (Mg), Phosphorus), Hepatic panel (Alkaline 
Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin), total protein, 
creatinine kinase, uric acid, lactate dehydrogenase
e. Calculated creatinine clearance
f. Fasting lipid panel, baseline and last visit only
g. Serum hormone levels (estradiol, estrone, and total and free testosterone), last 
visit only
h. Hemoglobin electrophoresis
i. Vital signs including weight
j. Reticulocyte Count on each patient visit
k. Urine or serum pregnancy (if applicable)
l. As clinically indicated anytime during the study


February 7, 2021
31
6.3
Research Sample Collection
6.3.1
Pharmacokinetics:


February 7, 2021
32

For subjects escalating to maximum AG-348 dose of 50 mg BID, blood samples 
will be collected at the following time points to measure plasma concentrations of 
AG-348:
o Visit 1
o Day 1, pre-dose, and 1, 2, 4, and 8 hours post-dose
o Day 14, ± 3 days: pre-dose, and 1, 2, 4, and 8 hours post-dose
o Day 28, ± 3 days: pre-dose, and 1, 2, 4, and 8 hours post-dose
o Day 42, ± 3 days: pre-dose, and 1, 2, 4, and 8 hours post-dose
o Day 54 , ± 3 days: After last dose of drug
o 4 weeks, ± 3 days post-last dose of AG-348

For subjects escalating to maximum AG-348 dose of 100 mg BID, blood samples 
will be collected at the following time points to measure plasma concentrations of 
AG-348:
o Visit 1
o Day 1, pre-dose, and 1, 2, 4, and 8 hours post-dose
o Day 14, ± 3 days: pre-dose, and 1, 2, 4, and 8 hours post-dose
o Day 28, ± 3 days: pre-dose, and 1, 2, 4, and 8 hours post-dose
o Day 42, ± 3 days: pre-dose, and 1, 2, 4, and 8  hours post-dose
o Day 56, ± 3 days: pre-dose, and 1, 2, 4, and 8  hours post-dose
o Day 71, ± 3 days: After last dose of drug
o 4 weeks, ± 3 days post-last dose of AG-348
6.3.2
Pharmacodynamics:

For subjects escalating to maximum AG-348 dose of 50 mg BID, blood samples 
will be collected for pharmacodynamics assessments at the following time points:
o
Visit 1
o
Day 1: pre-dose, and 1, 2, 4, and 8 hours post-dose
o
Day 14, ± 3 days: pre-dose, and 1, 2, 4, and 8 hours post-dose
o
Day 28, ± 3 days: pre-dose, and 1, 2, 4, and 8 hours post-dose
o
Day 42, ± 3 days: pre-dose, and 1, 2, 4, and 8 hours post-dose
o
Day 54, ± 3 days: After last dose of drug
o
4 weeks ± 3 days post-last dose of AG-348

For subjects escalating to maximum AG-348 dose of 100 mg BID, blood samples 
will be collected for pharmacodynamics assessments at the following time points:
o
Visit 1
o
Day 1: pre-dose, and 1, 2, 4, and 8 hours post-dose
o
Day 14, ± 3 days: pre-dose, and 1, 2, 4, and 8 hours post-dose
o
Day 28, ± 3 days: pre-dose, and 1, 2, 4, and 8 hours post-dose
o
Day 42, ± 3 days: pre-dose, and 1, 2, 4, and 8 hours post-dose
o
Day 56, ± 3 days: pre-dose, and 1, 2, 4, and 8 hours post-dose
o
Day 71, ± 3 days: After last dose of drug
o 4 weeks ± 3 days post-last dose of AG-348

The following research assessments will be performed on collected samples:
after signing consent, visit 1, 2, 3, 4, 5, 6, and 7 (and visit 8 only for subjects 
escalating to 100 mg BID AG-348 dose)


February 7, 2021
33
-
Determination of PK-R protein levels and concentrations of 2,3-DPG, and ATP 
in whole blood
-
Shift in p50 of hemoglobin from venous blood specimen (assessment of the 12- 
hour time point is optional for p50)
-
Percentage of sickled RBCs

Under normal ex vivo conditions

Under hypoxic ex vivo conditions
-
Investigation of the effects of AG348 on oxidation and proteomic changes in 
both RBC and RBC membrane
-
Glycated hemoglobin S fraction
6.4 Sample Storage: Research samples will be stored with subject identifiers in the secure 
laboratory of Dr. Swee Lay Thein using the NHLBI’s Biospecimen Inventory (BSI) tracking 
system in accordance with NHLBI DIR Biospecimen policy. The PI will be responsible for 
overseeing entry of data into an in-house password protected electronic system. Only those 
authorized can retrieve samples.
6.5 Intended Use: These specimens will not be read by a pathologist or used for diagnostic 
purposes. These studies will not be used in assessing the primary endpoint but are 
undertaken for descriptive or exploratory ancillary research.
6.6.1. Collaborative Efforts of Research
At the FDA laboratories, the team led by Dr. Abdu Alayash will focus on proteomic and 
metabolic analyses of blood taken from SCD subjects after treatment wth AG- 348.
Immunological and other biological assays will determine the effects of AG-348 treatment on 
oxygen reactive species (ROS), Adenosine Triphosphate (ATP) and 2,3-DPG.
6.6 How Specimens/Data will be Tracked: Samples collected at the NIH will be ordered and 
tracked through CRIS Research Screens. Should a CRIS screen not be available, the NIH 
form 2803-1 will be completed and will accompany the specimen and be filed in the medical 
record. All research samples collected at the NIH under this protocol will be coded with a 
specific patient number. Research Samples will be logged in on the NHLBI BSI tracking 
system with the sample code, date drawn, and location of storage.
6.7 End of Study Procedures: The study will remain open so long as sample or data analysis 
continues. Samples from consenting subjects will be stored until they are no longer of 
scientific value or if a subject withdraws consent for their continued use, at which time they 
will be destroyed.


February 7, 2021
34
6.8 Loss or Destruction of Samples: Should we become aware that a major breech in our plan 
for tracking and storage of samples has occurred, the IRB will be notified.
7.0 DRUG ADMINISTRATION – AG-348
On Day 1, two additional conditions must be met before a subject, considered eligible after 
completing screening, can be dosed:
1. The subject must not have received a transfusion since completing Screening 
(see Section 5.1, Inclusion Criterion).
2. The subject, if a female of reproductive potential (see Section 5.1.6, Inclusion Criterion) 
must have a negative urine or serum pregnancy test.
The first dose of study drug on Day 1 should be taken at the study site following all Day 1 
assessments.
Over the course of the study, subjects will attend visits on Day 1, Visit 2, Visit 3, Visit 4, Visit 5, 
Visit 6, and Visit 7 (and Visit 8 only for subjects escalating to 100 mg BID AG-348 dose). See 
Schedule and Events Tables 1A and 1B for laboratory assessments. Blood samples will be 
analyzed by Agios-approved laboratories for determination of PK-R protein levels and 
concentrations of ATP and 2,3-DPG in whole blood.
All subjects will receive an initial dose of 5 mg BID of AG-348 for 2 weeks followed by 2 dose 
increases, from 5 to 20 mg BID and from 20 to 50 mg BID, depending on safety and tolerability. 
At least 5 subjects will receive an additional dose increase from 50 mg BID to 100 mg BID; 
these will consist of subjects who are either undergoing dose escalation at the time of approval of 
amendment #5 and reconsented to escalate to the 100 mg BID dose or newly enrolled after the 
approval of amendment #5. Specifically, the treating clinician will assess the safety and 
tolerability of the current dose level before a decision is taken to escalate the dose to the next 
level.
7.1 Drug Accountability and Compliance:
Subjects will receive education on how to take the study medication. If they are hospitalized, for 
example for a pain crisis, they should take the bottle of study drug with them. Subjects will be 
given a diary, along with their take-home bottle of AG-348, to record when they take their study 
drug. The diary can also be used to record missed doses and any symptoms the subject 
experiences till the end of the study. Subjects will be instructed to bring back their study drug 
and diary at each visit.


February 7, 2021
35
When the study staff receives returned study medication, they will record the number of returned 
pills into the study drug accountability log. The remaining drug will be sent back to the NIH 
pharmacy.
Each subject will receive a list of NIH study contacts and phone numbers as well as a wallet card 
to refer to for questions or emergencies while in the study.
8.0 DATA AND BIOSPECIMEN MANAGEMENT PLAN
8.1 Data Collection:
Primary source data will be captured in the electronic medical record and case report forms 
(CRFs). The PI will be responsible for overseeing entry of this data into an in-house password 
protected electronic system and ensuring data accuracy, consistency and timeliness. The 
principal investigator and associate investigators, research nurses and/or a contracted data 
manager will assist with the data management efforts. Data will be abstracted from Clinical 
Center progress notes, as well as from progress notes forwarded from referring home physicians. 
Laboratory data from NIH will be imported electronically from CRIS into an in-house clinical 
trial database. Laboratory values from referring home physicians will be entered into the system.
All human subjects personally identifiable information (PII), eligibility and consent verification 
will be recorded. Primary data obtained during the conduct of the protocol will be kept in 
secure network drives or in approved alternative sites that comply with NIH security standards. 
Primary and final analyzed data will have identifiers so that research data can be attributed to an 
individual human subject participant.
All protocol data will be stored in the NHLBI secured network drive (P drive) with the following 
security measures: restricted access, password protection, and daily off-site back-up.
8.2 End of Study Procedures:
All medical information collected from study participants at the NIH will be kept in a locked file 
at the Clinical Center at the NIH. Unique patient identifiers will be used to label all data. Strict 
standards of confidentiality will be upheld at all times.
8.3 Data Sharing:
De-identified human data generated for use in future and ongoing research will be shared through 
a NIH-funded or approved repository (ClinicalTrials.gov) and BTRIS. At the completion


February 7, 2021
36
of data analysis, data will be submitted to ClinicalTrials.gov either before publication or at the 
time of publication or shortly thereafter.
Research data may be shared with qualified non-collaborator recipients following publication of 
the primary research results after removal of PII and IRB approval. Future research use of data 
not defined in the research protocol may occur only after IRB review and approval or an 
exemption from the NIH OHSRP. Refusal of a research subject participant to permit future use 
of data--other than required in the protocol or by the FDA--will be honored. Limitations in data 
sharing and future use of data due to contractual obligations (e.g., CRADAs) or intellectual 
property proceedings (such as patent filings) will be honored.
8.4 Future use of biospecimens:
Following analyses of biospecimens (blood) for primary research purposes, remaining samples 
suitable for future research will be stored in manner that conforms with DIR policy (such as BSI) 
or in a publicly accessible research biospecimen repository following IRB or OHSRP approval, 
as applicable. Biospecimens may be destroyed only when permitted by the clinical director and 
the IRB.
Any future research use of biospecimens not defined in the protocol in which NHLBI 
investigators are engaged in research (e.g., they are undertaking research activities and hold the 
key that identifies research subjects) requires IRB review and approval. Coded biospecimens 
(NHLBI investigators hold the key that identifies research subjects) to be shared outside of NIH 
for future research use requires an executed transfer agreement. Unlinked biospecimens (no key 
to identify research subjects exists) to be shared outside of NIH for future research use requires 
an executed transfer agreement. There are a few types of biospecimens  that do not require IRB 
or OHSRP approval for future research use outside of NIH, such as specimens from deceased 
individuals an executed transfer agreement is required in these special cases. Refusal of a 
research subject participant to allow for future use of identifiable biospecimens--other than 
required in the protocol or for appropriate regulatory purposes, e.g., by the FDA--will be honored 
by destroying the remining specimens collected under this protocol for the specific participant.
8.5 Loss or Destruction of Data:
Should we become aware that a major breach in our plan to protect subject confidentiality and 
trial data has occurred, the IRB will be notified.
8.6 Publication Policy:


February 7, 2021
37
Given the research mandate of the NIH, subject data including the results of testing and 
responses to treatment will be entered into an NIH-authorized and controlled research database. 
Any future research use will occur only after appropriate human subject protection institutional 
approval.
9.0 BIOSTATISTICAL CONSIDERATIONS
9.1
Primary Objective
To assess the clinical safety and tolerability of three escalating doses of AG-348, an allosteric 
activator of the enzyme pyruvate kinase, in subjects with stable sickle cell disease (SCD).
9.2
Endpoints
9.2.1
Primary Endpoint

