Pyruvate kinase (PK)

体外活性：Mitapivat，以前也称为 PKM2 激活剂 1020 是一种 PKM2 激活剂（丙酮酸激酶激活剂），具有治疗丙酮酸激酶缺陷的潜在用途。 Mitapivat 是一种有效的人 R 型丙酮酸激酶 (PKR) 抑制剂，对突变 PKR 也显示出效力，包括 R510Q PKR、R532W PKR、T384W PKR 等。丙酮酸激酶 M2 型，在多种肿瘤细胞类型中表达并发挥关键作用在有氧糖酵解中，具有非糖酵解功能，可以调节转录和细胞增殖。因此，据报道，丙酮酸激酶同工酶 M2 (PKM2) 的小分子激活剂可能会抑制肿瘤形成，但对已形成的肿瘤的影响尚不清楚。细胞检测：Mitapivat（0.1 nM-100 µM；16 小时）可激活健康捐献者红细胞中的 WT PK-R。 Mitapivat（0.01 nM-10 µM；16 小时）以剂量依赖性方式促进红细胞中 ATP 的产生[1]。

Mitapivat（50 mg/kg；口服；每天两次，持续 21 天）可改善 β-地中海贫血小鼠模型的贫血

将重组 PK-R 酶与不同浓度的 Mitapivat 孵育（PEP 设定为 0.065 mM），检测酶活性以评估 Mitapivat 的激活作用。进行技术重复实验，并记录平均值、标准差、范围和重复次数[1] - 重组 WT PK-R 酶经 PEP 刺激，分为预先用 5 μM Mitapivat 孵育组和未孵育组，检测酶活性，采用 3 次技术重复的平均值进行数据分析[1] - 多种重组 mtPK-R 酶用 10 μM Mitapivat 处理（PEP 浓度见补充表格），测定激活倍数和 AC₅₀ 值，以评估 Mitapivat 对突变酶的激活效果[1] - 重组 R532W mtPK-R 酶经 PEP 刺激，分为预先用 5 μM Mitapivat 孵育组和未孵育组，检测酶活性（3 次技术重复的平均值）[1] - 重组 R532W mtPK-R 酶与不同浓度的 FBP 或 Mitapivat 孵育（PEP = 0.05 mM），检测酶活性以比较两种化合物的激活作用[1] - WT 或 R510Q 重组酶与 5 μM Mitapivat 在 53°C 孵育（PEP = 2 mM），随时间检测残余活性，以评估 Mitapivat 对酶热稳定性的影响[1] - 测定 Mitapivat 或 FBP（终 assay 浓度均为 5 μM，PEP = 2 mM）从重组 R510Q 酶上的解离速率，以评估药物与突变酶的结合动力学[1] - 采用偶联酶分光光度法检测健康供体、PK 缺乏症患者和小鼠 RBCs 中的 PK 活性（患者 B 除外，其活性通过液相色谱-串联质谱法直接测定丙酮酸生成量来评估）。在特定 PEP 浓度（如 0.1 mM、0.5 mM）下进行实验，以确定 Mitapivat 对 PK 活性的影响[1, 3]

健康供体的 RBCs 与不同浓度的 Mitapivat 过夜孵育后，检测 PK-R 活性（PEP = 0.1 mM）和 ATP 水平，以评估药物对健康 RBCs 的体外作用[1] - PK 缺乏症患者的 RBCs 与 Mitapivat 孵育 24 小时后，检测 PK-R 活性（PEP = 0.5 mM）和 ATP 水平。此外，将 PK 缺乏症患者的 RBC 裂解液与 2 mM Mitapivat 孵育（或不孵育）2 小时（37°C），随后在 53°C 下热处理不同时间（5、10、20、40、60 分钟），检测残余 PK 活性，以评估 PK 热稳定性[3] - 采用渗透扫描曲线评估 PK 缺乏症患者的 RBC 变形能力，并确定 Mitapivat 体外处理（20 mM，24 小时）对 RBC 变形能力的影响[3] - PK 缺乏症患者和健康对照的红系细胞体外培养，分为加入 2 mM Mitapivat 组和未加入组。观察增殖和分化不同阶段的细胞形态，检测细胞增殖（细胞数量百分比），并测定 PK/己糖激酶（HK）比率，以评估 Mitapivat 对红系细胞功能的影响[3] - 用 Mitapivat 体外处理 β-地中海贫血患者的红系前体细胞，评估其对红细胞生成、红系成熟和凋亡的影响[2]

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.

Oncotarget.2017 Jul 26;8(53):90959-90968;Technol Cancer Res Treat.2018 Jan 1;17:1533034617749418.

Drug Indication
Pyrukynd is indicated for the treatment of pyruvate kinase deficiency (PK deficiency) in adult patients (see section 4. 4).

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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]

C48H60N8O13S3

1053.23

1052.344

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

268

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

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。