WO2015127173A1 - Therapeutically active compounds and their methods of use - Google Patents

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 solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), eac characterized by the presence of a mutant allele of IDH1. 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-difluorocyclobutyl) amino)-2-oxoethyl)-1-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)- 5-oxopyrrolidine-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. 61/942,172 filed February 20, 2014, 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 solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), each characterized by the presence of a mutant allele of IDHl .

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.

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 solid tumor, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), each characterized by the presence of a mutant allele of IDH1), lessen the severity of the disease/disorder (i.e., an advanced solid tumor, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non- small cell lung cancer (NSCLC), each characterized by the presence of a mutant allele of IDH1) or improve the symptoms associated with the

disease/disorder (i.e., an advanced solid tumor, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), each characterized by the presence of a mutant allele of IDH1.

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 solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), each characterized by the presence of a mutant allele of IDH1 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 of treating advanced solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), each characterized by the presence of a mutant allele of IDH1 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 1. 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 IDHl 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 solid tumor, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), 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 solid tumor, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), 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 solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), 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 solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), each characterized by the presence of a mutant allele of IDHl imparting such activity and in particular an IDHl R132H or R132C mutation.

In one embodiment, the efficacy of treatment of advanced solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), each characterized by the presence of a mutant allele of IDHl 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 solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non- small cell lung cancer (NSCLC), each characterized by the presence of a mutant allele of IDHl . 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

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 embodment 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 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 determend 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 embodment 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 solid tumor, such as glioma, intrahepatic

cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), 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 another embodiment, the advanced solid tumor to be treated is glioma, characterized by the presence of a mutant allele of IDHl. In another embodiment, the glioma has recurred following standard therapy. In another embodiment, the glioma has progressed following standard therapy. In another embodiment, the glioma has not responded to standard therapy.

In another embodiment, the advanced solid tumor to be treated is IHCC, characterized by the presence of a mutant allele of IDHl. In another embodiment, the IHCC has recurred following standard therapy. In another embodiment, the IHCC has progressed following standard therapy. In another embodiment, the IHCC has not responded to standard therapy.

In another embodiment, the advanced solid tumor to be treated is chondrosarcoma, characterized by the presence of a mutant allele of IDHl. In another embodiment, the chondrosarcoma has recurred following standard therapy. In another embodiment, the chondrosarcoma has progressed following standard therapy. In another embodiment, the chondrosarcoma has not responded to standard therapy.

In another embodiment, the advanced solid tumor to be treated is prostate cancer, characterized by the presence of a mutant allele of IDHl. In another embodiment, the prostate cancer has recurred following standard therapy. In another embodiment, the prostate cancer has progressed following standard therapy. In another embodiment, the prostate cancer has not responded to standard therapy.

In another embodiment, the advanced solid tumor to be treated is colon cancer, characterized by the presence of a mutant allele of IDHl. In another embodiment, the colon cancer has recurred following standard therapy. In another embodiment, the colon cancer has progressed following standard therapy. In another embodiment, the colon cancer has not responded to standard therapy.

In another embodiment, the advanced solid tumor to be treated is melanoma, characterized by the presence of a mutant allele of IDH1. In another embodiment, the melanoma) has recurred following standard therapy. In another embodiment, the melanoma has progressed following standard therapy. In another embodiment, the melanoma has not responded to standard therapy.

In another embodiment, the advanced solid tumor to be treated is non-small cell lung cancer (NSCLC), characterized by the presence of a mutant allele of IDH1. In another embodiment, the non- small cell lung cancer (NSCLC) has recurred following standard therapy. In another embodiment, the non-small cell lung cancer (NSCLC) has progressed following standard therapy. In another embodiment, the non- small cell lung cancer (NSCLC) has not responded to standard therapy.

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 solid tumor, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), each characterized by the presence of a mutant allele of IDH1.

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

pharmaceutically acceptable salt thereof, the method further comprises evaluating the IDH1 genotype of the advanced solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), each characterized by the presence of a mutant allele of IDH1. 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:

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 advanced solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), that harbor an IDH1 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 solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), that harbor an IDH1 mutation, characterization of the pharmacokinetics (PK) of compound 1, or a pharmaceutically acceptable salt thereof, in subjects with advanced solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), that harbor an IDH1 mutation, 3) evaluation of the PK/pharmacodynamic (PD) relationship of compound 1, or a pharmaceutically acceptable salt thereof, and 2-hydroxygluturate (2-HG), and 4) characterization of the clinical activity associated with compound 1, or a pharmaceutically acceptable salt thereof, in subjects with advanced solid tumors, such as glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), that harbor an IDH1 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 solid tumors, such as glioma, intrahepatic

cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC), 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, and changes in 2-HG concentration as detected by proton magnetic resonance spectroscopy (¹H-MRS) on 3 tesla (3T) magnetic resonance images (MRI) in glioma subjects, 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, or a pharmaceutically acceptable salt thereof, 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 2-HG 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

Expansion MTD⁴ 36⁵

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. Non-hematologic includes all clinically significant non-hematologic toxicities CTCAE >Grade 3. 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), 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 (e.g., with glioma, intrahepatic cholangiocarcinoma (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, or non-small cell lung cancer (NSCLC)) 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 2-HG 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 2-HG. 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 2-HG 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.

