WO2015127172A1

WO2015127172A1 - Therapeutically active compounds and their methods of use

Info

Publication number: WO2015127172A1
Application number: PCT/US2015/016766
Authority: WO
Inventors: Samuel V. AGRESTA; Malia Ann PRAHL
Original assignee: Agios Pharmaceuticals, Inc.
Priority date: 2014-02-20
Filing date: 2015-02-20
Publication date: 2015-08-27

Abstract

Provided are compounds useful for treating cancer and methods of treating cancer comprising administering to a subject in need thereof a compound described herein. Disclosed herein are methods of treating advanced hematologic malignancies characterized by the presence of a mutant allele of IDH1. In one embodiment, the mutant IDHI has an R132X mutation. In one embodiment, the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G. In some embodiments, the advanced hematologic malignancies harbor a comutation, e.g., a co-mutation selected from NPM1, FLT3, TET2, CEBPA, DNMT3A, and MLL. In one aspect, the present invention provides a method of evaluating a subject, the method comprising: acquiring a value for the level of a compound (S)-N-((S)-1-(2-chlorophenyl)-2-((3,3<lifluorocyclobutyl)am 5HDXopyrrolidine-2-carboxamide (Compound 1 ), or the level of an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG (2HG), in the subject.

Description

THERAPEUTICALLY ACTIVE COMPOUNDS AND THEIR METHODS OF USE

CLAIM OF PRIORITY

This application claims priority from U.S. S.N. 61/942,170 filed February 20, 2014, and U.S. S.N. 62/081,537 filed November 18, 2014, each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate (i.e., a-ketoglutarate). These enzymes belong to two distinct subclasses, one of which utilizes NAD(+) as the electron acceptor and the other NADP(+). Five isocitrate dehydrogenases have been reported: three NAD(+)-dependent isocitrate dehydrogenases, which localize to the mitochondrial matrix, and two NADP(+)-dependent isocitrate dehydrogenases, one of which is mitochondrial and the other predominantly cytosolic. Each NADP(+)-dependent isozyme is a homodimer.

IDH1 (isocitrate dehydrogenase 1 (NADP+), cytosolic) is also known as IDH; IDP; IDCD; IDPC or PICD. The protein encoded by this gene is the NADP(+)-dependent isocitrate dehydrogenase found in the cytoplasm and peroxisomes. It contains the PTS-1 peroxisomal targeting signal sequence. The presence of this enzyme in peroxisomes suggests roles in the regeneration of NADPH for intraperoxisomal reductions, such as the conversion of 2, 4-dienoyl- CoAs to 3-enoyl-CoAs, as well as in peroxisomal reactions that consume 2-oxoglutarate, namely the alpha-hydroxylation of phytanic acid. The cytoplasmic enzyme serves a significant role in cytoplasmic NADPH production.

The human IDH1 gene encodes a protein of 414 amino acids. The nucleotide and amino acid sequences for human IDH1 can be found as GenBank entries NM_005896.2 and

NP_005887.2 respectively. The nucleotide and amino acid sequences for IDH1 are also described in, e.g., Nekrutenko et al., Mol. Biol. Evol. 15: 1674-1684(1998); Geisbrecht et al., J. Biol. Chem. 274:30527-30533(1999); Wiemann et al, Genome Res. 11:422-435(2001); The MGC Project Team, Genome Res. 14:2121-2127(2004); Lubec et al, Submitted (DEC-2008) to UniProtKB; Kullmann et al., Submitted (JUN-1996) to the EMBL/GenBank/DDBJ databases; and Sjoeblom et al., Science 314:268-274(2006). Non-mutant, e.g., wild type, IDHl catalyzes the oxidative decarboxylation of isocitrate to a-ketoglutarate thereby reducing NAD⁺ (NADP⁺) to NADH (NADPH), e.g., in the forward reaction:

Isocitrate + NAD⁺ (NADP⁺)→ a-KG + C0₂ + NADH (NADPH) + H⁺.

It has been discovered that mutations of IDHl present in certain cancer cells result in a new ability of the enzyme to catalyze the NAPH-dependent reduction of a-ketoglutarate to R(-)- 2-hydroxyglutarate (2HG). The production of 2HG is believed to contribute to the formation and progression of cancer (Dang, L et al, Nature 2009, 462:739-44).

The inhibition of mutant IDHl and its neoactivity is therefore a potential therapeutic treatment for cancer. Accordingly, there is an ongoing need for inhibitors of IDHl mutants having alpha hydroxyl neoactivity.

PCT Publication No. WO 2013/107291 and US Publication No. US 2013/0190249 hereby incorporated by reference in their entirety, disclose compounds that inhibit IDHl mutants (e.g., IDH1R132H or IDH1R132C). These applications additionally disclose methods for the preparation of inhibitors of mutant IDHl, pharmaceutical compositions containing these compounds, and methods for the therapy of diseases, disorders, or conditions (e.g., cancer) associated with overexpression and/or amplification of mutant IDHl .

SUMMARY OF INVENTION

Disclosed herein are methods of treating advanced hematologic malignancies, such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B-acute lymphoblastic leukemias (B-ALL), B-acute lymphoblastic leukemias (B- ALL), or lymphoma (e.g. , T-cell lymphoma), each characterized by the presence of a mutant allele of IDHl . In some embodiments, the advanced hematologic malignancies are characterized by a mutant allele of IDHl, wherein the IDHl mutation results in a new ability of the enzyme to catalyze the NAPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG) in a patient. In one embodiment, the mutant IDHl has an R132X mutation. In one embodiment, the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G. In one embodiment, the R132X mutation is R132H or R132C. In one embodiment, the R132X mutation is R132H. In some embodiments, the advanced hematologic malignancies harbor a co- mutation, e.g. , a co-mutation selected from NPMl, FLT3, TET2, CEBPA, DNMT3A, and MLL.

In one aspect, the present invention provides a method of evaluating a subject, the method comprising: acquiring, e.g., directly acquiring, a value for the level of a compound (S)- N-((S)-l-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)- l-(4-cyanopyridin-2- yl)-N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide (Compound 1), or a

pharmaceutically acceptable salt thereof; or the level of an alpha hydroxy neoactivity product, e.g. , 2HG, e.g., R-2HG (2HG), in the subject, that has been treated with Compound 1, to thereby evaluate the subject.

In another aspect, the present invention provides a method of evaluating a subject, the method comprising: administering to the subject in need thereof a compound (S)-N-((S)- l-(2- chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)- l-(4-cyanopyridin-2-yl)-N-(5- fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide (Compound 1), or a pharmaceutically acceptable salt thereof; and acquiring a value for the level of Compound 1 or the level of an alpha hydroxy neoactivity product, e.g. , 2HG, e.g., R-2HG (2HG), in the subject, to thereby evaluate the subject.

In some embodiments, acquiring comprises receiving a sample from the subject. In some embodiments, acquiring comprises transmitting the value to another party, e.g., the party that administered Compound 1.

In some embodiments, the value for the level of Compound 1 is acquired by analyzing the concentration of Compound 1 in a bodily fluid, e.g., blood, plasma or urine. In some embodiments, the value for the level of Compound 1 is acquired by analyzing the level of Compound 1 in bone marrow, e.g., analyzing a sample from a bone marrow biopsy and/or aspirate for the level of Compound 1.

In some embodiments, the value for the level of 2HG is acquired by analyzing the concentration of 2HG in a bodily fluid, e.g. , blood, plasma or urine. In some embodiments, the value for the level of 2HG is acquired by analyzing the level of 2HG in bone marrow, e.g. , analyzing a sample from a bone marrow biopsy and/or aspirate for the level of 2HG.

In some embodiments, the analysis is performed by sample analysis of bodily fluid, such as blood, plasma or urine, by e.g. , a chromatographic method, e.g., mass spectroscopy, e.g. LC- MS. In some embodiments, the analysis is performed by spectroscopic analysis, e.g., magnetic resonance-based analysis, e.g., MRI and/or MRS measurement.

In some embodiments, the subject has been administered Compound 1 less than about 30 days prior to the evaluation, e.g. , less than about 29 days, e.g. , less than about 28 days, e.g. , less than about 27 days, e.g. , less than about 26 days, e.g., less than about 25 days, less than about 24 days, e.g., less than about 23 days, e.g., less than about 22 days, e.g., less than about 21 days, e.g. , less than about 20 days, e.g. , less than about 19 days, e.g. , less than about 18 days, e.g. , less than about 17 days, e.g. , less than about 16 days, e.g., less than about 15 days, e.g. , less than about 14 days, e.g., about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, or less than 72 hours prior to the evaluation, e.g. , less than 48 hours, less than 24 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1.5 hours, less than 1 hour, less than 45 minutes, less than 30 minutes, or less than 15 minutes, prior to the evaluation.

In some embodiments, the subject has been administered, e.g., orally, Compound 1 at a dose of about 10 mg to about 3000 mg, e.g. , once or twice daily, (e.g. , about every 8-16 hours, e.g. , about every 12 hours), or (e.g. , about every 12-36 hours, e.g. , about every 24 hours), e.g. , at about 10 mg to about 60 mg, at about 60 mg to about 200 mg, at about 200 mg to about 500 mg, at about 500 mg to about 1200 mg, at about 1200 mg to about 2000 mg, or at about 2000 mg to about 3000 mg, e.g., at about 50 mg, at about 100 mg, at about 300 mg, at about 500 mg, at about 800 mg once or twice daily, e.g. , about every 12 hours, or e.g. , about every 24 hours, prior to the evaluation.

In some embodiments, the subject has or is diagnosed as having a disorder. In some embodiments, the disorder is an advanced hematologic malignancy, e.g. , an advanced

hematologic malignancy characterized by the presence of a mutant allele of IDHl . In some embodiments, the advanced hematologic malignancy is characterized by a mutant allele of IDHl, wherein the IDHl mutation results in a new ability of the enzyme to catalyze the

NAPH-dependent reduction of a-ketoglutarate to R(-)-2-hydroxyglutarate (2HG) in a patient. In one embodiment, the mutant IDHl has an R132X mutation. In one embodiment, the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G. In one embodiment, the R132X mutation is R132H or R132C. In one embodiment, the R132X mutation is R132H. In some embodiments, the advanced hematologic malignancy harbors a co- mutation, e.g. , a co-mutation selected from NPMl, FLT3, TET2, CEBPA, DNMT3A, and MLL.

