Evaluation of the ex vivo effect of AG-348 treatment on pyruvate kinase-deficient cell metabolism and erythroid development

Pyruvate kinase deficiency (PKD) is an autosomal recessive enzymopathy that is
the most common cause of hereditary nonspherocytic hemolytic anemia (HNSHA). It
is a rare disease characterized by a life-long chronic hemolysis with severe
co-morbidities, in particular iron overload. The mature red blood cell is
completely dependent on glycolysis for it*s energy supply. Red blood cell
pyruvate kinase (PK-R) is a key regulatory enzyme of red cell glycolysis. PKD
red cells are characterized by changes in metabolism associated with defective
glycolysis, including a build-up of the PK-R substrate phosphoenolpyruvate
(PEP) and deficiency in the PK-R product adenosine triphosphate (ATP). It is
hypothesized that insufficient energy production affects red cell membrane
homeostasis, thereby promoting premature removal of the PK-deficient red blood
cell from the circulation. Clinical symptoms of PK deficiency are usually
limited to patients who are compound heterozygous or homozygous for a mutation
in PKLR. The phenotypic expression is highly variable, and we and others have
found that there is a poor correlation between the genotype and the phenotype.
Treatment of PKD patients is generally supportive, focusing on the resultant
anemia and iron overload state, and there are no approved drugs that directly
target mutated pyruvate kinase. Agios Pharmaceuticals recently developed and
reported on the mechanism of action and in vitro cellular effects of AG-348, an
allosteric activator of PK-R. They demonstrate that AG-348 can potently
activate a spectrum of recombinantly expressed PK-R mutant proteins, including
mutations that span distinct subdomains of the enzyme. The binding of AG-348
attenuates the thermostability defect of several mutant alleles of PK-R,
including the commonly observed p.(Arg510Gln) mutant.

Pilot ex vivo studies on PKD red cells exposed to AG-348 showed that these
cells had increased PK-R enzyme activity (up to 4-fold over control) and showed
consistent dose and time-dependent metabolic responses, including sharp
reductions in PEP (up to 70% compared to control) and increases in ATP levels
(up to 100% over control). In these ex-vivo settings, ATP levels in AG-348
treated cells can reach levels that are typical of normal, non-PKD red cells.

Altogether, these data support the hypothesis that drug intervention with
AG-348 in PKD red cells may restore glycolytic pathway activity and normalize
red cell metabolism in vivo. This therapeutic approach may therefore be an
effective way to correct the underlying pathology of PKD and, potentially,
provide clinical benefit to PKD patients. In this respect, Agios
Pharmaceuticals recently initiated two Phase 1, single-center, randomized,
double-blind, placebo-controlled clinical trials to assess the safety and
tolerability of AG-348 through dose escalation in healthy adult men and women
(http://www.agios.com/pipeline-pkr.php).

To extend and strengthen the initial pilot observations on PKD red blood cells,
and thereby further establish the activity of AG-348 treatment for PKD, UMC
Utrecht and Agios Pharmaceuticals will collaborate in this pre-clinical study.
In particular, the goal is to test the activity of AG-348 in restoring red
blood cell metabolism and cellular function in a range of different genetic
backgrounds. This is of great importance as there are only few relatively
common mutations in PKD. Hence, most patients are compound heterozygous for a
unique combination of two rare mutations. Depending on the type and location of
the mutations the response to AG-348 treatment may differ. The primary
objective of this study will therefore focus on the question if and how the PKD
genotype influences the response to AG-348 treatment. UMC Utrecht is a national
and international acknowledged Center of Expertise for PKD. We have diagnosed
more than 100 PKD patients in the last two decades, and have studied many of
them on the biochemical and genetic level. Our cohort of PKD patients
represents one of the biggest PKD cohorts in the world. All PKD patients have
been genotyped. This enables efficient selection of a variety of genotypes to
be study with regard to the primary objective.
In addition to studying these direct effects of AG-348 on PKD red blood cells,
the secondary objective of this study comprises the investigation of the effect
of AG-348 on ex vivo erythropoiesis in a PK-deficient background. Currently, it
is not known if, and how, AG-348 affects erythropoiesis in PKD patients. This
is of considerable importance, because ineffective erythropoiesis represents
one of pathophysiological features of PKD. It seems reasonable to assume that
any activation of mutant PK proteins by AG-348 may also occur on the level of
the developing red blood cell. Alternatively, PKD patients whose red blood
cells fail to respond to AG-348 treatment may show an effect of treatment on
the level of erythropoiesis, because ineffective erythropoiesis in PKD seems
not related to lack of metabolic energy but, rather, to increased levels of
apoptosis in PKD erythroid progenitor cells.

