IDH1R132H/isocitrate dehydrogenase 1 mutant

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].

---

Biochemical and cell biology profiling revealed that IvosidenibAG-120 inhibited several IDH1-R132 mutants with potency similar to that seen for R132H (Table 3) and was highly selective for IDH1 isoforms, showing no inhibition of IDH2 (WT or mutant) isoforms at micromolar concentrations (Table S2). Ivosidenib/AG-120 at 100 μM did not inhibit multiple dehydrogenases tested (Table S3).[1]

In vitro, AG-120 exhibited rapid-equilibrium inhibition against the mIDH1-R132 homodimer. Kinetic studies of binding to demonstrate mode of action were inconclusive due to persistent prebound NADP(H) in all soluble mIDH1 enzyme preparations (Supporting Information, Figures S1 and S2). Surprisingly, AG-120 demonstrated slow-tight binding inhibition against the IDH1-WT homodimer (Figure S3 and S4).[1]

AG-120/Ivosidenib also showed good cellular potency across multiple mIDH1-R132 endogenous and overexpressing cell lines (Table 3), indicating its potential for use across all mIDH1-R132 cancers. [1]

IDH mutations have been shown to block normal cellular differentiation via epigenetic and metabolic rewiring.1,3−5 To determine the effect of mIDH1 inhibition in primary human AML blast cells, mIDH1-R132H, mIDH1-R132C, and IDH1-WT, bone marrow or peripheral blood samples from patients (Table S5) were treated with Ivosidenib/AG-120 in an ex vivo assay. Living blast cells were sorted and cultured in medium containing cytokines (at a density of 0.5 × 106 cells/mL) in the presence or absence of AG-120. In mIDH1 samples, AG-120 reduced the level of intracellular 2-HG by 96% at the lowest tested dose (0.5 μM) and by 98.6% and 99.7%, respectively, at 1 and 5 μM (Figure 2). 2-HG was not measurable in multiple IDH1-WT patient samples assessed. AG-120 induced differentiation of primary mIDH1-R132H and mIDH1-R132C (but not IDH1-WT) blast cells from patients with AML treated ex vivo, as shown by enhanced ability to form differentiated colonies in methylcellulose assays, increased levels of cell-surface markers of differentiation, and increases in the proportion of mature myeloid cells [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].

---

PK studies of Ivosidenib performed in Sprague–Dawley rats, beagle dogs, and cynomolgus monkeys showed rapid oral absorption, low total body plasma clearance (CLp) and moderate to long half-life (t1/2) (Table S4). Although moderate exposure reduction was observed in a repeat-dose study in rodents (data not shown), no exposure reduction occurred in cynomolgus monkeys, and in patients with cancer a long t1/2 and accumulation of Ivosidenib/AG-120 following multiple doses were observed.

---

Following a single oral dose of 50 mg/kg to rats with an intact blood–brain barrier, Ivosidenib/AG-120 exhibited brain penetration of 4.1% (AUC0–8h [brain]/AUC0–8h [plasma]). However, brain penetration is likely to be higher in glioma patients who have a compromised blood–brain barrier. Given that AG-120 is very potent and well tolerated, it has the potential to achieve therapeutic concentration in the brain, and its therapeutic benefit in glioma is being evaluated in clinical trials.

---

AG-120/Ivosidenib showed robust tumor 2-HG reduction in female nude BALB/c mice inoculated with HT1080 cells. Each mouse received a single oral dose of vehicle or AG-120 at 50 or 150 mg/kg by gavage. Tumor 2-HG concentration declined rapidly, with maximum inhibition (92.0% and 95.2% at the 50 mg/kg and 150 mg/kg doses, respectively) achieved at ∼12 h post dose. Tumor 2-HG concentrations approached baseline levels 48–72 h following a single dose of AG-120 (Figure 1), consistent with the reversible nature of AG-120 inhibition [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

Absorption, Distribution and Excretion
Following oral administration, ivosidenib is rapidly absorbed. The Cmax following a single oral dose is 4503 ng/mL in patients with relapsed or refractory AML, 4820 ng/mL in patients with newly diagnosed AML who were also treated with azacitidine, and 4060 ng/mL in patients with cholangiocarcinoma. The steady-state was reached within 14 days. The steady-state Cmax is 6551 ng/mL in patients with relapsed or refractory AML, 6145 ng/mL in patients with newly diagnosed AML who were also treated with azacitidine, and 4799 ng/mL in patients with cholangiocarcinoma. The Tmax ranges from two to three hours. A high-fat meal increases ivosidenib exposure.
Following oral administration of ivosidenib, about 77% of the dose was eliminated in feces, where 67% was in the form of unchanged parent drug. About 17% of the dose was excreted in urine, where 10% was in the form of unchanged ivosidenib.
The apparent volume of distribution at steady state is 403 L in patients with relapsed or refractory AML, 504 L in patients with newly diagnosed AML who were also treated with azacitidine, and 706 L in patients with cholangiocarcinoma.
The apparent clearance at steady state is 5.6 L/h in patients with relapsed or refractory AML, 4.6 L/h in patients with newly diagnosed AML who were also treated with azacitidine, and 6.1 L/h in patients with cholangiocarcinoma.