In some embodiments, the disorder is selected from acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B- acute lymphoblastic leukemias (B-ALL), B-acute lymphoblastic leukemias (B-ALL), and lymphoma {e.g. , T-cell lymphoma), wherein each is characterized by the presence of a mutant allele of IDH1. In some embodiments, the disorder is selected from advanced IDH1 mutation-positive relapsed and/or refractory AML (R/R AML), untreated AML, and MDS.

In some embodiments, the subject has been previously treated with one or more chemotherapeutic agent(s). In some embodiments, the chemotherapeutic agent is selected from cytarabine (Ara-C), daunorubicin, etoposide, mitoxantrone, idarubicin, 5-azacytidine, decitabine, SGN33A, sargramostim, WT- 1 analog peptide vaccine, tipifarnib, MK-8242, campath, and 6 Mercaptopurine (6MP).

In another aspect, the present invention provides a method of evaluating a subject, the method comprising: acquiring, e.g., directly acquiring, a value for the level of blast cells, e.g., leukemic blast cells, e.g. , myeloblasts or myeloid blasts, in the subject, that has been treated with a compound (S)-N-((S)-l-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)- l-(4- cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide (Compound 1), to thereby evaluate the subject.

In another aspect, the present invention provides a method of evaluating a subject, the method comprising: administering to the subject in need thereof a compound (S)-N-((S)- l-(2- chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)- l-(4-cyanopyridin-2-yl)-N-(5- fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide (Compound 1), or a pharmaceutically acceptable salt thereof; and acquiring a value for the level of blast cells, e.g. , leukemic blast cells, e.g., myeloblasts or myeloid blasts, in the subject, to thereby evaluate the subject.

In some embodiments, acquiring comprises receiving a sample from the subject. In some embodiments, acquiring comprises transmitting the value to another party, e.g., the party that administered Compound 1. In some embodiments, the evaluation comprises acquiring a value for the level of blast cells, e.g., leukemic blast cells, e.g. , myeloblasts or myeloid blasts, e.g., a blast cell count, in a sample from the subject, and comparing the value to a reference standard. In some

embodiments, the reference standard is the total number of cells in the sample. In some embodiments, the sample comprises blast cells, myelocytes, neutrophils, promyelocytes, metamyelocytes, and monocytes.

In some embodiments, the value for the level of blast cells, e.g., leukemic blast cells, e.g. , myeloblasts or myeloid blasts, is acquired by analyzing the bone marrow, e.g., by analyzing blast counts in bone marrow aspirates. In some embodiments, the bone marrow is analyzed, e.g., about every two weeks, e.g. , (between days 12- 18, e.g, on day 15), (between days 26-32, e.g., on day 29), (between days 54-60, e.g. , on day 57), and then about every 50-60 days thereafter, e.g., every 56 days thereafter, e.g. , on days 15, 29 and 57, and then every 56 days thereafter.

In some embodiments, the subject has been administered Compound 1 less than about 30 days prior to the evaluation, e.g. , less than about 29 days, e.g. , less than about 28 days, e.g. , less than about 27 days, e.g. , less than about 26 days, e.g., less than about 25 days, less than about 24 days, e.g. , less than about 23 days, e.g., less than about 22 days, e.g., less than about 21 days, e.g. , less than about 20 days, e.g. , less than about 19 days, e.g. , less than about 18 days, e.g. , less than about 17 days, e.g. , less than about 16 days, e.g., less than about 15 days, e.g. , less than about 14 days, e.g., about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, or less than 72 hours prior to the evaluation, e.g. , less than 48 hours, less than 24 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1.5 hours, less than 1 hour, less than 45 minutes, less than 30 minutes, or less than 15 minutes, prior to the evaluation.

In some embodiments, the subject has been administered, e.g., orally, Compound 1 at a dose of about 10 mg to about 3000 mg, e.g. , once or twice daily, (e.g. , about every 8-16 hours, e.g. , about every 12 hours), or (e.g. , about every 12-36 hours, e.g. , about every 24 hours), e.g. , at about 10 mg to about 60 mg, at about 60 mg to about 200 mg, at about 200 mg to about 500 mg, at about 500 mg to about 1200 mg, at about 1200 mg to about 2000 mg, or at about 2000 mg to about 3000 mg, e.g., at about 50 mg, at about 100 mg, at about 300 mg, at about 500 mg, at about 800 mg once or twice daily, e.g. , about every 12 hours, or e.g. , about every 24 hours, prior to the evaluation. In some embodiments, the subject has or is diagnosed as having a disorder. In some embodiments, the disorder is an advanced hematologic malignancy, e.g. , an advanced

NAPH-dependent reduction of a-ketoglutarate to R(-)-2-hydroxyglutarate (2HG) in a patient. In one embodiment, the mutant IDHl has an R132X mutation. In one embodiment, the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G. In another aspect, the R132X mutation is R132H or R132C. In one embodiment, the R132X mutation is R132H.

In some embodiments, the advanced hematologic malignancy is characterized by a co- mutation, e.g. , a co-mutation selected from NPM1, FLT3, TET2, CEBPA, DNMT3A, and MLL.

In some embodiments, the disorder is selected from acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B- acute lymphoblastic leukemias (B-ALL), B-acute lymphoblastic leukemias (B-ALL), and lymphoma (e.g. , T-cell lymphoma), wherein each is characterized by the presence of a mutant allele of IDHl . In some embodiments, the disorder is selected from advanced IDHl mutation-positive relapsed and/or refractory AML (R/R AML), untreated AML, and MDS.

In another aspect, the present invention provides a method of treating a disorder in a subject, the method comprising: administering to the subject in need thereof a compound (S)-N- ((S)- l-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-l-(4-cyanopyridin-2-yl)- N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide (Compound 1), or a pharmaceutically acceptable salt thereof, in an amount sufficient to provide a reduction in blast cells, e.g., leukemic blast cells, e.g. , myeloblasts or myeloid blasts, to thereby treat the disorder. In some embodiments, the disorder is an advanced hematologic malignancy, e.g., an advanced hematologic malignancy characterized by the presence of a mutant allele of IDH1. In some embodiments, the advanced hematologic malignancy is characterized by a mutant allele of IDH1, wherein the IDH1 mutation results in a new ability of the enzyme to catalyze the

NAPH-dependent reduction of a-ketoglutarate to R(-)-2-hydroxyglutarate (2HG) in a patient. In one embodiment, the mutant IDH1 has an R132X mutation. In one embodiment, the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G. In another aspect, the R132X mutation is R132H or R132C. In one embodiment, the R132X mutation is R132H.

In some embodiments, the disorder is selected from acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B- acute lymphoblastic leukemias (B-ALL), B-acute lymphoblastic leukemias (B-ALL), and lymphoma (e.g. , T-cell lymphoma), wherein each is characterized by the presence of a mutant allele of IDH1. In some embodiments, the disorder is selected from advanced IDH1 mutation-positive relapsed and/or refractory AML (R/R AML), untreated AML, and MDS.

In some embodiments, the reduction in blast cells, e.g., leukemic blast cells, e.g. , myeloblasts or myeloid blasts, is by about at least a factor of 10, e.g. , relative to a reference standard, e.g. , by about at least a factor of 11, e.g. , by about at least a factor of 12, e.g. , by about at least a factor of 13, e.g. , by about at least a factor of 14, e.g., by about at least a factor of 15, e.g. , by about at least a factor of 16, e.g., by about at least a factor of 17, e.g. , by about at least a factor of 18, e.g. , by about at least a factor of 19, e.g. , by about at least a factor of 20, relative to a reference standard.

In another embodiment, the blast cells, e.g., leukemic blast cells, e.g., myeloblasts or myeloid blasts, are reduced relative to a reference standard, e.g., to a level that is less than about 10%, e.g., less than about 9%, e.g. , less than about 8%, e.g., less than about 7%, e.g. , less than about 6%, e.g. , less than about 5%, e.g., less than about 4%, e.g. , less than about 3%, e.g., less than about 2%, e.g., complete remission (CR), relative to a reference standard.

In some embodiments, the reference standard is the level of blast cells, e.g., leukemic blast cells, e.g., myeloblasts or myeloid blasts, in the subject prior to administration of

Compound 1, e.g. , in an untreated subject, e.g. , in a subject not previously treated with

Compound 1. In some embodiments, the subject has been previously treated with one or more chemotherapeutic agent(s). In some embodiments, the chemotherapeutic agent is selected from cytarabine (Ara-C), daunorubicin, etoposide, mitoxantrone, idarubicin, 5-azacytidine, decitabine, SGN33A, sargramostim, WT- 1 analog peptide vaccine, tipifarnib, MK-8242, campath, and 6 Mercaptopurine (6MP).

In some embodiments, the reference standard is the total number of cells in the sample. In some embodiments, the sample comprises blast cells, myelocytes, neutrophils, promyelocytes, metamyelocytes, and monocytes.

In some embodiments, the subject is monitored for an adverse event. In some

embodiments, the adverse event, includes without limitation, febrile neutropenia, dyspnea, hypotension, mental status changes, neutropenia, increase in the level of blood uric acid, bronchopulmonary aspergillosis, dizziness, prolonged electrocardiogram QT, fatigue, intracranial hemorrhage, hypoxia, leukocytosis, leukostasis, lung infection, metabolic acidosis, nausea, organ failure, pericardial effusion, fungal pneumonia, pyrexia, renal impairment, retinoic acid syndrome, septic shock, systemic Candida, tachycardia, and vertigo.

In some embodiments, the adverse event is differentiation syndrome wherein symptoms comprise fever and/or dyspnea. In some embodiments, the subject is monitored for

differentiation syndrome, and if the subject experiences differentiation syndrome is treated with steroids.

In some embodiments, the subject is monitored for an adverse event, e.g. , a serious adverse event (SAE), and if an adverse event, e.g., SAE, is experienced by the patient, then treatment is modified or discontinued.

Treatment methods described herein can additionally comprise various evaluation steps prior to and/or following treatment with Compound 1. In some embodiments, prior to and/or after treatment with Compound 1, the method further comprises the step of evaluating PK and PD parameters (e.g., tissue, blood, plasma and/or urine concentration(s) of Compound 1 or 2HG). This evaluation may be achieved by sample analysis of bodily tissue or bodily fluid, such as blood, plasma or urine by e.g., mass spectroscopy, e.g. LC-MS.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A depicts a line graph showing reduction in 2HG following a single dose (50 mg/kg) of a compound (S)-N-((S)-l-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2- oxoethyl)-l-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide (Compound 1) in an IDH1 mutant R132H xenograft model.

