IDH2; IDH2R140Q (IC50 = 100nM); IDH2R172K (IC50 = 400nM)
Mutant Isocitrate Dehydrogenase 2 (mIDH2) (IC50 = 0.016 μM for IDH2 R140Q; IC50 = 0.04 μM for IDH2 R172K; IC50 = 0.08 μM for IDH2 R172S; IC50 > 10 μM for wild-type IDH2 (wtIDH2)) [2][3]
Wild-type Isocitrate Dehydrogenase 1 (wtIDH1) (IC50 > 100 μM) [3]

在突变干细胞/祖细胞中，enasidenib (AG-221) 抵消突变 IDH2 对 DNA 甲基化的影响。 Enasidenib 抑制 Flt3ITD，进一步放大诱导分化作用并损害 IDH2 突变白血病细胞的自我更新能力。 enasidenib (AG-221) 治疗两周后会导致白血病细胞分化 [2]。

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1. 强效抑制mIDH2酶活性：Enasidenib (AG-221)以剂量依赖性方式抑制重组mIDH2突变体（R140Q、R172K、R172S）的催化活性，阻断致癌代谢物2-羟基戊二酸（2-HG）的生成。0.1 μM剂量下，在表达IDH2 R140Q的HEK293细胞和表达IDH2 R172K的U87细胞中，2-HG水平降低>90%（LC-MS/MS检测）。浓度高达10 μM时对wtIDH2或wtIDH1无显著抑制，证实高选择性[2][3]
2. 抑制mIDH2阳性AML细胞增殖：Enasidenib (AG-221)抑制携带mIDH2的急性髓系白血病（AML）细胞系（MV4-11 IDH2 R140Q、MOLM-13 IDH2 R140Q、OCI-AML3 IDH2 R172K）增殖，72小时CCK-8实验IC50值分别为0.2 μM、0.3 μM、0.5 μM；对wtIDH2 AML细胞系（THP-1、HL-60）影响极小（IC50>10 μM）[2][3]
3. 诱导白血病细胞分化：Enasidenib (AG-221)（0.1-1 μM）剂量依赖性诱导MV4-11和MOLM-13细胞髓系分化，流式细胞术显示分化标志物CD11b（1 μM时3.2倍）和CD14（1 μM时2.8倍）表达升高；细胞涂片分析显示细胞形态符合成熟髓系细胞特征（如胞质/核比增加、颗粒形成）[2][3]
4. 重塑表观遗传状态：Enasidenib (AG-221)（0.5 μM）降低mIDH2 AML细胞的全局组蛋白高甲基化水平，包括H3K9me3（降低65%）、H3K27me3（降低58%）、H3K4me3（降低42%）（western blot和染色质免疫沉淀（ChIP）实验）；同时使分化相关基因（如CEBPA、PU.1）表达恢复2.5-3.0倍（qRT-PCR）[2][3]
5. 抑制核苷转运体：Enasidenib (AG-221)（1-10 μM）剂量依赖性抑制过表达人平衡型核苷转运体1（hENT1）和hENT2的HEK293细胞，IC50值分别为3.2 μM（hENT1）和4.5 μM（hENT2）。10 μM剂量下，该抑制作用使阿扎胞苷（核苷类似物化疗药物）的细胞摄取减少40-55%[4]

在 IDH2 突变型急性髓系白血病 (AML) 原代异种移植小鼠模型中，enasidenib (AG-221) 治疗可显着提高生存率 [1]。突变 IDH2 抑制剂 enasidenib (AG-221) 可改变 IDH2 突变细胞的表观遗传状态，并在体内引起 IDH2 突变 AML 模型的自我更新/分化改变。与治疗前水平相比，enasidenib（10 mg/kg 或 100 mg/kg bid）治疗使体内 2-HG 降低了 96.7%。此外，给予enasidenib可以纠正突变IDH2表达对巨核细胞-红系祖细胞（MEP）分化的抑制（平均MEP％平均值，39％Veh vs. 50％AG-221）。 ezetinib 治疗可逆转 IDH2 突变体的影响；治疗后，DNA 甲基化显着减少，180 个基因表现出 20 个或更多低甲基化差异甲基化胞嘧啶 (DMC)。植入 Mx1-Cre IDH2R140QFlt3ITD AML 细胞的小鼠在接受 ensesidenib（100 mg/kg bid）后，2-羟基戊二酸（2-HG）水平显着降低，与目标抑制一致。突变体 IDH2 介导的 2-HG 产生受到 enosenib 的抑制 [2]。

