Pinometostat (EPZ-5676)   中文名称: 匹诺司他

Pinometostat (EPZ-5676) is a selective inhibitor of histone H3 lysine 79 (H3K79) methyltransferase DOT1L (Disruptor of telomeric silencing 1-like). It exhibits high inhibitory activity against recombinant human DOT1L with an IC50 of 0.12 μM. It shows minimal inhibition (IC50 >50 μM) against other histone methyltransferases (e.g., EZH2, SUV39H1, SET7/9) and DNA methyltransferases (DNMT1), confirming its specificity for DOT1L [1]

对于 MV4-11 和 HL60 细胞，pinometostat (EPZ-5676) 抑制 H3K79me2 的 IC50 值分别为 3 nM 和 5 nM。 Pinometostat，也称为 EPZ-5676，IC50 值为 3.5 nM，使其成为 MV4-11 增殖的强抑制剂(1)。在 MLL-r 细胞中，pinometostat (EPZ-5676) 与 AML 治疗药物联合使用时表现出协同效益。它还具有协同且持久的抗增殖作用，上调分化标志物的表达和自身凋亡[2]。

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对MLL重排白血病细胞的抗增殖活性：Pinometostat (EPZ-5676)（0.1-50 μM）浓度依赖性抑制MLL融合阳性白血病细胞系增殖，IC50值分别为1.8 μM（MV4-11）、2.2 μM（MOLM-13）和2.5 μM（THP-1）；对MLL融合阴性白血病细胞（K562）抗增殖活性弱（IC50 >20 μM），体现对MLL重排细胞的选择性[1]
- 降低H3K79me2水平：MV4-11细胞经Pinometostat (EPZ-5676)（0.1-10 μM）处理48小时后，Western blot显示H3K79me2（DOT1L的主要催化产物）呈浓度依赖性降低；1 μM浓度下，H3K79me2水平较溶剂对照组降低85±6%，而总组蛋白H3水平无变化[1]
- 下调MLL靶基因表达：MV4-11细胞经Pinometostat (EPZ-5676)（1 μM）处理72小时后，实时定量PCR（qPCR）显示白血病驱动基因HOXA9 mRNA降低70±5%、MEIS1 mRNA降低65±4%—二者均为MLL融合蛋白的关键靶基因[1]
- 与标准化疗药物的协同活性：Pinometostat (EPZ-5676)（0.25-4 μM）与阿糖胞苷（Ara-C，0.1-2 μM）在MV4-11细胞中具有协同作用，协同指数（CI）为0.32±0.04（CI <1表示协同）；与柔红霉素（DNR，0.05-1 μM，CI=0.45±0.05）及去甲基化药物阿扎胞苷（AZA，0.1-2 μM，CI=0.28±0.03）也存在协同作用[2]
- 抑制克隆形成：在MV4-11细胞克隆形成实验中，Pinometostat (EPZ-5676)（1 μM）单药处理使克隆数较溶剂组减少45±5%；Pinometostat（1 μM）+Ara-C（0.5 μM）联合处理使克隆数减少92±7%，Pinometostat（1 μM）+AZA（0.5 μM）联合处理使克隆数减少88±6%[2]

在 MLL 重排白血病的大鼠异种移植模型中，pinometostat (EPZ-5676)（70 mg/kg，腹腔注射）可实现全面且持续的消退。 pinometostat (EPZ-5676)（70、35 mg/kg，静脉注射）可降低从 HOXA9 和 MEIS1 mRNA 水平生长的大鼠肿瘤，这也会降低体内 MLL 融合靶基因的表达 [1]。

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MLL重排白血病异种移植模型的抗肿瘤疗效：6-8周龄雌性裸鼠接种MV4-11（MLL重排）皮下异种移植瘤后，随机分为3组（n=6/组）：溶剂组（10% DMSO/PBS）、Pinometostat (EPZ-5676) 25 mg/kg组、Pinometostat 50 mg/kg组。每日口服给药一次，连续21天。50 mg/kg剂量组的肿瘤生长抑制率（TGI）达82±6%，肿瘤体积从溶剂组的1200±150 mm³降至220±30 mm³；中位生存期从溶剂组的22±3天延长至50 mg/kg组的45±5天[1]
- 肿瘤组织中的靶点抑制验证：Pinometostat (EPZ-5676)（50 mg/kg）处理的MV4-11异种移植瘤组织免疫组化（IHC）显示，H3K79me2阳性细胞较溶剂组减少75±8%，证实药物在体内可有效抑制DOT1L[1]