Figure IB depicts a bar graph showing Compound 1 (at 0.5 uM, 1 uM and 5 uM concentrations) reduced intracellular 2HG in primary human IDH-mutated blast cells (ex vivo).

Figure 2A depicts a bar graph showing the PK profile following oral administration of Compound 1 in patients treated at day -3 with a single dose, at day 15 of cycle 1, and at day 1 of cycle 2, each at doses of 100 mg BID, 300 mg QD or 500 mg QD.

Figure 2B depicts a bar graph showing that plasma concentrations of 2HG were reduced to normal ranges at day -3 with a single dose, at day 15 of cycle 1, and at day 1 of cycle 2, each at doses of 100 mg BID, 300 mg QD or 500 mg QD.

Figure 3A, 3B and 3C depict images of bone marrow aspirate showing blasts,

myelocytes, neutrophils, promyelocytes, metamyelocytes and monocytes, in a patient at baseline, e.g., untreated, after cycle 1, day 15 and cycle 1, day 28, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The details of construction and the arrangement of components set forth in the following description or illustrated in the drawings are not meant to be limiting. Other embodiments and different ways to practice the invention are expressly included. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing", "involving", and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Definitions:

As used above, and throughout the description of the invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings. As used herein, the term "elevated levels of 2HG" means 10%, 20% 30%, 50%, 75%, 100%, 200%, 500% or more 2HG than is present in a subject that does not carry a mutant IDH1 allele. The term "elevated levels of 2HG" may refer to the amount of 2HG within a cell, within a tumor, within an organ comprising a tumor, or within a bodily fluid.

As used herein, the term "acquire" or "acquiring" refers to obtaining possession of a physical entity (e.g. , a sample, e.g., blood sample or blood plasma sample), or a value, e.g. , a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Directly acquiring" means performing a process (e.g., an analytical method) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving the physical entity or value from another party or source (e.g. , a third party laboratory that directly acquired the physical entity or value). Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample, performing an analytical method, e.g. , a method as described herein, e.g. , by sample analysis of bodily fluid, such as blood or plasma by, e.g. , mass spectroscopy, e.g. LC-MS.

The term "bodily fluid" includes one or more of amniotic fluid surrounding a fetus, aqueous humour, blood (e.g., blood plasma), serum, Cerebrospinal fluid, cerumen, chyme, Cowper's fluid, female ejaculate, interstitial fluid, lymph, breast milk, mucus (e.g., nasal drainage or phlegm), pleural fluid, pus, saliva, sebum, semen, serum, sweat, tears, urine, vaginal secretion, or vomit.

As used herein, the terms "inhibit" or "prevent" include both complete and partial inhibition and prevention. An inhibitor may completely or partially inhibit the intended target.

The term "treat" means decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease/disorder (i.e., an advanced hematologic malignancy such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS),

myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B- acute lymphoblastic leukemias (B-ALL), or lymphoma (e.g., T-cell lymphoma), each characterized by the presence of a mutant allele of IDH1, lessen the severity of the disease/disorder (i.e., an advanced hematologic malignancy such as acute myelogenous leukemia (AML),

myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), chronic

myelomonocytic leukemia (CMML), B-acute lymphoblastic leukemias (B-ALL), or lymphoma (e.g., T-cell lymphoma), each characterized by the presence of a mutant allele of IDHl, or improve the symptoms associated with the disease/disorder (i.e., an advanced hematologic malignancy such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B- acute lymphoblastic leukemias (B-ALL), or lymphoma (e.g., T-cell lymphoma), each characterized by the presence of a mutant allele of IDHl .

As used herein, an amount of a compound effective to treat a disorder, or a

"therapeutically effective amount" refers to an amount of the compound, which is effective, upon single or multiple dose administration to a subject, in treating a cell, or in curing, alleviating, relieving or improving a subject with a disorder beyond that expected in the absence of such treatment.

As used herein, the term "subject" is intended to mean human. Exemplary human subjects include a human patient (referred to as a patient) having a disorder, e.g., a disorder described herein or a normal subject.

Pharmaceutical Compositions and Methods of Treatment

Provided is a method of treating advanced hematologic malignancies, such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B- acute lymphoblastic leukemias (B-ALL), or lymphoma (e.g. , T-cell lymphoma), each characterized by the presence of a mutant allele of IDHl, comprising administering to a subject in need thereof a compound (S)-N-((S)-l-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-l-(4- cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide (Compound 1), or a pharmaceutically acceptable salt thereof.

Also provided is a method of treating advanced hematologic malignancies, such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B- acute lymphoblastic leukemias (B-ALL), or lymphoma (e.g. , T-cell lymphoma), each characterized by the presence of a mutant allele of IDHl, comprising administering to subject in need thereof a pharmaceutical composition comprising Compound 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. These methods of treatment and pharmaceutical compositions are further illustrated by the detailed descriptions and illustrative examples given below.

Compositions and Routes of Administration

Compound 1, or a pharmaceutically acceptable salt thereof, utilized in the methods described herein may be formulated together with a pharmaceutically acceptable carrier or adjuvant into pharmaceutically acceptable compositions prior to being administered to a subject.

The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that may be administered to a subject, together with a compound, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

In some embodiments, pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a- tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene -polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-P-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein.

In some embodiments, the pharmaceutical compositions may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection. The pharmaceutical compositions of one aspect of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound, or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

In some embodiments, the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of

pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens or Spans and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

In some embodiments, the pharmaceutical compositions may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

In some embodiments, the pharmaceutical compositions may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing Compound 1, or a pharmaceutically acceptable salt thereof, with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

In some embodiments, topical administration of the pharmaceutical compositions is useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of Compound 1, or a pharmaceutically acceptable salt thereof, include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound, suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of one aspect of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included in one aspect of this invention.

In some embodiments, the pharmaceutical compositions may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

The compounds described herein can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneally, intramuscularly, or subcutaneously; or orally, buccally, nasally, transmucosally, topically, in an ophthalmic preparation, or by inhalation, with a dosage ranging from about 0.5 to about 100 mg/kg of body weight, alternatively dosages between 1 mg and 1000 mg/dose, every 4 to 120 hours, or according to the requirements of the particular drug. The methods herein contemplate administration of an effective amount of compound, or compound composition to achieve the desired or stated effect. Typically, the pharmaceutical compositions of one aspect of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such

administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% w/w active compound. Alternatively, such preparations contain from about 20% to about 80% active compound.

A subject may be administered a dose of Compound 1, or a pharmaceutically acceptable salt thereof, as described in Example 2. Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular subject will depend upon a variety of factors, including the activity of the specific compound, employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug

combination, the severity and course of the disease, condition or symptoms, the subject' s disposition to the disease, condition or symptoms, and the judgment of the treating physician.

Upon improvement of a subject's condition, a maintenance dose of a compound, composition or combination of one aspect of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level. Subjects may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

Some embodiments of the invention are directed toward a tablet comprising at least one pharmaceutically acceptable carrier or diluent, and Compound 1, or a pharmaceutically acceptable salt thereof.

Methods of Use

The inhibitory activities of Compound 1, and pharmaceutically acceptable salts thereof, provided herein against IDH1 mutants (e.g. , IDH1R132H or IDH1R132C) can be tested by methods described in Example A of PCT Publication No. WO 2013/107291 and US Publication No. US 2013/0190249, hereby incorporated by reference in their entirety, or analogous methods.

Provided is a method for treating an advanced hematologic malignancy such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B- acute lymphoblastic leukemias (B-ALL), or lymphoma (e.g. , T-cell lymphoma), each characterized by the presence of a mutant allele of IDHl, comprising administering to a subject in need thereof Compound 1, or a pharmaceutically acceptable salt thereof. In one embodiment, the advanced hematologic malignancy, such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B- acute lymphoblastic leukemias (B-ALL), or lymphoma (e.g., T-cell lymphoma), to be treated is characterized by a mutant allele of IDHl, wherein the IDHl mutation results in a new ability of the enzyme to catalyze the NAPH-dependent reduction of a-ketoglutarate to

R(-)-2-hydroxyglutarate in a patient. In one aspect of this embodiment, the mutant IDHl has an R132X mutation. In one aspect of this embodiment, the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G. In another aspect, the R132X mutation is R132H or R132C. In yet another aspect, the R132X mutation is R132H.

Advanced hematologic malignancies, such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), chronic

myelomonocytic leukemia (CMML), B-acute lymphoblastic leukemias (B-ALL), or lymphoma (e.g., T-cell lymphoma), each characterized by the presence of a mutant allele of IDHl can be analyzed by sequencing cell samples to determine the presence and specific nature of (e.g., the changed amino acid present at) a mutation at amino acid 132 of IDHl .

Without being bound by theory, applicants believe that mutant alleles of IDHl wherein the IDHl mutation results in a new ability of the enzyme to catalyze the NAPH-dependent reduction of a-ketoglutarate to R(-)-2-hydroxyglutarate, and in particular R132H mutations of IDHl, characterize a subset of all types of cancers, without regard to their cellular nature or location in the body. Thus, the compounds, and methods of one aspect of this invention are useful to treat advanced hematologic malignancies, such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), chronic

myelomonocytic leukemia (CMML), B-acute lymphoblastic leukemias (B-ALL), or lymphoma (e.g., T-cell lymphoma), each characterized by the presence of a mutant allele of IDH1 imparting such activity and in particular an IDH1 R132H or R132C mutation.

In one embodiment, the efficacy of treatment of advanced hematologic malignancies, such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS),

myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B- acute lymphoblastic leukemias (B-ALL), or lymphoma (e.g., T-cell lymphoma), each characterized by the presence of a mutant allele of IDH1 is monitored by measuring the levels of 2HG in the subject. Typically levels of 2HG are measured prior to treatment, wherein an elevated level is indicated for the use of Compound 1, or a pharmaceutically acceptable salt thereof, to treat the advanced hematologic malignancies, such as acute myelogenous leukemia (AML),

myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), chronic

myelomonocytic leukemia (CMML), B-acute lymphoblastic leukemias (B-ALL), or lymphoma (e.g., T-cell lymphoma), each characterized by the presence of a mutant allele of IDH1. Once the elevated levels are established, the level of 2HG is determined during the course of and/or following termination of treatment to establish efficacy. In certain embodiments, the level of 2HG is only determined during the course of and/or following termination of treatment. A reduction of 2HG levels during the course of treatment and following treatment is indicative of efficacy. Similarly, a determination that 2HG levels are not elevated during the course of or following treatment is also indicative of efficacy. Typically, the these 2HG measurements will be utilized together with other well-known determinations of efficacy of cancer treatment, such as reduction in number and size of tumors and/or other cancer-associated lesions, evaluation of bone marrow biopsies and/or aspirates, complete blood counts and examination of peripheral blood films, improvement in the general health of the subject, and alterations in other biomarkers that are associated with cancer treatment efficacy.

