Agios Pharmaceuticals : Mitapivat Improves Transfusion Burden and Reduces Iron Overload in Thalassemic Mice

(MFI, mean fluorescence intensity)

Mitapivat Improves Transfusion Burden and Reduces Iron Overload in Thalassemic Mice

Alessandro Mattè1, Penelope A. Kosinski2, Enrica Federti1, Lenny Dang2, Antonio Recchiuti3, Roberta Russo4, Angela Siciliano1, Iana Iatcenko1, Anne Janin5, Christophe Leboeuf5, Achille Iolascon4 , Carlo Brugnara6, and Lucia DeFranceschi1

1. University of Verona and AOUI Verona, Verona, Italy; 2. Agios Pharmaceuticals, Inc.,Cambridge, MA; 3. University of Chieti, Chieti, Italy; 4. Dept. of Molecular Medicine and Medical Biotechnology and CEINGE, University of Naples Federico II , Naples, Italy; 5. University Diderot of Paris, Paris, France; 6. Department of Laboratory Medicine, Harvard Medical School, Boston Children's Hospital, Boston, MA.

Background

Results

• Mitapivat (AG-348) is an oral allosteric activator of red

blood cell pyruvate kinase1

Mitapivat reduces the need of transfusion in β-thal mice

Mitapivat treated transfused β-thal mice show reduced liver iron accumulation and

modulation of Dmt1 IRE expression in duodenum

• Recently, we showed that mitapivat improves anemia and

iron homeostasis in a mouse model of β-thalassemia

intermedia (Hbbth3/+) via a multi modal action2

a

b

				11
mitapivat		RBC	10.5
	(400 uL	(g/dl)Hb	10
50 mg/Kg BID	Hct ~40-45%)
10 days	Transfusion threshold 10.5 g/dL		9

Hb

8

determination

		c						Vehicle RBC Tr, Hbbth3/+
			(days)	20				Mitapivat RBC Tr, Hbbth3/+
			#	# #
Vehicle RBCs Tr, Hbbth3/+	1
Vehicle RBCs Tr, Hbb	th3/+	2		15
	interval
Vehicle RBCs Tr, Hbbth3/+	3
Mitapivat RBCs Tr, Hbbth3/+ 1		10
			Transfusion
Mitapivat RBCs Tr, Hbbth3/+ 2
Mitapivat RBCs Tr, Hbbth3/+ 3	5
Mitapivat RBCs Tr, Hbbth3/+ 4
				0

a

	20
Score	15
10
Iron
	5

Liver

#		tissue)	550
		500
	*	dry	450
	of
		400
		(ug/g
		350
		LIC	300

*

mHamp/LIC ratio

2.0

1.5

1.0

0.5

*

b

Transferrin saturation (%)

70

60

50

40

Plasma

#

*

Relative expression to GAPDH

Duodenum

c

Dmt1 IRE

2.5#

2.0

# *

1.5

1.0

0.5

Figure 4

a. Perls staining (left panel), non-heme liver iron
content (LIC- middle panel) and mHamp/LIC
ratio in the liver from transfused β-thal mice
treated with either vehicle or mitapivat (50
mg/Kg BID).
b. Serum	Transferrin	saturation	(%) in
untransfused and transfused β-thal mice
treated with either vehicle or mitapivat (50
mg/Kg BID).
c. mRNA	expression of	Dmt1-iron	response

0

10

20

30

40

50

Figure 1.	Time (days)

1. Design of the study. β-thal mice (Hbbth3/+), treated with vehicle or mitapivat (50 mg/Kg BID), were transfused with 400 µL washed heathy RBCs (RBC Tr.) at 40-45% hematocrit (Hct) (Park et al Blood. 2020). Hb 10.5 g/dL was chosen as transfusion threshold.

b. Overtime Hb changes in transfused β-thal mice treated with either vehicle or mitapivat (50 mg/Kg BID).

c. Mitapivat treated β-thal mice showed greater sustained rise in Hb from baseline compared to vehicle treated β-thal mice. This results in longer

0

		250
WT	b thal	bthal transf b thal transf mitapivat

0.0

30

0.0

element (IRE) by qRT-PCR on duodenums

from untransfused and transfused β-thal mice

treated with vehicle or mitapivat.

Data are mean ± SEM (N= 3 mice/group). # P<0.05 compared to untransfused β-thal mice. * P<0.05 compared to vehicle.

length of time between transfusion (mitapivat β-thal mice 13.8±1.0 days vs vehicle β-thal mice 10.5±1.0 days, n= 4 and n=3; # P<0.05 compared to vehicle treated animals). This was associated with a drop in both reticulocyte and peripheral circulating erythroblasts in both vehicle and mitapivat treated β-thal mice (data not shown).