The primary endpoint of this study will be safety and tolerability of AG-348 as assessed by 
frequency and severity of AEs, and changes in laboratory parameters, including levels in 
hemoglobin, reticulocyte counts, bilirubin and lactate dehydrogenase.
9.2.2
Secondary Endpoints

The secondary endpoints will be:
o
Dose to first achieve decrease in concentrations of 2,3-DPG from baseline.
o
Assessment of pyruvate kinase protein, 2,3-DPG, and ATP levels at different doses of 
AG-348 and change from baseline.
o
Change from baseline in oxygen binding p50 value at different doses of AG-348.
o
Percentage of sickled cells under normal and hypoxic ex vivo conditions at different 
doses of AG-348.
o
Safety endpoints, including: the type, incidence, severity, and relationship to study 
treatment of AEs and serious adverse events (SAEs); number of discontinuations due 
to AEs; results of clinical laboratory tests over time (e.g., serum chemistry, liver 
function test, hematology, lipids, sex steroids, urinalysis, coagulation); change from 
baseline in hemoglobin, reticulocyte count, LDH and serum bilirubin at week 7; 
physical examination findings; vital signs; 12-lead electrocardiogram (ECG) data.
o
Pharmacokinetic endpoints, including plasma concentrations over time and 
pharmacokinetic parameters of AG-348 (eg, area under the concentration × time curve 
[AUC], maximum [peak]concentration [Cmax], others as applicable).
o
Exposure-response relationship between safety parameters and AG-348 concentration


February 7, 2021
38
and relevant AG-348 pharmacokinetic parameters.
o
Exposure-response (or pharmacokinetic-pharmacodynamic) relationship between AG- 
348 levels and endpoints (e.g. 2,3-DPG, oxygen sickling assay, etc ) that are indicators 
of predisposition to HbS polymerization.
9.2.3
 Exploratory Endpoints
- Measuring the glycated hemoglobin S fraction as additional supportive evidence of an increase in 
RBC lifespan, glycated hemoglobin is known to increase in the setting of stable glycemia.
 
9.3
Sample Size
This is a study designed to assess the safety and activity of escalating multiple doses of AG-348 
in subjects with stable SCD. Approximately 20 - 25 patients will be enrolled to achieve 15 
patients who have completed all specified dose levels. Specifically, up to 10 subjects will 
complete the 3 dose levels, and at least 5 subjects will complete the 4 dose levels (see Section 3.0 
Study Design for details). The sample size is primarily driven by feasibility considerations and 
the ability to detect adverse events (Table 2).
Table 2. Probability of detecting at least one adverse event (AE) as a function of sample size and 
true underlying AE rate.
True Underlying AE Rate
Sample Size
15%
10%
5%
15
91%
79%
54%
20
96%
88%
64%
25
98%
93%
72%
9.4
Methods of statistical analysis:
The planned analyses will include descriptive statistics on the incidence and severity of adverse events. 
Of particular interest are four qualifying types of adverse events that are counted in evaluating whether a 
stopping rule has been met (see section 9.6). The proportion of patients with any qualifying adverse event 
will be summarized using sample proportion and confidence intervals for binomial distributions. In 
addition to this aggregate measure of any type of qualifying event, the proportions and confidence 
intervals of the four constituent events will be presented. Similar summaries will be produced for all 
treatment-emergent adverse events (TEAEs), related TEAEs (those considered by the Investigator as 
related to study treatment), SAEs, TEAEs leading to treatment discontinuation, TEAEs Grade ≥3 in 
severity, and adverse events of special interest (AESIs). Individual subject listings will be provided for 
any deaths, SAEs, TEAEs leading to interruption and/or reduction of study treatment dose, and TEAEs 
leading to treatment discontinuation. For clinical laboratory values, vital signs, and ECG 


February 7, 2021
39
assessments, both actual values and changes from baseline will be summarized by visit using 
summary statistics. The number and percentage of subjects with transaminase increases of >2.5× 
baseline or increases to Grade ≥2 (AESI of elevated transaminase is defined in the protocol), will 
be summarized. Changes in hemoglobin and reticulocyte count from baseline will be 
summarized.
Secondary endpoint analysis of dose-response modeling will employ linear mixed effects models 
with individual-specific random effects 76. This is similar to a repeated measures analysis of 
variance but allows for a potentially more complicated correlation patterns, has more flexibility 
when data are missing, and allows inclusion of other covariates that may increase power (e.g. age 
and gender). Correlation of residuals will be explored using an unstructured correlation matrix 
assuming the follow-up times are relatively similar across individuals. Average outcomes for the 
fixed dose levels will be represented non-parametrically by coefficients for indicator variables 
for the dose level. As some of the dose-response outcomes may be influenced by baseline age 
and gender these covariates will be included in these models. Data may be transformed, or a 
nonlinear mixed effects model may be considered if the distribution of outcome measures 
suggest the assumptions of normality or linearity are inappropriate.
9.5
Criteria for Study Termination
This study may be prematurely terminated if, in the opinion of the Sponsor, there is sufficient 
reasonable cause. In the event of such action, written notification documenting the reason for 
study termination will be provided to each Investigator.
Circumstances that may warrant termination include, but are not limited to the following:

Determination of unexpected, significant, or unacceptable risk to subjects, e.g., as 
determined by the Data Safety Monitoring Board [DSMB])

Plans to modify, suspend, or discontinue the development of the study treatment

Decisions of competent authorities or Institutional Review Board (IRB)/Independent 
Ethics Committee (IEC)

Stopping rules are met

Other administrative reasons
Should the study be closed prematurely, all study materials must be returned to the Sponsor or 
the Sponsor’s designee
9.6
Stopping Rules:
9.6.1 Stopping Rule for Qualifying Adverse Events
A safety committee (as described in section 11.0) will evaluate safety trial data based on the 
below triggers and if warranted, may recommend modification or even termination of this 
protocol.


February 7, 2021
40
Stopping rules follow a Bayesian design.77
The stopping rule is designed to halt enrollment for further evaluation of study data by the safety 
committee and determination whether enrollment can continue or the study should be modified 
or terminated. A Bayesian posterior probability will be calculated to determine the likelihood the 
probability of having a qualifying adverse event is 30% or more. Qualifying AEs/SAEs are any 
of the following four types of events:

Any Grade ≥3 treatment related Serious Adverse Events,

Grade 4 anemia, unexplained by underlying disease,

Any non-hematological AE Grade ≥ 3 assessed as related to AG-348, or

Any grade≥2 toxicity that does not resolve to Grade ≤1 in 14 days with appropriate
medical management.
When the Bayesian posterior probability reaches 85% or higher, it will trigger a meeting of the 
Safety Committee. The prior distribution for the probability of a qualifying adverse event is 
given by a beta distribution with parameters a = 1 and b = 7/3.
Table 3 summarizes the threshold numbers for the resulting boundary, which would lead to a 
meeting of the safety committee to evaluate stopping or modifying the study due to an excess 
number of qualifying AEs/SAEs.
Table 3
Number of subjects 
in the study
Hold enrollment for safety committee 
evaluation if the number of subjects 
who have developed a qualifying
AE/SAE is
≤5
3
≤7
4
≤10
5
≤13
6
≤15
7
We investigated the performance of the above stopping rule through simulation. In each 
simulation we generated a set of 15 independent Bernoulli trials; each representing a patient with 
probability p for having a qualifying AE/SAE. For each simulation we determined if the stopping 
boundary would have been reached with consideration for halting or suspending the study. We 
conducted this simulation 100,000 times for each different true value of probability p and 
recorded the average number of patients treated (it may be less than 15 if the study was stopped 
early) and the average number of qualifying AEs/SAEs observed. In addition, we show the 
proportion of the 100,000 simulations in which the stopping boundary was met. Table 4


February 7, 2021
41
summarizes the performance of the stopping rule under a number of different values for the 
qualifying AE/SAE probability p.
Table 4: Performance of the Stopping Rule under a number of scenarios for the qualifying 
AE/SAE Probability p
Probability of a qualifying 
AE/SAE
0.20
0.25
0.30
0.35
0.40
0.45
0.50
Proportion of Stopped 
Studies
9%
17%
28%
42%
56%
69%
80%
Average number of 
subjects
14.2
13.6
12.7
11.7
10.4
9.3
8.1
Average number of 
qualifying AEs/SAEs
2.9
3.4
3.8
4.1
4.2
4.2
4.1
These simulation results suggest that our stopping rule has a low probability stopping a study 
when the probability of a qualifying AE/SAE is below 30%, and the probability of stopping a 
study is high when the true probability of a qualifying event exceeds 35%. Thus, we believe that 
our Bayesian stopping rule has satisfactory statistical properties.
9.6.2 Stopping Rule for mortality
A separate stopping rule is proposed for deaths that are deemed to be possibly, probably, or 
definitely related to study medication. Should such an incident occur entry of other patients into 
the study will be suspended, and patients receiving study medication will not be dose escalated 
and will be closely monitored for occurrences of the same even whilst assessment of the event 
occurs.
The IRB/DSMB and the investigators and Agios will evaluate the full circumstances of the event 
and determine whether to terminate the study. Should it be determined the study be terminated, 
enrollment in the study will be halted permanently, subjects already on the study will undergo 
gradual or rapid dose taper to withdraw treatment.
10.0 OFF-STUDY CRITERIA

Subject choice

Subject non-compliance

Lost to follow-up

Study completion


February 7, 2021
42
11.0 DATA SAFETY AND MONITORING PLAN
11.1
Safety Monitoring
Principal Investigator: Accrual and safety data and conduct of the trial will be monitored by the 
PI and research team on an ongoing basis.
Safety Committee: A safety committee will evaluate safety data based on the triggers outlined in 
section 9.6. and if warranted, may recommend modification or even termination of this protocol. 
Minutes of the safety committee’s meeting will be placed in the trial master file. The safety 
committee contains members of the NIH study team and Agios Clinical Study team (quorum to 
include a minimum of Agios Clinical Development Lead, Agios Safety Lead and NIH PI).
NIH Intramural IRB: Adverse event recording and reporting will be conducted in accordance 
with the NIH IRB policy and procedures. ). Prior to implementation of this study, the protocol 
and the proposed subject consent and assent forms will be reviewed and approved by the 
properly constituted Institutional Review Board (IRB) operating according to 45 CFR 46 
(Protection of Human Subjects). This committee will approve all amendments to the protocol or 
informed consent, and conduct continuing annual review so long as the protocol is open to 
accrual or sample and/or data analysis continues.
NHLBI Hematology DSMB: The NHLBI Hematology Data Safety and Monitoring Board 
(DSMB) will review the protocol at 6 to 12 month intervals and the interval will be determined 
by DSMB. A progress report will be forwarded to the DSMB at these times and their 
recommendations will be expeditiously implemented. The DSMB may recommend early 
termination of the study for considerations of safety and efficacy.
Monitoring: As per ICH-GCP 5.18 and 21 CFR 312.50, clinical protocols are required to be 
adequately monitored by the study sponsor.  The monitoring of this study will be conducted by 
an independent contract organization working under an agreement with NHLBI to monitor 
aspects of the study in accordance with the appropriate regulations and the approved protocol. 
The objectives of a monitoring visit will be: 1) to verify the existence of signed informed consent 
form (ICF) and documentation of the ICF process for each monitored subject; 2) to verify the 
prompt and accurate recording of all monitored data points, and prompt reporting of all SAEs; 3) 
to compare abstracted information with individual subjects’ records and source documents 
(subject’s charts, laboratory analyses and test results, physicians’ progress notes, nurses’ notes, 
and any other relevant original subject information); and 4) to help ensure investigators are in 
compliance with the protocol. The monitors also will inspect the clinical site regulatory files to 
ensure that regulatory requirements (Office for Human Research Protections-OHRP) and 
applicable guidelines (ICH-GCP) are being followed. During the monitoring visits, the


February 7, 2021
43
investigator (and/or designee) and other study personnel will be available to discuss the study 
progress and monitoring visit.
12.0 ADVERSE EVENT REPORTING
12.1 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 (e.g., “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 non-serious) spontaneously reported by the subject and/or in response to an 
open question from study personnel or revealed by observation, physical examination, or other 
diagnostic procedures will be recorded on the appropriate page of the eCRF. Any clinically 
relevant deterioration in laboratory assessments or other clinical findings prior to initiating study 
drug is considered an AE and must be recorded on the appropriate pages of the eCRF. When 
possible, signs and symptoms indicating a common underlying pathology should be noted as one 
comprehensive event.
12.1.2
Adverse event management:
The following adverse event management guidelines are intended to ensure the safety of 
each patient while on the study. The AEs will be attributed (unrelated, unlikely, possibly, 
probably or definitely) to study medication and/or disease and graded by severity 
utilizing CTC version 5.0. A copy of the criteria can be down-loaded from the CTEP 
home page at http://ctep.cancer.gov/reporting/ctc.html.
Adverse event recording will start after consent. However, prior to drug administration, 
only procedure-specific AEs will be captured in the database.
All AEs, including abnormal findings on laboratory evaluations, regardless of 
severity, will be recorded and followed until satisfactory resolution. AEs should be 
reported up to 30 days following the last dose of study drug. AEs that are considered 
treatment related, expected, continuing, but not resolvable by 30 days after treatment 
completion will not be followed after the 30-day period.
It should be noted that hospitalizations occurring for safety monitoring following first dose and 
dose escalation are not considered new inpatient hospitalizations for safety reporting and will not 
be reported as SAEs unless an additional Medical condition occurs which meets any serious 
criteria.


