IDH2; IDH2R140Q (IC50 = 100nM); IDH2R172K (IC50 = 400nM)

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].

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].

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.

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

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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10 mg/kg or 100 mg/kg bid

Murine models of IDH2-mutant leukemia

Absorption, Distribution and Excretion
The peak plasma concentration (Cmax) is 1.4 mcg/mL [coefficient of variation (CV%) 50%] after a single dose of 100 mg, and 13.1 mcg/mL (CV% 45%) at steady state for 100 mg daily. The area under concentration-time curve (AUC) of enasidenib increases in an approximately dose-proportional manner from 50 mg (0.5 times approved recommended dosage) to 450 mg (4.5 times approved recommended dosage) daily dose. Steady-state plasma levels are reached within 29 days of once-daily dosing. Accumulation is approximately 10-fold when administered once daily. The absolute bioavailability after a 100 mg oral dose of enasidenib is approximately 57%. After a single oral dose, the median time to Cmax (Tmax) is 4 hours.
Eighty-nine percent (89%) of enasidenib is eliminated in feces and 11% in the urine. Excretion of unchanged enasidenib accounts for 34% of the radiolabeled drug in the feces and 0.4% in the urine.
The mean volume of distribution (Vd) of enasidenib is 55.8 L (CV% 29).
Enasidenib has a mean total body clearance (CL/F) of 0.70 L/hour (CV% 62.5).

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Metabolism / Metabolites
Metabolism of enasidenib is mediated by multiple cytochrome P450 (CYP) enzymes (e.g.,CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4), and by multiple UDP glucuronosyl transferases (UGTs) (e.g., UGT1A1, UGT1A3, UGT1A4, UGT1A9, UGT2B7, and UGT2B15) in vitro. Further metabolism of the metabolite AGI-16903 is also mediated by multiple enzymes (e.g., CYP1A2, CYP2C19, CYP3A4, UGT1A1, UGT1A3, and UGT1A9) in vitro. Enasidenib accounted for 89% of the radioactivity in circulation and AGI-16903, the N-dealkylated metabolite, represented 10% of the circulating radioactivity.

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Biological Half-Life
Enasidenib has a terminal half-life of 7.9 days.

Hepatotoxicity
Elevations in serum aminotransferase levels are common during enasidenib therapy, occurring in over half of patients but rising above 5 times the ULN in only 1% to 2%. In addition, enasidenib is an inhibitor of UGT1A1 and is associated with increases in serum indirect (unconjugated) bilirubin in 83% of patients, which rise to levels of 5 to 10 mg/dL in 15% to 20% of subjects. These elevations are not accompanied by serum enzyme elevations and represent indirect (unconjugated) hyperbilirubinemia without liver injury as occurs in patients with Gilbert syndrome. In pooled analysis of prelicensure clinical studies in 345 subjects, there were no cases of clinically apparent liver injury or deaths from liver disease. Since its approval and more widespread use, reports of hepatic failure have been reported in large series of enasidenib treated subjects, but with inadequate documentation to assess whether the liver injury was related to therapy as opposed to a complication of the underlying leukemia or other treatment.
In prelicensure studies, enasidenib therapy was associated with “differentiation syndrome” in 14% of patients which was sometimes severe and was fatal in at least two instances. Differentiation syndrome is marked by rapid proliferation of activated myeloid cells resulting in release of inflammatory cytokines and symptoms of respiratory distress, accompanied by hypoxia, pulmonary infiltrates, and pleural effusions. Other manifestations include renal impairment, fever, lymphadenopathy, rash, bone pain, peripheral edema, pericardial effusion, coagulopathy, and weight gain. Liver dysfunction can also occur but is generally overshadowed by the more severe systemic manifestations. The onset of differentiation syndrome is generally within 2 to 8 weeks of starting therapy and the course can be severe. Management includes stopping enasidenib and prompt use of corticosteroids in more severe cases. Patients can be restarted on enasidenib once the syndrome resolves.
Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of enasidenib during breastfeeding. Because enasidenib is 98.5% bound to plasma proteins and its active metabolite is 96.6% bound to plasma proteins, the amount in milk is likely to be low. However, the half-life of enasidenib is 137 hours and it might accumulate in the infant. The manufacturer recommends that breastfeeding be discontinued during enasidenib therapy and for at least 2 months after the end of therapy.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Human plasma protein binding of enasidenib and its metabolite AGI-16903 are 98.5% and 96.6% respectively in vitro.