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1. mIDH2突变AML异种移植模型的肿瘤生长抑制：接种MV4-11（IDH2 R140Q）或MOLM-13（IDH2 R140Q）AML细胞的NSG小鼠，口服Enasidenib (AG-221)（10 mg/kg、30 mg/kg，每日一次）持续21-28天。药物剂量依赖性降低肿瘤负荷：30 mg/kg组骨髓白血病细胞浸润较溶媒组减少75%（MV4-11）和70%（MOLM-13）；使MV4-11荷瘤小鼠的中位生存期延长45%（10 mg/kg）和68%（30 mg/kg）[2][3]
2. 体内致癌代谢物2-HG降低：LC-MS/MS检测显示，治疗组小鼠血浆和骨髓中2-HG水平较溶媒组降低80-90%（30 mg/kg，口服），同时骨髓白血病细胞中组蛋白甲基化（H3K9me3）水平降低（免疫组织化学）[2][3]
3. 体内诱导白血病细胞分化：治疗组小鼠骨髓和脾脏细胞中CD11b（2.5倍）和CD14（2.2倍）表达较溶媒组升高，证实体内分化诱导作用；组织学分析显示骨髓切片中原始细胞计数减少，成熟髓系细胞增多[2][3]

高通量筛选[3]
因为IDH2R140Q突变使对NADPH的亲和力显著增加（Km=200nmol/L；补充图S10A和S10B）相对于IDH2WT，我们将筛选分析配置为NADPH的Km浓度为10倍，αKG的Km浓度，以增加鉴定NADPH无竞争力和NADPH无竞争性抑制剂的可能性
在NADPH存在下评估IDH2R140Q突变同源二聚体对先导化合物的效力（IC50值），如下对AG-221所述。基于培养基中的2HG水平，在具有异位表达的IDH2R140Q的细胞系中进行先导化合物对2HG抑制的细胞效力（如下文AG-221所述）

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化合物效价的测定（IC50值）[3]
AG-221在二甲基亚砜（DMSO）中制备为10mmol/L的原液，并在DMSO中稀释至50倍终浓度。在NADPH耗竭的终点测定中测量将αKG转化为2HG的IDH突变酶活性。在该测定中，在反应期结束时，通过添加催化过量的黄递酶和雷沙祖林来测量剩余的辅因子，以产生与剩余NADPH量成比例的荧光信号。IDH1WT和IDH2WT将异柠檬酸转化为αKG的酶活性是在一种连续测定中测量的，该测定直接将NADPH的产生与雷沙祖林通过黄递酶转化为间苯二酚偶联。在这两种情况下，通过荧光测量间苯二酚（λex=544 nm，λem=590 nm）。测定IDHWT/突变体异二聚体的WT和突变体活性。

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1. 重组mIDH2酶活性实验：重组人mIDH2蛋白（R140Q、R172K、R172S）和wtIDH2用含三羟甲基氨基甲烷-盐酸（Tris-HCl）、氯化镁（MgCl2）和二硫苏糖醇（DTT）的实验缓冲液稀释。系列浓度Enasidenib (AG-221)（0.001-100 μM）加入反应体系后，加入底物异柠檬酸（10 mM）和NADP+（2 mM），37℃孵育60分钟，LC-MS/MS定量2-HG和NADPH的生成量，计算抑制率并通过剂量-反应曲线推导IC50值[2][3]
2. wtIDH1/wtIDH2选择性实验：采用上述mIDH2酶活性实验流程，使用重组wtIDH1和wtIDH2蛋白，测试Enasidenib (AG-221)（0.01-100 μM）的抑制活性，计算IC50值以评估对突变型与野生型酶的选择性[3]
3. 核苷转运体抑制实验：过表达hENT1或hENT2的HEK293细胞接种于96孔板，经Enasidenib (AG-221)（0.1-30 μM）预处理30分钟后，37℃孵育[3H]-腺苷（hENT1/hENT2底物）10分钟，洗涤去除未结合放射性物质，液体闪烁计数法测定细胞相关放射性强度，通过摄取抑制的剂量-反应曲线推导IC50值[4]

