A Phase 2, Open Label, Randomized, Dose Ranging, Safety, Efficacy, Pharmacokinetic and Pharmacodynamic Study of AG-348 in Adult Patients with Pyruvate Kinase Deficiency

Pyruvate Kinase (PK) deficiency with its attendant chronic hemolytic anemia,
transfusion therapy, frequent treatment in early childhood by splenectomy, and
iron overload is reasonably regarded as a serious medical condition. Any
therapy that could reduce the need for blood transfusion support, reduce the
frequency of splenectomy, and minimize iron overload would be a meaningful
benefit to affected patients.

The objectives of this study are to evaluate the safety, tolerability,
pharmacokinetics (PK) and pharmacodynamics (PD), and indicators of clinical
activity of AG-348 in patients with PK deficiency. The choice of dose and
schedule of administration of AG -348 for Arms 1 and 2 was based on the highest
safely tolerated dose (Arm 1: 300 mg BID) and the lowest dose with potentially
relevant PD activity (Arm 2: 50 mg BID) from the forerunner AG348-C-002
multiple ascending dose (MAD) study in healthy volunteers exposed to 14 day
dosing. Decisions regarding continuing enrollment and treatment in will be
based on Data Review Team (DRT; composed of treating investigators known for
their prominence and expertise in hematology and medicine) review of safety, PK
and PD data, and indicators of clinical activity collected from all patients.
The DRT will execute their duties every 6 weeks, and ad hoc as needed,
throughout the study. This design was chosen to minimize risk to patients while
allowing evaluation of safe and pharmacologically active dose levels of AG-348,
and to allow the necessary flexibility to adjust dose and schedule should the
safety, tolerability, PK, and/or PD be different in patients with PK deficiency
compared with healthy volunteers. Pre-testing of AG-348 in healthy adult
volunteers provided important safety and dosing information to minimize risk to
patients in the proposed study.

The present safety profile of AG-348 derived from two studies in healthy adult
volunteers; the significant salutary pharmacodynamic effects on the glycolytic
pathway observed in healthy volunteers at well tolerated doses of AG-348; and
the rigorous safety monitoring (DRT) in place for the proposed study are deemed
appropriate to mitigate and justify the potential risk of study participation
for patients with a disease as serious as PK deficiency

CORE PERIOD

Primary:
* Evaluate the safety and tolerability of up to 24 weeks of AG-348
administration in patients with pyruvate kinase deficiency (PK deficiency).

Secondary:
* Evaluate the pharmacokinetics (PK) of AG-348 and the metabolite AGI-8702.
* Evaluate the pharmacodynamic (PD) response of adenosine triphosphate (ATP)
and 2,3 diphosphoglycerate (2,3-DPG) after administration of AG-348.
* Evaluate indicators of clinical activity of AG-348 in patients with PK
deficiency, including changes in hemoglobin (Hb), hematocrit (HCT),
reticulocyte count, haptoglobin (Hp), carboxyhemoglobin (COHb), end tidal
carbon monoxide (ETCO), lactate dehydrogenase (LDH), total and indirect
bilirubin, erythropoietin (EPO), hepcidin, ferritin, and transferrin saturation
(serum iron/iron binding capacity).

Exploratory:
* Evaluate the relationship of additional PD biomarkers including pyruvate
kinase R (PKR) activity assay, glycolytic flux assays, and total PKR protein
levels in whole blood, after administration of AG 348.
* Evaluate the relationship of AG-348 exposure and PD effects with changes in
indicators of clinical activity.

EXTENSION PERIOD

Primary:
* Evaluate the long-term safety and tolerability of up to 4,5 years of AG-348
administration in patients with PK deficiency.

Secondary:
* Evaluate the PK of AG-348 and the metabolite AGI-8702.
* Evaluate the PD response of ATP and 2,3-DPG after administration of AG-348.
* Evaluate indicators of clinical activity of AG-348 in patients with PK
deficiency, including changes in Hb, HCT, reticulocyte count, Hp, COHb, LDH,
total and indirect bilirubin, EPO, ferritin, and transferrin saturation (serum
iron/iron binding capacity)
* Evaluate optimal maintenance dose of AG-348 for each individual subject
during the extension period.

Exploratory:
* Evaluate the relationship of total PKR protein levels in whole blood after
administration of AG-348.
* Evaluate the relationship of AG-348 exposure and PD effects with changes in
indicators of clinical activity.