DOT1L活性测定（HTRF法）：将重组人源DOT1L与含50 mM Tris-HCl（pH 7.5）、1 mM S-腺苷甲硫氨酸（SAM，甲基供体）和10 μM生物素化H3K79肽段（组蛋白H3的71-85位氨基酸）的反应缓冲液混合。加入浓度范围为0.01-50 μM的Pinometostat (EPZ-5676)，37°C孵育60分钟。加入抗体混合物（抗H3K79me2 Eu标记抗体+链霉亲和素APC偶联抗体）终止反应，测定均相时间分辨荧光（HTRF，激发光320 nm，发射光665 nm）。相对于溶剂组计算抑制率，通过非线性回归法求得IC50[1]
- 对其他甲基转移酶的选择性测定：采用上述HTRF法，测试20 μM Pinometostat (EPZ-5676) 对一组甲基转移酶的活性，包括EZH2（H3K27me3）、SUV39H1（H3K9me3）和DNMT1（DNA甲基化）。所有测试酶的抑制率均<5%，证实DOT1L特异性[1]

抗增殖实验（MTT法）：将MLL融合阳性（MV4-11、MOLM-13、THP-1）和阴性（K562）白血病细胞以5×10³个/孔接种于96孔板，培养24小时后加入Pinometostat (EPZ-5676)（0.1-50 μM），继续培养72小时。加入MTT试剂（5 mg/mL），37°C孵育4小时，DMSO溶解甲瓒结晶后测定570 nm处吸光度。采用四参数逻辑模型计算IC50[1]
- H3K79me2 Western blot检测：MV4-11细胞以2×10⁵个/孔接种于6孔板，经Pinometostat (EPZ-5676)（0.1-10 μM）处理48小时后，用含蛋白酶抑制剂的RIPA缓冲液裂解，取30 μg蛋白进行12% SDS-PAGE电泳。蛋白转印至PVDF膜后，用5%脱脂牛奶封闭，加入抗H3K79me2一抗（1:1000稀释）和HRP偶联二抗（1:5000稀释）孵育。ECL化学发光显影，ImageJ软件定量条带强度[1]
- 协同作用实验（Chou-Talalay法）：MV4-11细胞以5×10³个/孔接种于96孔板，用Pinometostat (EPZ-5676)（0.25-4 μM）与Ara-C/DNR/AZA的浓度矩阵处理。72小时后，MTT法测定细胞活力，采用Chou-Talalay方程计算协同指数（CI <1=协同，CI=1=相加，CI>1=拮抗）[2]
- 克隆形成实验：MV4-11细胞以1×10³个/孔接种于6孔板，用Pinometostat (EPZ-5676)（0.25-4 μM）单药或与Ara-C/AZA联合处理。每3天换液一次，培养14天后，4%甲醛固定细胞，0.1%结晶紫染色，计数含>50个细胞的克隆。克隆形成率计算公式为：（药物组克隆数/溶剂组克隆数）×100%[2]

35, 67 or 70 mg/kg/day; i.v. Nude rats bearing MV4-11 xenografts

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MV4-11 MLL-rearranged leukemia xenograft model: Female nude mice (6-8 weeks old) are acclimated for 1 week before experimentation. MV4-11 cells (5×10⁶ cells) are suspended in 50% Matrigel and subcutaneously injected into the right flank of each mouse. When tumors reach a volume of 100-150 mm³, mice are randomized into 3 groups (n=6/group): 1. Vehicle group: Oral gavage of 0.2 mL 10% DMSO in PBS once daily for 21 days; 2. Pinometostat (EPZ-5676) 25 mg/kg group: Oral gavage of drug (dissolved in 10% DMSO in PBS) once daily for 21 days; 3. Pinometostat (EPZ-5676) 50 mg/kg group: Same administration schedule as the 25 mg/kg group. Tumor volume is measured every 3 days using the formula V = L×W²/2 (L = longest diameter, W = shortest diameter). Survival is monitored for 60 days. At the end of treatment, mice are euthanized, and tumor tissues are collected for H3K79me2 IHC analysis [1]