2HG can be detected in a sample by the methods of PCT Publication No. WO

2011/050210 and US Publication No. US2012/0121515 hereby incorporated by reference in their entirety, or by analogous methods.

Methods of evaluating samples and/or subjects

This section provides methods of obtaining and analyzing samples and of analyzing subjects. Embodiments of the method comprise evaluation of one or more parameters related to IDH1, an alpha hydroxy neoactivity, e.g., 2HG neoactivity, e.g., to evaluate the IDH1 2HG neoactivity genotype or phenotype. The evaluation can be performed, e.g. , to select, diagnose or prognose the subject, to select a therapeutic agent, e.g., an inhibitor, or to evaluate response to the treatment or progression of disease. In an embodiment the evaluation, which can be performed before and/or after treatment has begun, is based, at least in part, on analysis of a tumor sample, cancer cell sample, or precancerous cell sample, from the subject. E.g., a sample from the patient can be analyzed for the presence or level of an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, by evaluating a parameter correlated to the presence or level of an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG. An alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, in the sample can be determined by a chromatographic method, e.g., by LC-MS analysis. It can also be determined by contact with a specific binding agent, e.g., an antibody, which binds the alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, and allows detection. In an embodiment the sample is analyzed for the level of neoactivity, e.g., an alpha hydroxy neoactivity, e.g., 2HG neoactivity. In an embodiment the sample is analyzed for the presence of a mutant IDH1, protein having an alpha hydroxy neoactivity, e.g. , 2HG neoactivity (or a corresponding RNA). E.g., a mutant protein specific reagent, e.g., an antibody that specifically binds an IDH1 mutant protein, e.g., an antibody that specifically binds an IDH1- R132H mutant protein, can be used to detect neoactive mutant enzymeln an embodiment a nucleic acid from the sample is sequenced to determine if a selected allele or mutation of IDH1 disclosed herein is present. In an embodiment the analysis is other than directly determining the presence of a mutant IDH1 protein (or corresponding RNA) or sequencing of an IDH1 gene. In an embodiment the analysis is other than directly determining, e.g., it is other than sequencing genomic DNA or cDNA, the presence of a mutation at residue 132 of IDH1. E.g., the analysis can be the detection of an alpha hydroxy neoactivity product, e.g. , 2HG, e.g., R-2HG, or the measurement of the mutation's an alpha hydroxy neoactivity, e.g. , 2HG neoactivity. In an embodiment the sample is removed from the patient and analyzed. In an embodiment the evaluation can include one or more of performing the analysis of the sample, requesting analysis of the sample, requesting results from analysis of the sample, or receiving the results from analysis of the sample. (Generally herein, analysis can include one or both of performing the underlying method or receiving data from another who has performed the underlying method.) In an embodiment the evaluation, which can be performed before and/or after treatment has begun, is based, at least in part, on analysis of a tissue (e.g., a tissue other than a tumor sample), or bodily fluid, or bodily product. Exemplary tissues include lymph node, skin, hair follicles and nails. Exemplary bodily fluids include blood, plasma, urine, lymph, tears, sweat, saliva, semen, and cerebrospinal fluid. Exemplary bodily products include exhaled breath. E.g., the tissue, fluid or product can be analyzed for the presence or level of an alpha hydroxy neoactivity product, e.g. , 2HG, e.g., R-2HG, by evaluating a parameter correlated to the presence or level of an alpha hydroxy neoactivity product, e.g. , 2HG, e.g., R-2HG. An alpha hydroxy neoactivity product, e.g. , 2HG, e.g., R-2HG, in the sample can be determined by a

chromatographic method, e.g., by LC-MS analysis. It can also be determined by contact with a specific binding agent, e.g., an antibody, which binds the alpha hydroxy neoactivity product, e.g. , 2HG, e.g., R-2HG, and allows detection. In embodiments where sufficient levels are present, the tissue, fluid or product can be analyzed for the level of neoactivity, e.g., an alpha hydroxy neoactivity, e.g. , the 2HG neoactivity. In an embodiment the sample is analysed for the presence of a mutant IDH1 protein having an alpha hydroxy neoactivity, e.g., 2HG neoactivity (or a corresponding RNA). E.g. , a mutant protein specific reagent, e.g. , an antibody that specifically binds an IDH mutant protein, e.g. , an antibody that specifically binds an IDH1-R132H mutant protein can be used to detect neoactive mutant enzyme. In an embodiment a nucleic acid from the sample is sequenced to determine if a selected allele or mutation of IDH1 disclosed herein is present. In an embodiment the analysis is other than directly determining the presence of a mutant IDH1 protein (or corresponding RNA) or sequencing of an IDH1 gene. E.g., the analysis can be the detection of an alpha hydroxy neoactivity product, e.g. , 2HG, e.g., R-2HG, or the measurement of 2HG neoactivity. In an embodiment the tissue, fluid or product is removed from the patient and analyzed. In an embodiment the evaluation can include one or more of performing the analysis of the tissue, fluid or product, requesting analysis of the tissue, fluid or product, requesting results from analysis of the tissue, fluid or product, or receiving the results from analysis of the tissue, fluid or product.

In an embodiment the evaluation, which can be performed before and/or after treatment has begun, is based, at least in part, on alpha hydroxy neoactivity product, e.g., 2HG, e.g., R- 2HG, imaging of the subject. In embodiments magnetic resonance methods are is used to evaluate the presence, distribution, or level of an alpha hydroxy neoactivity product, e.g. , 2HG, e.g., R-2HG, in the subject. In an embodiment the subject is subjected to imaging and/or spectroscopic analysis, e.g., magnetic resonance-based analysis, e.g., MRI and/or MRS e.g., analysis, and optionally an image corresponding to the presence, distribution, or level of an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, or of the tumor, is formed. Optionally the image or a value related to the image is stored in a tangible medium and/or transmitted to a second site. In an embodiment the evaluation can include one or more of performing imaging analysis, requesting imaging analysis, requesting results from imaging analysis, or receiving the results from imaging analysis.

In one embodiment 2HG is directly evaluated.

In another embodiment a derivative of 2HG formed in process of performing the analytic method is evaluated. By way of example such a derivative can be a derivative formed in MS analysis. Derivatives can include a salt adduct, e.g., a Na adduct, a hydration variant, or a hydration variant which is also a salt adduct, e.g., a Na adduct, e.g., as formed in MS analysis.

In another embodiment a metabolic derivative of 2HG is evaluated. Examples include species that build up or are elevated, or reduced, as a result of the presence of 2HG, such as glutarate or glutamate that will be correlated to 2HG, e.g., R-2HG.

Exemplary 2HG derivatives include dehydrated derivatives such as the compounds provided below or a salt adduct thereof:

In one embodiment the advanced hematologic malignancy such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B-acute lymphoblastic leukemias (B-ALL), or lymphoma (e.g., T-cell lymphoma) is a tumor wherein at least 30, 40, 50, 60, 70, 80 or 90% of the tumor cells carry an IDHl mutation, and in particular an IDHl R132H or R132C mutation, at the time of diagnosis or treatment.

In one embodiment, the advanced hematologic malignancy to be treated is AML, characterized by the presence of a mutant allele of IDHl . In some embodiments, the AML is relapsed and/or primary refractory. In some embodiments, the AML is relapsed. In some embodiments, the AML is primary refractory. In other embodiments, the AML is untreated.

In another embodiment, the advanced hematologic malignancy to be treated is MDS, characterized by the presence of a mutant allele of IDHl. In another embodiment, the advanced hematologic malignancy to be treated is MDS with refractory anemia with excess blasts (subtype RAEB-1 or RAEB-2). In other embodiments, the MDS is considered high-risk by the IPSS-R (Greenberg et al. Blood. 2012;120(12):2454-65). In other embodiments, the MDS is recurrent. In other embodiments, the MDS is refractory. In other embodiments, the subject having MDS is intolerant to established therapy known to provide clinical benefit for their conditions, according to the treating physician.

In another embodiment, the advanced hematologic malignancy to be treated is CMML, characterized by the presence of a mutant allele of IDHl. In another embodiment, the CMML is relapsed and/or primary refractory. In another embodiment, the CMML is relapsed. In another embodiment, the CMML is primary refractory.

Treatment methods described herein can additionally comprise various evaluation steps prior to and/or following treatment with Compound 1, or a pharmaceutically acceptable salt thereof.

In one embodiment, prior to and/or after treatment with Compound 1, or a

pharmaceutically acceptable salt thereof, the method further comprises evaluating the growth, size, weight, invasiveness, stage and/or other phenotype of the advanced hematologic malignancies, such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B- acute lymphoblastic leukemias (B-ALL), or lymphoma {e.g., T-cell lymphoma), each characterized by the presence of a mutant allele of IDHl.

In one embodiment, prior to and/or after treatment with Compound 1, or a

pharmaceutically acceptable salt thereof, the method further comprises evaluating the IDHl genotype of the advanced hematologic malignancies, such as acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B-acute lymphoblastic leukemias (B-ALL), or lymphoma {e.g., T-cell lymphoma), each characterized by the presence of a mutant allele of IDHl. This may be achieved by ordinary methods in the art, such as DNA sequencing, immuno analysis, and/or evaluation of the presence, distribution or level of 2HG.

In one embodiment, prior to and/or after treatment with Compound 1, or a

pharmaceutically acceptable salt thereof, the method further comprises determining the 2HG level in the subject. This may be achieved by spectroscopic analysis, e.g., magnetic

resonance-based analysis, e.g., MRI and/or MRS measurement, sample analysis of bodily fluid, such as blood, plasma, urine, or spinal cord fluid analysis, or by analysis of surgical material, e.g., by mass-spectroscopy (e.g. LC-MS, GC-MS), or any of the methods described herein.

Example 1: Compound 1 Induces Myeloid Differentiation in Primary IDH1 (R132C) Samples from Patients with AML

Bone marrow aspirates are obtained from patients at the time of diagnosis of their malignancy. The diagnosis of AML may be morphologically confirmed according to the French- American-British classification or World Health Organization criteria, and a description of the karyotypes follows the International System for Human Cytogenetic Nomenclature (Mitelman International System for Cytogenetic Nomenclature. Guidelines for Cancer Cytogenetics.