February 7, 2021
44
An abnormal laboratory value will be considered an AE if the laboratory abnormality is 
characterized by any of the following:

Results in discontinuation from the study

Is associated with clinical signs or symptoms

Requires treatment or any other therapeutic intervention

Is associated with death or another serious adverse event, including 
hospitalization

Is judged by the Investigator to be of significant clinical impact

If any abnormal laboratory result is considered clinically significant, the 
investigator will provide details about the action taken with respect to the test 
drug and about the patient’s outcome.
Exclusions to data reporting:
The following Adverse Events will be captured only in the source documents and will not 
be reported to the IRB.

Laboratory values that do not meet the definition of AE.

All grade 1 events listed as expected in the investigator’s brochure.
12.1.3.
Dose modifications due to Adverse Events
The Investigator will monitor all subjects for safety. It is important that, as much as possible, a 
subject does not abruptly discontinue study drug, due to the risk of withdrawal hemolysis or 
VOC. After careful consideration of the relative risk of withdrawal hemolysis or VOC when 
stopping study drug abruptly versus reducing the dose rapidly, the Investigator should determine 
if a gradual dose taper (GDT Table 6) for the management of non-acute AEs (e.g., insomnia), 
rapid dose taper (RDT; Table 7 ) for the management of acute AEs or potential
SAEs (e.g., Grade 4 transaminase increase), or abrupt discontinuation of study drug to receive 
emergency medical treatment is necessary.
Table 5:
Dose Modification for Adverse Events (Except Transaminase Increases) 
Considered Related to the Study Drug
Related Adverse 
Event(s) Severity
Dose Modification
Grade 1

None required.
Grade 2

None required.


February 7, 2021
45
Grade 3

First Occurrence: Perform a dose taper1 to stop the study drug until 
event resolution to Grade ≤1 or baseline, whichever is higher, within 
21 days of suspension, and then restart AG-348 at the next lowest dose 
level. If event not resolved after 21 days of study drug suspension, 
consider discontinuation of subject from the study.

Second occurrence: Perform a dose taper1 to discontinue the study 
drug
Grade 4

Perform a dose taper1 to discontinue the study drug
Abbreviations: BID = twice daily.
1 All subjects who are discontinuing or reducing study drug should undergo a dose taper unless an emergency 
situation justifies interrupting the study drug abruptly. Dose tapers should be conducted as detailed in Section12.1.4.
12.1.4
Dose Taper Regimens
Subjects undergoing a dose taper should be monitored for signs of hemolysis and worsening of 
anemia. Investigators should carefully consider the relative risk of withdrawal hemolysis or VOC 
when rapidly reducing the dose versus gradually reducing the dose.
In subjects undergoing a gradual dose taper, the regimen detailed in Tables 6A and 6B will be 
followed.
Table 6A: Gradual Dose Taper Regimen for subjects escalating to maximum AG-348 dose of 50 
mg BID.
Current Dose of 
AG-348
First Step
×5 days
Second Step
×5 days
Third Step
x 5 days
Fourth Step
×5 days
5 mg BID
5 mg QD
n/a
n/a
n/a
20 mg BID
20 mg QD
5 mg BID
5 mg QD
n/a
50 mg BID
50 mg QD
20 mg BID
20 mg QD
5 mg QD
Abbreviations: BID = twice daily; n/a = not applicable; QD = once daily.
Table 6B: Gradual Dose Taper Regimen for subjects escalating to maximum AG-348 dose of 
100 mg BID.
Abbreviations: BID = twice daily; n/a = not applicable; QD = once daily; AM = daily in the morning; PM = daily at 
night.


February 7, 2021
46
Current Dose 
of AG-348
First Step
×5 days
Second Step
×5 days
Third Step 
x 5 days
Fourth Step
×5 days
Fifth Step
×5 days
5 mg BID
5 mg QD
n/a
n/a
n/a
n/a
20 mg BID
20 mg QD
5 mg BID
5 mg QD
n/a
n/a
50 mg BID
50 mg QD
20 mg BID
20 mg QD
5 mg QD
n/a
100 mg BID
50 mg BID
50 mg QD
20 mg BID
20 mg QD
5 mg QD
In subjects undergoing a Rapid Dose Taper, the regimen in Tables 7A and 7B will be followed.
Table 7A: Rapid Dose Taper Regimen for subjects escalating to maximum AG-348 dose of 50 
mg BID.
Current Dose of 
AG-348
First Step
×3 days
Second Step
×3 days
Third Step
×3 days
Fourth Step
x3 days
5 mg BID
5 mg QD
n/a
n/a
n/a
20 mg BID
20 mg QD
5 mg QD
n/a
n/a
50 mg BID
50 mg QD
20 mg BID
20 mg QD
5 mg QD
Abbreviations: BID = twice daily; n/a = not applicable; QD = once daily.
Table 7B: Rapid Dose Taper Regimen for subjects escalating to maximum AG-348 dose of 100 
mg BID.
Current Dose 
of AG-348
First Step
×3 days
Second Step
×3 days
Third Step
x3 days
Fourth Step
×3 days
Fifth Step
×3 days
5 mg BID
5 mg QD
n/a
n/a
n/a
n/a
20 mg BID
20 mg QD
5 mg BID
5 mg QD
n/a
n/a
50 mg BID
50 mg QD
20 mg BID
20 mg QD
5 mg QD
n/a
100 mg BID
100 mg AM / 
50 mg PM
(Day 1)
50 mg BID
(Day 2-3)*
50 mg QD
20 mg BID
20 mg QD
5 mg QD
Abbreviations: BID = twice daily; n/a = not applicable; QD = once daily; AM = daily in the morning; PM = daily at 
night.
* For the first step of the taper, subjects will receive 100 mg of AG-348 for the first AM dose only, then reduce dose 
to 50 mg BID for the remainder of the 3 days.


February 7, 2021
47
12.1.5
Adverse Events of Special Interest
An AESI can be serious or nonserious. Ongoing monitoring and rapid communication (within 
24 hours) by the Investigator to Agios is required to allow for further characterization and 
reporting to regulatory authorities.
Transaminase Increase
Transaminase increase is an AESI for AG-348. In the event of a transaminase increase of
>2.5 × baseline or an increase in transaminase to ≥Grade 2 in severity, whichever is lower, the 
Investigator will report this occurrence to Agios, using the SAE form, within 24 hours of their 
first knowledge of the event.
An LFT panel should then be monitored weekly until the transaminases have decreased to
<2.5 × baseline (defined as the mean of the Screening and Day 1 values). Additionally, the 
following should be taken to gain further information on the possible cause of the transaminase 
increase.
1. Rule out biliary causes by Liver Imaging, Liver MRI, Liver ultrasound, or Magnetic 
resonance cholangiopancreatography as clinically indicated.
2. Viral screen for EBV and CMV using the central laboratory.
3. Autoimmune hepatitis panel consisting of: Serum antinuclear antibody, Anti–smooth 
muscle antibody, Liver-kidney microsomal type 1, Antibodies to soluble liver antigen, 
and Anti-mitochondrial antibodies all at first alert level of transaminase increase and 
repeated 4 weeks later using the central laboratory, if it was negative the first time.
Table 9:
Dose Modification for Transaminase Increases
Transaminase 
Increase Severity
Dose Modification
Grade 1 where result 
is not >2.5 x baseline

None required. Follow closely.


February 7, 2021
48
Transaminase 
Increase Severity
Dose Modification
Grade 2
or Grade 1 with
>2.5 x baseline

First occurrence: none required but consider performing a dose taper1 
to stop the study drug, if deemed necessary by the Investigator and 
Agios. If dose is held, hold until event resolution to Grade ≤1 or 
baseline, whichever is higher, within 21 days of suspension, and then 
restart AG-348 at the same dose.

Second occurrence: perform a dose taper1 to stop the study drug until 
event resolution to Grade ≤1 or baseline, whichever is higher, within 
21 days of suspension, and then restart AG-348 at the next lowest dose 
level (ie, 50 mg BID to 20 mg BID).

Third occurrence perform a dose taper1 to stop the study drug 
permanently.
Grade 3

First occurrence: perform a dose taper1 to stop the study drug until
event resolution to Grade ≤1 or baseline, whichever is higher,
within 21 days of suspension, and then restart (with confirmation from 
Agios) AG-348 at the next lowest dose level (ie, 50 mg BID to 20 mg 
BID).

Second occurrence: perform a dose taper1 to stop the study drug 
permanently.
Grade 4

Perform a dose taper1 to stop the study drug permanently.
Abbreviations: BID = twice daily.
1 All subjects who are discontinuing or reducing study drug should undergo a dose taper unless an emergency 
situation justifies interrupting the study drug abruptly. Dose tapers should be conducted as detailed in Section 12.1.4
12.2 Grading of Adverse Events
Table 10
Grade
Category
Description
1
Mild
Mild; asymptomatic; clinical or diagnostic observations only;
intervention not indicated
2
Moderate
Moderate; minimal, local or noninvasive intervention indicated;
limiting age- appropriate instrumental ADL


February 7, 2021
49
3
Severe
Severe or medically significant but not immediately life-
threatening; hospitalization or prolongation of hospitalization 
indicated; disabling; limiting selfcare ADL
4
Life
threatening
Life- threatening consequences; urgent intervention indicated
5
Death
Death related to AE
12.3 Attribution of Adverse Events
Table 11
Relationship
Attribution
Description
Unrelated
The AE is clearly NOT
related to the intervention
Unrelated to investigational 
agent/intervention1
Unlikely
The AE is doubtfully related
to the intervention
Possibly
The AE may be related to the
intervention
Probably
The AE is likely related to
the intervention
Related to investigational 
agent/intervention1
Definitely
The AE is clearly related to
the intervention
1NOTE: AEs listed as ‘possibly, probably, or definitely’ related to the investigational 
agent/intervention are considered to have a suspected ‘reasonable causal relationship’ to the 
investigational agent/intervention (ICH E2A).
Attribution of causality must be signed by the principal investigator
13.0 NIH INTRAMURAL IRB AND NHLBI CD REPORTING
13.1
Expedited Reporting
Events requiring expedited reporting will be submitted to the IRB per Policy 801 “Reporting 
Research Events”.
13.2 Reports to the IRB at the time of Continuing Review:
The PI or designee will refer to HRPP Policy 801 “Reporting Research Events” to determine 
IRB reporting requirements and timelines.


