Pyruvate kinase (PK), including wild-type (WT) PK-R and various mutant PK-R (mtPK-R) enzymes encoded by PKLR genotypes
[1]
Pyruvate kinase (PK), including wild-type (WT) PK and mutant PK enzymes[3]

In healthy donor erythrocytes, mitapivat (0.1 nM-100 μM; 16 h) activates WT PK-R[1]. In red blood cells, mitapivat (0.01 nM-10 μM; 16 h) stimulates ATP generation in a dose-dependent way [1].

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Treatment of recombinant WT PK-R enzyme with Mitapivat (5 μM) resulted in enhanced activity when stimulated with PEP. The cocomplex crystal structure of PK-R tetramer bound to Mitapivat showed key interactions in the allosteric-binding pocket, with PEP and FBP also present in the structure[1]
- Mitapivat activated a spectrum of recombinant mtPK-R enzymes. At a concentration of 10 μM, it exhibited varying fold activation and AC₅₀ values for different mtPK-R enzymes. For example, preincubation of recombinant R532W mtPK-R enzyme with 5 μM Mitapivat enhanced its activity when stimulated with PEP, and the enzyme showed increased activity when incubated with Mitapivat compared to FBP at the same concentration (PEP = 0.05 mM). Additionally, Mitapivat (5 μM) increased the residual activity of WT or R510Q recombinant enzymes following incubation at 53°C and had a specific off-rate from recombinant R510Q enzyme (5 μM final assay concentration, PEP = 2 mM)[1]
- Ex vivo treatment of red blood cells (RBCs) from healthy donors with Mitapivat at various concentrations for overnight incubation increased WT PK-R activity (PEP = 0.1 mM) and ATP levels[1]
- Ex vivo treatment of RBCs from patients with PK deficiency with Mitapivat for 24 hours increased PK-R activity (PEP = 0.5 mM) in most patient samples (mean increase 1.8-fold, range 1.2-3.4) and ATP levels (mean increase 1.5-fold, range 1.0-2.2), which was similar to the ATP increase in control cells (mean increase 1.6-fold, range 1.4-1.8). PK thermostability was strongly reduced in PK-deficient RBCs, but Mitapivat treatment increased residual activity 1.4 to >10-fold compared to vehicle-treated samples. In half of the patients, this treatment was associated with an increase in RBC deformability. However, treatment effects were minimal in patient cells with very low or undetectable levels of PK-R protein[3]
- Ex vivo treatment of erythroid precursors from patients with β-thalassemia with Mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis[2]

In a mouse model of beta-thalassemia, mitapivat (50 mg/kg; oral; twice daily for 21 days) improves anemia [2].

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Administration of Mitapivat to mice twice daily for 7 days at different dose levels resulted in detectable plasma concentrations of the drug, with calculated AUC₀₋₁₂ hours at each dose level. PK-R activity (PEP = 0.1 mM) and ATP levels in RBCs, as well as 2,3-DPG levels in whole blood from the mice, were measured, and the AUC₀₋₁₂ hours was calculated for each parameter (with the 150 mg/kg dose level data shown clearly)[1]
- Oral administration of Mitapivat (50 mg/kg twice daily) to Hbbᵗʰ³/⁺ mice (a β-thalassemia mouse model) ameliorated ineffective erythropoiesis and anemia. It increased ATP levels, reduced reactive oxygen species (ROS) production, and improved mitochondrial clearance, thereby reducing markers of mitochondrial dysfunction. The treatment also enhanced responsiveness to erythropoietin, leading to reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Additionally, Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2-HIF2α axis, which, together with Hamp, controlled iron absorption and prevented liver iron overload. The treatment also improved red cell survival, reduced hemolysis, and increased the glutathione/glutathione disulfide ratio in the mice[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]

Cell Viability Assay[1]
Cell Types: RBC cells
Tested Concentrations: 0.1 nM-100 µM
Incubation Duration: 16 h (incubate overnight)
Experimental Results: Increased PK-R activity in a dose-dependent manner to ~2.5-fold of DMSO control with an AC50 of 62 nM.

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Cell Viability Assay[1]
Cell Types: RBC cells
Tested Concentrations: 0.01 nM-10 µM
Incubation Duration: 16 h (incubate overnight)
Experimental Results: Consistently increased ATP levels in a dose-dependent manner by an average of 60% over DMSO control with an AC50 of 10.9 nM.

