| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
|
||
| 10mg |
|
||
| 25mg |
|
||
| 50mg |
|
||
| 100mg |
|
||
| 250mg |
|
||
| Other Sizes |
|
Purity: ≥98%
Enasidenib (formerly AG221; CC90007; AG-221; CC-90007; Idhifa) is an orally bioactive, first-in-class, and selective inhibitor of IDH2 (Isocitrate dehydrogenase 2) with potential anticancer activity. As of 2017, it has been approved to treat relapsed or refractory acute myeloid leukemia in people with specific mutations of the IDH2 gene, determined by an FDA-approved IDH2 companion diagnostic test. It inhibits IDH2R140Q and IDH2R172K with IC50s of 100 and 400 nM, respectively.
| Targets |
IDH2; IDH2R140Q (IC50 = 100nM); IDH2R172K (IC50 = 400nM)
|
||
|---|---|---|---|
| ln Vitro |
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].
|
||
| ln Vivo |
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].
|
||
| Enzyme Assay |
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). 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 Assay |
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 |
||
| Animal Protocol |
|
||
| References |
[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. |
||
| Additional Infomation |
Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are observed in patients with acute myeloid leukemia (AML). Leukemia-associated IDH1/2 mutations result in aberrant accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). The observation that IDH1/2 mutations are mutually exclusive with TET2 mutations led to the finding that IDH1/2-mutant production of 2-HG inhibits TET2 function and induces changes in DNA methylation. These data suggested that small molecule inhibition of mutant IDH enzymes might reverse the aberrant epigenetic remodeling of IDH-mutant leukemia cells and restore normal hematopoietic differentiation.
We therefore investigated the in vivo efficacy of AG-221, a potent and selective mutant IDH2 inhibitor in early-phase clinical trials, in murine models of IDH2-mutant leukemia. We first assessed the impact of AG-221 on 2-HG production in hematopoietic cells expressing mutant IDH2-R140Q. AG-221 treatment (10mg/kg or 100mg/kg bid) led to a reduction in 2-HG in vivo (96.7% below pre-treatment levels). Moreover, AG-221 treatment restored megakaryocyte-erythroid progenitor (MEP) differentiation that is suppressed by mutant IDH2 expression in vivo (mean MEP% mean, 39% Veh vs 50% AG-221).
View MoreWe next investigated the impact of mutant IDH2 inhibition with AG-221 on DNA methylation in vivo. We used eRRBS, a bisulfite-based next-generation sequencing platform, to assess the effect of AG-221 therapy on DNA methylation. AG-221 or vehicle therapy treated LSK stem cells (lin- Sca+ c-Kit+) were sorted from mice expressing IDH2-R140Q and evaluated by eRRBS. AG-221 therapy reversed the effects of mutant IDH2; we observed a significant reduction in DNA methylation, including 180 genes that had 20 or more hypomethylated differentially methylated cytosines (DMCs) following treatment. 84 of these genes had reduced methylation at 10 or more DMCs in the gene promoter with AG-221 therapy compared to vehicle. Mutant IDH2 inhibition with AG-221 reversed aberrant methylation at many genes with a known role in hematopoietic proliferation and differentiation, including the master transcriptional factor RUNX1. We next assessed in vivo effects of the small-molecule IDH2-R140Q inhibitor in a mouse model of IDH2-mutant leukemia. We generated mice that simultaneously expressed a constitutive Flt3ITD knock-in allele and a conditional mutant IDH2R140Q knock-in allele. As reported recently using retroviral/transgenic models, Mx1-Cre IDH2R140QFlt3ITD developed fully penetrant, transplantable AML with expansion of c-Kit+ positive blasts in the peripheral blood, and widespread leukemic infiltration. AG-221 inhibited the serial replating capacity of IDH2R140QFlt3ITD expressing cells in vitro. We competitively transplanted IDH2R140QFlt3ITD AML cells and normal bone marrow cells into secondary recipients, and then assessed the effect of AG-221 therapy on leukemia in vivo and on disease burden. AG-221 (100mg/kg bid) treatment of mice engrafted with Mx1-Cre IDH2R140QFlt3ITD AML cells markedly reduced 2HG levels consistent with on target inhibition in vivo. AG-221 therapy induced differentiation of leukemic cells, with an increase in the