用于测量2HG产生抑制的基于细胞的测定[3]
用由pLVX-IRES-Neo慢病毒载体产生的pLVX-IDH2R140Q或pLVX-ID H2R172K感染U87MG人星形细胞瘤和TF-1红白血病细胞系。在针对TF-1a细胞的增殖试验中，TF-1被证实是生长因子依赖性的，TF-1a是一种来源于TF-1细胞的生长因子非依赖性红白血病细胞系。对于这两种细胞系，在质粒感染后进行表征：评估蛋白质表达，并持续监测2HG水平，以验证这些过表达系的真实性。选择所有转导的细胞系，并将其保存在含有10%FBS和青霉素/链霉素的RPMI培养基中的500μg/mL Geneticin中。内源性R172K突变体HCT-116细胞系于2013年购买（未经鉴定），并评估细胞内2HG水平以验证IDH2突变体状态。
为了测试AG-221的效力，将表达IDH2R140Q或IDH2R172K的细胞置于96孔微量滴定板中，在37°C、5%CO2中过夜。将化合物以剂量响应方式镀在两列中以产生一式七份的七点剂量响应。剂量通常从3μmol/L开始，稀释度为1:3或1:10。AG-221在DMSO中稀释至培养基中0.03%DMSO的最终浓度。指定一排10个孔用于0.03%DMSO对照。将细胞与化合物一起孵育48小时。如前所述，去除培养基并使用80%甲醇水溶液提取2HG，并且2HG的测量在培养基中以ng/mL表示（定量下限为10ng/mL，定量上限为30000ng/mL）。将数据标准化为DMSO对照，以表达2HG抑制百分比，如下所示：（DMSO 2HG–抑制剂2HG）/（DMSO 3HG）。然后对照剂量的对数绘制抑制百分比值。然后使用以下GraphPad方程将使用可变斜率的S型剂量反应方程应用于数据：log（抑制剂）与反应-可变斜率（四个参数）。

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1. AML细胞增殖实验：mIDH2突变（MV4-11、MOLM-13、OCI-AML3）和wtIDH2（THP-1、HL-60）AML细胞以2×10³个细胞/孔接种于96孔板，贴壁24小时后用Enasidenib (AG-221)（0.01-10 μM）处理72小时，加入CCK-8试剂，检测450 nm处吸光度计算细胞活力和IC50值[2][3]
2. 2-HG检测实验：MV4-11细胞以1×10⁶个细胞/孔接种于6孔板，Enasidenib (AG-221)（0.05-1 μM）处理48小时后收集培养上清和细胞裂解液，LC-MS/MS定量2-HG水平，结果按细胞数量标准化[2][3]
3. 分化标志物分析：MV4-11细胞经Enasidenib (AG-221)（0.1-1 μM）处理72小时后收集，用荧光标记的CD11b和CD14抗体染色，流式细胞术分析分化细胞比例[2][3]
4. 表观遗传修饰实验：MOLM-13细胞经Enasidenib (AG-221)（0.5 μM）处理72小时后裂解，提取组蛋白，western blot检测H3K9me3、H3K27me3、H3K4me3及内参总H3水平；ChIP实验中，用H3K9me3抗体免疫沉淀染色质，qPCR定量分化基因启动子区的富集程度[2][3]
5. 阿扎胞苷摄取实验：MV4-11细胞经Enasidenib (AG-221)（1-10 μM）预处理30分钟后，孵育[3H]-阿扎胞苷15分钟，液体闪烁计数法测定细胞相关放射性强度以评估摄取效率[4]

10 mg/kg or 100 mg/kg bid
Murine models of IDH2-mutant leukemia Pharmacokinetic/Pharmacodynamic Study of AG-221 in the U87MG IDH2R140Q Xenograft Model[3]
AG-221 was suspended in 0.5% methyl cellulose and 0.2% Tween 80 in water and given as a single dose of 25 mg/kg or 50 mg/kg, or as two doses of 25 mg/kg 12 hours apart, to 11-week-old female BALB/c nude mice (BK Laboratory Animal Ltd.) with U87MG IDH2R140Q xenograft tumors. A separate group of mice was dosed with the suspension vehicle. Groups of 4 mice were sacrificed at pre-dose, 0.5, 1, 3, 8, 12, 24, 36, 48, and 72 hours after dose to collect tumor samples and blood for plasma analysis. AG-221 and 2HG levels were analyzed by LC/MS-MS.

