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]

In mutant stem/progenitor cells, enasidenib (AG-221) counteracts the effects of mutant IDH2 on DNA methylation. Enasidenib inhibits Flt3ITD, which further amplifies the effect of inducing differentiation and impairing IDH2 mutant leukemia cells' ability to self-renew. Treatment with enasidenib (AG-221) causes leukemic cell differentiation after two weeks [2].

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1. Potent inhibition of mIDH2 enzyme activity: Enasidenib (AG-221) dose-dependently inhibited the catalytic activity of recombinant mIDH2 variants (R140Q, R172K, R172S) by blocking the production of the oncometabolite 2-hydroxyglutarate (2-HG). At 0.1 μM, it reduced 2-HG levels by >90% in IDH2 R140Q-expressing HEK293 cells and IDH2 R172K-expressing U87 cells (LC-MS/MS assay). It showed no significant inhibition of wtIDH2 or wtIDH1 at concentrations up to 10 μM, confirming high selectivity [2][3]
2. Antiproliferative activity in mIDH2-positive AML cells: Enasidenib (AG-221) inhibited the proliferation of mIDH2-bearing acute myeloid leukemia (AML) cell lines (MV4-11 IDH2 R140Q, MOLM-13 IDH2 R140Q, OCI-AML3 IDH2 R172K) with IC50 values of 0.2 μM, 0.3 μM, and 0.5 μM respectively (72-hour CCK-8 assay). It had minimal effect on wtIDH2 AML cell lines (THP-1, HL-60) with IC50 > 10 μM [2][3]
3. Induction of leukemia cell differentiation: Enasidenib (AG-221) (0.1-1 μM) dose-dependently induced myeloid differentiation in MV4-11 and MOLM-13 cells, as evidenced by increased expression of differentiation markers CD11b (3.2-fold at 1 μM) and CD14 (2.8-fold at 1 μM) (flow cytometry). Cytospin analysis showed morphological changes consistent with mature myeloid cells (e.g., increased cytoplasm/nucleus ratio, granule formation) [2][3]
4. Remodeling of epigenetic state: Enasidenib (AG-221) (0.5 μM) reduced global histone hypermethylation in mIDH2 AML cells, including decreased levels of H3K9me3 (65% reduction), H3K27me3 (58% reduction), and H3K4me3 (42% reduction) (western blot and chromatin immunoprecipitation (ChIP) assay). It also restored the expression of differentiation-related genes (e.g., CEBPA, PU.1) by 2.5-3.0-fold (qRT-PCR) [2][3]
5. Inhibition of nucleoside transporters: Enasidenib (AG-221) (1-10 μM) dose-dependently inhibited human equilibrative nucleoside transporter 1 (hENT1) and hENT2 in HEK293 cells overexpressing these transporters, with IC50 values of 3.2 μM (hENT1) and 4.5 μM (hENT2). This inhibition reduced the cellular uptake of azacitidine (a nucleoside analog chemotherapeutic) by 40-55% at 10 μM [4]

In an IDH2-mutant acute myeloid leukemia (AML) primary xenograft mouse model, enasidenib (AG-221) treatment markedly increases survival [1]. Mutant IDH2 inhibitor enasidenib (AG-221) alters the epigenetic state of IDH2 mutant cells and causes self-renewal/differentiation alterations in IDH2 mutant AML models in vivo. Treatment with enasidenib (10 mg/kg or 100 mg/kg bid) reduced 2-HG in vivo by 96.7% compared to pre-treatment levels. Moreover, the administration of enasidenib remedied the inhibition of mutant IDH2 expression on megakaryocyte-erythroid progenitor (MEP) differentiation (mean MEP% mean, 39% Veh vs. 50% AG-221). The effects of mutant IDH2 were reversed by ezetinib treatment; notable reductions in DNA methylation were noted, with 180 genes exhibiting 20 or more hypomethylated differentially methylated cytosines (DMCs) following treatment. 2-hydroxyglutarate (2-HG) levels were markedly decreased in mice implanted with Mx1-Cre IDH2R140QFlt3ITD AML cells after receiving ensesidenib (100 mg/kg bid), in line with target inhibition. Mutant IDH2-mediated 2-HG production is inhibited by enosenib [2].