Mitapivat induced improvement of anemia is maintained in beta-thal mice

treated with deferiprone (DFP)

• Ad interim results from the extension period of a phase 2

trial of a phase-2 trial with mitapivat in non transfusion

dependent thalassemic (NTDT) patients demonstrates

sustained long-term increase in Hb (≥ 1 g/dL) with

improvement of hemolysis and ineffective erythropoiesis

(Kuo et al.abstract # 576; ASH 2021).

In β-thal mice, mitapivat beneficially impacts spleen iron-overload by reduction

of transfusion burden and amelioration of β-thal ineffective erythropoiesis

a		b			c
					WT	Hbbth3/+	Vehicle RBC Tr, Hbbth3/+	Mitapivat RBC Tr, Hbbth3/+
Plasmatic EPO (pg/ml)	2000		30
1500
#		20	#	#
1000	#
500	weight/mouseSpleen weight(mg/g)ratio	10
0	0

a

Deferiprone (DFP)

(1.25 mg/mL)

mitapivat

7 days	28 days

b

(g/dl)Hb

	Mitapivat Hbb	th3/+	1
	Mitapivat Hbb	th3/+	2
11
10
9
8
7
Mitapivat DFP	7	14	21
	time (days)

Mitapivat DFP Hbb	th3/+	1
Mitapivat DFP Hbb	th3/+	2
Mitapivat DFP Hbb	th3/+	3
		50
	(%)	40
	Erythroblasts	30
	20
	10
		0
28		Mitapivat DFP	7	14	21	28
				time (days)

Figure 5

1. Design of the study. Mitapivat (50 mg/Kg BID) was administrated in combination with deferiprone (DFP), an oral iron chelator (1.25 mg/ml ) to β-thal (Hbbth3/+) mice.
2. Hemoglobin (Hb, left panel) and circulating erythroblasts (%, right panel) in β-thal mice treated with either mitapivat or mitapivat+DFP. Data are presented for single mouse at the different time points.

•	Mitapivat might be a potential therapeutic option also for
	TDT patients since transfusion burden severely impact
	patients' quality of life.3
•	To address this question, we exposed Hbbth3/+ mice to
	chronic transfusion with or without mitapivat treatment.
	We also evaluated the effect of mitapivat associated with
	deferiprone (DFP), an oral iron chelator, on hematologic
	parameters.

d

Figure 2

a. Plasma erythropoietin (EPO) in Hbbth3/+ untransfused and in Hbbth3/+ exposed to chronic RBC transfusion and treated with either vehicle or mitapivat (50 mg/Kg BID). Data are presented as mean ± SEM n= 3-4 mice/group, # p<0.05 vs untransfused Hbbth3/+ mice.

b. Spleen weight/mouse weight ratio in untransfused Hbbth3/+ and in Hbbth3/+ exposed to chronic RBC transfusion and treated with either vehicle or mitapivat (50 mg/Kg BID) Data are presented as mean ± SEM, n= 3-4 mice/group, # p<0.05 vs untransfused Hbbth3/+ mice.

	Bone Marrow	Spleen
(%)	50	45
	#
High	45	40
Fsc	#	35	#	#
Ter119+
40
CD44+		30
35	25
	30	20

Bone Marrow

	0.25
	0.20
II/IV	0.15
pop
Ratio	0.10
	0.05
	0.00

Spleen

0.3

0.2

0.1

0.0

Conclusions

1. We generated a model for chronic RBC transfusion in β-thal mice.

o In this model, we show that mitapivat:

• Increases the length of time between transfusion, resulting in decreased

spleen and liver iron overload.

Methods

Mouse strains and design of the study. 3-4 months old female mice of C57BL6/J, as wild-type controls (WT), and Hbbth3/+ mice (β-thal), as mouse model of β-thalassemia intermedia, were used in the present study.2 Whenever indicated mice were treated by oral gavage with Mitapivat (AG-348) or vehicle at the dosage of 50 mg/Kg twice daily (BID) up to 71 days. For the transfusion study, Hbbth3/+ mice, treated with mitapivat (50 mg/Kg BID) or vehicle for 10 days, were transfused with 400 µL washed heathy RBCs at 40-45% Hematocrit (Hct). 4 We chose Hb 10.5 g/dL as transfusion threshold for our β-thal mouse model. Whenever indicated deferiprone (DFP) was administered to Hbbth3/+ mice treated with mitapivat (50 mg/Kg BID) in drinking water at the dosage of 1.25 mg/ml. 5 For nonterminal and terminal blood collection, mice were anesthetized by isoflurane inhalation and blood was collected by retro-orbital venipuncture using heparinized microcapillary tubes. Hemoglobin was manually determined by staining with Drabkin's reagent (Sigma-Aldrich, St. Louis, MO) followed by spectrophotometric analysis at 540 nm. Reticulocytes and circulating erythroblasts were measured either by Sysmex XN-1000 Hematology Analyzer (Sysmex Corporation, Japan) or by flow cytometric analysis using CD71-PE (Thermo Fisher Scientific, Waltham, USA) staining as previously reported.