CD11b+ population and a decrease in the c-Kit+ population in the peripheral blood at 2wks. We next assessed the impact of treatment with both AG-221 therapy with AC220, a potent, specific Flt3 inhibitor in late phase clinical trials. Combined IDH2R140Q and Flt3ITD inhibition resulted in a marked decrease in leukemic burden to vehicle-treated mice, with a significant reduction in leukemic cell chimerism in vivo in the setting of combined inhibition at 2 wks, (mean 45.2 fraction 88% veh, 73% AG-221, p<.01). These data demonstrate that AG-221 inhibits mutant IDH2-mediated 2-HG production in vivo and reverses the effects of mutant IDH2 on DNA methylation in mutant stem/progenitor cells. AG-221 induces differentiation and impairs self-renewal of IDH2-mutant leukemia cells, effects that are further enhanced by simultaneous inhibition of Flt3ITD. Clinical trials combining IDH2 inhibitors with other targeted AML therapies are warranted in order to increase therapeutic efficacy[1]. Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate–dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation–positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation–positive advanced hematologic malignancies[2]. 1. The present study investigated inhibitory effects of enasidenib and its metabolite AGI-16903 on (a) recombinant human nucleoside transporters (hNTs) in hNT-producing Xenopus laevis oocytes, and (b) azacitidine uptake in a normal B-lymphoblast peripheral blood cell line (PBC) and acute myeloid leukemia (AML) cell lines. 2. Enasidenib inhibited hENT1, hENT2, hENT3, and hENT4 in oocytes with IC50 values of 7, 63, 27, and 76 μM, respectively, but exhibited little inhibition of hCNT1-3. AGI-16903 exhibited little inhibition of any hNT produced in oocytes. 3. Azacitidine uptake was more than 2-fold higher in AML cells than in PBC. Enasidenib inhibited azacitidine uptake into OCI-AML2, TF-1 and PBC cells in a concentration-dependent manner with IC50 values of 0.27, 1.7, and 1.0 µM in sodium-containing transport medium, respectively. 4. IC50 values shifted approximately 100-fold higher when human plasma was used as the incubation medium (27 µM in OCI-AML2, 162 µM in TF-1, and 129 µM in PBC), likely due to high human plasma protein binding of enasidenib (98.5% bound). 5. Although enasidenib inhibits hENTs and azacitidine uptake in vitro, plasma proteins attenuate this inhibitory effect, likely resulting in no meaningful in vivo effects in humans.[3] References: [1]. https://ashpublications.org/blood/article/124/21/437/91960/AG-221-a-Small-Molecule-Mutant-IDH2-Inhibitor. [2]. https://aacrjournals.org/cancerdiscovery/article/7/5/478/6259/AG-221-a-First-in-Class-Therapy-Targeting-Acute. [3]. https://pubmed.ncbi.nlm.nih.gov/30394160/ |
| Molecular Formula |
C19H17F6N7O
|
|
|---|---|---|
| Molecular Weight |
473.38
|
|
| Exact Mass |
473.13986
|
|
| Elemental Analysis |
C, 48.21; H, 3.62; F, 24.08; N, 20.71; O, 3.38
|
|
| CAS # |
1446502-11-9
|
|
| Related CAS # |
Enasidenib mesylate;1650550-25-6; Enasidenib-d6;2095569-76-7; 1446502-11-9; 1650550-25-6 (mesylate)
|
|
| PubChem CID |
89683805
|
|
| Appearance |
Typically exists as solids (or liquids in special cases) at room temperature
|
|
| Density |
1.5±0.1 g/cm3
|
|
| Boiling Point |
581.0±60.0 °C at 760 mmHg
|
|
| Melting Point |
NA
|
|
| Flash Point |
305.2±32.9 °C
|
|
| Vapour Pressure |
0.0±1.7 mmHg at 25°C
|
|
| Index of Refraction |
1.573
|
|
| LogP |
4.24
|
|
| tPSA |
108.74
|
|
| SMILES |
CC(O)(C)CNC1=NC(C2=NC(C(F)(F)F)=CC=C2)=NC(NC3=CC(C(F)(F)F)=NC=C3)=N1
|
|
| InChi Key |
DYLUUSLLRIQKOE-UHFFFAOYSA-N
|
|
| InChi Code |
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)
|
|
| Chemical Name |
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
|
|
| Synonyms |
|
|
| HS Tariff Code |
2934.99.9001
|
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
|
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
|
|||
|---|---|---|---|---|
| Solubility (In Vivo) |
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. 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. View More
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. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1125 mL | 10.5623 mL | 21.1247 mL | |
| 5 mM | 0.4225 mL | 2.1125 mL | 4.2249 mL | |
| 10 mM | 0.2112 mL | 1.0562 mL | 2.1125 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
COA