Pyruvate kinase (PK)

Mitapivat (50 mg/kg; p.o.; twice daily for 21 days) improves anemia in a mouse model for β-thalassemia
In β-thalassemia, anemia results from ineffective erythropoiesis and shortened red blood cell survival. The presence of excess free heme and the accumulation of unpaired α-globin chains impose considerable oxidative stress on both erythroblasts and erythrocytes, thereby disrupting cellular metabolism. We hypothesized that mitapivat (AG-348)-induced activation of pyruvate kinase in the Hbbth3/+ mouse model of β-thalassemia would alleviate chronic hemolysis and ineffective erythropoiesis by enhancing red cell glycolytic metabolism. Oral administration of mitapivat improved ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP levels, reduced reactive oxygen species production, decreased markers of mitochondrial dysfunction, and enhanced mitochondrial clearance indicated that mitapivat promotes improved metabolism in β-thalassemia. Enhanced responsiveness to erythropoietin led to reduced soluble erythroferrone levels, increased hepatic Hamp expression, and decreased liver iron overload. Additionally, mitapivat reduced duodenal Dmt1 expression, potentially via activation of the pyruvate kinase M2-HIF2α axis, representing a mechanism beyond Hamp for regulating iron absorption and preventing β-thalassemia-related liver iron overload. In ex vivo studies using erythroid precursors from β-thalassemia patients, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and reduced apoptosis. Collectively, pyruvate kinase activation as a therapeutic strategy for β-thalassemia in preclinical models demonstrated multiple beneficial effects within and beyond the erythropoietic compartment, providing a strong scientific foundation for further clinical investigation.[2]

Recombinant PK-R enzyme was incubated with different concentrations of Mitapivat (with PEP set at 0.065 mM), and the activity of the enzyme was measured to assess the activating effect of Mitapivat. Technical replicates were performed, and the mean, standard deviation, range, and number of replicates were recorded[1] - Recombinant WT PK-R enzyme was stimulated with PEP either with or without preincubation with 5 μM Mitapivat, and the enzyme activity was measured. The average of 3 technical replicates was used for data analysis[1] - Various recombinant mtPK-R enzymes were treated with 10 μM Mitapivat (with PEP concentrations as listed in the supplemental table), and the fold activation and AC₅₀ values were determined to evaluate the activating effect of Mitapivat on mutant enzymes[1] - Recombinant R532W mtPK-R enzyme was stimulated with PEP with or without preincubation with 5 μM Mitapivat, and the enzyme activity was measured (average of 3 technical replicates)[1] - Recombinant R532W mtPK-R enzyme was incubated with different concentrations of FBP or Mitapivat (PEP = 0.05 mM), and the enzyme activity was measured to compare the activating effects of the two compounds[1] - WT or R510Q recombinant enzymes were incubated at 53°C with 5 μM Mitapivat (PEP = 2 mM), and residual activity was measured over time to assess the effect of Mitapivat on enzyme thermostability[1] - Off-rate measurement of Mitapivat or FBP (both at 5 μM final assay concentration, PEP = 2 mM) from recombinant R510Q enzyme was performed to evaluate the binding kinetics of Mitapivat to the mutant enzyme[1] - PK activity in RBCs from healthy donors, PK-deficient patients, and mice was measured using a coupled enzyme spectrometric assay (except for patient B, for whom activity was assessed by direct measurement of pyruvate formation using liquid chromatography followed by tandem mass spectrometry). The assay was conducted under specific PEP concentrations (e.g., 0.1 mM, 0.5 mM) to determine the effect of Mitapivat on PK activity[1, 3]