February 7, 2021
50
13.3
Reports to the CD:
The PI or designee will refer to NHLBI DIR guidelines to determine CD reporting requirements 
and timelines.
14.0
IND SPONSOR REPORTING CRITERIA
The PI or designee will refer to NHLBI DIR guidelines to determine CD (study sponsor) 
reporting requirements and timelines.
Expedited IND reporting to FDA will be performed in accordance with Guidance for Industry 
and Investigators Safety Reporting Requirements for INDs and BA/BE Studies December 2012.
14.1
Reporting Pregnancy
If a patient becomes pregnant during the course of the study, the study treatment should be 
discontinued immediately, and the pregnancy reported to the Sponsor. 
Pregnancy itself is not regarded as an SAE. However, if a patient becomes pregnant while on study, 
risk intrauterine exposure of the fetus to the agents which may be teratogenic. Pregnancy should be reported 
in an expedited manner to the Sponsor and manufacturer as Grade 3 “Pregnancy, puerperium and perinatal 
conditions - Other (pregnancy)” under the Pregnancy, puerperium and perinatal conditions SOC.
Congenital abnormalities or birth defects and spontaneous miscarriages should be reported and 
handled as SAEs. Elective abortions without complications should not be handled as AEs. The outcome of 
all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or 
congenital abnormality) should be followed up and documented.
If any pregnancy occurs in the course of the study, then the investigator should inform the Sponsor 
within 1 day, i.e., immediately, but no later than 24 hours of when he or she becomes aware of it.
The same timelines apply when outcome information is available.
All subjects, male and female, must agree to use effective contraception during the entire study 
and for 28 days (female subjects) and 90 days (male subjects) following the last dose of AG-
348. Abstinence is an acceptable method only


February 7, 2021
51
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 (e.g., calendar, symptothermal, and post- 
ovulation methods) and withdrawal are not acceptable methods of contraception.
14.2
Expedited Adverse Event Reporting Criteria to the IND Drug Manufacturer 
Copies of all Safety Reports will be provided to Agios concurrently with their submission to the 
FDA, and with any other information affecting the safety of Human Subjects in research 
conducted under the executed CRADA with Agios Pharmaceuticals Inc.
15.0 HUMAN SUBJECT PROTECTION
15.1
Rationale for Subject Selection
Study Population: All patients 18 years old and over will be considered for this protocol. No 
patient will be excluded from participation based on gender, race, or ethnicity. Patients may self- 
refer, be recruited through the NIH office of recruitment and may include subjects participating 
on NIH Clinical Center Protocols.
15.2
Informed Consent Processes and Procedures
Informed consent shall be documented using the current IRB‐approved consent form, which 
should be downloaded from the NIH Clinical Center active consent website. Each participant will 
receive an oral and written explanation of the goals, procedures, and risks of this study. When 
consent is obtained, the consent document(s) must be signed and dated by the subject, and the 
person obtaining consent. The original, signed informed consent document will be placed in the


February 7, 2021
52
medical record, and the subject will receive a signed copy of the informed consent document. 
Documentation of informed consent and the signed consent form will be maintained in CRIS. 
The objectives of this research, the procedure and its attendant risks and discomforts will be 
carefully explained to the subject and a signed informed consent document will be obtained prior 
to entry onto this study.
At any time during participation in the protocol, should new information become available 
relating to risks, adverse events, or toxicities, this information will be provided orally or in 
writing to all enrolled or prospective patient participants. Documentation will be provided to the 
IRB and if necessary the informed consent amended to reflect relevant information.
15.3
Patient Advocate
A patient’s rights representative is available to patients on this protocol. The representative is 
located in Building 10 and can be reached by phone at 301-496-2626. Patients may ask any 
questions about the study and may withdraw their consent at any time.
15.4
Privacy and Confidentiality
All efforts, within reason, will be made to keep subjects’ private identifiable information (PII) 
private. Using or sharing (“disclosure”) such data must follow federal privacy rules. Under 
certain circumstances, the United States Office of Human Research Protections (OHRP), The US 
Food and Drug Administration (FDA), and the NIH Institutional Review Board (IRB), will be 
able to inspect and copy confidential study-related records which identify participants by name. 
Therefore, absolute confidentiality cannot be guaranteed.
15.5
Risks and Discomforts
15.5.1 Summary of safety findings from clinical trials of AG-348
Overall, AG-348 has been generally well tolerated among healthy adult subjects and adult 
subjects with PK deficiency. Safety data are available as of 27 March 2017 from 36 healthy 
subjects treated with AG-348 at single doses ranging from 30 to 2,500 mg (Study AG348-C-001) 
78, 36 healthy adult subjects treated with multiple doses ranging from 15 to 700 mg BID or 120 
mg QD for 14 days (Study AG348-C-002), and 52 subjects with PK deficiency randomized to 
initial treatment with 50 or 300 mg BID (Study AG348-C-003). Many of the adult subjects with 
PK deficiency have actually received doses lower than 50 or 300 mg, as per protocol-allowed 
dose reductions.
After a single AG-348 dose in healthy adult subjects, AEs 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 in 
healthy subjects, AEs that occurred in >5% of all AG-348-treated subjects across all cohorts (i.e.,
>1 subject) included headache and nausea (13.9% each); vomiting, decreased appetite, feeling


February 7, 2021
53
hot, and restlessness (8.3% each); 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.
All but 1 AE reported in healthy adult subjects was Grade 1 or 2 in severity. The only Grade 3 
AE, occurring in 1 subject treated with AG-348 700 mg in the MAD study, was elevated liver 
function tests (alanine aminotransferase [ALT] and aspartate aminotransferase [AST]), which 
was considered to be study drug-related and led to study drug discontinuation. Overall, 4 subjects 
discontinued Study AG348-C-002 prematurely; 2 subjects discontinued due to withdrawal by 
subject (each experienced vomiting) and 2 subjects were discontinued due to AEs (vomiting, 
liver function test abnormal, and decreased appetite in 1 subject and drug eruption in the other 
subject).
No deaths or other serious adverse events (SAEs) were reported in healthy adult subjects. Only 1 
DLT was documented in Study AG348-C-002 for the event of Grade 3 elevated liver function 
tests described above.
In Study AG348-C-003, of the 52 adult subjects with PK deficiency who received AG-348, 52 
subjects (100%) experienced at least 1 TEAE. The AEs that occurred in >10% of AG-348- 
treated subjects across both cohorts included headache (24 [46.2%] subjects); insomnia
(22 [42.3%] subjects); nausea (21 [40.4%] subjects); viral upper respiratory tract infection (16 
[30.8%] subjects); influenza, arthralgia, fatigue, hot flush, vomiting, diarrhoea (89 [17.3%] 
subjects each); cough, oropharyngeal pain, dizziness, pyrexia (8 [15.4%] subjects each); 
gastroenteritis, back pain, dysmenorrhoea (7 [13.5%] subjects each) and dyspepsia (6 [11.5%] 
subjects each).
There were no deaths in Study AG348-C-003, and 20 SAEs were reported in 16 subjects, 
including pharyngitis and hemolytic anemia (2 subjects); and cellulitis, gastroenteritis, influenza, 
hemolysis, colitis, enteritis, inguinal hernia, mesenteric vein thrombosis, cholelithiasis, post 
procedural hemorrhage, increased alanine aminotransferase, hypertriglyceridemia, osteoporosis, 
renal cyst, ovarian cyst and unintended pregnancy (1 subject each). The majority of these SAEs 
were assessed as unrelated to AG-348 treatment.
Important identified risks associated with administration of AG-348 in clinical studies include 
bone mineral density decrease (including osteoporosis and osteopenia due to aromatase 
inhibition), withdrawal hemolysis, and insomnia (not clinically serious, i.e., not Grade 3 or 
Grade 4). Potential risks associated with AG-348 administration include anaphylactoid reaction, 
aromatase inhibition, gastrointestinal disturbances, photosensitivity, transaminase increases, and 
triglyceride increase. Transaminase elevations are adverse events of special interest (AESIs) for 
AG-348.
The following risks are assessed by Agios as being related to AG-348


February 7, 2021
54
Risk of Withdrawal Hemolysis
In Study AG348-C-003 conducted in adult subjects with PK deficiency, 2 of 52 subjects (3.8%) 
experienced hemolysis upon sudden withdrawal of the drug, including 1 SAE of withdrawal 
hemolysis. In both subjects, a rapid Hb increase during AG-348 treatment was followed by a 
sudden discontinuation of AG-348 without taper, resulting in withdrawal hemolysis and 
anaemia. By contrast, subjects who missed only a few doses of AG-348, or for whom the dose 
was reduced, did not experience TEAEs indicative of hemolysis, and their Hb levels were either 
not recorded immediately after the short interruption or decreased gradually after the dose 
reduction. Refer to the study protocol for specific procedures for dose modifications to prevent 
withdrawal hemolysis in subjects with PK deficiency. When subjects experience an AE, the 
Investigator should consider the relative risk of a potential withdrawal hemolysis versus 
maintaining the subject on study treatment. The Investigator should consider the severity of the 
AE and the need to remove the subject from the drug.
Risk of Insomnia
In Study AG348-C-003 conducted in adult subjects with PK deficiency, insomnia was reported 
in 22 of 52 subjects (42.3%). In the AG-348 50 mg BID initial treatment group, insomnia was 
reported in 6 of 27 subjects (22.2%) (5 subjects with TEAEs of insomnia and 1 subject with a 
TEAE of initial insomnia), and 2 of the TEAEs of insomnia required dose modification. In the 
AG-348 300 mg BID initial treatment group, insomnia was reported in 16 of 25 subjects (64%). 
Dose reduction was required in 5 subjects in the 300 mg BID initial treatment group due to 
TEAEs of insomnia. One of these subjects and 1 additional subject had a temporary dose 
interruption of AG-348 because of insomnia. None of the insomnia TEAEs were SAEs. Two 
TEAEs of insomnia (1 TEAE each in the initial AG-348 50 mg and 300 mg BID initial 
treatment groups) were Grade 3 in severity.
Risk of Bone Mineral Density Decrease
AG-348 inhibits human aromatase activity based on studies in human placental microsomes and 
rat ovarian microsomes. In Study AG348-C-003, 1 subject from 52 treated subjects in Study 
AG348-C-003 experienced a medically important event of osteoporosis (Grade 2), assessed as 
likely due to aromatase
inhibition. Subjects in clinical studies are monitored for potential aromatase inhibition by serial 
assessments of serum hormone levels and bone density (DXA) scans. As this has occurred in a 
single subject, this event is not considered expected for reporting purposes.