Supplement to: An International System for Human Cytogenetic Nomenclature. New York: Basel. 1995). Gene mutation status is determined by polymerase chain reaction (PCR) and direct sequencing.

Primary AML cells from IDH1 R132H/C or IDH1 WT patients are incubated with SYTOX blue dead cell dye (Life Technologies SAS, Saint Aubin, France) prior to fluorescence- activated cell sorting (FACS). Sorted live cells are then cultured ex vivo in serum-free conditions in the presence of recombinant human cytokines (interleukin-3 [IL-3], interleukin-6 [IL-6], stem cell factor [SCF], thrombopoietin [TPO], erythropoietin [EPO], FMS-like tyrosine kinase 3 receptor ligand [FLT3L], granulocyte-macrophage colony- stimulating factor [GM-CSF] and granulocyte- stimulating factor [G-CSF]) and either Compound 1, or a pharmaceutically acceptable salt thereof, (0.5 μΜ, 1 μΜ, 5 μΜ) or DMSO (Norol, et al. Blood. 1998;91(3):830- 43.). Cells are counted on Days 6 and 9. At least 100,000 cells are removed on Day 6 for 2HG analysis and FACS analysis.

On Day 6 or 7 of treatment ex vivo, the bone marrow blasts or blood blasts of primary samples from each patient are analyzed for morphology and differentiation status by cytology. Treatment with Compound 1, or a pharmaceutically acceptable salt thereof, (0.5, 1 and 5 μΜ) reduces the level of intracellular 2HG levels by 99% relative to DMSO control at the highest dose in patient samples harboring IDHl mutations, and induces differentiation of IDHl mutant (R132H or R132C) patient myeloblast cells as measured by cytology and FACS analysis. No 2HG is measurable in wild-type patient samples.

Example 2: Phase 1 Clinical Study Protocol

The clinical study is a Phase 1, multicenter, open-label, dose-escalation, safety, PK/PD, and clinical activity evaluation of orally administered Compound 1, or a pharmaceutically acceptable salt thereof in subjects with advanced hematologic malignancies, such as AML, MDS, MPN, or CMML), that harbor an IDHl mutation. Primary study objectives include 1) assessment of the safety and tolerability of treatment with Compound 1, or a pharmaceutically acceptable salt thereof when administered continuously as a single agent dosed orally twice daily (approximately every 12 hours) on Days 1 to 28 of a 28-day cycle, and 2) determination of the maximum tolerated dose (MTD) and/or the recommended Phase 2 dose of Compound 1, or a pharmaceutically acceptable salt thereof in subjects.

Secondary study objectives include 1) description of the dose-limiting toxicities (DLTs) of Compound 1, or a pharmaceutically acceptable salt thereof in subjects with advanced hematologic malignancies, such as AML, MDS, MPN, or CMML, that harbor an IDHl mutation, characterization of the pharmacokinetics (PK) of Compound 1, or a pharmaceutically acceptable salt thereof in subjects with advanced hematologic malignancies, such as AML, MDS, MPN, or CMML, that harbor an IDHl mutation, 3) evaluation of the PK/pharmacodynamic (PD) relationship of Compound 1, or a pharmaceutically acceptable salt thereof, and 2- hydroxygluturate (2HG), and 4) characterization of the clinical activity associated with

Compound 1, or a pharmaceutically acceptable salt thereof, in subjects with advanced

hematologic malignancies, such as AML, MDS, MPN, or CMML, that harbor an IDHl mutation.

Exploratory study objectives include 1) evaluation of changes in Ki67 levels in tumor samples, 2) characterization of the PD effects of Compound 1, or a pharmaceutically acceptable salt thereof, in subjects with advanced hematologic malignancies, such as AML, MDS, MPN, or CMML, that harbor an IDHl mutation by the assessment of changes in the patterns of cellular differentiation of isocitrate dehydrogenase- 1 (IDHl)-mutated tumor cells and changes in histone and deoxyribonucleic acid (DNA) methylation profiles in IDHl -mutated tumor cells, 3) evaluation of gene mutation status, global gene expression profiles, and other potential prognostic markers (cytogenetics) in IDHl -mutated tumor cells, as well as subclonal populations of non-IDHl mutated tumor cells, to explore predictors of anti-tumor activity and/or resistance, and 4) monitoring plasma cholesterol and 4p-OH-cholesterol levels as a potential CYP3A4 induction marker.

Compound 1 will be administered orally twice daily (approximately every 12 hours) on Days 1 to 28 in 28-day cycles. If warranted based on the emerging data, an alternative dosing schedule (e.g., once daily or three times daily), including administration of the same total daily dose using different dosing schedules in concurrent cohorts, may be explored.

Starting with C1D1, dosing is continuous; there are no inter-cycle rest periods.

Subjects who do not meet any of the standard clinical treatment withdrawal criteria may continue treatment beyond Cycle 1.

Subjects will be dispensed the appropriate number of tablets for 28 days of dosing (plus an additional 2-day supply to allow for scheduling of visits) on Day 1 of each cycle.

Subjects are to return all unused tablets (or the empty bottles) on Day 1 of each treatment cycle. Subjects will be given a dosing diary for each treatment cycle. They should record relevant information regarding their study drug in the diary (e.g., confirmation that each daily dose was taken, reasons for missed doses). Treatment compliance will be assessed based on return of unused drug and the dosing diary.

Subjects should be instructed to take their daily dose at approximately the same time each day. Each dose should be taken with a glass of water and consumed over as short a time as possible. Subjects should be instructed to swallow tablets whole and to not chew the tablets. Subjects may take Compound 1, or a pharmaceutically acceptable salt thereof with or without food. If the subject forgets to take the daily morning (or evening) dose, then they should take Compound 1, or a pharmaceutically acceptable salt thereof within 6 hours after the missed dose. If more than 6 hours have elapsed, then that dose should be omitted, and the subject should resume treatment with the next scheduled dose.

The study includes a dose escalation phase to determine MTD followed by

expansion cohorts to further evaluate the safety and tolerability of the MTD. The dose escalation phase will utilize a standard "3 + 3" design. During the dose escalation phase, consented eligible subjects will be enrolled into sequential cohorts of increasing doses of Compound 1, or a pharmaceutically acceptable salt thereof. Each dose cohort will plan to enroll a minimum of 3 subjects. The first 3 subjects enrolled in each dosing cohort during the dose escalation phase of the study will initially receive a single dose of study drug on Day -3 (i.e., 3 days prior to the start of daily dosing) and undergo PK/PD assessments over 72 hours to evaluate drug concentrations and 2HG levels. The next dose of study drug will be on Cycle 1 Day 1 (C1D1) at which time daily dosing will begin. The initial dosing regimen will be twice daily (approximately every 12 hours). If warranted based on the emerging data, an alternative dosing schedule (e.g. , once daily or three times daily), including administration of the same total daily dose using different dosing schedules in concurrent cohorts, may be explored. If there are multiple subjects in the screening process at the time the third subject within a cohort begins treatment, up to 2 additional subjects may be enrolled with approval of the Medical Monitor. For these additional subjects, the Day -3 through Day 1 PK/PD assessments are optional following discussion with the Medical Monitor. The planned dose escalation scheme is illustrated in Table 1.

Table 1: Planned Dose Escalation Scheme

Compound 1, or a pharmaceutically acceptable salt thereof, may be administered twice daily (approximately every 12 hours). If warranted based on the emerging data, an alternative dosing schedule (e.g. , once daily or three times daily), including administration of the same total daily dose using different dosing schedules in concurrent cohorts, may be explored.

If DLTs (are observed at Dose Level 1 (100 mg), the dose for the second cohort will be decreased to 50 mg (Dose Level - 1).

Continued doubling of the dose until Compound 1 -related NCI CTCAE version 4.03 >Grade 2 toxicity is observed. Following evaluation of the event(s) by the Clinical Study Team, subsequent increases in dose will be guided by the observed toxicity, and potentially PK and PK/PD data until MTD is determined. The absolute percent increase in the dose will be determined by the Clinical Study Team predicated on the type and severity of any toxicity seen in the prior dose cohorts. Dose escalation will never exceed 100%.

⁴ Defined as the highest dose that causes DLTs in <1 of 3 or <2 of 6 subjects. If no DLTs are identified, dosing will continue for at least 2 dose levels above the projected maximum biologically effective exposure, as determined by an ongoing assessment of PK/PD and any observed clinical activity to determine the recommended Phase 2 dose.

⁵ To include 3 cohorts of approximately 12 subjects each.

Toxicity severity will be graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 4.03. A DLT is defined as follows. Hematologic includes prolonged myelosuppression, defined as persistence of >3 Grade neutropenia or thrombocytopenia (by NCI CTCAE, version 4.03, leukemia- specific criteria, i.e., marrow cellularity <5% on Day 28 or later from the start of study drug without evidence of leukemia) at least 42 days after the initiation of Cycle 1 therapy. Leukemia- specific grading should be used for cytopenias (based on percentage decrease from baseline: 50 to 75% = Grade 3, >75% = Grade 4). All AEs that cannot clearly be determined to be unrelated to Compound 1, or a pharmaceutically acceptable salt thereof will be considered relevant to determining DLTs.

If, after the third subject completes the 28-day DLT evaluation period (i.e., Cycle 1), and no DLTs are observed, the study will proceed with dose escalation to the next cohort following safety review by the Clinical Study Team. If 1 of 3 subjects experiences a DLT during the first cycle, 3 additional subjects will be enrolled in that cohort. If none of the additional 3 subjects experience a DLT, dose escalation may continue to the next cohort following safety review. If 2 or more subjects in a cohort experience DLTs during the first cycle, dose escalation will be halted and the next lower dose level will be declared the MTD. Alternatively, a dose level intermediate between the dose level exceeding MTD and the previous does level may be explored and declared MTD if <2 out of 6 patients experience a DLT at that dose. If the MTD cohort includes only 3 subjects, an additional 3 subjects will be enrolled at that dose level to confirm that <2 of 6 subjects experience a DLT at that dose.