RBCs from healthy donors were incubated overnight with various concentrations of Mitapivat, and then PK-R activity (PEP = 0.1 mM) and ATP levels were measured to assess the in vitro effect of the drug on healthy RBCs[1] - RBCs from patients with PK deficiency were incubated with Mitapivat for 24 hours, and PK-R activity (PEP = 0.5 mM) and ATP levels were measured. Additionally, PK thermostability of RBC lysates from PK-deficient patients was evaluated by pre-incubating the lysates with or without 2 mM Mitapivat for 2 hours at 37°C, followed by heat treatment at 53°C for different time periods (5, 10, 20, 40, 60 minutes) and measurement of residual PK activity[3] - RBC deformability of PK-deficient patients was assessed using osmoscan curves, and the effect of Mitapivat treatment (20 mM for 24 hours ex vivo) on RBC deformability was determined[3] - Ex vivo cultured erythroid cells from PK-deficient patients and healthy controls were incubated with or without 2 mM Mitapivat. Cell morphology was observed at different stages of proliferation and differentiation, cell proliferation (cell numbers in %) was measured, and the PK/Hexokinase (HK) ratio was determined to evaluate the effect of Mitapivat on erythroid cell function[3] - Erythroid precursors from patients with β-thalassemia were treated with Mitapivat ex vivo, and the effects on erythropoiesis, erythroid maturation, and apoptosis were assessed[2]

Absorption, Distribution and Excretion
Following a single dose, the absolute bioavailability of mitabiva is approximately 73%. Mitabiva exposure increases in a dose-dependent manner. After twice-daily oral administration of mitabiva at doses of 5 mg, 20 mg, and 50 mg, the mean steady-state (CV%) Cmax was 101.2 (17%) ng/mL, 389.9 (18%) ng/mL, and 935.2 (18%) ng/mL, respectively. The mean (CV%) AUC was 450.4 (28%) ng·h/mL, 1623.8 (28%) ng·h/mL, and 3591.4 (28%) ng·h/mL, respectively. Within the dose range of 5 mg to 50 mg twice daily, the median steady-state Tmax was 0.5 to 1.0 hours after administration. In healthy subjects, a high-fat diet did not affect drug exposure but reduced the absorption of mitabiva, with a 42% decrease in Cmax and a 2.3-hour delay in Tmax compared to fasting administration. Mitabiva is primarily eliminated via hepatic metabolism. Following a single oral dose of radiolabeled mitabiva in healthy subjects, the total recovery rate of the radiopharmaceutical was 89.2%. Approximately 49.6% of the radiopharmaceutical was recovered in urine, and 2.6% was excreted unchanged. Approximately 39.6% of the radiopharmaceutical was recovered in feces, of which less than 1% was unchanged. The steady-state mean volume of distribution (V_ss) was 42.5 L. The population pharmacokinetic-derived steady-state median clearance/fecal volume (CL/F) was 11.5 L/h for the 5 mg twice-daily group, 12.7 L/h for the 20 mg twice-daily group, and 14.4 L/h for the 50 mg twice-daily group.

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Metabolism/Metabolites
According to in vitro studies, mitabiva is primarily metabolized by CYP3A4. It is also a substrate of CYP1A2, CYP2C8 and CYP2C9. After a single oral administration of 120 mg of radiolabeled mitabiva to healthy subjects, the main circulating component in plasma was unmetabolized mitabiva.

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Biological Half-Life
In patients with pyruvate kinase deficiency, the mean effective half-life (t_1/2) of mitabiva was 3 to 5 hours after twice-daily administration of 5 mg to 20 mg mitabiva.

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In mice, plasma concentrations were determined after twice-daily administration of different doses of mitabiva for 7 days, and the AUC_0.5 hours at each dose level was calculated [1].

Protein Binding
Miltapivaca showed a 97.7% binding rate to plasma proteins, with a red blood cell to plasma ratio of 0.37.

[1]. AG-348 enhances pyruvate kinase activity in red blood cells from patients with pyruvate kinase deficiency. Blood. 2017 Sep 14;130(11):1347-1356.

[2]. The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a β-thalassemia mouse model. J Clin Invest. 2021 May 17;131(10):e144206.