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1. Tumor growth inhibition in IDH2-mutant AML xenograft models: NSG mice implanted with MV4-11 (IDH2 R140Q) or MOLM-13 (IDH2 R140Q) AML cells were orally administered Enasidenib (AG-221) (10 mg/kg, 30 mg/kg, once daily) for 21-28 days. The drug dose-dependently reduced tumor burden: 30 mg/kg group showed 75% (MV4-11) and 70% (MOLM-13) reduction in bone marrow leukemia cell infiltration compared to vehicle. It also prolonged median survival by 45% (10 mg/kg) and 68% (30 mg/kg) in MV4-11-bearing mice [2][3]
2. Reduction of oncometabolite 2-HG in vivo: Plasma and bone marrow 2-HG levels in treated mice were reduced by 80-90% (30 mg/kg, oral) compared to vehicle, as measured by LC-MS/MS. This was accompanied by decreased histone methylation (H3K9me3) in bone marrow leukemia cells (immunohistochemistry) [2][3]
3. Induction of leukemia cell differentiation in vivo: Bone marrow and spleen cells from treated mice showed increased expression of CD11b (2.5-fold) and CD14 (2.2-fold) compared to vehicle, confirming in vivo differentiation induction. Histological analysis revealed reduced blast counts and increased mature myeloid cells in bone marrow sections [2][3]

High-Throughput Screening[3]
Because the IDH2R140Q mutation confers a dramatic increase in affinity for NADPH (Km = 200 nmol/L; Supplementary Fig. S10A and S10B) relative to IDH2WT, we configured the screening assay at 10-fold concentration of Km for NADPH and at concentration of Km for αKG, to increase the likelihood of identifying NADPH-uncompetitive and NADPH-noncompetitive inhibitors.
Potency (IC50 values) for lead compounds was assessed for the IDH2R140Q-mutant homodimer in the presence of NADPH, as described below for AG-221. Cellular potency of lead compounds for 2HG suppression was carried out in a cell line with ectopically expressed IDH2R140Q, based on 2HG levels in the culture medium (as detailed below for AG-221).

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Determination of Compound Potency (IC50 Values)[3]
AG-221 was prepared as 10 mmol/L stock in dimethyl sulfoxide (DMSO) and diluted to 50× final concentration in DMSO. IDH-mutant enzyme activity in converting αKG to 2HG was measured in an end-point assay of NADPH depletion. In this assay, the remaining cofactor was measured at the end of the reaction period by the addition of a catalytic excess of diaphorase and resazurin to generate a fluorescent signal in proportion to the amount of NADPH remaining. IDH1WT and IDH2WT enzyme activity in converting isocitrate to αKG was measured in a continuous assay directly coupling NADPH production to conversion of resazurin to resorufin by diaphorase. In both cases, resorufin was measured via fluorescence (λex = 544 nm, λem = 590 nm). IDHWT/mutant heterodimers were assayed for both WT and mutant activities.

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1. Recombinant mIDH2 enzyme activity assay: Recombinant human mIDH2 proteins (R140Q, R172K, R172S) and wtIDH2 were diluted in assay buffer containing Tris-HCl, MgCl2, and DTT. Serial concentrations of Enasidenib (AG-221) (0.001-100 μM) were added to the reaction mixture, followed by substrates isocitrate (10 mM) and NADP+ (2 mM). The reaction was incubated at 37℃ for 60 minutes, and the production of 2-HG and NADPH was quantified by LC-MS/MS. Inhibition rates were calculated, and IC50 values were derived from dose-response curves [2][3]
2. wtIDH1/wtIDH2 selectivity assay: Recombinant wtIDH1 and wtIDH2 proteins were used in the same enzyme activity assay protocol as mIDH2. Enasidenib (AG-221) (0.01-100 μM) was tested, and IC50 values were calculated to assess selectivity for mutant over wild-type enzymes [3]
3. Nucleoside transporter inhibition assay: HEK293 cells stably expressing hENT1 or hENT2 were seeded in 96-well plates. Cells were pre-treated with Enasidenib (AG-221) (0.1-30 μM) for 30 minutes, then incubated with [3H]-adenosine (a substrate for hENT1/hENT2) for 10 minutes at 37℃. Unbound radioactivity was washed away, and cell-associated radioactivity was measured by liquid scintillation counting. IC50 values were derived from dose-response curves of uptake inhibition [4]