February 7, 2021
55
15.5.2 Related to blood draws & IV
No major risks are involved with blood draws. Minor complications including bleeding, pain, 
and hematoma formation at the site of blood draws, vasovagal reactions or infections may rarely 
occur.
15.5.3 Related to cardiac monitoring (during screening)
Echocardiogram (ECHO): An echocardiogram uses sound waves to visualize and evaluate the 
function of the heart. There are no risks associated with obtaining an echocardiogram.
Electrocardiogram (ECG): An electrocardiogram is a test that measures the electrical activity 
of the heartbeat. A technician will place electrodes on the chest, arms, and legs. The electrodes 
are soft and do not cause discomfort when placed or removed by the technician. There are no 
risks associated with obtaining an electrocardiogram.
15.6
Conflict of Interest
A Guide to Avoiding Financial and Non-Financial Conflicts of Interest or Perceived Conflicts of 
Interest in Clinical Research at NIH (September 2015) has been distributed to all NIH 
investigators who have reviewed the guide and indicated no conflicts of interest exist. No 
investigators hold patents or patents pending, or have any financial conflict of interest associated 
with this study.
This protocol has no associated patents or CTAs.
A CRADA between Agios and the NHLBI is in the process of being established.
16.0 COMPENSATION & REIMBURSEMENT
Reimbursement for local travel, US travel, food, and lodging will be in accordance with NHLBI 
travel policy reimbursement will be consistent with NIH and NHLBI guidelines.
Financial Compensation


February 7, 2021
56
Subjects will receive monetary compensation for their time and inconvenience in accordance 
with present NIH guidelines.
Table 12A: Compensation for subjects escalating to maximum dose of 50 mg BID.
Procedure(s)/Test(s)
IU*
Amount
Frequency
Total amount
EKG
1
$10
2
$20
Oxygen monitoring during inpatient
stay
1
$10
4
$40
Medical History and Physical Exam
2.5
$25
8
$200
Urinalysis
1
$10
2
$20
Blood Draw
1
$10
8
$80
Blood Draw through an IV
1
$10
20
$200
IV placement
1
$10
4
$40
Diary
1
$10
6
$60
Drug Administration, General
2
$20
4
$80
Inpatient visit $40 per night
N/A
$40
4
$160
OUTPATIENT- 1st HOUR
NA
$20
4
$80
OUTPATIENT TIME-
Not to Exceed More than 4 Hours
3
$30
3
$90
TOTAL
$1,070
Table 12B: Compensation for subjects escalating to maximum dose of 100 mg BID.
Procedure(s)/Test(s)
IU*
Amount
Frequency
Total amount
EKG
1
$10
2
$20
Oxygen monitoring during inpatient
stay
1
$10
5
$50
Medical History and Physical Exam
2.5
$25
9
$225
Urinalysis
1
$10
2
$20
Blood Draw
1
$10
9
$90
Blood Draw through an IV
1
$10
25
$250
IV placement
1
$10
5
$50
Diary
1
$10
7
$70
Drug Administration, General
2
$20
5
$100
Inpatient visit $40 per night
N/A
$40
5
$200
OUTPATIENT- 1st HOUR
NA
$20
4
$80
OUTPATIENT TIME-
Not to Exceed More than 4 Hours
3
$30
3
$90
TOTAL
$1245
17.0 PHARMACEUTICALS – AG-348
Supply: AG-348 will be supplied by Agios Pharmaceuticals, Inc. 88 Sidney Street Cambridge, 
MA 02139-4169
Pharmacology: Investigational drug AG-348 (formerly known as AGI-1480 and AGX-0841) is a 


February 7, 2021
57
potent, broad-spectrum activator of alleles of the red blood cell (RBC)-specific form of pyruvate 
kinase (PKR). PKR is 1 of 4 pyruvate kinase isoenzymes expressed in human tissues from 2 
distinct genes. PKR and liver-type pyruvate kinase (PKL) are generated from the PKLR gene by 
2 separate tissue-specific promoters, while PKM1 and PKM2 are from the PKM gene via 
differential splicing of the ribonucleic acid. AG-348 is an allosteric activator of PKR, PKL, and 
PKM2, with similar potency against each.
In clinical studies, AG-348 has been administered orally at the dose and regimen prescribed in 
the study protocols.
Product description: AG-348 is currently supplied for oral administration as tablets to all 
subjects. Tablets are supplied in dose strengths of 5, 20, or 50 mg of mitapivat. The tablets 
contain the following inactive ingredients: microcrystalline cellulose, croscarmellose sodium, 
sodium stearyl fumarate, and mannitol. Tablets are coated with a nonfunctional film coat 
composed of generally recognized as safe (GRAS) excipients.
Storage and Stability: The recommended storage condition is stated on the product label to ensure the 
stability and proper product identification of the mitapivat tablets, the drug should be dispensed from the 
packaging in which it is supplied.
Route of Administration: Oral tablets.
Administration: AG-348 should be taken orally and swallowed whole with water. The tablets 
are not to be crushed, chewed, or dissolved in water. Doses of study drug may be taken with or 
without food.
If a subject misses a scheduled dose by 4 hours or less, they should still take that dose. If a 
subject misses a scheduled dose by more than 4 hours, they should skip that dose. If a dose
is skipped, the next dose should then be taken approximately 24 hours from the previous dose 
taken. If a subject experiences a dose interruption for non-safety reasons, they should be restarted 
on the prescribed dose as soon as possible.
Toxicities: The most common adverse events experienced by subjects receiving study drug are:

Headache (44.3% of patients)

Nausea (34.2% of patients)

Insomnia (34.2% of patients)

Nasopharyngitis (The common cold) (27.8% of  patients)

Fatigue (24.1% of patients)

Vomiting (17.7% of patients)

Alanine aminotransferase increased (16.5% of patients)

Oropharyngeal pain (15.2% of patients)

Back pain (15.2% of patients)

Diarrhea (13.9% of patients)

Influenza (13.9% of patients)

Cough (12.7% of patients)


February 7, 2021
58

Hot flush (12.7% of patients)

Dizziness (12.7% of patients)

Upper respiratory tract infection (12.7% of patients)

Pyrexia (11.4% of patients)

Arthralgia (11.4% of patients)

Hypertriglyceridemia (11.4% of patients) 

Dyspepsia (11.4% of patients)

Asthenia (10.1% of patients)

Gastroenteritis (10.1% of patients)

Dysmenorrhea (10.1% of patients)


February 7, 2021
59
Potential Drug Interactions
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 (see also Appendix 1- Investigator’s Brochure detached from this protocol) for lists 
of medications to be avoided or carefully monitored).
Since AG-348 exhibits pH-dependent solubility, proton-pump inhibitors and H2-receptor 
antagonists may decrease the absorption of AG-348.
The following therapies are contraindicated during the study:
Strong inhibitors of CYP3A4
Products known to inhibit CYP3A4 such as grapefruit or grapefruit juice
Strong inducers of CYP3A4
The following therapies should be avoided and replaced with alternative treatments. If this is not 
possible, subjects receiving these drugs should be adequately monitored.
Moderate inhibitors of CYP3A4
Moderate inducers of CYP3A4
Corticosteroids (sensitive substrates of CYP3A4 and weak CYP3A4 inducers)
Sensitive substrates of CYP3A4.
Proton-pump inhibitors and H2-receptor antagonists
AG-348, as a potential CYP3A4 inducer, has the potential to reduce the effectiveness of 
hormonal contraceptives. Therefore, female subjects using hormonal contraceptives must also 
utilize a barrier method while enrolled in the study and until at least 30 days after their last dose 
of study drug.
Oral penicillin are not expected to interact with AG-348.


February 7, 2021
60
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ICF_002.pdf: 
Informed Consent Cover Page for FDAAA consent posting: 
 
 
Official Title: A Pilot Study to Evaluate the Safety, Tolerability, Pharmacokinetics, and 
Pharmacodynamics of escalating multiple oral doses of AG-348 in Subjects with Stable Sickle Cell 
Disease 
 
 
NCT Number: NCT04000165 
 
 
Document Date: 09/04/2020 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY 
 
 
PRINCIPAL INVESTIGATOR: 
Swee Lay Thein, M.D. 
STUDY TITLE: 
A 
Pilot 
Study 
to 
Evaluate 
the 
Safety, 
Tolerability, 
Pharmacokinetics, and Pharmacodynamics of escalating multiple 
oral doses of AG-348 in Subjects with Stable Sickle Cell Disease 
STUDY SITE: 
NIH CLINICAL CENTER (CC), NHLBI 
 
Cohort: Standard 
Consent Version: 09/01/2020 
 
 
WHO DO YOU CONTACT ABOUT THIS STUDY? 
Principal Investigator, Swee Lay Thein, M.D.; Building 10-CRC, Room 5E-5142, 
Telephone: 301- 402-6699; E-mail: sweelay.thein@nih.gov 
Study Coordinator, Ingrid Frey, RN; Building 10-CRC, Room 5E-1424 Telephone: 301- 
402- 6674; E-mail: Ingrid.frey@nih.gov. 
KEY INFORMATION ABOUT THIS RESEARCH 
This consent form describes the research study and is designed to help you decide if you 
would like to be a part of the research study. 
You are being asked to take part in a research study at the National Institutes of Health (NIH). 
This section provides the information we believe is most helpful and important to you to in 
making your decision about participating in this study. Additional information that may help 
you make a decision can be found in other sections of the document. Taking part in research at 
the NIH is your choice. It is completely voluntary. 
The purpose of this research study is to test the tolerability and safety of an investigational 
medication called AG-348 in people with sickle cell disease. “Investigational” means that the 
drug has not been approved by any authority that regulates new medicines, including the US 
Food and Drug administration (FDA). 
The study will last approximately 14 weeks and includes 8 visits to the NIH Clinical Center 
(CC) Visit 1 will be determining your eligibility 
• At visits 2, 3, 4, 5, and 6, you will receive study medication and you will have to stay at the 
NIH for 2 days (1 night) at a time. This way we can watch you closely -to see if you develop 
any reaction to the study drug when increasing or decreasing your dose. You will receive a 
study diary and bottle of study pills to take home at each visit. You will take the study drug 
every 12 hours (for example, 9 AM and 9PM) and record in the diary when you take the study 
drug as well as any symptoms you experience. You will bring back your diary and study drug 
bottle at each of these visits, even if the bottle is empty. 
 
 
 
 
PATIENT IDENTIFICATION 
Consent to Participate in a Clinical Research Study 
NIH-2977 (4-17) 
File in Section 4: Protocol Consent (1) 
Version Date: 9/1/2020 
 
CC pre rCR ICF template v. 05.05.2020 
Page 1 of 19 
lRB NUMBER: 19H0097 
lRB APPROVAL DATE: 09/04/2020 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY 
PATIENT IDENTIFICATION 
Consent to Participate in a Clinical Research Study 
NIH-2977 (4-17) 
File in Section 4: Protocol Consent (1) 
Version Date: 9/1/2020 
lRB NUMBER: 19H0097 
lRB APPROVAL DATE: 09/04/2020 
Page 2 of 19 
CC pre rCR ICF template v. 05.05.2020 
 
 
 
 
• 
You will stop medication at visit 7 
• 
At visit 8, you will be followed up to see if you are doing okay 
• 
Lab tests will be done on all the visits 
• 
Side Effects of Study Drug 
There are many side effects and risks related to this drug, but this drug has been used in other 
studies before and most of these risks are known and can be addressed. 
• 
The most common side effects are: 
headache 
insomnia 
nausea. 
• 
At the highest dose level, you may experience in addition to the above vomiting hot 
flashes and fever 
• 
Other possible risks that may be related to the study drug are: 
the decrease in bone mineral density (decrease in bone mass) 
withdrawal hemolysis (stopping the drug quickly may destroy your red blood cells) 
changes in liver enzymes (ALT and AST) 
changes in levels of certain fats (triglycerides) 
changes in certain hormone levels (testosterone-related and estrogen-related hormones) 
acute pain crisis caused by withdrawal of study drug (stopping the drug quickly may 
destroy your red blood cells) 
• 
Compensation for your time and inconvenience is offered. 
• 
You can discuss with your doctor to see if there are other alternative studies that you can 
participate in if you decide not to participate in this one. You will not be able to continue any 
other medication should you take part in this study 
 
 
 
The remaining document will now describe this research study in more detail. The 
additional information should be considered before you make your choice. Members of 
the study team will talk with you about all the information described in this document. 
Some people have personal, religious, or ethical beliefs that may limit the kinds of medical 
or research treatments they would want to receive (such as blood transfusions). Take the 
time needed to ask any questions and discuss this study with NIH staff, and with your 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY 
PATIENT IDENTIFICATION 
Consent to Participate in a Clinical Research Study 
NIH-2977 (4-17) 
File in Section 4: Protocol Consent (1) 
Version Date: 9/1/2020 
lRB NUMBER: 19H0097 
lRB APPROVAL DATE: 09/04/2020 
Page 3 of 19 
CC pre rCR ICF template v. 05.05.2020 
 
 
family, friends, and personal health care providers. 
IT IS YOUR CHOICE TO TAKE PART IN THE STUDY 
You may choose not to take part in this study for any reason. If you join this study, you may 
change your mind and stop participating in the study at any time and for any reason. In 
either case, you will not lose any benefits to which you are otherwise entitled. However, to 
receive care at the NIH, you must be taking part in a study or are being considered for a 
study. If you do choose to leave the study, please inform your study team to ensure a safe 
withdrawal from the research. 
WHY THIS STUDY IS BEING DONE? 
This is a research study. The purpose of this research study is to test the tolerability and 
safety of a new medication called AG-348 in people with sickle cell disease. We also want 
to evaluate the amount of study drug that gets into the blood and how long it stays in the 
blood; what the study drug does in the body; and look for signs as to whether the study 
drug is working. Laboratory research has shown that AG-348 will help with speeding up 
the activities in the red blood cell that helps transport oxygen throughout the body. 
We are asking you to join this research study because you have SCD. If you are a NIH 
employee, you will be given the NIH Information Sheet on Employee Research 
Participation. 
AG-348 is considered investigational, which means that it has not been approved by the 
U.S. Food and Drug Administration (FDA) or other regulatory authorities around the 
world to treat alone or in combination with any drug in this group of people. The drug 
AG-348, is also known as AGI- 1480 and AGX-0841. This drug activates an enzyme 
called pyruvate kinase in the red blood cell that is very necessary for its function in 
transporting oxygen as well as maintaining its structure. We would like to see if the study 
medication is safe to give to subjects with sickle cell disease and how this medication 
works in their bodies. AG-348 is a capsule taken by mouth. 
So far, AG-348 has been studied in in healthy volunteers and currently under study in 
people who lack the pyruvate kinase protein. In both healthy subjects and people with 
pyruvate kinase deficiency, the drug is generally well tolerated and shown to be safe. 
AG-348 is manufactured by Agios Pharmaceuticals Inc. We have a research agreement 
with this company that allows them to give us this drug. We will not accept any money 
from Agios Pharmaceuticals Inc. to conduct this study. 
WHAT WILL HAPPEN DURING THE STUDY? 
If you decide to take part in this study, you will be asked to come to the Clinical Center 
of the National Institutes of Health for Screening and visits 1 through 8. You will receive 
4 different doses of the study drug, 5, 20, 50, and 100 milligrams at different time points. 
We will give you a list of study contacts and phone numbers to keep with your diary and 
a wallet card, in case you have any questions or have an emergency while you are in the 
study. 