[3]. AG-348 (Mitapivat), an allosteric activator of red blood cell pyruvate kinase, increases enzymatic activity, protein stability, and ATP levels over a broad range of PKLR genotypes. Haematologica. 2021 Jan 1;106(1):238-249.

Drug Indication
Pyrukynd is indicated for the treatment of pyruvate kinase deficiency (PK deficiency) in adult patients (see Section 4.4). Pharmacodynamics Mitabiva is a pyruvate kinase activator that increases the activity of erythrocyte pyruvate kinase, an enzyme responsible for energy production and survival in erythrocytes. It effectively upregulates the activity of both wild-type and mutant erythrocyte pyruvate kinase. Notably, mitabiva is a mild to moderate inhibitor of aromatase (CYP19A1), an enzyme involved in the synthesis of estrogen from androgen precursors. Aromatase inhibition is associated with decreased bone mineral density because estrogen has an inhibitory and anti-resorption effect on osteoclasts, generally promoting bone formation rather than resorption. Therefore, low estrogen levels increase bone turnover and osteoclast activity, leading to net bone loss and decreased bone quality. The inhibitory effect of mitabiva on aromatase may have some clinical significance because patients with pyruvate kinase deficiency have a relatively high incidence of osteopenia and osteoporosis. The long-term effects of mitapivat on bone mineral density require further investigation. One study suggests that this off-target effect may have little clinical impact on adults, but may have some clinical significance in developing children. Mitapivat (AG-348) is a pyruvate kinase allosteric activator [1, 2, 3] - Pyruvate kinase deficiency is a rare genetic disorder that causes chronic hemolytic anemia for which there is currently no targeted treatment. Mitapivat can restore glycolytic pathway activity in patients with pyruvate kinase deficiency by increasing pyruvate kinase activity, thus providing clinical benefit [1] - Anemia in β-thalassemia is associated with ineffective hematopoiesis and reduced erythrocyte survival. Excessive accumulation of free heme and unpaired α-globin chains causes significant oxidative stress on erythroblasts and erythrocytes in β-thalassemia. Mitapivat reduces chronic hemolysis and ineffective erythropoiesis by stimulating erythrocyte glycolysis.[2] Mitapivat is currently undergoing clinical trials for the treatment of pyruvate kinase deficiency (ClinicalTrials.gov: NCT02476916, NCT03853798, NCT03548220, NCT03559699).[3]

C48H60N8O13S3

1053.23

1052.344

C, 57.70; H, 5.45; N, 11.21; O, 16.01; S, 9.63

2151847-10-6

2151847-10-6 (sulfate hydrate);1260075-17-9 (free);2329710-91-8 (sulfate); 2559738-69-9 (HCl); 2559738-74-6

134693700

Typically exists as solid at room temperature

7

19

12

72

831

0

C1CC1CN2CCN(CC2)C(=O)C3=CC=C(C=C3)NS(=O)(=O)C4=CC=CC5=C4N=CC=C5.C1CC1CN2CCN(CC2)C(=O)C3=CC=C(C=C3)NS(=O)(=O)C4=CC=CC5=C4N=CC=C5.O.O.O.OS(=O)(=O)O

DMRIPASJCJRBMV-UHFFFAOYSA-N

InChI=1S/2C24H26N4O3S.H2O4S.3H2O/c2*29-24(28-15-13-27(14-16-28)17-18-6-7-18)20-8-10-21(11-9-20)26-32(30,31)22-5-1-3-19-4-2-12-25-23(19)22;1-5(2,3)4;;;/h2*1-5,8-12,18,26H,6-7,13-17H2;(H2,1,2,3,4);3*1H2

N-[4-[4-(cyclopropylmethyl)piperazine-1-carbonyl]phenyl]quinoline-8-sulfonamide;sulfuric acid;trihydrate

AG-348 sulfate hydrate, Mitapivat sulfate; AG348; PKR-IN-1; trade name Pyrukynd

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)