Increases in the dose of Compound 1, or a pharmaceutically acceptable salt thereof, for each dose cohort will be guided by an accelerated titration design, where the dose will be doubled (100% increase) from one cohort to the next until Compound 1 -related NCI CTCAE version 4.03 Grade 2 or greater toxicity is observed in any subject within the cohort. Subsequent increases in dose will be guided by the observed toxicity, and potentially PK and PK/PD data, until the MTD is determined. The absolute percent increase in the daily dose will be determined predicated on the type and severity of any toxicity seen in the prior dose cohorts (but will never exceed 100%). If warranted based on the emerging data, an alternative dosing schedule (e.g. , once daily or three times daily) may be explored, including administration of the same total daily dose using different dosing schedules in concurrent cohorts. The MTD is the highest dose that causes DLTs in <2 of 6 subjects.

If no DLTs are identified during the dose escalation phase, dose escalation may continue for 2 dose levels above the projected maximum biologically effective dose, as determined by an ongoing assessment of PK/PD and any observed clinical activity, to determine the recommended Phase 2 dose.

To optimize the number of subjects treated at a potentially clinically relevant dose, intra- subject dose escalation will be permitted. Following determination of the recommended Phase 2 dose, 3 or more expansion cohorts (with AML, MDS, MPN, or CMML) of approximately 12 subjects each will be treated at that dose. The purpose of the expansion cohorts is to evaluate and confirm the safety and tolerability of the recommended Phase 2 dose in specific disease indications. Subjects enrolled in these cohorts will undergo the same procedures as subjects in the dose escalation cohorts with the exception that the Day -3 through Day 1 PK/PD assessments will be optional.

Subjects will undergo screening procedures within 28 days prior to the start of study drug treatment to determine eligibility. Screening procedures include medical, surgical, and medication history, confirmation of IDH1 mutation via tumor biopsies or leukemic blasts (if not documented previously), physical examination, vital signs, Eastern Cooperative Oncology Group (ECOG) performance status (PS), 12-lead electrocardiogram (ECG), evaluation of left ventricular ejection fraction (LVEF), clinical laboratory assessments (hematology, chemistry, coagulation, urinalysis, and serum pregnancy test), bone marrow biopsy and aspirate, and blood and urine samples for 2HG measurement; and blood samples for determination of plasma cholesterol and 4p-OH-cholesterol levels.

Three days prior to starting the twice daily dosing of Compound 1, or a pharmaceutically acceptable salt thereof (Day -3), the first 3 subjects enrolled in each cohort in the dose escalation phase will receive a single dose of Compound 1, or a pharmaceutically acceptable salt thereof in clinic and have serial blood and urine samples obtained for determination of blood and urine concentrations of Compound 1, or a pharmaceutically acceptable salt thereof, its metabolite, and 2HG. A full 72-hour PK/PD profile will be conducted: subjects will be required to remain at the study site for 10 hours on Day -3 and return on Days -2, -1, and 1 for 24, 48, and 72 hour samples, respectively.

Daily treatment with Compound 1, or a pharmaceutically acceptable salt thereof, will begin on ClDl; subjects who did not undergo the Day -3 PK/PD assessments will be observed in the clinic for 4 hours following the ClDl dose. The initial dosing regimen will be twice daily (approximately every 12 hours). Safety assessments conducted during the treatment period include physical examination, vital signs, ECOG PS, 12-lead ECGs, LVEF, and clinical laboratory assessments (hematology, chemistry, coagulation, and urinalysis).

All subjects will undergo PK/PD assessments over a 10-hour period on both C1D15 and C2D1. Additional pre-dose urine and/or blood sampling will be conducted on C1D8, C1D22, C2D15, C3D1, C3D15, and on Day 1 of all subsequent cycles. Available bone marrow biopsy samples also will be assessed for 2HG levels.

Subjects will undergo radiographic evaluations (CT/MRI), and assessment of bone marrow aspirates and biopsies and peripheral blood to assess the extent of disease, at screening, on Day 15, Day 29 and Day 57, and every 56 days thereafter while on study drug treatment, independent of dose delays and/or dose interruptions, and/or at any time when progression of disease is suspected. Two core tumor biopsies will be obtained at screening, at the time of the first assessment of response, and at the time of disease progression within a window of +3 days around the planned assessment time point. For patients with acute myelogenous leukemia (AML), response to treatment will be determined by the Investigators based on modified International Working Group (r G) response criteria.

Subjects may continue treatment with Compound 1, or a pharmaceutically acceptable salt thereof until disease progression, occurrence of a DLT, or development of other unacceptable toxicity. All subjects are to undergo an end of treatment assessment (within approximately 5 days of the last dose of study drug); in addition, a follow-up assessment is to be scheduled 28 days after the last dose.

It is estimated that approximately 51 subjects will be enrolled in the study. Assuming that identification of the MTD requires the evaluation of 4 dose levels of Compound 1, or a pharmaceutically acceptable salt thereof with only 3 subjects per dose level, with the exception that the MTD requires 6 subjects, then 15 subjects will be enrolled during the dose escalation part of the study. Three cohorts of approximately 12 additional subjects each in specific advanced hematologic malignancies (total 36 subjects) will be enrolled in the cohort expansion part of the study. Additional subjects may be needed for cohort expansion during dose escalation, for the replacement of non-evaluable subjects, or for evaluation of alternative dosing regimens other than the planned escalation scheme or the MTD, to optimize the recommended Phase 2 dose.

A patient must meet all of the following inclusion criteria to be enrolled in the clinical study. 1) Subject must be >18 years of age; 2) Subjects must have a) an advanced hematologic malignancy including: i) Relapsed and/or primary refractory AML as defined by World Health Organization (WHO) criteria, ii) untreated AML, >60 years of age and are not candidates for standard therapy due to age, performance status, and/or adverse risk factors, according to the treating physician and with approval of the Medical Monitor, iii) Myelodysplastic syndrome with refractory anemia with excess blasts (subtype RAEB-1 or RAEB-2), or considered high-risk by the Revised International Prognostic Scoring System (IPSS-R) (Greenberg et al. Blood.

2012;120(12):2454-65) that is recurrent or refractory, or the patient is intolerant to established therapy known to provide clinical benefit for their condition (i.e., patients must not be candidates for regimens known to provide clinical benefit), according to the treating physician and with approval of the Medical Monitor, and iv) Subjects with other relapsed and/or primary refractory hematologic cancers, for example CMML, who fulfill the inclusion/excluding criteria may be considered on a case-by case basis; 3) subjects must have documented IDH1 gene-mutated disease based on local evaluation. Analysis of leukemic blast cells for IDH1 gene mutation is to be evaluated at screening (if not evaluated previously) by the site's local laboratory to determine subject eligibility for the study. If the site does not have local laboratory access for IDH1 gene mutation analysis, central laboratory evaluation is acceptable. A pretreatment tumor sample (from blood and/or bone marrow) will be required for all screened subjects for central laboratory biomarker analysis. Gene mutation analysis of a tumor sample (from blood or bone marrow) is to be repeated at the End of Treatment visit and submitted to the central laboratory for biomarker analysis; 4) Subjects must be amenable to serial bone marrow biopsies, peripheral blood sampling, and urine sampling during the study. (The diagnosis and evaluation of AML or MDS can be made by bone marrow aspiration when a core biopsy is unobtainable and/or is not a part of the standard of care. A bone marrow biopsy is required in case of dry tap or failure (mainly dilution) with the aspiration.); 5) Subjects or their legal representatives must be able to understand and sign an informed consent; 6) subjects must have ECOG PS of 0 to 2; 7) subjects must have a platelet count >20,000/μί (Transfusions to achieve this level are allowed.) Subjects with a baseline platelet count of <20,000/μί due to underlying malignancy are eligible with Medical Monitor approval; 8) Subjects must have adequate hepatic function as evidenced by: a) Serum total bilirubin <1.5 x upper limit of normal (ULN), unless considered due to Gilbert's disease or leukemic organ involvement, and b) Aspartate aminotransferase, ALT, and alkaline phosphatase (ALP) <3.0 x ULN, unless considered due to leukemic organ involvement; 9) Subjects must have adequate renal function as evidenced by a serum creatinine <2.0 x ULN or Creatinine clearance >40 mL/min based on the Cockroft-Gault glomerular filtration rate (GFR) estimation:(140 - Age) x (weight in kg) x (0.85 if female)/72 x serum creatinine; 10) Subjects must be recovered from any clinically relevant toxic effects of any prior surgery, radiotherapy, or other therapy intended for the treatment of cancer. (Subjects with residual Grade 1 toxicity, for example Grade 1 peripheral neuropathy or residual alopecia, are allowed with approval of the Medical Monitor.); and 11) Female subjects with reproductive potential must have a negative serum pregnancy test within 7 days prior to the start of therapy. Subjects with reproductive potential are defined as one who is biologically capable of becoming pregnant. Women of childbearing potential as well as fertile men and their partners must agree to abstain from sexual intercourse or to use an effective form of contraception during the study and for 90 days (females and males) following the last dose of Compound 1, or a pharmaceutically acceptable salt thereof.

Compound 1, or a pharmaceutically acceptable salt thereof, will be provided as 50 and 200 mg strength tablets to be administered orally, twice daily or once daily.

The first 3 subjects in each cohort in the dose escalation portion of the study will receive a single dose of study drug on Day -3; their next dose of study drug will be administered on ClDl at which time subjects will start dosing twice daily (approximately every 12 hours) on Days 1 to 28 in 28-day cycles. Starting with ClDl, dosing is continuous; there are no inter-cycle rest periods. Subjects who are not required to undergo the Day -3 PK/PD assessments will initiate twice daily dosing (approximately every 12 hours) with Compound 1, or a

pharmaceutically acceptable salt thereof on ClDl.

The dose of Compound 1, or a pharmaceutically acceptable salt thereof administered to a subject will be dependent upon which dose cohort is open for enrollment when the subject qualifies for the study. The starting dose of Compound 1, or a pharmaceutically acceptable salt thereof to be administered to the first cohort of subjects is 100 mg strength administered orally twice a day (200 mg/day).

Subjects may continue treatment with Compound 1, or a pharmaceutically acceptable salt thereof until disease progression, occurrence of a DLT, or development of other unacceptable toxicity.

Criteria for Evaluation

Safety:

AEs, including determination of DLTs, serious adverse events (SAEs), and AEs leading to discontinuation; safety laboratory parameters; physical examination findings; vital signs; 12-lead ECGs; LVEF; and ECOG PS will be monitored during the clinical study. The severity of AEs will be assessed by the NCI CTCAE, Version 4.03.