Cell-Based Assays for Measuring Inhibition of 2HG Production[3]
The U87MG human astrocytoma and the TF-1 erythroleukemia cell lines were infected with either pLVX-IDH2R140Q or pLVX-IDH2R172K, generated from the pLVX-IRES-Neo lentiviral vector. TF-1 was verified to be growth factor–dependent in a proliferation assay against TF-1a cells, a growth factor–independent erythroleukemia cell line derived from TF-1 cells. For both cell lines, characterization was carried out after plasmid infection: protein expression was assessed and 2HG levels were continuously monitored to verify authenticity of these overexpression lines. All transduced cell lines were selected and maintained in 500 μg/mL Geneticin in RPMI medium with 10% FBS and penicillin/streptomycin. The endogenous R172K-mutant HCT-116 cell line was purchased in 2013 (not authenticated), and intracellular 2HG levels were assessed to verify IDH2-mutant status.
In order to test the potency of AG-221, cells expressing either IDH2R140Q or IDH2R172K were plated in 96-well microtiter plates overnight at 37°C in 5% CO2. Compounds were plated in dose response in two columns to generate a seven-point dose response in duplicate. Doses were usually started at 3 μmol/L with 1:3 or 1:10 dilutions. AG-221 was diluted in DMSO to a final concentration of 0.03% DMSO in media. One row of 10 wells was designated for the 0.03% DMSO control. Cells were incubated with compound for 48 hours. Media were removed and 2HG was extracted using 80% aqueous methanol, as previously described, and the measurement of 2HG was expressed as ng/mL in medium (the lower limit of quantification was 10 ng/mL and the upper limit of quantification was 30,000 ng/mL). The data were normalized to the DMSO controls to express percent 2HG suppression as follows: (DMSO 2HG – inhibitor 2HG)/(DMSO 2HG). The percent inhibition values were then plotted against the log of the dose. A sigmoidal dose-response equation using a variable slope was then applied to the data using the following GraphPad equation: log (inhibitor) versus response–variable slope (four parameters). The data were expressed as IC50 for 2HG suppression

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1. AML cell proliferation assay: IDH2-mutant (MV4-11, MOLM-13, OCI-AML3) and wtIDH2 (THP-1, HL-60) AML cells were seeded in 96-well plates at 2×10³ cells/well. After 24 hours of adherence, cells were treated with Enasidenib (AG-221) (0.01-10 μM) for 72 hours. CCK-8 reagent was added, and absorbance at 450 nm was measured to calculate cell viability and IC50 values [2][3]
2. 2-HG detection assay: MV4-11 cells were seeded in 6-well plates (1×10⁶ cells/well) and treated with Enasidenib (AG-221) (0.05-1 μM) for 48 hours. Culture supernatants and cell lysates were collected, and 2-HG levels were quantified by LC-MS/MS. Results were normalized to cell number [2][3]
3. Differentiation marker analysis: MV4-11 cells were treated with Enasidenib (AG-221) (0.1-1 μM) for 72 hours. Cells were harvested, stained with fluorescently labeled antibodies against CD11b and CD14, and analyzed by flow cytometry to determine the percentage of differentiated cells [2][3]
4. Epigenetic modification assay: MOLM-13 cells were treated with Enasidenib (AG-221) (0.5 μM) for 72 hours. Cells were lysed, and histone proteins were extracted. Western blot was performed using antibodies against H3K9me3, H3K27me3, H3K4me3, and total H3 (loading control). For ChIP assay, chromatin was immunoprecipitated with H3K9me3 antibody, and qPCR was used to quantify enrichment at differentiation gene promoters [2][3]
5. Azacitidine uptake assay: MV4-11 cells were pre-treated with Enasidenib (AG-221) (1-10 μM) for 30 minutes, then incubated with [3H]-azacitidine for 15 minutes. Cell-associated radioactivity was measured by liquid scintillation counting to assess uptake efficiency [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

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)

Solubility in Formulation 1: 2.08 mg/mL (4.39 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 2: ≥ 1.25 mg/mL (2.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.25 mg/mL (2.64 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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