Pyruvate kinase (PK)

Absorption, Distribution and Excretion
The absolute bioavailability of mitapivat after a single dose is approximately 73%. Mitapivat exposure increases dose-proportionally. Following twice-daily oral administration of mitapivat at the dose of 5 mg, 20 mg, and 50 mg, the mean (CV%) Cmax at steady state were 101.2 (17%) ng/mL, 389.9 (18%) ng/mL, and 935.2 (18%) ng/mL, respectively. The mean (CV%) AUC were 450.4 (28%) ng x h/mL, 1623.8 (28%) ng x h/mL, and 3591.4 (28%) ng x h/mL, respectively. The median Tmax values at steady state were 0.5 to 1.0 hour post-dose across the dose range of 5 mg to 50 mg twice daily. In healthy subjects, a high-fat meal did not affect the drug exposure but reduced the rate of mitapivat absorption, with a 42% reduction in Cmax and a delay in Tmax of 2.3 hours when compared to dosing under fasted conditions.
Mitapivat is primary eliminated via hepatic metabolism. After a single oral administration of radiolabeled mitapivat in healthy subjects, the total recovery of administered radioactive dose was 89.2%. About 49.6% of radioactivity was recovered in the urine with 2.6% excreted as unchanged mitapivat. About 39.6% of radioactivity was recovered in the feces with less than 1% being the unchanged drug.
The mean volume of distribution at steady state (V_ss) was 42.5 L.
Population pharmacokinetics derived median CL/F at steady state was 11.5, 12.7, and 14.4 L/h for the 5 mg twice daily, 20 mg twice daily, and 50 mg twice daily regimens, respectively.

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Metabolism / Metabolites
According to _in vitro_ studies, mitapivat is primarily metabolized by CYP3A4. It is also a substrate of CYP1A2, CYP2C8, and CYP2C9. Following a single oral dose administration of 120 mg of radiolabeled mitapivat in healthy subjects, unchanged mitapivat was the major circulating component in plasma.

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Biological Half-Life
In patients with pyruvate kinase deficiency receiving multiple doses of 5 mg mitapivat twice daily to 20 mg twice daily, the mean effective half-life (t_1/2) of mitapivat ranged from 3 to 5 hours.

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Plasma concentrations of Mitapivat were measured in mice after twice-daily dosing for 7 days at different dose levels, and AUC₀₋₁₂ hours was calculated for each dose level[1]

Protein Binding
Mitapivat is 97.7% bound to plasma proteins, with an RBC-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
Mitapivat is a pyruvate kinase activator that works to increase the activity of erythrocyte pyruvate kinase, an enzyme responsible for energy production for and survival of red blood cells. It is effective in upregulating the activity of both wild-type and mutant forms of erythrocyte pyruvate kinase. Interestingly, mitapivat is a mild-to-moderate inhibitor of the aromatase enzyme (CYP19A1), which is an enzyme involved in biosynthesis of estrogens from androgen precursors. Inhibition of aromatase is associated with bone density loss, as estrogen mediates suppressive, antiresorptive effects on osteoclasts and generally favours bone formation over resorption. Thus, low estrogen levels can increase bone turnover and osteoclast activity, resulting in net bone loss and decreased bone quality. Inhibition of aromatase by mitapivat may have some clinical implications, as patients with pyruvate kinase deficiency have considerably high rate of osteopenia and osteoporosis. The long-term effect of mitapivant on bond mineral density requires further investigation. One study suggests that this off-target effect may have negligible clinical effects on adults, but may potentially have some clinical implications in developing children.

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Mitapivat (AG-348) is an allosteric activator of pyruvate kinase[1, 2, 3]
- PK deficiency is a rare genetic disease that causes chronic hemolytic anemia, and there are currently no targeted therapies for this condition. Mitapivat has the potential to restore the glycolytic pathway activity in patients with PK deficiency by increasing PK enzyme activity, thereby leading to clinical benefit[1]
- Anemia in β-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired α-globin chains impose substantial oxidative stress on β-thalassemic erythroblasts and erythrocytes. Mitapivat reduces chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism[2]
- Mitapivat is currently in clinical trials for the treatment of PK 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

265 Ų

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。