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The next section describes what will happen at each visit: 
SCREENING -VISIT 1: 
A member of the study team will tell you about the study and ask you some questions to 
determine if you are eligible for this study. After you agree to be in the study and sign this 
consent form, we will do the following: 
x 
Medical History and Physical Examination 
x 
Blood Collection: We will take blood from a vein in your arm using a needle. 
The amount of blood drawn is considered a safe amount for adults per NIH 
guidelines. We will use your blood to test or confirm if you have SCD. We will 
also use your blood to run some standard clinical labs. 
x 
Urine Collection: We will use your urine to test for standard laboratory tests and 
pregnancy if you are a female of child bearing age. 
STUDY VISITS: 
Lab tests will be performed at all visits and the study medication will be given to you at 
the following doses at each visit. You will stay at the NIH for 1 night each for visits 2-6. 
The lab tests will show if it is safe for you to continue to the next increased dose of the 
drug. 
Visit 2 (inpatient) – You will have an IV placed in your arm and a blood draw. We will 
provide you with 5 milligrams of AG-348 to take by mouth. After taking the study drug 
you will have a blood draw through your IV after 1, 2, 4, and 8 hours. After the last blood 
draw, you will be discharged home and will take the next dose of 5 milligrams of AG-348 
at home. Before you leave, we will provide you with 2-weeks’ worth of study medication 
and a study diary to take home with you. 
About Taking the Study Medication: 
AG-348 comes in the form of a tablet that you will take twice a day, every 12 hours (for 
example, 9AM and 9PM). The study tablets are to be taken by mouth and swallowed whole 
with water. The tablets are not to be crushed, chewed, or dissolved in water. The study 
drug may be taken with or without food. Keep the study medication in its original bottle. 
If you miss a dose by 4 hours or less, you should still take that dose. If a you miss a dose 
by more than 4 hours, you should skip that dose and take your next dose at the usual time. 
If you do not tolerate the drug and it is not safe for you to continue, we will gradually reduce the 
study drug and continue to follow you to monitor your safety until the end of the study. 
Bring the bottle of study medication with you to every visit until Visit 8. 
About the Study Diary: 
We will give you a study diary to record when you take the study medication. You will 
also keep track of any symptoms until the end of the study. Bring your study diary to 
every visit until the end of the study. 
Visit 3 (inpatient) – Bring your bottle of study medication and study diary. You will have 


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an IV placed in your arm and a blood draw. We will provide you with 20 milligrams of 
AG-348 to take by mouth. After taking the study drug you will have a blood draw through 
your IV after 1, 2, 4, and 8 hours. After the last blood draw you will be discharged home 
and will take the next dose of 20 milligrams of AG-348 at home. You will spend the night 
and be discharged the following morning. Before you leave we will provide you with 2- 
weeks’ worth of study medication and return your study diary to you to take home. 
Visit 4 (inpatient) - Bring your bottle of study medication and study diary. You will have 
an IV placed in your arm and a blood draw. We will provide you with 50 milligrams of 
AG-348 to take mouth. After taking the study drug you will have a blood draw through 
your IV after 1, 2, 4, and 8 hours. After the last blood draw you will be discharged home 
and will take the next dose of 50 milligrams of AG-348 at home. Before you leave we 
will provide you with 2-weeks’ worth of study medication and return your study diary to 
you to take home. 
Visit 5 (inpatient) - Bring your bottle of study medication and study diary. You will have 
an IV placed in your arm and a blood draw. We will provide you with a total of 100 
milligrams of AG-348 (2 tablets of 50 milligrams) to take mouth. After taking the study 
drug you will have a blood draw through your IV after 1, 2, 4, and 8 hours. After the last 
blood draw, you will be discharged home and will take the next dose of 100 milligrams 
of AG-348 at home. Before you leave we will provide you with 2-weeks’ worth of study 
medication and return your study diary to you to take home. 
Visit 6 (inpatient)- Bring your bottle of study medication and study diary. You will have 
an IV placed in your arm and a blood draw. We will provide you with 100 milligrams of 
AG-348 to take mouth on the morning of your taper. After taking the study drug you will 
have a blood draw through your IV after 1, 2, 4, and 8 hours. After the last blood draw you 
will be discharged home, where you will begin taking 50 milligrams twice a day. Before 
you leave we will provide you with sufficient study medication for your drug taper and 
return your study diary to you to take home. 
The morning after your inpatient stay you will begin to gradually reduce the dose over 15 
days (dose tapering) until you are no longer taking the study medication. The schedule for 
the dose taper will be as follows: 100 mg in the morning (provided inpatient) and 50 mg 
in the evening for the first day, followed by 50 mg twice a day for 2 days, 50 mg once a 
day for 3 days, 20 mg twice a day for 3 days, then 20 mg once a day for 3 days, then 5 mg 
once a day for 3 days until you come in for Visit 7. You will be instructed to contact the 
study team right away if you develop any changes in your symptoms or overall health, or 
if you feel that you may be experiencing a sickle cell pain crisis. If this happens, we may 
change the schedule for your dose taper. 
Visit 7 - Lab tests 
Visit 8 – (4 week follow-up) Lab tests 
The approximate amounts of blood to be collected at each visit at pre-dose, 1 hour, 2nd 
hour, 4th hour, and 8th hour for each study visit (except during the 1st visit/screening, 
and visits 7 and 8) as follows: 


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Visit 
Amount of blood 
Screening/Visit 1 
 
2 tbsp 
Visit 2 
5 tbsp 
Visit 3 
5 tbsp 
Visit 4 
5 tbsp 
Visit 5 
5 tbsp 
Visit 6 
5 tbsp 
Visit 7 
5 tbsp 
Visit 8 
5 tbsp 
Total 
2.3 cups 
 
tbsp=tablespoon; tsp=teaspoon 
Genetic Testing for Research: 
We will take DNA from the blood samples you provide. We will test your DNA for the PKLR 
gene which is inherited that can cause differences in response to the study drug. We may also test 
one, many or other of your genes to see if we can find any genes that interact with the PKLR 
variant and SCD. Methods for looking at genes are constantly changing and we plan to continue 
to use new and improved techniques as they become available to look for all possible changes in 
genes that may help explain the differences in response to the drug, and possibly in symptoms of 
SCD. We may also test your DNA for other genes we know that may affect symptoms of SCD. 
Because we don’t know much about how these genes can affect sickle cell disease, the results of 
our research studies will not be provided to you or your referring doctor. 
HOW LONG WILL THE STUDY TAKE? 
If you agree to take part in this study, your involvement will last for about 14 weeks in total. 
Please refer to the previous section to review the study visits. 3 outpatient visits usually 
will range from 4 to 6 hours in length. 5 inpatient visits require a 24-hour hospital stay. 
 
HOW MANY PEOPLE WILL PARTICIPATE IN THIS STUDY? 
Up to 25 people may participate in this study at the NIH Clinical Center. 
 
WHAT ARE THE RISKS AND DISCOMFORTS OF BEING IN THE STUDY? 
a) 
Blood collection: 
There may be some discomfort on your arm when we collect your blood with a needle. 
There is a small chance that you will get a bruise, feel lightheaded, faint, or have an 
infection at the place where you were pricked. 
b) Genetic and DNA analysis: 
Issues related to your confidentiality: Some people are concerned that information about 


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them from their medical records could be given out without them knowing about it. 
Possible problems might include insurance or employment discrimination. Steps to 
protect your information are detailed below. These problems may also occur if you give 
out information yourself or agree to have your research records given out. Information 
from this study will be identified with a code number instead of your name. The key for 
this code will be stored in a locked file cabinet. 
There may be a risk that genetic information taken as part of participation in research 
could be misused for discriminatory purposes. However, state and federal laws provide 
some protections against genetic discrimination. 
We may publish results of this research study in scientific journals and public databases, 
including your medical history and other medical information but the information 
provided in the publications will be anonymous. It is possible but unlikely that you and/or 
a family member could be identified because of such publications. 
Researchers who will have access to genetic information about you will take measuresto 
maintain the confidentiality of your genetic information. 
c) 
AG-348 side effects: 
You may have side effects while on the study. Everyone taking part in the study will be 
watched carefully for any side effects. However, doctors don’t know all the side effects 
that may happen. Side effects may be mild or very serious. Your health care team may 
give you medicines to help lessen side effects. Your doctor may reduce or delay dosing 
with the study drug to ensure your safety. Many side effects go away soon after you stop 
taking an experimental drug. In some cases, side effects can be serious, long lasting, or 
may never go away. You should talk to your study doctor about any side effects that you 
have while taking part in the study. 
The following table shows safety data from an ongoing clinical study of patients with 
pyruvate kinase deficiency (PKD). In this study a total of 52 patients were treated with 
doses of study drug up to 300 mg twice a day (higher than the doses that will be used in 
this study) and 42 of the patients were treated for at least 6 months. The list below shows 
the adverse events that were seen in more than 10 % of patients (most of them were not 
serious). 
At the highest doses it is possible that these side effects may be more frequent or more 
severe. 
Side effects seen in more than 10% of the 79 patients with PKD receiving the study drug 
x Headache (44.3% of patients) 
x Nausea (34.2% of patients) 
x Insomnia (Difficulty falling asleep or staying asleep) (34.2% of patients) 
x Nasopharyngitis (The common cold) (27.8% of patients) 
x Fatigue (Feeling tired) (24.1% of patients) 


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x Vomiting (Throwing up) (17.7% of patients) 
x Alanine aminotransferase increased (Increased liver enzymes “ALT”) (16.5% of 
patients) 
x Oropharyngeal pain (Sore throat) (15.2% of patients) 
x Back pain (15.2% of patients) 
x Diarrhea (Loose stool) (13.9% of patients) 
x Influenza (The “flu”) (13.9% of patients) 
x Cough (12.7% of patients) 
x Hot flush (Sudden feeling of warmth) (12.7% of patients) 
x Dizziness (12.7% of patients) 
x Upper respiratory tract infection (A common viral infection that affects the nose, 
throat, and airways) (12.7% of patients) 
x Pyrexia (Fever) (11.4% of patients) 
x Arthralgia (Joint Pain) (11.4% of patients) 
x Hypertriglyceridemia (High levels of a type of fat found in blood) (11.4% of 
patients) 
x Dyspepsia (indigestion/heartburn) (11.4% of patients) 
x Asthenia (Weakness) (10.1% of patients) 
x Gastroenteritis (the “stomach flu”, an inflammation of the lining of the intestines 
caused by a virus, bacteria, or parasites) (10.1% of patients) 
x Dysmenorrhea (painful menstrual periods) (10.1% of patients) 
 
