The Pyruvate Kinase Activator Mitapivat Ameliorates Anemiaand Prevents Iron Overload in a Mouse Model of

Hereditary SpherocytosisMatte A,1 Wilson AB,1 Federti E,1 Kung C,2 Kosinski P, 2 Riccardi V, 1 Iatcenko I,1 Dang L,2 Lebouef C,3

Janin A,3 Brugnara C,3 Mohandas N,4 De Franceschi L 1

1Dept of Medicine, University of Verona &AOUI Verona; Italy; 2Agios Pharmaceuticals Inc., Cambridge, MA; 3Universtity Diderot of Paris, Paris, France; 4Dept of Laboratory Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, MA; 5 Laboratory of Red Cell Physiology, New

York Blood Center, New York, NY.

Hereditary Spherocytosis (HS)

• Hereditary Spherocytosis (HS) is the most common hemolytic anemia due to inherited RBC membrane defects with a prevalence estimated from 1: 2,000 to 1: 5,000 births.

• HS is due to mutation on genes encoding for red cell membrane or cytoskeleton proteins such as ankyrin, band 3, band 4.2 or α-, β-spectrin.

• HS clinical presentation is characterized by hemolytic anemia, reticulocytosis, jaundice, cholelithiasis and splenomegaly.

Iolascon A et al. BJH, 2019; Mohandas N et al. Blood, 2008

HS red cells are characterized by membrane mechanical and metabolic instability

• In HS, the absence/reduction in one of the membrane/cytoskeleton key proteins promotes membrane mechanical instability, resulting in:– red cell membrane exposure of phosphatidylserine (PS), – release of erythroid microvesicles,– generation of spherocytes.

• HS red cells display decrease ATP content when exposed to oxidation or after 24hr incubation. Iolascon A et al. BJH, 2019; Bozzi A et al Biochem Mol Biol Int 32: 95-103, 1994; De Jong K et al Blood 94: 319,

1999; Mohandas N et al. Blood 112: 3939, 2008

The oral PK activator Mitapivat improves anemia in PK deficiency and thalassemia

• Mitapivat (AG-348) is an oral small-molecule activator of pyruvate kinase.

• Two phase 2 clinical trials of mitapivat, one in adult patients with pyruvate kinase deficiency and the other in non transfusion-dependent thalassemia, demonstrated rapid and sustained increase in Hb levels

• Mitapivat ameliorates murine β-thalassemic anemia with a beneficial effect on iron homeostasis.

Grace RF et al. NEJM 381: 933, 2019; Yang H et al. Clin Pharmacol Dev 8: 246, 2019; Kung C et al Blood 130: 1347-1356, 2017; Matte A et al. Haematologica 101:18, 2016; Kuo KHM, et al. HemaSphere. 2020;4(S1):Abstract S297.

Aim of the study

To Investigate the effects of the PK activator, Mitapivat, on red cell metabolism and hematologic phenotype of

band 4.2-/- mice, a model of HS.

Study design• Two to eight months-old female Band 4.2-/- and C57BL6/Jmice were used (n=6-11 animals in each group);

• Mitapivat (100 mg/Kg/d) was administrated for 6 months;

• CBC and reticulocytes were determined with a Sysmex Hematology Analyzer;

• Erythroid Annexin-V positivity was evaluated by FACS;

• Perls’ staining was carried out on fixed spleen and liver;

• Spleen and liver iron concentration were measured;

• Immunoblot analysis was carried out on mouse red cells.

0 2 4 6 80.6

0.7

0.8

0.9

1.0

1.1

Hb/R

DW ra

tio

**

*° *°*°

0 2 4 6 80

4

8

10

12

Ret

ics

(%)

0 2 4 6 835

40

45

50

()

Mitapivat ameliorates the anemia of band 4.2-/- mice

*° *°*°

* * **

0 2 4 6 88

10

12

14

16

Hb

(g/d

l)

** *

*

°° *

* *

*° *°*°

* *

Band 4.2-/- Mitapivat

Band 4.2-/- Vehicle

WT

Hct

(%)

*

Time (months)

*p<0.05 compared to WT mice°p<0.05 compared to vehicle treated mice

0

500

1,000

1,500

Mitapivat reduces hemolysis in band 4.2-/- mice

EPO

(pg/

ml)

*°

*

CD

44+T

er11

9+Fs

c H

igh (

cell*

107 )

0

1

2

3

4

5

0

2

4

6

8

10

Bone marrow Spleen

*

°

CD

44+T

er11

9+Fs

c H

igh (

cell*

107 )

Tota

l Bili

rubi

n(m

g/dL

)

*°

*

LDH

(U

/L)

Indices of Hemolysis EPO

*°

*

*p<0.05 compared to WT mice°p<0.05 compared to vehicle treated mice

Mitapivat improves 4.2-/- mouse red cell membrane mechanical stability

Anne

xin

V+ R

BC

s (%

)

0

1

2

3

*°

*

D

TWb:

β-Spectrin

KCC-1

Blanc L et al Biochemistry 49: 4516, 2010; An X et al. JBC 277: 31796, 2002; Lutz HU et al Frontiers Physioldoi:org/10.3389/fphs.2013.00387, 2013; Zaninoni A et al. Frontiers Immunol doi org 10.3389/fimmu.2020.01389, 2020; Reliene R et

al Blood 100: 2208, 2002

0

1

2

3

4

Nat

ural

occ

urrin

g Ab

s

posi

tive

RB

Cs

(%)

*°

*

*p<0.05 compared to WT mice°p<0.05 compared to vehicle treated mice

In 4.2-/- mice, Mitapivat reduces the release of erythroid microvesicles

0

1

2

4

3

Eryt

hroi

d m

icro

vesi

cles

(Fol

d of

WT)

75~

135~

Kda245~

25~35~

48~

63~

20~

Wb: Band-3

Prx-2

Erythroid microvesicles and protein composition

In band 4.2-/- mice, Mitapivat reduces splenomegaly and spleen iron-overload

Band 4.2-/- Vehicle

Band 4.2-/- Mitapivat

H&

EH

&E

Perls

Perls

Sple

nic

Iron

Con

tent

(n

g/m

g of

dry

tiss

ue)

0

500

1,000

1,500

2,000

*°*

SIC

0

2

4

6

8

10

Sple

en w

eigh

t/Mou

se w

eigh

t Rat

io

*°

*

*p<0.05 compared to WT mice°p<0.05 compared to vehicle treated mice

Band 4.2-/- Vehicle

Band 4.2-/- Mitapivat

H&

EH

&E

Perls

Perls

In band 4.2-/- mice, Mitapivat decreases liver iron-overload

LIC

Live

r Iro

n C

onte

nt

(ng/

mg

of d

ry ti

ssue

)

0

50

100

150

*°

*

*p<0.05 compared to WT mice°p<0.05 compared to vehicle treated mice

Conclusions

• Band 4.2-/- mice treated with Mitapivat show:– Reduced hemolysis and amelioration of anemia– Improved red cell membrane mechanical stability– Reduction in spleen and liver iron overload

• Mitapivat might represent an interesting and novel therapeutic option for HS patients