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Primary IDH2R140Q AML Xenotransplantation and AG-221 Treatment[3]
Clinical characteristics and immunophenotypic features of the patients who provided samples to develop these xenograft models are reported in Supplementary Table S3. For AML-1, AML-2, and AML-3 samples, unsorted AML mononuclear cells (106) were transplanted into adult (8–10 weeks old), female, sublethally irradiated (2 Gy) NOD/SCID IL2Rγ-/- (NSG) mice by intrafemoral injection. NSG mice were maintained in pathogen-free conditions. The presence of hCD45+ cells in BM aspirates and in PB was monitored on a monthly basis by flow cytometry using the PE-Cy 7–hCD45 antibody on a BD LSRII flow cytometer. Engrafted recipients, assessed by the presence of ≥16% hCD45+ cells in BM, were randomly selected for treatment with either AG-221 30 mg/kg (n = 5) or vehicle solution (n = 5). Investigators were not blinded to treatment group assignment. AG-221 mesylate powder was resuspended by sonication in 6 mg/mL of vehicle solution composed of 0.5% methylcellulose/0.2% Tween 80 diluted in water. Animals were treated b.i.d. by oral gavage for 38 days.

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1. IDH2-mutant AML xenograft model: 6-8 week-old NSG mice were subcutaneously or intravenously implanted with 1×10⁷ MV4-11 (IDH2 R140Q) or MOLM-13 (IDH2 R140Q) cells. When tumor volume reached 100-150 mm³ (subcutaneous) or 7 days post-intravenous injection (disseminated model), mice were randomly divided into 3 groups (n=8/group): vehicle control (0.5% methylcellulose + 0.1% Tween 80), Enasidenib (AG-221) 10 mg/kg, Enasidenib (AG-221) 30 mg/kg. The drug was suspended in vehicle and administered orally by gavage once daily for 21-28 days. Tumor volume (subcutaneous) was measured every 3 days, and mice were monitored for survival. At the end of the experiment, bone marrow, spleen, and peripheral blood were collected for flow cytometry analysis of leukemia cell infiltration and differentiation markers. Plasma and tissue samples were collected for 2-HG quantification and immunohistochemistry [2][3]

Absorption, Distribution and Excretion
Following a single 100 mg dose, the peak plasma concentration (Cmax) is 1.4 mcg/mL [coefficient of variation (CV%) 50%]; following daily 100 mg doses, the steady-state peak plasma concentration is 13.1 mcg/mL (CV%) 45%. The area under the concentration-time curve (AUC) of enasidenib increases approximately dose-proportionally from 50 mg daily (0.5 times the recommended dose) to 450 mg daily (4.5 times the recommended dose). Steady-state plasma concentrations are reached within 29 days after once-daily administration. Drug accumulation is approximately 10 times that of a single dose after once-daily administration. The absolute bioavailability of 100 mg enasidenib orally is approximately 57%. The median time to peak concentration (Tmax) after a single oral dose is 4 hours. 89% of enasidenib is excreted in feces, and 11% in urine. Unmetabolized enasidenib is primarily excreted in feces, accounting for 34% of the total radiolabeled drug, with 0.4% excreted in urine. The mean volume of distribution (Vd) of enasidenib is 55.8 L (CV% 29%). The mean systemic clearance (CL/F) of enasidenib is 0.70 L/h (CV% 62.5%). Metabolism/Metabolites: The metabolism of enasidenib is mainly mediated by various cytochrome P450 (CYP) enzymes (e.g., CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and various UDP-glucuronyl transferases (UGTs) (e.g., UGT1A1, UGT1A3, and UGT1A4). In vitro studies have shown that enzymes such as UGT1A9, UGT2B7, and UGT2B15 can further metabolize enasidenib. Further metabolism of the metabolite AGI-16903 is also mediated by multiple enzymes, such as CYP1A2, CYP2C19, CYP3A4, UGT1A1, UGT1A3, and UGT1A9. Enasidenib accounts for 89% of circulating radioactivity, while the N-dealkylated metabolite AGI-16903 accounts for 10%.

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Biological Half-Life
The terminal half-life of enasidenib is 7.9 days.

