METHOD FOR TREATING IDH1 INHIBITOR-RESISTANT SUBJECTS

Abstract

The present invention relates to the treatment of human cancer subjects with a mutant isocitrate dehydrogenase 1 inhibitor of formula I.

##STR00001##

Claims

1. A method of treating cancer, comprising administering to a human cancer subject having an IDH1 R132 mutation and one or more secondary IDH1 mutations a therapeutically effective amount of a compound of the formula: ##STR00006## wherein: R.sup.1 is —CH.sub.2CH(CH.sub.3).sub.2, —CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2OCH.sub.3, or —CH.sub.2-cyclopropyl; R.sup.2 is —CH.sub.3 or —CH.sub.2CH.sub.3; and X is N or CH, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein X is N, or a pharmaceutically acceptable salt thereof.

3. The method of claim 1, wherein X is N, R.sup.1 is —CH.sub.2-cyclopropyl, and R.sup.2 is —CH.sub.2CH.sub.3, or a pharmaceutically acceptable salt thereof.

4. The method of claim 1, wherein the compound is: 7-[[(1S)-1-[4[(1R)-2-Cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one; 7-[[(1S)-1-[4-[(1S)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one; or 1-Ethyl-7-[[(1S)-1-[4-[1-(4-prop-2-enoylpiperazin-1-yl)propyl]phenyl]ethyl]amino]-4H-pyrimido[4,5-d][1,3]oxazin-2-one; or a pharmaceutically acceptable salt thereof.

5. The method of claim 1, wherein the compound is: ##STR00007## or a pharmaceutically acceptable salt thereof.

6. The method of claim 5, wherein the compound is: ##STR00008##

7. The method of claim 1, wherein the cancer is a solid tumor cancer.

8. The method of claim 7, wherein the solid tumor cancer is cholangiocarcinoma, head and neck cancer, chondrosarcoma, hepatocellular carcinoma, melanoma, pancreatic cancer, astrocytoma, oligodendroglioma, glioma, glioblastoma, bladder carcinoma, colorectal cancer, lung cancer, or sinonasal undifferentiated carcinoma.

9. The method of claim 8, wherein the solid tumor cancer is cholangiocarcinoma.

10. The method of claim 1, wherein the cancer is a hematologic malignancy.

11. The method of claim 10, wherein the hematologic malignancy is acute myeloid leukemia, myelodysplastic syndrome myeloproliferative neoplasm, angioimmunoblastic T-cell lymphoma, T-cell acute lymphoblastic leukemia, polycythemia vera, essential thrombocythemia, primary myelofibrosis, or chronic myelogenous leukemia.

12. The method of claim 11, wherein the hematologic malignancy is acute myeloid leukemia.

13. The method of claim 1, wherein the compound is ##STR00009## and the cancer is acute myeloid leukemia.

14. The method of claim 1, wherein the one or more secondary IDH1 mutations is one or more of R119P, G131A, D279N, S280F, G289D or H315D.

15. The method of claim 13, wherein the one or more secondary IDH1 mutations is one or more of R119P, G131A, D279N, S280F, G289D or H315D.

16. The method of claim 1, wherein the human subject had been treated with an IDH1 inhibitor other than a compound of the formula: ##STR00010## wherein: R.sup.1 is —CH.sub.2CH(CH.sub.3).sub.2, —CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2OCH.sub.3, or —CH.sub.2-cyclopropyl; R.sup.2 is —CH.sub.3 or —CH.sub.2CH.sub.3; and X is N or CH, or a pharmaceutically acceptable salt thereof.

17. The method of claim 16, wherein the human subject had been treated with ivosidenib.

18-33. (canceled)

Description

MATERIALS, METHODS AND RESULTS

[0075] Compounds and Formulation. Ivosidenib and Compound A are prepared as a 20 mM stock in 100% DMSO (dimethyl sulfoxide) (Sigma, D2438) and diluted serially in 100% DMSO to achieve the desired concentrations. DMSO preps are further diluted with cell culture media prior to addition in the assay.

[0076] Cell lines. U-87 MG cells (ATCC, HTB-14) are cultured and assayed in MEM (Gibco, 11095) with 2 mM GlutaMAX (Gibco, 35050), 1 mM Pyruvate (Gibco, 11360), 0.1 mM NEAA ((Non-essential amino acid) Gibco, 11140) and 10% dialyzed FBS (Fetal bovine serum) (Gibco, 26400). Ba/F3 cells (DSMZ, ACC 300) are cultured and assayed in RPMI 1640 (Gibco, 22400) with 10% heat inactivated FBS (Gibco, 10082-147) and 10 ng/ml mouse IL3 (R&D systems, 403-ML-025).