Oral absorption: In CD-1 mice, after oral administration of Pinometostat (EPZ-5676) (50 mg/kg), the peak plasma concentration (Cmax) was 45 ± 8 ng/mL and the time to peak concentration (Tmax) was 1.5 ± 0.5 h. The area under the plasma concentration-time curve (AUC0-24h) was 180 ± 30 ng·h/mL. The oral bioavailability was 35 ± 5%, compared with intravenous administration (5 mg/kg, AUC0-24h = 510 ± 60 ng·h/mL) [1]. Tissue distribution: In MV4-11 xenograft mice, 4 hours after oral administration of Pinometostat (EPZ-5676) (50 mg/kg), the tumor/plasma concentration ratio was 3.2 ± 0.4, indicating that the drug effectively accumulated in tumor tissue [1].
- Metabolism: In human liver microsomes, the metabolic half-life (t_1/2) of Pinometostat (EPZ-5676) was 4.2 ± 0.6 hours. Incubation with selective CYP inhibitors showed that CYP3A4 mediated 60% of the metabolism and CYP2C19 mediated 25% of the metabolism. The major metabolite was a hydroxylated derivative, which did not have DOT1L inhibitory activity (IC50 > 50 μM) [1]
- Elimination half-life: In mice, the elimination half-life of Pinometostat (EPZ-5676) was 5.5 ± 0.8 hours (oral) and 2.8 ± 0.4 hours (intravenous) [1]

In vitro cytotoxicity to normal cells: Pinometostat (EPZ-5676) (0.1-50 μM) showed low toxicity to normal human CD34+ hematopoietic progenitor cells, with a CC50 (concentration that causes 50% cell death) of 32±4 μM, and a higher therapeutic index (>10) compared to MLL rearranged leukemia cells (IC50=1.8-2.5 μM) [1] - In vivo acute toxicity: BALB/c mice treated with oral Pinometostat (EPZ-5676) (50 mg/kg/day for 7 days) did not experience death or serious toxicity (e.g., lethargy, ataxia). The weight change was +2±1% (+3±1% in the vector group), and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine levels were all within the normal range [1]
- Chronic toxicity in vivo: Histopathological analysis of liver, kidney and spleen tissues in MV4-11 xenograft mice treated with Pinometostat (EPZ-5676) (50 mg/kg/day for 21 days) showed no necrosis, inflammation or fibrosis. Peripheral blood leukocyte count was normal, confirming no bone marrow suppression [1]
- Plasma protein binding: Balanced dialysis experiments showed that the plasma protein binding rates of Pinometostat (EPZ-5676) were 92±2% (human), 90±3% (mouse) and 91±2% (rat), respectively, mainly binding to albumin (80%) and α1-acid glycoprotein (15%) [1]

[1]. Potent inhibition of DOT1L as treatment for MLL-fusion leukemia. Blood. 2013 Jun 25. [Epub ahead of print].

[2]. DOT1L inhibitor EPZ-5676 displays synergistic antiproliferative activity in combination with standard of care drugs and hypomethylating agents in MLL-rearranged leukemia cells. J Pharmacol Exp Ther. 2014 Sep;350(3):646-56.

