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