CERTAIN RISKS ARE KNOWN TO BE CAUSED BY THE USE OF THE STUDY DRUG: 
Bone mineral density decrease was reported in 3.8% of PKD patients: Bone mineral 
density is a measure of how strong your bones are. A decrease in bone mineral density 
could lead to an increased risk of bone fractures. A serious decrease in bone mineral density 
has been reported in one patient who took the study drug for more than six months. 
Withdrawal hemolysis was reported in 2.5% of PKD patients: PKD causes your red blood 
cells to be destroyed (hemolysis). If the study drug works very well for you (increases your 
hemoglobin level), and you quickly stop taking the study drug, the hemolysis may come 
back quickly and you could feel sick. Hemolysis also occurs in sickle cell disease (SCD), 
and it is possible that stopping the study drug too quickly could lead to an increase in 
hemolysis or an acute pain crisis (see below). It is important that you do not stop taking 


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the study drug without checking with your study doctor first unless it is absolutely 
necessary for emergency medical care and you are instructed to stop treatment by an 
emergency medical care professional. If you think you may be running out of study drug, 
you should contact the study site immediately. 
Insomnia, trouble falling asleep and/or staying asleep, was reported in 34.2% of PKD 
patients and 44.4% of Thalassemia patients. In the ongoing study described above, 
insomnia and trouble falling asleep were reported. These events were seen more often in 
patients receiving 300 mg twice a day, a higher dose of the study drug than the doses (5, 
20, 50, and 100 mg) that will be used in this study. 
Changes in liver enzymes (ALT and AST) in blood have been seen in some patients 
receiving the study drug. These increases may be early signs of liver damage. Your 
doctor will use blood tests to check your liver enzymes throughout the study. If you 
experience an increase of liver enzymes (ALT or AST) which meets certain criteria the 
study doctor will prescribe additional tests. These tests may include: 
x Blood tests for viruses that can infect the liver and cause increases in liver 
enzymes. Examples of such viruses include Cytomegalovirus (CMV), Epstein 
Barr Virus (EBV), Hepatitis A, B and C, and Human Immunodeficiency Virus 
(HIV). 
x Blood tests for markers of autoimmunity. Autoimmunity is when the body has 
an immune response that causes the body to attack itself. 
x Imaging tests to see if there are any blockages or other problems with your liver: 
such as liver ultrasound, Magnetic Resonance Imaging (MRI) of liver, Computed 
Tomography (CT) scan of liver, /or Magnetic resonance 
cholangiopancreatography. 
Changes in levels of certain fats (triglycerides) in blood have been seen in some patients 
receiving the study drug. Long term exposure to very large increases in certain fats may 
lead to inflammation of your pancreas (pancreatitis) which may be serious. Your doctor 
will check the level of fat in your blood throughout the study. 
Changes in certain hormone levels (testosterone-related and estrogen-related 
hormones) in blood have been seen in some patients receiving the study drug. These 
hormone changes can contribute to osteoporosis, which is a weakening of the bones. Your 
doctor will check your hormone levels throughout the study (blood tests). 
Changes in hemoglobin levels. We anticipate an increase in hemoglobin when you take 
the study drug, and will monitor the increase very carefully as excessive increase may not 
be beneficial. As the response to the study drug and tolerance to the level of increased 
hemoglobin varies between individuals, we will monitor your symptoms in response to 
the increase and may have to reduce the dose of study drug to suit you. 


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In addition, the following risk may be caused by use of the drug: 
 
Acute pain 
It is possible that you may have an acute pain crisis, particularly when going through the 
dose taper portion of the study. This severe pain is known as acute vaso-occlusive crises 
(VOC). To reduce this possible risk, we will decrease the dose of the study drug over time 
during the drug taper. If you start experiencing acute pain or symptoms of a VOC, you 
should let your study doctor know immediately. If this happens, we may decrease the 
study drug dose even slower in order to prevent the development of a VOC. 
d) 
Interactions with other drugs 
Sometimes different drugs can interact with each other in the body and cause side effects 
when used together, which do not occur when each drug is used alone. It is important that 
you discuss all medications, including prescribed medications, over-the-counter 
medications, and supplements (like vitamins), as well as grapefruit products that you are 
taking before you start the study and also tell your doctor of any changes in your 
medications during the study. Your study doctor will let you know if you need to stop 
taking certain medications or switch to another medication while you are taking the study 
drug. 
What are the risks related to pregnancy? 
You should not become pregnant or father a baby while in this study because the effects 
on an unborn baby are unknown. If you are a woman who can have children, you will 
have a pregnancy test before you can be in this study. If you are pregnant or breast feeding, 
you cannot be in this study. Tell one of the study staff members right away if you suspect 
that you have become pregnant while in the study or, if you are a man, you suspect that 
your partner has become pregnant. If you are a female and become pregnant during the 
study you will be required to discontinue study drug, in consultation with your study 
doctor. 
You may be able to remain in the study and off of study drug during the pregnancy and 
while you are breast feeding, and be allowed to restart the drug at the end of the pregnancy 
and when you have completed breast feeding, if the study is still ongoing. 
Whether you are a man or a woman, you need to either remain abstinent from sexual 
intercourse, (if this is part of your usual lifestyle), or use highly effective methods of birth 
control while in this study and for at least 28 days after the last dose of study drug for 
women and 90 days following the last does of study drug for men. Check with your study 
doctor about the methods of birth control to use. 
Based on animal studies, the study drug may affect fertility in males and females. In 
animals, these effects were reversible after discontinuation of the study drug. The study 
drug may also affect the ability to maintain pregnancy. 
If you are a female and become pregnant during the study you will be required to 


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discontinue study drug, in consultation with your study doctor. 
 
WHAT ARE THE BENEFITS OF BEING IN THE STUDY? 
You might not benefit from being in this study. The purpose of this study is to find doses of the 
drug AG-348 that are safe and tolerable in patients with SCD. Even if you benefit from taking 
the study drug, we will need to take you off of the drug at the end of the study. 
However, in the future, other people might benefit from this study because once we find 
a dose that can be tolerated and safe, we will start a clinical study of AG-348 to evaluate 
its efficacy in improving the anemia and reducing the hemolysis that is related to SCD. 
The knowledge acquired from the study will also help understand the sickling process 
itself, and perhaps provide information on future treatments for targeting the sickling 
process. 
WHAT OTHER OPTIONS ARE THERE FOR ME? 
You do not have to participate in this study if you do not want to. You may withdraw 
from this study at any time. If you decide to withdraw from the study, we would like to 
keep your test results to properly analyze this research study. If you have concerns about 
this, please speak with members of your research team. 
If you decide not to participate in this research study, other treatments or medications 
may be available for the treatment of SCD. There may also be other clinical research 
studies in which you may choose to participate. You may discuss these alternatives with 
your doctor and decide whether to participate in this study. Please note that some 
treatments are forbidden during the course of this study. Therefore, before you start any 
new medication you must notify your doctor responsible for the study at the NIH. Your 
doctor will tell you if anynew information about AG- 348 become available during the 
course of the study 
DISCUSSION OF FINDINGS 
New information about the study 
If we find out any new information that may affect your choice to participate in this 
study, we will get in touch with you to explain what we have learned. This may be 
information we have learned while doing this study here at the NIH or information we 
have learned from other scientists doing similar research in other places. 
Return of research results 
You will be provided with the results of the standard clinical labs but the results of our 
research studies will not be provided to you or your referring doctor. 
EARLY WITHDRAWAL FROM THE STUDY 
Discontinuing Participation: 
- 
If you decide to stop participating in this study, you may request this by either 


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informing the investigators or by writing to the research team to the address at the 
end of this consent (under Problems and Questions on last page of consent). You 
will not be asked for further information or samples. 
- 
If we are unable to contact you or reach you. 
- 
When the study has completed. 
Discontinuing Treatment (You can still be part of the study and will be followed until end 
of study): 
- 
If the study drug affects the safety of your health or if the study doctor decides 
that it is in your best interest for you to be off the drug. 
- 
Pregnancy or unwillingness to use acceptable forms of contraception. 
Research Subject’s Rights: You are free to refuse to undergo any of these tests. You may 
withdraw from the study at any time. If you decide to participate and later change your 
mind, you are free to stop participation at any time. You will not be penalized or lose 
benefits to which you are otherwise entitled. Refusal to participate will not affect your 
legal rights or the quality of health care that you may receive at this center. 
Explanation of Conditions for Early Withdrawal: If new previously undisclosed 
information emerges during the study that would exclude you from the study, the 
investigators looking after you will discuss these with you. 
WHAT WILL HAPPEN WHEN THE RESEARCH STUDY IS OVER? 
The data from this study will be preserved for future use until it is no longer of scientific 
value. Future research use of data not defined in the research protocol may occur only 
after IRB review and approval or an exemption from the NIH Office of Human Subjects 
Research Protections (OHSRP). Refusal of a research subject participant to permit future 
use of data will be honored. 
CONFIDENTIALITY PROTECTIONS PROVIDED IN THIS STUDY 
When results of an NIH research study are reported in medical journals or at scientific 
meetings, the people who take part are not named and identified. In most cases, the NIH 
will not release any information about your research involvement without your written 
permission. However, if you sign a release of information form, for example, for an 
insurance company, the NIH will give the insurance company information from your 
medical record. This information might affect (either favorably or unfavorably) the 
willingness of the insurance company to sell you insurance. 
Will my medical information be kept private? 
We will do our best to make sure that the personal information in your medical record 
will be kept private. However, we cannot guarantee total privacy. Organizations that may 
look at and/or copy your medical records for research, quality assurance, and data analysis 
include: 
• 
The Food and Drug Administration (FDA) and the Office for Human Research 


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Protections, which are involved in keeping research safe for people. 
• 
National Institutes of Health Intramural Institutional Review Board 
This study is protected by a Certificate of Confidentiality. The researchers with this 
certificate may not release or use data or information about you under certain 
circumstances. Please refer to the OTHER IMPORTANT INFORMATION section near 
the end of this consent form for more information on this type of protection. 
WILL YOU SAVE MY SAMPLES OR DATA FOR USE IN OTHER RESEARCH 
STUDIES? 
As part of this study, we are obtaining specimens and data from you. We plan to use these 
specimens and data for studies going on right now, as well as studies in the future. These 
studies may provide additional information that will be helpful in understanding sickle 
cell disease or other diseases or conditions. This could include studies to develop other 
research tests, treatments, drugs, or devices, that may lead to development of a 
commercial product by the NIH and/or its research or commercial partners. There are no 
plans to provide financial compensation to you if this happens. Also, it is unlikely that we 
will learn anything from these studies that may directly benefit you. By agreeing to let us 
use your specimens and data, you give the NIH any rights you may have in the specimens 
and data. 
We may share your specimens and data with other researchers. They may be doing 
research in areas similar to this research or in other unrelated areas. These researchers 
may be at NIH, other research centers and institutions, or industry sponsors of research. 
We may put your research data in a large database which will be freely available to the 
public that anyone can use. These data repositories might or might not be located at the 
NIH. This data is intended for other researchers to use and learn from but anyone can gain 
access to it, including law enforcement. The information in this database could include 
but is not limited to genetic information, ethnicity and sex. This public information will 
not be labeled with your name or other information that could be used to easily identify 
you. This information when combined with information from other public sources could 
be used to identify you, though we believe it is unlikely that this will happen. 
In addition to the use and sharing of your specimens and data described above, we might 
remove any information from your specimens and data that can identify you such as name, 
address, or medical record number, and then use the specimens and data for additional 
research studies at the NIH or other places. If we do this, we might not contact you to ask 
your permission or otherwise inform you. 
If you change your mind and do not want us to store and use your specimens and data for 
future research, you should contact the research team member identified at the top ofthis 
document. We will do our best to comply with your request but cannot guarantee that we 
will always be able to destroy your samples. For example, if some research with your 
specimens and data has already been completed, the information from that research may 
still be used. Also, for example, if the specimens and data have been shared already with 
other researchers, it might not be possible to withdraw the specimens and data. 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY 
PATIENT IDENTIFICATION 
Consent to Participate in a Clinical Research Study 
NIH-2977 (4-17) 
File in Section 4: Protocol Consent (1) 
Version Date: 9/1/2020 
lRB NUMBER: 19H0097 
lRB APPROVAL DATE: 09/04/2020 
Page 14 of 19 
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How Long Will My Samples and Data be Stored by the NIH? 
Your blood samples will be stored at NIH indefinitely. 
Risks of Storage and Sharing of Samples and Data 
When we store your samples and data, we take precautions to protect your information 
from others that should not have access to it. When we share your samples and data, we 
will do everything we can to protect your identity by removing information that can 
identify you. Even with the safeguards we put in place, we cannot guarantee that your 
identity will never become known or someone may gain unauthorized access to your 
information. New methods may be created in the future that could make it possible to re- 
identify your data or samples. 
COMPENSATION, REIMBURSEMENT, AND PAYMENT 
Will I receive compensation for participation in the study? 
You will receive the following compensation your participation in this study for your time 
and inconvenience. The amount of compensation, if any, is guided by NIH policies and 
guidelines. 
 