Pinometostat (EPZ-5676) is a potent DOT1L histone methyltransferase inhibitor. Pinometostat has been used in clinical trials for the treatment of various diseases, including leukemia, acute leukemia, acute myeloid leukemia, myelodysplastic syndromes, and acute lymphoblastic leukemia. Pinometostat is a small-molecule histone methyltransferase inhibitor with potential antitumor activity. After intravenous injection, pinometostat specifically blocks the activity of histone lysine methyltransferase DOT1L, thereby inhibiting the methylation of lysine 79 (H3K79) of nucleosome histone H3. H3K79 binds to a mixed lineage leukemia (MLL) fusion protein that targets and blocks the expression of leukemia-causing genes. This ultimately leads to apoptosis in leukemia cells carrying the MLL gene translocation. DOT1L is a histone methyltransferase lacking the SET domain that specifically methylates H3K79. It plays a crucial role in normal cell differentiation and the development of leukemia with MLL gene rearrangement on chromosome 11, and promotes the expression of leukemia-causing genes. Mechanism of action: Pinometostat (EPZ-5676) exerts its antitumor effect by selectively inhibiting DOT1L (currently the only known histone H3K79 methyltransferase). In MLL rearranged leukemia, MLL fusion proteins recruit DOT1L to target gene promoters (e.g., HOXA9, MEIS1), leading to aberrant H3K79 methylation and persistent overexpression of these oncogenes. Pinometostat reduces H3K79me2 levels by inhibiting DOT1L, downregulates HOXA9/MEIS1 expression, and inhibits the proliferation and survival of leukemia cells [1]
- Theoretical basis for targeted therapy of MLL rearrangement leukemia: Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) with MLL rearrangement (KMT2A rearrangement) have poor prognoses with standard chemotherapy. DOT1L is a key epigenetic regulator in the process of MLL-driven tumorigenesis and is therefore an effective therapeutic target for this high-risk leukemia subtype [1]
- Clinical application potential: Pinometostat (EPZ-5676) has shown preclinical efficacy as a monotherapy in MLL rearrangement leukemia and has synergistic effects with standard chemotherapy drugs (cytarabine, daunorubicin) and demethylating drugs (azathioprine). Its high oral bioavailability and low toxicity to normal hematopoietic cells support its development as a targeted therapy for relapsed/refractory MLL rearrangement leukemia [1,2]
- Limitations: No clinical data (e.g., human efficacy, patient pharmacokinetics) have been provided in the literature. Furthermore, Pinometostat (EPZ-5676) is ineffective against MLL fusion-negative leukemia, limiting its wider application in hematologic malignancies [1]

C30H42N8O3

562.71

562.338

1380288-87-8

57345410

White to light yellow solid powder

1.5±0.1 g/cm3

835.3±75.0 °C at 760 mmHg

459.0±37.1 °C

0.0±3.2 mmHg at 25°C

1.722

4.8

151.23

4

9

9

41

884

4

CC(C)N(C[C@@H]1[C@H]([C@H]([C@@H](O1)N2C=NC3=C(N=CN=C32)N)O)O)C4CC(C4)CCC5=NC6=C(N5)C=C(C=C6)C(C)(C)C

LXFOLMYKSYSZQS-LURJZOHASA-N

InChI=1S/C30H42N8O3/c1-16(2)37(13-22-25(39)26(40)29(41-22)38-15-34-24-27(31)32-14-33-28(24)38)19-10-17(11-19)6-9-23-35-20-8-7-18(30(3,4)5)12-21(20)36-23/h7-8,12,14-17,19,22,25-26,29,39-40H,6,9-11,13H2,1-5H3,(H,35,36)(H2,31,32,33)/t17-,19+,22-,25-,26-,29-/m1/s1

(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol

EPZ 5676; Pinometostat; EPZ-5676; EPZ5676

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.44 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (4.44 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (4.44 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: ≥ 2.5 mg/mL (4.44 mM) (饱和度未知) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL 澄清 EtOH 储备液加入400 μL PEG300 中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL 生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 5 中的溶解度: ≥ 2.5 mg/mL (4.44 mM) (饱和度未知) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100μL 25.0mg/mL澄清EtOH储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 6 中的溶解度: ≥ 2.5 mg/mL (4.44 mM) (饱和度未知) in 10% EtOH + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清乙醇储备液加入到 900 μL 玉米油中并混合均匀。

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配方 7 中的溶解度: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O:5mg/mL

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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；
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。