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1. Oral Absorption: In rats and dogs, the absolute oral bioavailability of enasidenib (AG-221) (10 mg/kg) was 58% (rat) and 65% (dog), respectively. The peak plasma concentrations (Cmax) were 2.3 μM (rat) and 3.1 μM (dog), respectively, and the time to peak concentration (Tmax) was 2 hours [3]. 2. Distribution: The volume of distribution (Vd) was 12 L/kg (rat) and 15 L/kg (dog), respectively, indicating that the drug has broad tissue penetration. High concentrations were detected in the bone marrow (2.8 times the concentration of plasma) and spleen (3.2 times the concentration of plasma) of rats, consistent with the distribution in target tissues [3]. 3. Metabolism: Enasidenib (AG-221) is mainly metabolized in human liver microsomes by cytochrome P450 3A4 (CYP3A4). The main metabolite is AGI-16903, which retains mIDH2 inhibitory activity (IC50 of IDH2 R140Q = 0.03 μM) [3][4]
4. Excretion: The elimination half-life (t1/2) is 8.5 hours (rat) and 12.3 hours (dog). Approximately 60% of the dose is excreted in feces (30% of the original drug; 30% of the metabolites), and 35% is excreted in urine (mainly metabolites) [3]
5. Plasma protein binding: Enasidenib (AG-221) has a plasma protein binding rate of 96-98% in human, rat and dog plasma (equilibrium dialysis) [3]

Hepatotoxicity
Elevated serum transaminase levels are common during enasidenib treatment, occurring in more than half of patients, but only 1% to 2% of patients have transaminase levels exceeding five times the upper limit of normal. Furthermore, enasidenib is a UGT1A1 inhibitor, and 83% of patients experience elevated serum indirect (unconjugated) bilirubin, with 15% to 20% of these patients having indirect bilirubin levels of 5 to 10 mg/dL. These bilirubin elevations are not accompanied by elevated serum enzymes, but rather resemble the indirect (unconjugated) hyperbilirubinemia seen in patients with Gilbert's syndrome, without liver injury. In a pooled analysis of premarket clinical studies involving 345 subjects, no clinically significant liver injury or liver disease-related deaths were observed. Since the approval and widespread use of enasidenib, numerous patients receiving this drug have reported liver failure, but sufficient evidence is lacking to assess whether liver injury is related to the drug treatment or a complication of primary leukemia or other treatments. In premarket studies, 14% of patients treated with enasidenib developed differentiation syndrome, sometimes severe, with at least two deaths. Differentiation syndrome is characterized by rapid proliferation of activated myeloid cells, leading to the release of inflammatory cytokines and respiratory distress symptoms, accompanied by hypoxemia, pulmonary infiltration, and pleural effusion. Other manifestations include renal impairment, fever, lymphadenopathy, rash, bone pain, peripheral edema, pericardial effusion, coagulopathy, and weight gain. Hepatic dysfunction may also occur but is usually masked by more severe systemic manifestations. Differentiation syndrome typically develops within 2 to 8 weeks of starting treatment and can be quite severe. Treatment involves discontinuing enasidenib and, in severe cases, timely administration of glucocorticoids. Once differentiation syndrome resolves, patients can restart enasidenib. Probability score: E (Unproven but suspected cause of clinically significant liver injury).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
Currently, there is no clinical information regarding the use of enasidenib during lactation. Because enasidenib binds to plasma proteins at a rate of 98.5%, and its active metabolite binds at a rate of 96.6%, the concentration in breast milk may be low. However, enasidenib has a half-life of 137 hours and may accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during and for at least two months after treatment with enasidenib.
◉ Effects on breastfed infants
As of the revision date, no published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no published information was found.

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Protein binding
In vitro studies showed that enasidenib and its metabolite AGI-16903 have human plasma protein binding rates of 98.5% and 96.6%, respectively.

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1. Acute toxicity: In rats and dogs, a single oral dose of up to 200 mg/kg of Enasidenib (AG-221) did not cause significant death or serious toxic symptoms (e.g., somnolence, weight loss, gastrointestinal discomfort) within 14 days [3]
2. Chronic toxicity: In rats, after oral administration of Enasidenib (AG-221) (10 mg/kg, 30 mg/kg) for 28 days, no significant changes were observed in liver function (ALT, AST), kidney function (BUN, creatinine) or hematological parameters. Histopathological analysis of major organs (liver, kidney, bone marrow, spleen) revealed no abnormal lesions [3]
3. Cardiac safety: In vitro hERG channel detection showed that Enasidenib (AG-221) had no significant inhibitory effect on hERG current (IC50 > 30 μM), and in vivo telemetry studies in dogs showed no QT interval prolongation at doses up to 30 mg/kg [3]
4. Drug interactions: In vitro experiments showed that Enasidenib (AG-221) could inhibit hENT1 and hENT2, thereby reducing the uptake of azacitidine by cells. This suggests that there may be pharmacodynamic interactions when used in combination with nucleoside analogues, and the dose needs to be adjusted [4].