Study AG348-C-003 is a Phase 2, open label, two arm, multicenter, randomized,
dose-ranging study in adult patients with PK deficiency;the study will be
divided in to a Core Period and an Extension Period. During the Core Period,
patients will receive multiple doses of AG 348 for up to 24 weeks; patients
who are eligible can enter the Extension Period to receive AG-348 for up to 4
years following the end of the Core Period. Patients with PK deficiency
confirmed by red blood cell (RBC) PK enzymatic assay performed at Screening
will be eligible to participate in this study. At Week 25, patients who safely
tolerate AG 348 and demonstrate clinical activity of AG 348 may be eligible to
immediately roll over to the Extension Period for continued treatment. Patients
who complete treatment at the end of the Core Period (24 weeks) will undergo
follow-up assessment 4 weeks after the last dose of study drug. If a patient
discontinues at any other time (including early discontinuation or
discontinuation during the Core or Extension Period), the follow-up assessments
will be conducted 4 weeks after discontinuation.
Patients with toxicity suspected to be related to study drug will continue
follow-up until the adverse event (AE) resolves, is declared chronic by the
Investigator, or the patient is lost to follow-up.
For the Core Period, up to 25 patients will be initially randomized on an
open-label 1:1 basis to each of two twice-daily (BID) doses of AG-348 (up to 50
patients total; see Figure 1, Study Schema). The dose of Arm 1 is 300 mg of
AG-348 administered orally (PO) every 12 hours (q12h, BID). The dose of Arm 2
is 50 mg of AG 348 administered PO q12h (BID). Randomization will be stratified
by PKR mutation in order to maintain balance as much as possible across the
dose arms for the specific mutations expected to be most frequently enrolled.
The PKR mutation stratification factor will consist of 4 levels (R510Q, R486W,
and R479H) and all other mutations (*other*). Mutation status is defined by the
presence of at least one of the indicated mutations; patients with more than
one stratified mutation will be assigned based on Sponsor*s discretion.
The doses for each arm of the Core Period have been selected from the
AG348-C-001 single ascending dose (SAD) and AG348-C-002 multiple ascending dose
(MAD) studies in healthy adult volunteers to represent the range of
doses/exposures that were safely tolerated and resulted in maximal or
near-maximal PD effects on 2,3-DPG and ATP.
Because PK deficiency is a rare disease with a limited eligible patient
population and because the underlying pathophysiology and clinical phenotype of
affected patients is heterogeneous due to the wide variety of mutations in PKR
that cause the disease, it is important to focus closely on dose findings in
this first-in-patient study. Therefore, in addition to initiating this study
with 2 different doses of AG-348 administered q12h, a Data Review Team (DRT)
will be established to review study data on a regular basis and adapt the study
design, dose and schedule of AG-348.
The DRT will monitor safety on an on-going basis and meet at regular intervals
of approximately every 6 weeks, or ad hoc, as necessary, for as long as any
patients are still in the Core Period to review AEs, vital signs (VS), clinical
laboratory assessments (hematology, clinical chemistry, coagulation, and
urinalysis), and electrocardiograms (ECGs). The DRT will also review available
PK/PD data and indicators of clinical activity (e.g., changes from baseline in
Hb). These DRT meetings will also include data review for all patients that may
be under treatment in the Extension Period. If there are no patients still
being treated in the Core Period, and the only patients being treated are those
in the Extension Period, then the frequency of the DRT meetings will reduce to
approximately every 3 months in order to match the frequency of patient visits
(and new data collection) in the Extension Period.
The DRT will be comprised of the study Coordinating Investigator, treating
Investigators, Medical Monitor, Clinical Pharmacologist, Statistician, and
Sponsor*s Responsible Medical Officer.
Beginning 6 weeks after the first patient is dosed in the Core Period or ad hoc
as necessary, and proceeding according to the schedule indicated above
(approximately every 6 weeks during the Core Period, and approximately every 3
months during the Extension Period once all patients have completed the Core
Period), the DRT will review cumulative safety data, available PK/PD data, and
clinical activity data. Based on the DRT*s recurring reviews, the DRT may
exercise one or more of the following options during the Core Period:
* Continue treatment and enrollment in existing arms without change.
* Add 1 new dose arm (Arm 3) to enroll up to 25 patients at a dose to be
determined; the dose for Arm 3 may be lower or higher than Arm 1 and Arm 2
doses, but will not exceed 360 mg q12h; and the dose regimen may be less
frequent than q12h.
* Terminate or suspend enrollment to allow further review of clinical data in
Arm 1 and/or Arm 2 (and/or potential Arm 3). Enrollment in an arm could be
terminated or suspended to allow further review, for example, for unacceptable
safety/tolerability, poor PD response, or lack of signs of clinical activity.
* Re-assign patients* doses and schedule in a terminated arm to match the dose
and schedule of another arm of the study. In this case, the patients in the
terminated arm will remain in their original arm, i.e., they will not count
towards the enrollment quota of the arm whose dose and schedule is being
adopted.
* Implement specific genotype restrictions to enrollment in one or more arms to
ensure representation of patients with genotypes of greatest clinical relevance.

The DRT may exercise one or more of the following options during the Extension
Period:
* Continue treatment without change;
* Re-assign patients* doses and schedule to an existing dose and schedule that
has been determined to be safer, and/or have a better PD response, or produce
signs of clinical activity;
* Terminate or suspend treatment to allow further review of clinical data (eg,
for unacceptable safety/tolerability, poor PD response, or lack of signs of
clinical activity).