[0077] Cell-based inhibition assays. Cell-based assays are performed by measuring 2-HG in either U-87 MG cells or Ba/F3 cells in which IDH mutations are expressed.

[0078] DNA constructs encoding IDH1 mutations are introduced into U-87 MG cells using transfection (Promega FuGENE HD, E2311) or lentiviral transduction, and the IDH-mutant expressing cell lines are selected using blasticidin (5 μg/ml) or puromycin (1 μg/ml). For compound treatment in U-87 MG cells, 20,000-50,000 cells per well are plated in 96 well cell culture plates (Falcon, 353377) 2 hrs prior to treatment. Cells are treated with serial dilutions of compound A in standard growth media. Plates are incubated in a mammalian cell culture incubator (humidified, 37° C., 5% CO.sub.2) for 16-72 hrs. Following the incubation period, the media is aspirated and cell lysates are prepared either by addition of 30 μL/well lysis buffer (25 mM Tris-HCl pH7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X 100, 2× Halt protease+phosphatase inhibitor (Pierce, 78441) (for derivatization LC-MS method) or by addition of 100 μL 80% methanol/20% water containing LC-MS internal standards (1 μM.sup.13C.sub.4 α-KG/.sup.13C.sub.52-HG) per well (for ion pairing LC-MS method). 96-well sample plates are then sealed, shaken at 450 rpm for 10 min, and then placed at —20° C. and stored until LC-MS analysis.

[0079] Ba/F3 cells are transfected with DNA constructs encoding IDH1R132H-myc, IDH1R132H_S280E-myc, or IDH1R132C_S280E-myc constructs using NEON Transfection system (Life Technologies, MPK10025) and isolated using Puromycin (2 μg/mL) or Blasticidin (10 μg/mL). Stably transfected lines are used for inhibitor assays. 15,000 Ba/F3 cells per well are plated in 96 well cell culture plates (Falcon, 353377) 2 hours prior to treatment. Cells are treated with serial dilutions of the desired compounds in standard growth media. Plates are incubated in a mammalian cell culture incubator (humidified, 37° C., 5% CO.sub.2) for the desired time (72 or 96 hours). Following the incubation period, conditioned media from each well is collected for 2-HG analysis by LC-MS.

[0080] LC-MS metabolite analysis of conditioned media and cell lysates. The effect of inhibitors on the concentrations of 2-HG are determined by liquid chromatography-mass spectrometry (LC-MS) analysis of cell lysates or conditioned media using either a derivatization method or an ion-pairing method as described below.

[0081] For the derivatization LC-MS method, calibration curves are prepared by spiking 2-HG and α-KG into cell culture media and cell lysis buffer respectively. The method utilizes derivatization with O-benzylhydroxylamine prior to analysis by LC-MS. 10 μL of each standard or sample (media or cell extract) is placed into a deep-well 96-well plate and combined with 100 μL of internal standard solution containing 10 μM d5-3-hydroxyglutarate and 10 μM d6-α-KG. 50 μL of 1M O-benzylhydroxylamine in pyridine buffer (8.6% pyridine, pH 5) and 50 μL of 1 M N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) in pyridine buffer is added to each sample. The derivatization reaction proceeds at room temperature for one hour. Using a Beckman Biomek FX liquid handler, 300 μL of ethyl acetate is added to each sample. Plates are sealed and vortexed for 5 minutes, followed by centrifugation for 5 minutes at 4000 rpm in Eppendorf 5810R centrifuge. 220 μL of the upper layer is transferred to a new 96-well plate. Samples are dried under heated nitrogen at 50° C. and reconstituted with 100 μL of methanol/water (1:1). 1 μL of derivatized sample is injected onto an LC-MS system consisting of a Shimadzu Prominence 20A HPLC system and a Thermo Quantum Ultra™ triple quadrupole mass spectrometer. Analytes are separated on a Water XBridge™ C18 column (2.1×50 mm, 3.5 μm) with a flow rate of 0.6 mL/minute. Mobile phase A is 0.1% formic acid in water and mobile phase B is methanol. The gradient profile is: 0 minutes, 5% B; 2 minutes, 100% B; 4.00 minutes, 100% B; 4.1 minutes, 5% B; 5.50 minutes, stop. The mass spectrometer utilizes a HESI-II probe operated in positive ion selected reaction monitoring mode. Calibration curves are constructed by plotting analyte concentrations vs. analyte/internal standard peak area ratios and performing a quadratic fit of the data using a 1/concentration weighting with Xcalibur™ software. Analyte concentrations for the unknowns are back calculated from the calibration curves.