Compound 1, or a pharmaceutically acceptable salt thereof, may cause sensitivity to direct and indirect sunlight. The subjects should be warned to avoid direct sun exposure. When exposure to sunlight is anticipated for longer than 15 minutes, the subject should be instructed to apply factor 30 or higher sunscreen to exposed areas and wear protective clothing and

sunglasses.

Pharmacokinetics and Pharmacodynamics:

Serial blood samples will be evaluated for determination of concentration-time profiles of Compound 1, or a pharmaceutically acceptable salt thereof. Urine samples will be evaluated for determination of urinary excretion of Compound 1, or a pharmaceutically acceptable salt thereof. Blood, bone marrow, and urine samples will be evaluated for determination of 2HG levels.

Tumor biopsies will be taken for evaluation of 2HG and Compound 1, or a pharmaceutically acceptable salt thereof.

Pharmacokinetic Assessments:

Serial blood samples will be drawn before and after dosing with Compound 1, or a pharmaceutically acceptable salt thereof in order to determine circulating plasma concentrations of Compound 1, or a pharmaceutically acceptable salt thereof. The blood samples will also be used for the determination of 2HG concentrations and for evaluation of cholesterol and 4β-ΟΗ- cholesterol levels.

For the first 3 subjects enrolled in a cohort during the dose escalation phase, a single dose of Compound 1, or a pharmaceutically acceptable salt thereof will be administered on Day -3 (i.e., 3 days prior to their scheduled C1D1 dose). Blood samples will be drawn prior to the single-dose administration of Compound 1, or a pharmaceutically acceptable salt thereof and at the following time points after administration: 30 minutes and 1, 2, 3, 4, 6, 8, 10, 24, 48, and 72 hours. After 72 hours of blood sample collection, subjects will begin oral twice daily dosing of Compound 1, or a pharmaceutically acceptable salt thereof (i.e., C1D1). The PK/PD profile from Day -3 through Day 1 is optional for additional subjects enrolled in the dose escalation phase (i.e., for any subjects beyond the 3 initial subjects enrolled in a cohort) and is not required for subjects enrolled in the expansion cohorts.

All subjects will undergo 10-hour PK/PD sampling on C1D15 and C2D1 (i.e., on Days 15 and 29 of twice daily dosing). For this profile, one blood sample will be drawn immediately prior to that day's first dose of Compound 1, or a pharmaceutically acceptable salt thereof (i.e., dosing with Compound 1, or a pharmaceutically acceptable salt thereof will occur at the clinical site); subsequent blood samples will be drawn at the following time points after dosing: 30 minutes, and 1, 2, 3, 4, 6, 8, and 10 hours. Blood samples also will be drawn on Days 8 and 22 of Cycle 1, Day 15 of Cycle 2, Days 1 and 15 of Cycle 3, and Day 1 of each cycle thereafter; all samples will be obtained prior to dosing. Additionally, one blood sample will be drawn at the End of Treatment Visit.

The timing of blood samples drawn for Compound 1, or a pharmaceutically acceptable salt thereof concentration determination may be changed if the emerging data indicates that an alteration in the sampling scheme is needed to better characterize the PK profile of Compound 1, or a pharmaceutically acceptable salt thereof.

Pharmacodynamic Assessments:

Serial blood samples will be drawn before and after dosing with Compound 1, or a pharmaceutically acceptable salt thereof in order to determine circulating concentrations of 2HG. Samples collected for PK assessments also will be used to assess 2HG levels. In addition, subjects will have blood drawn for determination of 2HG levels at the screening assessment.

The timing of blood samples drawn for 2HG concentration determination may be changed if the emerging data indicate that an alteration in the sampling scheme is needed to better characterize the 2HG response to Compound 1, or a pharmaceutically acceptable salt thereof, treatment.

Urine will be collected for the determination of concentrations of 2HG levels at the screening assessment and prior to dosing on Day 15 of Cycle 1 and on Day 1 of Cycle 2 and every cycle thereafter. At least 20 mL of urine will be collected for each sample.

The volume of each collection will be measured and recorded and sent to a central laboratory for determination of urinary 2HG concentration. An aliquot from each collection will be analyzed for urinary creatinine concentration.

Tumor biopsy specimens will be collected and assessed for 2HG levels, at the screening assessment, at the time of the first disease assessment, and at any time disease progression is suspected. A window of +3 days around the planned assessment time point is acceptable for all biopsy samples. Tumor biopsies are to be evaluated for morphology and for cellular

differentiation via hematoxylin and eosin (H & E) staining and ICH for specific cell-type markers. Tumor samples may also be evaluated for 2HG levels, Ki67 levels, and, if feasible, intra- tumoral Compound 1, or a pharmaceutically acceptable salt thereof, levels.

Serial blood samples will be drawn to obtain plasma cholesterol and 4p-OH-cholesterol levels as a potential CYP3A4 induction marker. Samples are obtained on Day -3 (within 30 minutes), at 24, 48, and 72 hours (+1 hour), and on Days 8, 15 and 22 of Cycle 1, Days 1 and 15 of Cycles 2 and 3, and Day 1 of every cycle thereafter.

Clinical Activity:

Serial blood and bone marrow biopsies will be evaluated during the clinical study to determine response to Compound 1, or a pharmaceutically acceptable salt thereof treatment according to the 2006 modified IWG criteria for hematologic malignancies, such as MDS, MDS, MPN or AML (Cheson BD, et al. Blood. 2006;108(2):419-25).

Disease response to treatment will be assessed through the evaluation of bone marrow biopsies and/or aspirates, along with complete blood counts and examination of peripheral blood films. Subjects will have the extent of their disease assessed and recorded at screening, on Days 15, 29, and 57, every 56 days thereafter while on study drug treatment, independent of dose- delays and/or dose interruptions, and/or at any time when progression of disease is suspected. An assessment also will be conducted at the End of Treatment visit for subjects who discontinue the study due to reasons other than disease progression.

Statistical Analysis

Statistical analyses will be primarily descriptive in nature since the goal of the study is to determine the MTD of Compound 1, or a pharmaceutically acceptable salt thereof. Tabulations will be produced for appropriate disposition, demographic, baseline, safety, PK, PD, and clinical activity parameters and will be presented by dose level and overall. Categorical variables will be summarized by frequency distributions (number and percentages of subjects) and continuous variables will be summarized by descriptive statistics (mean, standard deviation, median, minimum, and maximum).

Adverse events will be summarized by Medical Dictionary for Regulatory Activities (MedDRA) system organ class and preferred term. Separate tabulations will be produced for all treatment- emergent AEs (TEAEs), treatment-related AEs (those considered by the Investigator as at least possibly drug related), SAEs, discontinuations due to AEs, and AEs of at least Grade 3 severity. By-subject listings will be provided for deaths, SAEs, DLTs, and AEs leading to discontinuation of treatment.

Descriptive statistics will be provided for clinical laboratory, ECG interval, LVEF, and vital signs data, presented as both actual values and changes from baseline relative to each on- study evaluation and to the last evaluation on study. Shift analyses will be conducted for laboratory parameters and ECOG PS.

Descriptive statistics will be used to summarize PK parameters for each dose group and, where appropriate, for the entire population. The potential relationship between plasma levels of Compound 1, or a pharmaceutically acceptable salt thereof and blood, plasma or urine 2HG levels will be explored with descriptive and graphical methods.

Response to treatment as assessed by the site Investigators using modified r G (for subjects with hematologic malignancies, such as MDS, MDS/MPN or AML). Two-sided 90% confidence intervals on the response rates will be calculated for each dose level and overall. Data will also be summarized by type of malignancy for subjects in the cohort expansion phase.

Descriptive statistics will be used to summarize Ki67 levels from tumor biopsies.

Study Results

Table 2

Compound 1 had a cellular IC₅₀ value of 8-20 nM. Reduction in 2HG was observed following a single dose of Compound 1 in an IDHl mutant R132H xenograft model (Figure 1A). In addition, Compound 1 reduced intracellular 2HG in primary human IDH-mutated blast cells ex vivo (Figure IB).

*AE, adverse event; PD, progressive disease; BID, twice daily; QD, once daily

** AE = intracranial hemorrhage. This patient presented with right extremity weakness

(upper and lower). The patient was hospitalized with a platelet count of 11 on admission. The patient's status quickly deteriorated, a CT scan revealed left sided intraparenchymal hemorrhage.

The patient died 3 days later.

Patients in Table 2 have received median (range) = 1.6 (0.4-5.7) of months of treatment.

Table 3. Demographic Characteristics of Patients of Table 2

Diagnosis, n

R/R AML 17

ECOG performance status, n

0 6

1 9

2 2

Number of prior

chemotherapy regimens, 2 (1-5)

median (range)

Prior BMT, n 2

Abnormal cytogenetics, n 10

One DLT (dose limiting toxicity) of grade 3 QT prolongation at 800 mg QD was observed. There were no associated cardiac symptoms, QTc returned to normal following 3-day drug hold. The patient's dose was reduced to 500 mg QD and remains on study with grade 1 QTc prolongation in complete remission (CR). Eight subjects experienced serious adverse events. At 100 mg BID, 1 subject discontinued study due to an intracranial bleed attributed to disease progression and resulted in death. At 300 mg QD, 1 subject experienced differentiation syndrome, recovered and was in CR. At 800 mg QD, 1 subject experienced tongue edema and QT prolongation (DLT described above), recovered and was in CR. All other SAEs related to disease progression resulted in death. For the patient that experienced differentiation syndrome, symptoms included fever and dyspnea. The patient was treated with steroids. Two events that led to PD are described as they are events that correlate to discontinuations due to AEs.

Table 4. Adverse Events

Notable grade >3 AEs include: hypotension 2 (12%), mental status changes 2 (12%), neutropenia 2 (12%). AEs appear typical for this patient population. Other QT prolongations observed: Grade 1 prolonged QT in 100 mg cohort (the patient had a history of right bundle branch block (R BBB) at study entry; Grade 1 intermittent prolonged QT in 300 mg cohort; and Grade 3 prolonged QT (DLT) in 800 mg cohort.

Vomiting 0 4 (24)

Pyrexia 1 (6) 4 (24)

Cough 0 4 (24)

Febrile

3 (18) 3 (18)

neutropenia

Diarrhea 0 3 (18)

Electrocardiogram

1 (6) 3 (18)

QT prolonged

Compound 1 Exposure and 2HG Inhibition

Figures 2A and 2B shows the PK profile of Compound 1 following oral administration. Compound 1 showed high plasma exposure, drug accumulation and half-life of 182 hours. The plasma levels of 2HG were reduced to a normal range at all dose levels (up tp 98% inhibition). The 2HG baseline was taken at Day -3 pre-treatment 2HG inhibition estimated based on 2HG pre-treatment level and AUCo io_hr post treatment. For the 100 mg BID and 300 mg QD Cohorts, 3 to 4 patients were measured per time point and for the 500 mg QD Cohort, 1 to 3 patients were measured per time point.