---

Metabolism / Metabolites
Ivosidenib is predominantly metabolized by CYP3A4 via oxidation. The exact chemical structures of the metabolites formed from CYP3A4-mediated oxidation have not been fully characterized. Ivosidenib can also undergo N-dealkylation and hydrolysis as minor metabolic pathways.

---

Biological Half-Life
The terminal half-life at steady state is 58 hours in patients with relapsed or refractory AML, 98 hours in patients with newly diagnosed AML who were also treated with azacitidine, and 129 hours in patients with cholangiocarcinoma.

Hepatotoxicity
Elevations in serum aminotransferase levels are common during ivosidenib therapy occurring in 15% to 20% of patients but rising above 5 times the upper limit of the normal range in only 1% to 2%. Ivosidenib has had limited clinical use but has not been linked to instances of acute liver injury with symptoms or jaundice. Because of the limited clinical experience with the use of IDH inhibitors, their potential for causing liver injury is not well defined.
In prelicensure studies, ivosidenib therapy was associated with "differentiation syndrome" in 5% to 20% of patients, which was sometimes severe and life-threatening. Differentiation syndrome is marked by rapid proliferation of myeloid cells and symptoms of respiratory distress, accompanied by hypoxia, pulmonary infiltrates and pleural effusions. Other manifestations include renal impairment, fever, lymphadenopathy, bone pain, peripheral edema 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 ivosidenib and use of corticosteroids and hydroxyurea in more severe cases. Patients can be restarted on ivosidenib once the syndrome resolves.
Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury).

---

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of ivosidenib during breastfeeding. Because ivosidenib is 92 to 96% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 93 hours and it might accumulate in the infant. The manufacturer recommends that breastfeeding be discontinued during ivosidenib therapy and for 1 month after the dose.
◉ 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.

---

Protein Binding
_In vitro_, ivosidenib is 92-96% bound to plasma proteins.

[1]. 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.

Ivosidenib is a tertiary carboxamide resulting from the formal condensation of the carboxy group of (2S)-1-(4-cyanopyridin-2-yl)-5-oxopyrrolidine-2-carboxylic acid with the secondary amino group of (2S)-2-(2-chlorophenyl)-N-(3,3-difluorocyclobutyl)-2-[(5-fluoropyridin-3-yl)amino]acetamide. It is approved by the FDA for the treatment of acute myeloid leukemia (AML) in patients with an isocitrate dehydrogenase-1 (IDH1) mutation. It has a role as an antineoplastic agent and an EC 1.1.1.42 (isocitrate dehydrogenase) inhibitor. It is a member of monochlorobenzenes, a cyanopyridine, a member of pyrrolidin-2-ones, an organofluorine compound, a tertiary carboxamide and a secondary carboxamide.

Ivosidenib is a first-in-class isocitrate dehydrogenase-1 (IDH1) inhibitor. IDH1 is an enzyme that is often mutated and overexpressed in some cancers, leading to aberrant cell growth and proliferation. Ivosidenib inhibits mutated IDH1, blocking the enzymatic activity and further differentiation of cancer cells. Ivosidenib was granted accelerated approval by the FDA in July 2018 for the treatment of relapsed of refractory acute myeloid leukemia in adults. It is currently approved to also treat newly diagnosed acute myeloid leukemia in older adults in combination [azacitidine] or as monotherapy, as well as locally advanced or metastatic cholangiocarcinoma and relapsed or refractory myelodysplastic syndromes in adults. The drug is only effective in patients with a susceptible IDH1 mutation. In February 2023, the EMA's Committee for Medicinal Products for Human Use (CHMP) adopted a positive opinion of ivosidenib and recommended it be granted marketing authorization for the treatment of acute myeloid leukemia and cholangiocarcinoma. It was fully approved by the EMA in May 2023.

Ivosidenib is an Isocitrate Dehydrogenase 1 Inhibitor. The mechanism of action of ivosidenib is as an Isocitrate Dehydrogenase 1 Inhibitor, and Cytochrome P450 3A4 Inducer, and Cytochrome P450 2C9 Inducer.
Ivosidenib is an orally available small molecule inhibitor of mutated isocitrate dehydrogenase-1 that is used as an antineoplastic agent in the treatment of adults of acute myelogenous leukemia (AML). Ivosidenib is associated with a moderate rate of serum aminotransferase elevations during therapy and is suspected to be the cause of rare instances of clinically apparent acute liver injury.