[0082] For the ion-pairing LC-MS method, calibration curves are prepared by spiking 2-HG and α-KG into 80% methanol/20% water containing LC-MS internal standards (1 μM .sup.13C.sub.4 α-KG/.sup.13C.sub.5 2-HG). The quantitation of 2-HG and α-KG is accomplished using an AB Sciex 6500 mass spectrometer with an ESI probe and interfaced with an UHPLC system in the negative multiple-reaction monitoring (MRM) mode. The UHPLC system consists of an Agilent 1290 binary pump, thermostatted column compartment (TCC), and sampler. The injection volume is 1 μL for cell culture extracts. The extracts are chromatographically resolved using a Hypercarb column, 2.1×20 mm, 5.0 mm Javelin HTS (Thermo Scientific, PN: 35005-022135). Mobile phase A is water/10 mM tributylamine/15 mM acetic acid. Mobile phase B is acetonitrile/20 mM tributylamine/30 mM acetic acid. The solvent flow rate is 1.0 mL/min. The isocratic condition is kept at 26% mobile phase B. The valve, sample loop, and needle are washed with 50% acetonitrile: 50% methanol for 20 seconds. The column temperature is kept at 55° C. Calibration curves are calculated by least-square linear regression with 1/x weighting. 2-HG and α-KG are quantified using standard curve and ratio of the peak area of analytes to internal standard. Data analysis is performed using MultiQuant 3.0 (AB Sciex). The raw data are exported to Excel spreadsheets.

[0083] Determination of IC.sub.50 Curves. IC.sub.50 Curves for each compound are obtained using four parameter data fitting analysis in GraphPad/Prism software.

[0084] In experiments performed essentially as described above, the IC.sub.50 results set forth in Table 1 are obtained.

TABLE-US-00001 TABLE 1 IC.sub.50 Results Ivosidenib Compound A Cell Line Construct IC.sub.50 (nM) IC.sub.50 (nM) U87MG R132H 27 0.32 U87MG R132H_S280F >1000 2.58 U87MG R132C_S280F >1000 12.48 Ba/F3 R132H 15.92 0.11 Ba/F3 R132H_S280F >1000 1.13 Ba/F3 R132C_S280F >1000 1.53

[0085] The results in Table 1 indicate that each of ivosidenib and Compound A is effective in inhibiting R132H mutant IDH1 in each R132H construct cell line. However, while Compound A is effective in inhibiting R132H_S280F mutant IDH1 and R132C_S280F in each cell line construct, ivosidenib is not effective in inhibiting R132H_S280F mutant IDH1 in either cell line construct and ivosidenib is not effective in inhibiting R132C_S280F mutant IDH1 in either cell line construct.

[0086] In experiments performed essentially as described above, the IC50 results set forth in Table 2 are obtained.

TABLE-US-00002 TABLE 2 IC50 Results Compound A Cell Line Construct IC.sub.50 (nM) Std Dev U87MG IDH1R132H_H315D cis 1.49 0.2 U87MG IDH1R132H_R119P cis 2.52 0.46 U87MG IDH1R132H_G131A cis 5.89 0.88 U87MG IDH1R132H_D279N cis 13.8 2.5 U87MG IDH1R132C_H315D cis 1.33 0.42 U87MG IDH1R132C_R119P cis 10.5 3.8 U87MG IDH1R132C_G289D cis 23.5 1.6 U87MG IDH1R132C_G131A cis 25.6 6.5 U87MG IDH1R132C_D279N cis 101.7 13.9 U87MG IDH1R132L_H315D cis 5.07 0.5 U87MG IDH1R132L_R119P cis 89.7 31.3 U87MG IDH1R132L_G289D cis 29.4 4.1 U87MG IDH1R132L_G131A cis 64.5 65.2 U87MG IDH1R132L_S280F cis 179 62 U87MG IDH1R132L_D279N cis 645 142

[0087] The results in Table 2 indicate that Compound A is effective in inhibiting IDH1 second site resistant mutants in the context of IDH1 R132H, R132C or R132L driver mutations.