The data that the DRT will review to make these decisions is expected to
include, but are not necessarily limited to, the following:
* Safety Observations: all AEs; VS, clinical laboratory (hematology, clinical
chemistry, coagulation, and urinalysis), and ECG;
* PK and PD Observations: including changes in 2,3-DPG and ATP;
* Indicators of Clinical Activity: including changes in Hb, HCT, reticulocyte
count, Hp, COHb, ETCO (Core Period only), LDH, EPO, total and indirect
bilirubin, hepcidin, ferritin, and transferrin saturation.
If a third dose arm is implemented, the dose of AG-348 selected will not exceed
360 mg BID, as this was the highest dose that demonstrated acceptable safety
and tolerance in the 14-day multiple BID dosing study in healthy volunteers.
The PK/PD sampling schedule in a potential third dosing arm will be determined
by Sponsor*s discretion and may follow either the extensive or limited PK/PD
sampling schedules as specified.
The DRT will monitor the data in an ongoing manner as described and may make a
decision to terminate enrollment in an arm if unacceptable AEs are observed, PD
activity is limited, and/or markers of clinical activity indicate no effect.
Unacceptable AEs are defined as * Grade 3 AEs (using National Cancer Institute
Common Terminology Criteria for Adverse Events [NCI CTCAEv4.03]) that are
assessed as at least possibly related to AG 348, with the exception of Grade 3
headache, hot flash/flushing, nausea, vomiting, and/or diarrhea that is
transient in duration (< 24 hours) or able to be medically managed to * Grade 2
within 24 hours. The DRT may also exercise discretion to terminate enrollment
in an arm if it observes a frequency of Grade 2 AEs that would make it
difficult to support long-term dosing.

Patients in the Extension Period will continue on the dose they were randomized
to in the Core Period, unless the DRT had reason to establish a different
dose/schedule during the course of the Core Period (the DRT will not propose a
dose higher than 360 mg q12h), or unless the treating Investigator exercises
the option for intra-patient dose escalation (see Section 9.7.2).

Due to the potential for AG-348-mediated aromatase inhibition, dual-energy
x-ray absorptiometry (DXA) scan (hip and spine) will be performed at Screening
(if patient has not had prior DXA scan within 3 months of Day 1) to obtain T
and Z scores. These data are intended to serve as a baseline measure of bone
mineral density for all enrolling patients, and are deemed of particular
importance for those who enter the longer term Extension Period after
completing 24 weeks of treatment (Core Period). All patients will have a second
DXA scan in the interval between Weeks 24 and 28 for theCore Period. Patients
in the Extension Period will have additional DXA scans at Months 12,18, 24, 30,
42, 54 and 55.
As the number of enrolling arms changes in the study (for example, from 2 to
3), the randomization scheme will adjust to enable balanced randomization into
each actively accruing arm. Randomization and stratification will cease in the
event that only a single arm is left enrolling.
Depending on possible early termination of 1 or both of the initial 2 arms, or
the addition of a third arm, the study could enroll up to a maximum of 75
patients.

Gradual dose taper period
At the 1st scheduled visit of the patient, the study doctor will explain the
gradual dose taper process and start the 1st step.
One week and 2 weeks after the patient have started on a lower dose, blood
collection for blood count and haptoglobin will be done at the study site, or,
if the patient wishes and the study doctor agrees, at patient's home by a
visiting nurse, or at the local doctor*s office, or at a local laboratory.
Three weeks after thet patient have started on a lower dose, the patient will
come back to the study to allow the doctor to assess how the patient is doing
and decide whether the dose can be decreased further or should be increased
back to its previous level.

AG-348 sulfate hydrate capsules will be provided as 5 mg, 25 mg or 100 mg
(free-base equivalent) of AG-348 sulfate hydrate without excipients in dark
green opaque (5 mg), Swedish orange (25 mg) or white opaque (100 mg) gelatin
capsules (size 2 capsules for all dose strengths). AG-348 will be administered
PO BID. The number of capsules per dose will vary by assigned dose group.
AG-348 will be administered with water and may be administered with or without
food.

There are now 2 different formulations of AG-348, capsules and tablets. While
this study (AG348-C-003) was started with the capsule formulation, the tablet
formulation will be introduced to gradually replace the capsule formulation.

No dose adjustment is required following a switch to the tablet formulation.
When the patient will switched from receiving AG-348 capsules to AG-348
tablets, the same dose of tablet as the capsule dose should be administered
(f.e. if the patient is taking 25 mg capsules, for each 25 mg capsule the
patient was taking before, he/she will take one 20 mg tablet and one 5 mg
tablet). The patient will be switched from receiving the capsule to the tablet
formulation of AG 348 when the supply of the capsule formulation is exhausted
or sooner as deemed appropriate by the Investigator and Medical Monitor.

Patients who are eligible can enter the Extension Period to receive AG-348 for
up to 4 years following the end of the Core Period.

Patients are asked to undergo procedures described in the flowchart of the
study protocol. These procedures
include physical examination, vital signs, urine pregnancy tests
(female;chidbearing patients, ECG, DXA scan, extended lab tests,completing
questionnaire, diaries and adminsitration of study drug (oral). Additionally,
fertile patients who are sexually active must agree to use an effective form of
contraception with their sexual partners throughout participation in the study.
Patients are also asked to inform their study doctor on their medication use
and change in health status.
For side effects see section E9.