Flow Cytometric Analysis of Mouse Erythroid Precursors from bone marrow and spleen was carried out using the CD44-Ter119 gating strategy as previously described. 2 The following antibodies were used: anti-CD16/CD32 blocking agent, anti-CD44-FITC,CD71-PE,Ter119-APC, CD45 APC-eFluor 780, GR1 APC-Cy7, and CD11b APC-Cy7 (all from eBiosciences, Thermo Fisher Scientific, USA). Samples were acquired using the FACSCanto II flow cytometer (Becton Dickinson) and analyzed with FlowJo software version 10 (Tree Star). 2

In vivo Erythrophagocytosis was determined by flow cytometry as previously described 6. Spleen macrophages (MΦ) from WT and Hbbth3/+ mice were stained with anti-F4/80 antibody and anti-Ter-119 antibody to detect ingested RBCs. Phagocytosis was assessed as the percentage of double positive (F4/80+/Ter-119+) cells. Macrophage M1 and M2 polarization was determined using the specific M1 marker CD80 and M2 CD206 antibodies on spleen MΦ.6

Liver and spleen iron content were analyzed either using the Perls prussian blue staining or measured using the bathophenanthroline method as previously described.2

Statistical Analysis: Data were analyzed using either the one-way analysis of variance (ANOVA) and the Two-tailed unpaired Student t test. A difference with a p<0.05 was considered significant.

c. Perl's stained sections of spleens from WT, untransfused and transfused Hbbth3/+ mice treated with either vehicle or mitapivat (50 mg/Kg BID), showing a reduced spleen iron overload in mitapivat treated transfused β-thal mice compared to vehicle transfused β-thal animals. One representative image from 6 with similar results.

d. Flow-cytometric analysis of the erythropoietic activity and the maturation pattern of the erythroid precursors (immature-Pop II/mature -Pop IV erythroblasts) from the bone marrow (left panel) and spleen (right panel) in transfused β-thal mice treated with vehicle (Hbbth3/+ RBC Tr. vehicle) or mitapivat (50 mg/Kg BID, Hbbth3/+ RBC Tr. mitapivat) compared to non transfused mice (Hbbth3/+). Data are mean ± SEM (N= 3-4 mice/group). # P < 0.05 compared to untransfused β-thal mice.

	In transfused β-thal mice, Mitapivat induces a pro-resolving profile of splenic
a	b	macrophages	Figure 3
	a. Flow cytometric analysis of the in vivo splenic
			erythrophagocytosis,	assessed	as	the
	expression (MFI)	expression (MFI)	percentage of F4/80+/Ter-119+ double positive
Ter-119+ MΦ	cells in	spleen from WT, untransfused or
transfused β-thal mice treated with mitapivat (50
mg/Kg BID) or vehicle. Data are shown as mean
± SEM	(n= 3-4 mice/group). No cumulative
effect of the two treatments was observed.
%	CD80	CD206
b. Flow	cytometric	analysis of	the	surface
expression of the M1 marker CD80 and the M2
			marker CD206 on spleen macrophages (MΦ)
			data are	mean ± SEM (n= 3-4	mice/group).

•	Induces a pro-resolving profile of spleen macrophages
•	Improves iron homeostasis by targeting Dmt1 expression

In conclusion, mitapivat reduces the transfusion burden in a mouse model of transfused β-thalassemia, which could potentially impact the management of

iron chelation and anemia in TDT.

References

1. Kung C et al. Blood. 2017 Sep 14; 130(11): 1347-1356	4.	Park SY et al. Blood. 2020 Jun 4; 135(23): 2071-2084
2. Matte' A et al. J Clin Invest. 2021 May 17; 131(10): e144206	5.	Casu C et al. Haematologica. 2016 Jun;101(1): e8-e11
3. Thaler AT et al. Exper review of Hematology. 2021 Sept 15; 14(10): 897-909	6.	Mattè A et al Blood. 2019 Jan 17;133(3): 252-265.

Disclosures

PAK and LD - AgiosPharmaceuticals,Inc- Current Employment and Current holder of stock options in a privately-held company	CONTACT: [e-mail]
LDF - F. Hoffmann-La Roche Ltd - Consultancy; Novartis - Consultancy