IDH1R132H/isocitrate dehydrogenase 1 mutant (12 nM)

Ivosidenib (AG-120) (0-13 μM; 48 hours) suppresses many IDH1-R132 mutants with comparable potency and IC50 values: IDH1-R132H (IC50=12 nM); IDH1-R132C (IC50=13 nM); IDH1-R132G (IC50=8 nM); IDH1-R132L (IC50=13 nM); and IDH1-R132S (IC50=12 nM), respectively [1].

Twelve hours after therapy, AG-120 (gavage delivery; 50 mg/kg and 150 mg/kg) produced maximal inhibition (92.0% and 95.2% at the 50 mg/kg and 150 mg/kg dosages, respectively) and rapidly decreased tumor 2-HG concentrations [1].

Determination of compound inhibition potency against the mIDH1-R132H enzyme reaction using a diaphorase/resazurin coupled system [1]
In the primary reaction, the reduction of α-KG acid to D-2-hydroxyglutarate (2-HG) is accompanied by a concomitant oxidation of NADPH to NADP. The amount of NADPH remaining at the end of the reaction time is measured in a secondary diaphorase/resazurin reaction in which the NADPH is consumed in a 1:1 molar ratio with the conversion of resazurin to the highly fluorescent resorufin. Uninhibited reactions exhibit a low fluorescence at the end of the assay, while reactions in which the consumption of NADPH by mIDH1- R132H has been inhibited by a small molecule show a high fluorescence. The primary reaction was performed in a volume of 50 L 1X Buffer (150 mM NaCl, 20 mM Tris 7.5, 10 mM MgCl2, 0.05% w/v bovine serum albumin [BSA]), contained 2 nM mIDH1-R132H, 1 mM α-KG, and 4 M NADPH, and was conducted for 60 minutes at 25°C. To perform the secondary reaction, 25 L of 1X buffer containing 36 g/mL diaphorase and 30 mM 10 resazurin was added to the primary reaction and incubated for a further 10 minutes at 25°C. Florescence was read on a Spectramax plate reader at Ex 544 Em 590. Recombinant protein was expressed and purified as previously described. 5 Compounds or compound dilutions were prepared in 100% dimethyl sulfoxide (DMSO) concentration and diluted 1:100 into the final reaction. mIDH1-R132C was assayed under similar conditions, with the exception that the 1X Buffer was 50 mM K2HPO4 pH 6.5, 40 mM NaHCO3, 5 mM MgCl2, 10% glycerol, 0.03% w/v BSA.

---

Assay of the IDH1-WT enzyme reaction for determination of inhibitor potency [1]
IDH1-WT enzyme was assayed in a modified version of the assay used for mIDH1-R132H. Since this enzyme converts NADP to NADPH stoichiometrically with the conversion of isocitrate to α-KG, NADPH product can be continuously assayed by direct coupling to the diaphorase/resazurin system and reading resorufin production at Ex 544 Em 590. Assays were conducted in 50 L of 1X Buffer (150 mM NaCl, 20 mM Tris pH 7.5, 10 mM MgCl2, 0.05% (w/v) BSA, 2 mM beta-mercaptoethanol [B-ME]) containing 50 M NADP, 70 M DL-isocitrate, and 31.2 ng/mL IDH1-WT enzyme (reaction time 1 or 16 hours). The direct coupling system comprised 20 g/mL diaphorase and 40 M resazurin.

---

Assay of the IDH2-WT enzyme reaction for determination of inhibitor potency[1]
Inhibitory potency of compounds against the IDH2-WT enzyme was determined in a coupled assay to diaphorase. In this assay, production of NADPH by IDH2-WT was linked to a concomitant reduction of resazurin to the highly fluorescent resorufin. Enzyme was diluted to 0.06 g/mL in 40 L 1X Assay Buffer (150 mM NaCl, 50 mM potassium phosphate pH 7, 10 mM MgCl2, 10% glycerol, 2 mM B-ME, 0.03% BSA), to which 1 L of compound was added in DMSO. The mixture was incubated for 16 hours at room temperature (RT). The reaction was started with the addition of 10 L of Substrate Mix (200 M isocitrate, 175 M NADP, 60 g/mL diaphorase, 200 M resazurin, in 1X Assay Buffer), and run for 30 minutes at RT. The reaction was halted with the addition of 25 L of 6% sodium dodecyl sulfate and read on a Spectramax Plate Reader at Ex544/Em590.