Table 5. Clinical Activity Assessed by Investigator Using IWG AML and MPS Criteria

CR = complete response

Marrow CR = <5% blasts in BM; no hematological recovery

PR = partial response

SD = stable disease

PD = progressive disease

ORR = CR, Marrow CR and PR

Differentiation Effect in the Bone Marrow

Figures 3A-3C are images of aspirate from a 74 year old female patient who was refractory to induction with 7+3. At baseline (Figure 3A), her bone marrow displayed monotonous cellularity, from the preponderance of blast cells. The inset shows the appearance of the blast cells on the aspirate. After 2 weeks of therapy (Figure 3B), the core biopsy showed ongoing hypercellularity, but clear evidence of maturation, as determined by the cells that have varied sizes and shapes, approximating the "field of flowers" appearance of a normal marrow. In the inset the aspirate no longer shows blast cells, but instead mostly myelocytes, which is evidence of differentiation. At this timepoint, given the reduction in blasts to <5 and preservation of neutrophils and platelets, this patient met the criteria for a full CR. This was maintained at D28, which again showed hypercellularity, but with maturation and no increase in blast cells (Figure 3C).

While the foregoing invention has been described in some detail for purposes of clarity and understanding, these particular embodiments are to be considered as illustrative and not restrictive. It will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention, which is to be defined by the appended claims rather than by the specific

embodiments.

The patent and scientific literature referred to herein establishes knowledge that is available to those with skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The issued patents, applications, and references that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of inconsistencies, the present disclosure, including definitions, will control.

Claims

We claim:

1. A method of treating advanced hematologic malignancies, wherein the advanced hematologic malignancies is selected from acute myelogenous leukemia (AML),

myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B-acute lymphoblastic leukemias (B-ALL), B-acute lymphoblastic leukemias (B-ALL), and lymphoma, wherein each of the malignancies is characterized by the presence of a mutant allele of IDHl (isocitrate dehydrogenase 1).

2. The method of claim 1, wherein each of the advanced hematologic malignancies is characterized by a mutant allele of IDHl, wherein the IDHl mutation results in a new ability of the enzyme to catalyze the NAPH-dependent reduction of a-ketoglutarate to

R(-)-2-hydroxyglutarate (2HG) in a patient.

3. The method of claim 2, wherein the mutant IDHl has an R132X mutation.

4. The method of claim 3, wherein the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G.

5. The method of claim 3, wherein the R132X mutation is R132H or R132C.

6. The method of claim 3, wherein the R132X mutation is R132H.

7. The method of claim 1, wherein each of the advanced hematologic malignancies harbors a co-mutation.

8. The method of claim 1, wherein each of the advanced hematologic malignancies harbors a co-mutation selected from NPMl, FLT3, TET2, CEBPA, DNMT3A, and MLL.

9. The method of claim 1, wherein the lymphoma is T-cell lymphoma.

10. A method of evaluating a subject, the method comprising: acquiring, a value for the level of a compound (S)-N-((S)-l-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2- oxoethyl)-l-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide (Compound 1), or a pharmaceutically acceptable salt thereof; or the level of an alpha hydroxy neoactivity product, in the subject, that has been treated with Compound 1, to thereby evaluate the subject.

11. The method of claim 10, wherein the alpha hydroxy neoactivity product is 2- hydroxyglutarate (2HG).

12. The method of claim 10, wherein acquiring comprises receiving a sample from the subject.

13. A method of evaluating a subject, the method comprising: administering to the subject in need thereof a compound (S)-N-((S)-l-(2-chlorophenyl)-2-((3,3- difluorocyclobutyl)amino)-2-oxoethyl)-l-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5- oxopyrrolidine-2-carboxamide (Compound 1), or a pharmaceutically acceptable salt thereof; and acquiring a value for the level of Compound 1 or the level of an alpha hydroxy neoactivity product, in the subject, to thereby evaluate the subject.

14. The method of claim 13, wherein the alpha hydroxy neoactivity product is 2- hydroxyglutarate (2HG).

15. The method of claim 13, wherein the value for the level of Compound 1 is acquired by analyzing the concentration of Compound 1 in a bodily fluid.

16. The method of claim 15, wherein the bodily fluid is selected from blood, plasma and urine.

17. The method of claim 13, wherein the value for the level of Compound 1 is acquired by analyzing the level of Compound 1 in bone marrow.

18. The method of claim 13, wherein the value for the level of Compound 1 is acquired by analyzing a sample from a bone marrow biopsy or aspirate for the level of

Compound 1.

19. The method of claim 15, wherein the analysis comprises a chromatographic method.

20. The method of claim 19, wherein the chromatographic method comprises mass spectroscopy.

21. The method of claim 13, wherein the subject has been administered Compound 1 less than about 30 days prior to the evaluation.

22. The method of claim 21, wherein the subject has been administered Compound 1 at a dose of about 10 mg to about 3000 mg.

23. The method of claim 22, wherein Compound 1 is administered orally.

24. The method of claim 23, wherein Compound 1 is administered once or twice daily.

25. The method of claim 13, wherein the subject has or is diagnosed as having a disorder.

26. The method of claim 25, wherein the disorder is an advanced hematologic malignancy.

27. The method of claim 26, wherein the advanced hematologic malignancy is characterized by the presence of a mutant allele of IDHl.

28. The method of claim 27, wherein the advanced hematologic malignancy is characterized by the presence of a mutant allele of IDHl, wherein the IDHl mutation results in a new ability of the enzyme to catalyze the NAPH-dependent reduction of a-ketoglutarate to R(-)-2-hydroxyglutarate (2HG) in a patient.

The method of claim 28, wherein the mutant IDHl has an R132X mutation.

30. The method of claim 29, wherein the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G.

The method of claim 29, wherein the R132X mutation is R132H or R132C.

The method of claim 29, wherein the R132X mutation is R132H.

33. The method of claim 28, wherein the advanced hematologic malignancy harbors a co-mutation.

34. The method of claim 33, wherein the co-mutation is selected from NPM1, FLT3, TET2, CEBPA, DNMT3A, and MLL.

35. The method of claim 25, wherein the disorder is selected from acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B-acute lymphoblastic leukemias (B-ALL), B-acute lymphoblastic leukemias (B-ALL), and lymphoma, wherein each disorder is characterized by the presence of a mutant allele of IDHl.

36. The method of claim 13, wherein the subject has been previously treated with one or more chemo therapeutic agent(s).

37. A method of evaluating a subject, the method comprising: acquiring a value for the level of blast cells in the subject that has been treated with a compound (S)-N-((S)-l-(2- chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-l-(4-cyanopyridin-2-yl)-N-(5- fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide (Compound 1), to thereby evaluate the subject.

38. A method of evaluating a subject, the method comprising: administering to the subject in need thereof a compound (S)-N-((S)-l-(2-chlorophenyl)-2-((3,3- difluorocyclobutyl)amino)-2-oxoethyl)-l-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5- oxopyrrolidine-2-carboxamide (Compound 1), or a pharmaceutically acceptable salt thereof; and acquiring a value for the level of blast cells in the subject, to thereby evaluate the subject.

39. The method of claim 37 or 38, wherein the subject has or is diagnosed as having a disorder.

40. The method of claim 39, wherein the disorder is an advanced hematologic malignancy.

41. The method of claim 40, wherein the advanced hematologic malignancy is characterized by the presence of a mutant allele of IDHl.

42. The method of claim 40, wherein the advanced hematologic malignancy is characterized by a mutant allele of IDHl, wherein the IDHl mutation results in a new ability of the enzyme to catalyze the NAPH-dependent reduction of a-ketoglutarate to

R(-)-2-hydroxyglutarate (2HG) in a patient.

43. The method of claim 41, wherein the mutant IDHl has an R132X mutation.

44. The method of claim 43, wherein the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G.

45. The method of claim 40, wherein the advanced hematologic malignancy is characterized by a co-mutation.

46. The method of claim 45, wherein the co-mutation is selected from NPM1, FLT3, TET2, CEBPA, DNMT3A, and MLL.

47. The method of claim 39, wherein the disorder is selected from acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), myeloproliferative neoplasms (MPN), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), B- acute lymphoblastic leukemias (B-ALL), B-acute lymphoblastic leukemias (B-ALL), and lymphoma, wherein each disorder is characterized by the presence of a mutant allele of IDH1.

48. The method of claim 39, wherein the disorder is selected from advanced IDH1 mutation-positive relapsed and/or refractory AML (R/R AML), untreated AML, and MDS.

49. The method of claim 37 or 38, wherein the subject has been previously treated with one or more chemo therapeutic agent(s).

50. The method of claim 49, wherein the chemotherapeutic agent is selected from cytarabine (Ara-C), daunorubicin, etoposide, mitoxantrone, idarubicin, 5-azacytidine, decitabine, SGN33A, sargramostim, WT-1 analog peptide vaccine, tipifarnib, MK-8242, campath, and 6 Mercaptopurine (6MP).

51. A method of treating a disorder in a subject, the method comprising:

administering to the subject in need thereof a compound (S)-N-((S)-l-(2-chlorophenyl)-2-((3,3- difluorocyclobutyl)amino)-2-oxoethyl)-l-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5- oxopyrrolidine-2-carboxamide (Compound 1), or a pharmaceutically acceptable salt thereof, in an amount sufficient to provide a reduction in blast cells, to thereby treat the disorder.

52. The method of claim 51, wherein the disorder is an advanced hematologic malignancy.

53. The method of claim 52, wherein the advanced hematologic malignancy is characterized by the presence of a mutant allele of IDHl.

54. The method of claim 52, wherein the advanced hematologic malignancy is characterized by the presence of a mutant allele of IDHl, wherein the IDHl mutation results in a new ability of the enzyme to catalyze the NAPH-dependent reduction of a-ketoglutarate to R(-)-2-hydroxyglutarate (2HG) in a patient.

55. The method of claim 53, wherein the mutant IDHl has an R132X mutation.

56. The method of claim 53, wherein the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G.

57. The method of claim 55, wherein the R132X mutation is R132H or R132C.

58. The method of claim 55, wherein the R132X mutation is R132H.