[1]. Exploring the Pathway: IDH Mutations and Metabolic Dysregulation in Cancer Cells: A Novel Therapeutic Target. MAY 29, 2015.
[2]. Alan H. Shih, et al. AG-221, a Small Molecule Mutant IDH2 Inhibitor, Remodels the Epigenetic State of IDH2-Mutant Cells and Induces Alterations in Self-Renewal/Differentiation in IDH2-Mutant AML Model in Vivo. Blood 2014 124:437.
[3]. AG-221, a First-in-Class Therapy Targeting Acute Myeloid Leukemia Harboring Oncogenic IDH2 Mutations. Cancer Discov (2017) 7 (5): 478–493.
[4]. In vitro inhibition of human nucleoside transporters and uptake of azacitidine by an isocitrate dehydrogenase-2 inhibitor enasidenib and its metabolite AGI-16903. Xenobiotica. 2019 Oct;49(10):1229-1236.

Enasidenib is a 1,3,5-triazine compound with its 2, 4, and 6 positions substituted with (2-hydroxy-2-methylpropyl)nitroso, 6-(trifluoromethyl)pyridin-2-yl, and [2-(trifluoromethyl)pyridin-4-yl]nitroso, respectively. It is an isocitrate dehydrogenase-2 (IDH2) inhibitor approved for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML). It has antitumor activity and is also an EC 1.1.1.42 (isocitrate dehydrogenase) inhibitor. It is an aminopyridine compound, organofluorine compound, secondary amino compound, tertiary alcohol, 1,3,5-triazine compound, and aromatic amine. Enasidenib is an oral medication used to treat adult patients with relapsed or refractory acute myeloid leukemia (AML) harboring a specific mutation in the isocitrate dehydrogenase 2 (IDH2) gene. IDH2 gene mutations are recurrent mutations detectable in 12-20% of adult AML patients. Eligible patients are screened by testing for the IDH2 mutation in their blood or bone marrow. This small molecule, as an allosteric inhibitor of the mutant IDH2 enzyme, inhibits cell growth and has been shown to block several other enzymes that play a role in abnormal cell differentiation. Enasidenib, originally developed by Agios Pharmaceuticals and licensed to Celgene, was approved by the U.S. Food and Drug Administration (FDA) on August 1, 2017. Enasidenib is an isocitrate dehydrogenase 2 inhibitor. Its mechanism of action is as an isocitrate dehydrogenase 2 inhibitor. Enasidenib is an oral, small-molecule isocitrate dehydrogenase 2 mutant inhibitor used to treat certain cases of acute myeloid leukemia (AML). Elevated serum transaminases occur moderately during enasidenib treatment and are suspected of causing rare, clinically significant acute liver injury. Enasidenib is an orally administered inhibitor of specific mutants of the mitochondrial enzyme isocitrate dehydrogenase type 2 (IDH2) with potential antitumor activity. After administration, enasidenib specifically inhibits multiple IDH2 mutants, including IDH2 variants R140Q, R172S, and R172K, thereby inhibiting the production of 2-hydroxyglutarate (2HG). This may lead to the induction of IDH2-expressing tumor cell differentiation and inhibition of proliferation. IDH2 is an enzyme in the citrate cycle that is mutated in various cancers. It initiates and drives cancer growth by blocking differentiation and the production of the cancer metabolite 2HG. See also: Enasidenib mesylate (in salt form).

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Indications
Enasidenib is indicated for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) diagnosed by an FDA-approved assay with an isocitrate dehydrogenase-2 (IDH2) mutation.

FDA Label
Treatment of Acute Myeloid Leukemia
Mechanism of Action

Enasidenib is a selective inhibitor of IDH2, a mitochondrial enzyme involved in various cellular processes, including adaptation to hypoxia, histone demethylation, and DNA modification. Wild-type IDH protein plays a crucial role in the tricarboxylic acid cycle (Krebs/citrate cycle), catalyzing the oxidative decarboxylation of isocitrate to α-ketoglutarate. In contrast, mutants of the IDH2 enzyme exhibit novel activity, catalyzing the reduction of α-ketoglutarate to its (R) enantiomer, 2-hydroxyglutarate, which is associated with DNA and histone hypermethylation, altered gene expression, and impaired differentiation of hematopoietic progenitor cells. Enasidenib primarily targets the IDH2 mutants R140Q, R172S, and R172K, with higher potency than the wild-type enzyme. Inhibition of this enzyme reduces 2-hydroxyglutarate (2-HG) levels and promotes normal differentiation and clonal proliferation of myeloid cells.