Ultimately, this pre-clinical study will provide a broad dataset by which to
evaluate the question of which aspects of AG-348 ex-vivo activity translate to
clinical efficacy. This study will substantially increase the understanding of
the mode of action of AG-348 treatment for PKD red blood cells, both on the
cellular level, as well as on the level of erythropoiesis. We anticipate that
the results will support the hypothesis that AG-348 treatment represents an
effective and attractive way to correct the underlying pathologies of PKD,
ultimately providing clinical benefit to PKD patients.


Primary Objective: To evaluate the ex vivo effect of AG-348 on cell metabolism
and erythropoiesis in red cells from PKD patients.

Secondary Objective(s): To establish baseline parameters in PKD patients
compared to healthy control individuals.

This study will be conducted as an observational case-control study, consisting
of 2 stages.

Stage 1.
Blood samples (36 mL) will be collected from 15 PKD patients, and an equal
number of healthy human volunteers as control. They latter will be recruited by
the Minidonordienst of the UMC Utrecht. After collection, baseline values will
be obtained from both patients and controls. This includes the measurement of
general haematological laboratory parameters (e.g. hemoglobin, hematocrit,
reticulocyte count), red blood cell morphology, osmotic gradient ektacytometry
(to measure red blood cell deformability: a measure of overall cellular
integrity and functionality), metabolite profile (including 2,3-DPG and ATP
levels), PK antigen levels, and levels of PK-R and PK-M2 protein expression.
After baseline evaluation, samples will be incubated either with or without
AG-348 for a total of 24 hours. At specific time points measurements will be
performed to study the effect of AG-348 on red blood cell metabolism, red cell
integrity, and the PK protein itself. Certain experiments will use whole blood
whereas the more in depth analysis of red blood cell PK and its characteristics
will require purified erythrocytes as a source because of the presence of
pyruvate kinase activity in white cells.

Stage 2.
Based on their response to AG-348 treatment, 5-10 PKD patients will be selected
for stage 2 of this study. Patients whose red blood cells strongly respond to
AG-348 treatment, as well as PKD patients that show little or no response at
all will be included in this stage which comprises the investigation of any
effect AG-348 treatment may have on erythropoiesis in PKD. Both patient groups
are of interest for this part of the study, because AG-348 treatment may have
an effect on red blood development in PKD that is independent (and perhaps
different) from the direct effect AG-348 treatment has on PKD red blood cells.
Furthermore, it is unclear which aspects of AG-348 ex-vivo activity will
ultimately correlate with in vivo clinical efficacy. For stage 2, a single
amount of blood (70 mL) will be collected on a separate occasion from selected
patients as well as an equal number of healthy human volunteers. Peripheral
blood mononuclear cells will be isolated and cultured ex vivo into erythroid
cells (i.e. reticulocytes) in presence or absence of AG-348. Erythroid
development will be monitored by FACS analysis of erythroid differentiation
markers. After completion of the culture (approximately 21 days), PK activity
and stability will be measured, and PK-M2 and PK-R levels will be evaluated by
Western blot analysis.

The duration of the study is approximately 18 months. It will be conducted by
the Laboratory for Clinical Chemistry and Haematology of UMC Utrecht. All
measurements will be performed at this Department, except for red cell
metabolite analyses. These will be performed at the laboratory of Agios
Pharmaceuticals (Cambridge, MA, USA). For this, concerning aliquots of
processed blood samples of PKD patients will be shipped to Agios
Pharmaceuticals for the purpose of being analysed and used for this study only.

Patient participation comprises the donation of a limited amount of blood.
According to the EMA (European Medicines Agency) and WHO (World Health
Organization) guidelines and standards it is safe to withdraw a general maximum
of 2.4 ml of blood / kg. Even when considering very conservative mean average
weights of adult males and females (64 and 51 kg, respectively) the associated
maximum volumes (154 and 122 ml, respectively) are well above the maximum
volume of blood to be collected for this study (36 ml in stage 1 and 70 ml in
stage 2). Physical discomfort is limited but may include bruising.
Due to the fact that participation in this study requires only either a single
donation of blood, or two donations of blood on separate occasions (separated
by at least 2 months) we consider the participation of patients and human
healthy volunteers in this study to be without risks.