Pyruvate kinase (PK)

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.

---

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.

---

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.

---

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]

C48H54N8O10S3

999.18

998.3125

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

2329710-91-8

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

139593419

Typically exists as solids at room temperature

4

16

12

69

831

0

C1C=C2C(N=CC=C2)=C(S(NC2C=CC(=CC=2)C(=O)N2CCN(CC3CC3)CC2)(=O)=O)C=1.S(O)(O)(=O)=O.C1C=C2C(N=CC=C2)=C(S(NC2C=CC(=CC=2)C(=O)N2CCN(CC3CC3)CC2)(=O)=O)C=1

XNNUNDNGUZFBHS-UHFFFAOYSA-N

InChI=1S/2C24H26N4O3S.H2O4S/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)

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

AG-348 hemisulfate; Mitapivat hemisulfate; Mitapivat sulfate anhydrous; KM3KSE3QH9; UNII-KM3KSE3QH9; 2329710-91-8;

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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