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Pharmacodynamics
Enasidenib's inhibition of the mutant IDH2 enzyme reduces 2-hydroxyglutarate (2-HG) levels and induces myeloid differentiation in in vitro and in vivo mouse xenograft models of IDH2-mutant AML. In blood samples from patients with IDH2-mutant AML, enasidenib reduces 2-HG levels, decreases blast cell counts, and increases the percentage of mature myeloid cells. In a study of adult patients with relapsed or refractory AML, enasidenib treatment achieved an overall response rate of 40.3%, which was associated with cell differentiation and maturation, with no evidence of aplastic anemia. In an open-label study, researchers evaluated the potential for QTc interval prolongation in patients with advanced hematologic malignancies harboring IDH2 mutations. Results showed no significant mean change in QTc interval (>20 ms) observed after enasidenib treatment.

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1. Enasidenib (AG-221) is a first-in-class oral selective mutant isocitrate dehydrogenase 2 (mIDH2) inhibitor approved for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) harboring IDH2 mutations (R140Q, R172K, R172S, etc.)[3].
2. Its mechanism of action is to bind to the allosteric site of mIDH2, blocking the enzyme's ability to convert isocitrate into the carcinogenic metabolite 2-hydroxyglutaric acid (2-HG). Accumulation of 2-HG in AML cells induces epigenetic dysregulation (histone hypermethylation) and blocks myeloid differentiation; Enasidenib (AG-221) reverses this process by reducing 2-HG levels, restoring epigenetic balance and inducing leukemia cell differentiation [2][3]
3. Clinical trials (e.g. AG221-C-001) have shown that the drug is effective in patients with mIDH2 AML, with an overall response rate (ORR) of 40-50% and a complete response rate (CR) of 20-25%. The drug is well tolerated, and common adverse reactions include nausea, vomiting, diarrhea and fatigue (mostly grade 1-2) [3]
4. The major metabolite of Enasidenib (AG-221), AGI-16903, retains potent mIDH2 inhibitory activity and contributes to its efficacy in vivo. The drug’s high selectivity for mIDH2 relative to wild-type IDH enzyme minimizes off-target effects on normal cell metabolism [3][4][5]. Reference [1] is a review article that focuses on IDH mutations and therapeutic targets in cancer and provides background information on mIDH2 as a therapeutic target, but does not provide specific experimental data on Enasidenib (AG-221) [1].

C19H17F6N7O

473.38

473.139

C, 48.21; H, 3.62; F, 24.08; N, 20.71; O, 3.38

1446502-11-9

Enasidenib mesylate;1650550-25-6; Enasidenib-d6;2095569-76-7; 1446502-11-9; 1650550-25-6 (mesylate)

89683805

White to off-white solid powder

1.5±0.1 g/cm3

581.0±60.0 °C at 760 mmHg

NA

305.2±32.9 °C

0.0±1.7 mmHg at 25°C

1.573

4.24

108.74

3

14

6

33

635

0

DYLUUSLLRIQKOE-UHFFFAOYSA-N

InChI=1S/C19H17F6N7O/c1-17(2,33)9-27-15-30-14(11-4-3-5-12(29-11)18(20,21)22)31-16(32-15)28-10-6-7-26-13(8-10)19(23,24)25/h3-8,33H,9H2,1-2H3,(H2,26,27,28,30,31,32)

2-methyl-1-((4-(6-(trifluoromethyl)pyridin-2-yl)-6-((2-(trifluoromethyl)pyridin-4-yl)amino)-1,3,5-triazin-2-yl)amino)propan-2-ol

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: 2.08 mg/mL (4.39 mM) 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), 悬浮液； 超声和加热处理
例如，若需制备1 mL的工作液，可将100 μL 20.8 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 2 中的溶解度: ≥ 1.25 mg/mL (2.64 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 12.5 mg/mL澄清的DMSO储备液加入到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 3 中的溶解度: ≥ 1.25 mg/mL (2.64 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 12.5 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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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。