Ivosidenib is an orally available inhibitor of isocitrate dehydrogenase type 1 (IDH1), with potential antineoplastic activity. Upon administration, AG-120 specifically inhibits a mutated form of IDH1 in the cytoplasm, which inhibits the formation of the oncometabolite, 2-hydroxyglutarate (2HG). This may lead to both an induction of cellular differentiation and an inhibition of cellular proliferation in IDH1-expressing tumor cells. IDH1, an enzyme in the citric acid cycle, is mutated in a variety of cancers; it initiates and drives cancer growth by both blocking cell differentiation and catalyzing the formation of 2HG.
IVOSIDENIB is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2018 and has 3 approved and 7 investigational indications. This drug has a black box warning from the FDA. Pharmacodynamics
Ivosidenib is an antineoplastic agent that is effective in cancers with a susceptible IDH1 mutation, which indicates increased levels of oncometabolite D-2-hydroxyglutarate (D-2HG) in cancer cells. Ivosidenib decreases D-2HG levels in a dose-dependent manner by inhibiting the IDH1 enzyme. Ivosidenib inhibits both the mutant and wild-type IDH1 but does not inhibit IDH2.

---

Somatic point mutations at a key arginine residue (R132) within the active site of the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) confer a novel gain of function in cancer cells, resulting in the production of d-2-hydroxyglutarate (2-HG), an oncometabolite. Elevated 2-HG levels are implicated in epigenetic alterations and impaired cellular differentiation. IDH1 mutations have been described in an array of hematologic malignancies and solid tumors. Here, we report the discovery of AG-120 (ivosidenib), an inhibitor of the IDH1 mutant enzyme that exhibits profound 2-HG lowering in tumor models and the ability to effect differentiation of primary patient AML samples ex vivo. Preliminary data from phase 1 clinical trials enrolling patients with cancers harboring an IDH1 mutation indicate that AG-120 has an acceptable safety profile and clinical activity.
Together, these compelling preclinical data provided the rationale to advance AG-120 into clinical development. The discovery of enasidenib, which is active against mIDH2, and now AG-120 (ivosidenib) against mIDH1 as described here, presents a novel class of cancer therapy based on cellular differentiation. AG-120 is a potent mIDH1 inhibitor with favorable nonclinical and clinical safety profiles that has shown promising clinical activity in phase 1 clinical trials for both solid and hematologic malignancies. In patients with relapsed/refractory mIDH1 AML, interim results from the ongoing phase 1 trial have demonstrated an overall response rate of 42% and a complete response rate of 22% (median duration of complete response 9.3 months).15 Long-term stable disease has been observed in patients with previously treated nonenhancing mIDH1 gliomas,16 and in heavily pretreated patients with mIDH1 cholangiocarcinoma, where the median progression-free survival was 3.8 months and the 6-month progression-free survival rate was 40%.17 In these two single arm, phase 1 studies, AG-120 has demonstrated an acceptable safety profile to date.15−18 AG-120 is currently in late-stage clinical development in adults with mIDH1 AML (ClinicalTrials.gov NCT03173248), and with previously treated advanced mIDH1 cholangiocarcinoma (NCT02989857).[1]

C28H22CLF3N6O3

582.96

582.139

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

2070009-31-1

Ivosidenib;1448347-49-6;IDH1 Inhibitor 8;1448346-63-1

121230976

White to off-white solid

1.5±0.1 g/cm3

854.3±65.0 °C at 760 mmHg

470.4±34.3 °C

0.0±3.2 mmHg at 25°C

1.651

0.38

1

9

7

41

1050

2

N1(C2=NC=CC(C#N)=C2)C(=O)CC[C@H]1C(N([C@H](C1=CC=CC=C1Cl)C(NC1CC(F)(F)C1)=O)C1=CC(F)=CN=C1)=O

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

(R,S)-Ivosidenib; 2070009-31-1; (S)-N-((R)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-1-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide; Ivosidenib, (R,S)-; NDE4ELL9MX; (2R)-2-(2-chlorophenyl)-2-{1-[(2S)-1-(4-cyanopyridin-2-yl)-5-oxopyrrolidin-2-yl]-N-(5-fluoropyridin-3-yl)formamido}-N-(3,3-difluorocyclobutyl)acetamide; (2S)-N-[(1R)-1-(2-chlorophenyl)-2-[(3,3-difluorocyclobutyl)amino]-2-oxoethyl]-1-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide; (R,S)-AG-120;

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)

DMSO: 3.33 mg/mL (5.71 mM)

Solubility in Formulation 1: ≥ 0.33 mg/mL (0.57 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 3.3 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.

---

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