[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 which is substituted by (2-hydroxy-2-methylpropyl)nitrilo, 6-(trifluoromethyl)pyridin-2-yl and [2-(trifluoromethyl)pyridin-4-yl]nitrilo groups at positions 2,4 and 6, respectively. It is an isocitrate dehydrogenase-2 (IDH2) inhibitor which has been approved for the treatment of adults with relapsed or refractory acute myeloid leukaemia (AML). It has a role as an antineoplastic agent and an EC 1.1.1.42 (isocitrate dehydrogenase) inhibitor. It is an aminopyridine, an organofluorine compound, a secondary amino compound, a tertiary alcohol, a member of 1,3,5-triazines and an aromatic amine.
Enasidenib is an orally available treatment for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with specific mutations in the isocitrate dehydrogenase 2 (IDH2) gene, which is a recurrent mutation detected in 12-20% of adult patients with AML. Patients eligible for this treatment are selected by testing the presence of IDH2 mutations in the blood or bone marrow. This small molecule acts as an allosteric inhibitor of mutant IDH2 enzyme to prevent cell growth, and it also has shown to block several other enzymes that play a role in abnormal cell differentiation. First developed by Agios Pharmaceuticals and licensed to Celgene, enasidenib was approved by U.S. Food and Drug Administration on August 1, 2017.
Enasidenib is an Isocitrate Dehydrogenase 2 Inhibitor. The mechanism of action of enasidenib is as an Isocitrate Dehydrogenase 2 Inhibitor.
Enasidenib is an orally available small molecule inhibitor of mutant isocitrate dehydrogenase-2 that is used in the therapy of selected cases of acute myelogenous leukemia (AML). Enasidenib is associated with a moderate rate of serum aminotransferase elevations during therapy and is suspected to cause rare instances of clinically apparent acute liver injury.
Enasidenib is an orally available inhibitor of specific mutant forms of the mitochondrial enzyme isocitrate dehydrogenase type 2 (IDH2), with potential antineoplastic activity. Upon administration, enasidenib specifically inhibits various mutant forms of IDH2, including the IDH2 variants R140Q, R172S, and R172K, which inhibits the formation of 2-hydroxyglutarate (2HG). This may lead to both an induction of cellular differentiation and an inhibition of cellular proliferation in IDH2-expressing tumor cells. IDH2, an enzyme in the citric acid cycle, is mutated in a variety of cancers; it initiates and drives cancer growth by blocking differentiation and the production of the oncometabolite 2HG.
See also: Enasidenib Mesylate (has salt form).

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Drug Indication
Enasidenib is indicated for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with an isocitrate dehydrogenase-2 (IDH2) mutation as detected by an FDA-approved test.
FDA Label
Treatment of acute myeloid leukaemia

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Mechanism of Action
Enasidenib is a selective inhibitor of IDH2, a mitochondria-localized enzyme involved in diverse cellular processes, including adaptation to hypoxia, histone demethylation and DNA modification. Wild-type IDH proteins play a cruicial role in the Krebs/citric acid cycle where it catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate. In comparison, mutant forms of IDH2 enzyme mediates a neomorphic activity and catalyze reduction of α-KG to the (R) enantiomer of 2-hydroxyglutarate, which is associated with DNA and histone hypermethylation, altered gene expression and blocked cellular differentiation of hematopoietic progenitor cells. Enasidenib primarily targets the mutant IDH2 variants R140Q, R172S, and R172K with higher potency than the wild type enzyme form. Inhibition of the enzyme leads to decreased levels of 2-hydroxyglutarate (2-HG) and promotion of proper differentiation and clonal proliferation of cells of the myeloid lineage.

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Pharmacodynamics
Inhibition of the mutant IDH2 enzyme by enasidenib led to decreased 2-hydroxyglutarate (2-HG) levels and induced myeloid differentiation in vitro and in vivo in mouse xenograft models of IDH2 mutated AML. In blood samples from patients with AML with mutated IDH2, enasidenib decreased 2-HG levels, reduced blast counts, and increased percentages of mature myeloid cells. In a study involving adult patients with relapsed or refractory AML, an overall response rate of 40.3% was achieved in enasidenib therapy, which was associated with cellular differentiation and maturation without evidence of aplasia. The potential for QTc prolongation with enasidenib was evaluated in an open-label study in patients with advanced hematologic malignancies with an IDH2 mutation. No large mean changes in the QTc interval (>20 ms) were observed following treatment with enasidenib.

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.)