---

Assay of the mIDH2-R140Q and mIDH2-R172K enzyme reaction for determination of inhibitor potency[1]
Inhibitory potency against the mIDH2-R140Q and mIDH2-R172K enzymes was determined in an endpoint assay in which the amount of NADPH remaining at the end of the reaction was measured by the addition of a large excess of diaphorase and resazurin. mIDH2-R140Q 11 was diluted to 0.25 g/mL in 40 L 1X Assay Buffer (150 mM NaCl, 50 mM potassium phosphate pH 7.5, 10 mM MgCl2, 10% glycerol, 2 mM B-ME, 0.03% BSA) and incubated for 16 hours at 25°C in the presence of 1 L of compound in DMSO. The reaction was started with the addition of 10 L of Substrate Mix (20 M NADPH, 8 M α-KG, in 1X Assay Buffer) and incubated for 1 hour at 25°C. Then, remaining NADPH was measured by the addition of 25 L of Detection Mix (36 g/mL diaphorase, 18 M resazurin in 1X Assay Buffer), incubated for 5 minutes at 25°C, and read as described above. mIDH2-R172K was assayed as for mIDH2-R140Q with the following modifications: 1.25 g/mL of protein was used, the Substrate Mix contained 50 M NADPH and 6.4 M α-KG, and the compound was incubated for 1 hour before starting the reaction.

Cells were seeded in their respective growth media at a density of 5000 (U87MG, HCCC9810, COR-L 105) or 2500 (HT1080) cells/well into 96-well microtiter plates and incubated overnight at 37°C and 5% CO2. The next day, AG-120 was prepared in 100% DMSO as a 10 mM stock and then diluted in media for a final concentration of 0.1% DMSO. Highest concentration dose was 3 µM. Medium was removed from the cell plates and 200 µL of the compound dilutions were added to each well. For neurospheres, compounds and cells (40,000/well) were plated together at the same time. After 48 hours of incubation with compound at 37°C, 100 µL of media was removed from each well and analyzed as described below. The cell plates were then allowed to incubate another 24 hours. At 72 hours post compound addition, a 10 mL/plate of Promega Cell Titer Glo reagent was thawed and mixed. The cell plate was removed from the incubator and allowed to equilibrate to RT. Then 100 µL of reagent was added to each well of media. The cell plate was placed on an orbital shaker for 10 minutes and then allowed to sit at RT for 20 minutes. The plate was then read for luminescence with an integration time of 500 ms to determine any compound effects on growth inhibition (half maximal inhibition of cell proliferation, GI50).[1]

Animal/Disease Models: Female BALB/c nude mice were inoculated with HT1080 cells [1]
Doses: 50 mg/kg and 150 mg/kg
Route of Administration: intragastric (po) (po)administration; 50 mg/kg and 150 mg/kg
Experimental Results: Mouse tumors were shown 2-HG was Dramatically diminished.

---

Generation of HT1080 mIDH1-R132C xenografts[1]
All animal studies were approved by the Institutional Animal Care and Use Committee and conducted in compliance with all national and local guidelines and regulations. HT1080 mIDH1-R132C cells were grown and 3 × 106 cells were inoculated subcutaneously on the flank of female BALB/c mice. When tumors reached approximately 200 mm3 the mice were randomized into dosing groups according to tumor size and treated with AG-120. Mice were dosed orally by gavage with a single dose of AG-120 at 50 or 150 mg/kg (n = 21 per dose group). Blood and tumor tissue samples were collected at 1, 3, 6, 12, 24, 48, and 72 hours following the dose (n = 3 at each time point) and were analyzed for AG-120 and 2-HG via LC-MS/MS

[1]. Popovici-Muller J, et al. Discovery of AG-120 (Ivosidenib): A First-in-Class Mutant IDH1 Inhibitor for the Treatment of IDH1Mutant Cancers. ACS Med Chem Lett. 2018 Jan 19;9(4):300-305.

C28H22CLF3N6O3

582.97

582.13940

C, 57.69; H, 3.80; Cl, 6.08; F, 9.78; N, 14.42; O, 8.23

1448347-49-6

(R,S)-Ivosidenib;2070009-31-1;IDH1 Inhibitor 8;1448346-63-1 (racemate)

White to off-white solid

0.38

O=C([C@H](CC1)N(C2=NC=CC(C#N)=C2)C1=O)N([C@@H](C3=CC=CC=C3Cl)C(NC4CC(F)(F)C4)=O)C5=CC(F)=CN=C5

WIJZXSAJMHAVGX-DHLKQENFSA-N

InChI=1S/C28H22ClF3N6O3/c29-21-4-2-1-3-20(21)25(26(40)36-18-11-28(31,32)12-18)37(19-10-17(30)14-34-15-19)27(41)22-5-6-24(39)38(22)23-9-16(13-33)7-8-35-23/h1-4,7-10,14-15,18,22,25H,5-6,11-12H2,(H,36,40)/t22-,25-/m0/s1

(S)-N-((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-1-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide

RG-120; AG120; trade name: Tibsovo®, AG-120; AG 120; RG 120; RG120

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 (3.57 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with 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 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.

---

Solubility in Formulation 2: 2.08 mg/mL (3.57 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 ultrasonication.
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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (3.57 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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