 
Procedure(s)/Test(s) 
IU* 
Amount 
Frequency 
Total amount 
EKG 
1 
$10 
2 
$20 
Oxygen monitoring during inpatient stay 
1 
$10 
5 
$50 
Medical History and Physical Exam 
2.5 
$25 
8 
$200 
Urinalysis 
1 
$10 
2 
$20 
Blood Draw 
1 
$10 
9 
$90 
Blood Draw through an IV 
1 
$10 
30 
$300 
IV placement 
1 
$10 
5 
$50 
Diary 
1 
$10 
7 
$70 
Drug Administration, General 
2 
$20 
5 
$100 
Inpatient visit $40 per night 
N/A 
$40 
5 
$200 
OUTPATIENT- 1st HOUR 
NA 
$20 
4 
$80 
OUTPATIENT TIME- 
Not to Exceed More than 4 Hours 
 
3 
 
$30 
 
3 
 
$90 
TOTAL 
 
 
 
$1,270 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY 
PATIENT IDENTIFICATION 
Consent to Participate in a Clinical Research Study 
NIH-2977 (4-17) 
File in Section 4: Protocol Consent (1) 
Version Date: 9/1/2020 
lRB NUMBER: 19H0097 
lRB APPROVAL DATE: 09/04/2020 
Page 15 of 19 
CC pre rCR ICF template v. 05.05.2020 
 
 
 
The NIH has guidelines for the participation of its staff in research protocols. If you are a 
NIH staff member, please refer to the NIH Manual Chapter 2300-630-3 which details the 
NIH leave policy for NIH employees who participate in NIH protocols. Participants who 
are NIH staff members should be aware of these guidelines and discuss any questions they 
have with the research team. 
If you are unable to finish the study, you will receive compensation for the parts you 
completed as indicated in the table above. 
With few exceptions, study compensation is considered taxable income that is reportable 
to the Internal Review Service (IRS). A “Form 1099-Other Income” will be sent to you if 
your total payments for research participation are $600 or more in a calendar year. If you 
have unpaid debt to the federal government, please be aware that some or all of your 
compensation may be automatically reduced to repay that debt on your behalf. 
Will I receive reimbursement or direct payment by NIH as part of my participation? 
Some NIH Clinical Center studies offer reimbursement or payment for travel, lodging or 
meals while participating in the research. The amount, if any, is guided by NIH policies 
and guidelines. 
This study does not offer reimbursement for, or payment of, travel, lodging or meals. 
Will taking part in this research study cost me anything? 
NIH does not bill health insurance companies or participants for any research or clinical 
care that you receive at the NIH Clinical Center. 
• 
You will receive study treatment at no charge to you. This may include surgery, 
medicines, laboratory testing, x-rays or scans done at the Clinical Center, National 
Institutes of Health (NIH), or arranged for you by the research team to be done outside 
the Clinical Center, NIH if the study related treatment is not available at the NIH. 
• 
There are limited funds available to cover the cost of some tests and procedures 
performed outside the Clinical Center, NIH. You may have to pay for these costs if they 
are not covered by your insurance company. 
• 
Medicines that are not part of the study treatment will not be provided or paid for 
by the Clinical Center, NIH. 
• 
Once you have completed taking part in the study, medical care will no longer be 
provided by the Clinical Center, NIH. 
CONFLICT OF INTEREST 
The National Institutes of Health reviews NIH staff researchers at least yearly for 
conflicts of interest. The following link contains details on this process 
http://ethics.od.nih.gov/forms/Protocol-Review-Guide.pdf . You may ask your research 
team for additional information or a copy of the Protocol Review Guide. 
This protocol may have investigators who are not NIH employees. Non-NIH investigators 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY 
PATIENT IDENTIFICATION 
Consent to Participate in a Clinical Research Study 
NIH-2977 (4-17) 
File in Section 4: Protocol Consent (1) 
Version Date: 9/1/2020 
lRB NUMBER: 19H0097 
lRB APPROVAL DATE: 09/04/2020 
Page 16 of 19 
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are expected to adhere to the principles of the Protocol Review Guide but are not required 
to report their personal financial holdings to the NIH. 
Cooperative Research and Development Agreement (CRADA) 
None of the researchers involved with this study have a direct financial interest related to 
this study. The NIH and members of the research team are making products to be usedin 
research. This means that it is possible that the results of this study could lead to payments 
to the NIH and to NIH scientists. By law, the NIH and government scientists are required 
to receive such payments for their inventions. You will not receive any money from the 
development of these products. 
Agios Pharmaceuticals Inc. is providing the drug AG-348 for this study to NIH without 
charge. No NIH employee involved in this study receives any payment or other benefits 
from Agios Pharmaceuticals Inc. 
CLINICAL TRIAL REGISTRATION AND RESULTS REPORTING 
A description of this clinical trial will be available on http://www.clinicaltrials.gov, as 
required by U.S. Law. This website will not include information that can identify you. At 
most, the website will include a summary of the results. You can search this website at 
any time. 
OTHER IMPORTANT INFORMATION 
Confidentiality 
When results of an NIH research study are reported in medical journals or at scientific 
meetings, the people who take part are not named and identified. In most cases, the NIH 
will not release any information about your research involvement without your written 
permission. However, if you sign a release of information form, for example, for an 
insurance company, the NIH will give the insurance company information from your 
medical record. This information might affect (either favorably or unfavorably) the 
willingness of the insurance company to sell you insurance. 
If we share your specimens or data with other researchers, in most circumstances we will 
remove any information that shows your identity before sharing them. You should be 
aware that there is a slight possibility that someone could figure out the information is 
about you. 
To help us protect your privacy, the NIH Intramural Program has received a Certificate 
of Confidentiality (CoC). NIH researchers must use this Certificate to legally refuse to 
disclose information that may identify you in any federal, state, or local civil, criminal, 
administrative, legislative, or other proceedings, for example, if there is a court subpoena. 
NIH researchers will use the Certificate to resist any demands for information that would 
identify you, except as explained below. 
There are several circumstances in which the Certificate does not provide protection. 
These include when information: 
1. 
is disclosed to people connected with the research, for example, information may 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY 
PATIENT IDENTIFICATION 
Consent to Participate in a Clinical Research Study 
NIH-2977 (4-17) 
File in Section 4: Protocol Consent (1) 
Version Date: 9/1/2020 
lRB NUMBER: 19H0097 
lRB APPROVAL DATE: 09/04/2020 
Page 17 of 19 
CC pre rCR ICF template v. 05.05.2020 
 
 
be used for auditing or program evaluation internally by the NIH; or 
2. 
is required to be disclosed by Federal, State, or local laws, for example, when 
information must be disclosed to meet the legal requirements of the federal Food and Drug 
Administration (FDA); 
3. 
is necessary for your medical treatment and you have consented to this disclosure; 
4. 
is for other research; 
5. 
is disclosed with your consent 
In addition, identifiable, sensitive information collected or compiled during research and 
protected by this Certificate cannot be admissible as evidence or used for any purpose in 
any action, suit, or proceeding without your consent. You should understand that a CoC 
does not prevent you from voluntarily releasing information about yourself or your 
involvement in this research. If an insurer, medical care provider, or other person obtains 
your written consent for us to disclose the research information, then the researchers will 
not use the Certificate to withhold that information. 
The Certificate of Confidentiality will not be used to prevent disclosure to state or local 
authorities of harm to self or others including, for example, child abuse and neglect, and 
by signing below you consent to those disclosures. 
The protections of the Certificate apply to all copies of the identifiable, sensitive 
information collected or compiled during the research. Therefore, if an NIH investigator 
shares a copy of your identifiable, sensitive information with any other investigator or 
institution (whether a collaborator on this study, or researcher conducting secondary 
research in the future) that party must agree to comply with the disclosure restrictions 
under the Certificate of Confidentiality described above. 
Additionally, the Federal Privacy Act generally protects the confidentiality of your NIH 
medical records. In some cases, the Privacy Act protections differ from the Certificate of 
Confidentiality. For example, sometimes the Privacy Act allows release of information 
from your medical record without your permission, for example, if it is requested by 
Congress. However, NIH will only release information from your medical record if it is 
permitted by both the Certificate of Confidentiality and the Privacy Act. 
POLICY REGARDING RESEARCH-RELATED INJURIES 
The NIH Clinical Center will provide short-term medical care for any injury resulting 
from your participation in research here. In general, no long-term medical care or financial 
compensation for research-related injuries will be provided by the NIH, the NIH Clinical 
Center, or the Federal Government. However, you have the right to pursue legal remedy 
if you believe that your injury justifies such action. 
PROBLEMS OR QUESTIONS 
If you have any problems or questions about this study, or about your rights as a research 
participant, or about any research-related injury, contact the Principal Investigator, Swee 
Lay Thein, MD; sweelay.thein@nih.gov, 301-402-6699. You may also call the NIH 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY 
PATIENT IDENTIFICATION 
Consent to Participate in a Clinical Research Study 
NIH-2977 (4-17) 
File in Section 4: Protocol Consent (1) 
Version Date: 9/1/2020 
lRB NUMBER: 19H0097 
lRB APPROVAL DATE: 09/04/2020 
Page 18 of 19 
CC pre rCR ICF template v. 05.05.2020 
 
 
Clinical Center Patient Representative at 301-496-2626, or the NIH Office of IRB 
Operations at 301-402-3713, if you have a research-related complaint or concern. 
CONSENT DOCUMENT 
Please keep a copy of this document in case you want to read it again. 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY 
 
Adult Research Participant: I have read the explanation about this study and have been given the opportunity 
to discuss it and to ask questions. I consent to participate in this study. 
 
 
 
Signature of Research Participant 
Print Name of Research Participant 
Date 
 
 
Investigator: 
 
 
 
Signature of Investigator 
Print Name of Investigator 
Date 
 
 
Witness to the oral short-form consent process only: This section is only required if you are doing the oral 
short-consent process with a non-English speaking subject and this English consent form has been approved 
by the IRB for use as the basis of translation. 
 
Witness: 
 
 
 
Signature of Witness* 
Print Name of Witness 
Date 
 
 
 
 
*NIH ADMINISTRATIVE SECTION TO BE COMPLETED REGARDING THE USE OF AN 
INTERPRETER: 
 
    An interpreter, or other individual, who speaks English and the participant’s preferred language facilitated 
the administration of informed consent and served as a witness. The investigator obtaining consent may not 
also serve as the witness. 
 
  
 An interpreter, or other individual, who speaks English and the participant’s preferred language facilitated 
the administration of informed consent but did not serve as a witness. The name or ID code of the person 
providing interpretive support is:  
. 
 
 
 
 
 
 
 
 
 
 
PATIENT IDENTIFICATION 
 
 
 
CC ICF template v. 09.27.19 
Consent to Participate in a Clinical Research Study 
NIH-2977 (4-17) 
File in Section 4: Protocol Consent (1) 
Version Date: 09/01/2020 
Page 19 of 19 
lRB NUMBER: 19H0097 
lRB APPROVAL DATE: 09/04/2020