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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]

Animal/Disease Models: WT C57B6 and Hbbth3/+ mice (both are 2-month-old female mice; β-thalassemia model)[2].
Doses: 50 mg/kg
Route of Administration: In animal feedings; single daily for 3 weeks.
Experimental Results: Increased the expression of pyruvate kinase isoforms in both red cells and erythroid precursors from Hbbth3/+ mice. Elevated pyruvate kinase activity in cells from Hbbth3/+ mice, and markedly increased ROS level in erythrocytes. Increased the expression of PKM2 in polychromatic and orthochromatic erythroblasts of Hbbth3/+ mice.

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Animal/Disease Models: WT C57B6 and Hbbth3/+ mice (both are 2-month-old female mice; β-thalassemia model)[2].
Doses: 50 mg/kg
Route of Administration: po (oral gavage), twice (two times) daily for 21 days.
Experimental Results: Ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice and increased ATP, decreased ROS production, as well as decreased markers of mitochondrial dysfunction associated with improved mitochondrial clearance.

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Mice were dosed with Mitapivat twice daily for 7 days at different dose levels. Blood samples were collected at specific time points to measure plasma concentrations of Mitapivat and calculate AUC₀₋₁₂ hours. RBCs and whole blood were also collected to determine PK-R activity, ATP levels, and 2,3-DPG levels[1]
- WT and Hbbᵗʰ³/⁺ mice (β-thalassemia model) were treated with vehicle or Mitapivat at 50 mg/kg twice daily via oral administration. The treatment duration was 21 days. Various parameters were measured, including erythrocyte morphology, hemoglobin (Hb) levels, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), reticulocyte count, α-globin/β-globin ratio, total and soluble Hb, ROS levels, glutathione/glutathione disulfide ratio, red cell survival (using CFSE-labeled red cells), plasma erythropoietin (EPO) levels, spleen weight/mouse weight ratio, flow cytometry analysis of bone marrow and spleen erythroid cells, ATP content in sorted erythroblasts, mRNA expression of Erfe, soluble plasma ERFE levels, mitochondrial content (using MitoTracker), mRNA expression of mitochondrial genes (Atp6, Mtco1, Cytb, Pgc1a, Yme1l), liver iron staining (Perl’s Prussian blue), liver protein oxidation (OxyBlot), mRNA expression of hepcidin (Hamp) and Id1, Western blot analysis of liver proteins (phospho NF-κB p65, NF-κB p65, pSTAT3, STAT3, pNRF2, NRF2), duodenal iron staining, Western blot analysis of duodenal proteins (PKR, PKM2, HIF2α, pNF-κB p65, NF-κB p65), and mRNA expression of Dmt1-IRE[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.

Pharmacodynamics
Mitabivat is a pyruvate kinase activator that increases the activity of erythrocyte pyruvate kinase. Erythrocyte pyruvate kinase is 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, mitapivat is a mild to moderate inhibitor of aromatase (CYP19A1). Aromatase is an enzyme involved in the synthesis of estrogen from androgen precursors. Inhibition of aromatase 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 mitapivat 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 for developing children. Mitapivat (AG-348) is an allosteric activator of pyruvate kinase [1, 2, 3] Pyruvate kinase deficiency is a rare genetic disorder that causes chronic hemolytic anemia, and there is currently no targeted treatment for the disease. Mitapivat is expected to restore glycolytic pathway activity in patients with PK deficiency by increasing pyruvate kinase (PK) enzyme activity, thereby bringing clinical benefits [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]

C24H26N4O3S

450.56

450.173

C, 63.98; H, 5.82; N, 12.44; O, 10.65; S, 7.12

1260075-17-9

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

59634741

White to off-white solid powder

4.233

1

6

6

32

750

0

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

XAYGBKHKBBXDAK-UHFFFAOYSA-N

InChI=1S/C24H26N4O3S/c29-24(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/h1-5,8-12,18,26H,6-7,13-17H2

N-{4-[4-(cyclopropylmethyl)piperazine-1-carbonyl]phenyl}quinoline-8-sulfonamide

PKM2 activator 1020; AG348; Mitapivat; PKM2 activator; 1260075-17-9; PKR-IN-1; AG-348; 2WTV10SIKH; PKR-IN-1; AG-348; 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)

Solubility in Formulation 1: 2.5 mg/mL (5.55 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.55 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.55 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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