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 with IHCC, chondrosarcoma, and glioma that has recurred or progressed following standard therapy (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 histologically or

cytologically confirmed solid tumor, including glioma, that has recurred or progressed following standard therapy, or that have not responded to standard therapy; 3) subjects must have documented IDH1 gene-mutated disease based on local evaluation. Analysis of tumor 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 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 have evaluable disease by RECIST vl.l for subjects without glioma or by RANO criteria for subjects with glioma; 5) Subjects must be amenable to serial peripheral blood sampling, urine sampling, and biopsies during the study; 6) Subjects or their legal representatives must be able to understand and sign an informed consent; 7) subjects must have ECOG PS of 0 to 1 and expected survival if at least 3 months; 8) subjects must have adequate bone marrow function (Absolute neutrophil count >1.5 xl09/L; Hemoglobin >9 g/dL; Platelets >75 x 109/L (Transfusions to achieve these levels are allowed)); 9) Subjects must have adequate bone marrow function as evidenced by: a) Absolute neutrophil count >1.5 x 109/L; b)

Hemoglobin >9 g/dL (Subjects are allowed to be transfused to this level) and c)Platelets >75 x 109/L; 10) 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; 11) 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; 12) 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 13) 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 2-HG levels. Tumor biopsies will be taken for evaluation of 2-HG 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 2-HG 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.

Pharmakodynamic 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 2- HG. Samples collected for PK assessments also will be used to assess 2-HG levels. In addition, subjects will have blood drawn for determination of 2-HG levels at the screening assessment.

The timing of blood samples drawn for 2-HG concentration determination may be changed if the emerging data indicate that an alteration in the sampling scheme is needed to better characterize the 2-HG response to compound 1, or a pharmaceutically acceptable salt thereof treatment. Urine will be collected for the determination of concentrations of 2-HG 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 2-HG concentration. An aliquot from each collection will be analyzed for urinary creatinine concentration.

Tumor biopsy specimens will be collected and assessed for 2-HG 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 2-HG 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:

Radiographic assessments (CT or MRI) to obtain tumor measurements will be evaluated during the clinical study to determine response to treatment according to by assessing response to compound 1, or a pharmaceutically acceptable salt thereof treatment according to RECIST vl.l (Eisenhauer, et al. Eur J Cancer. 2009;45(2):228-47) for subjects without glioma, or by modified RANO criteria for subjects with glioma (Wen, et al. J Clin Oncol. 2010;28(11): 1963-72).

Radiographic assessments (CT or MRI) to obtain tumor measurements will be conducted at screening and every 56 days thereafter while on compound 1, or a pharmaceutically acceptable salt thereof 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. For subjects with glioma, 1H-MRS will also be performed as a part of an exploratory analysis on the same schedule as CT/MRI scans with an additional scan on Day 29; results of 1H-MRS scans will not be used to make decisions regarding treatment continuation status.

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 2-HG levels will be explored with descriptive and graphical methods.

Response to treatment as assessed by the site Investigators using, RECIST (for subjects without glioma), or modified RANO criteria (for subjects with glioma) will be tabulated. 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. 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 solid tumors in a subject, the method 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.

2. The method of claim 1, wherein the advanced solid tumors is selected from glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, and non- small cell lung cancer (NSCLC).

3. The method of claim 1, wherein the advanced solid tumor is characterized by the presence of a mutant allele of IDH1.

4. The method of claim 3, wherein the mutant allele of IDH1 has an R132X mutation.

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

6. The method of claim 4, wherein the R132X mutation is R132H or R132C.

7. The method of claim 4, wherein the R132X mutation is R132H.

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

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

10. The method of claim 1, wherein Compound 1 is administered twice daily.

11. The method of claim 1, wherein Compound 1 is administered at a dose of about 50 mg to about 800 mg.

12. The method of claim 1, wherein the method comprises analyzing the advanced solid tumors by sequencing cell samples to determine the presence of the mutant allele of IDHl.

13. The method of claim 12, wherein the sequencing comprises DNA sequencing.

14. The method of claim 1, wherein the subject is evaluated prior to or after treatment with Compound 1.

15. The method of claim 14, wherein the subject is evaluated prior to and after treatment with Compound 1.

16. The method of claim 14 or 15, wherein the evaluating comprises determining the 2-hydroxyglutarate (2HG) level in the subject.

17. The method of claim 16, wherein the evaluating comprises spectroscopic analysis of a sample obtained from the subject.

18. The method of claim 17, wherein the spectroscopic analysis comprises magnetic resonance-based analysis.

19. The method of claim 18, wherein the spectroscopic analysis comprises MRI or MRS measurements.

20. The method of claim 17, wherein the sample comprises bodily fluid or surgical material.

21. The method of claim 20, wherein the bodily fluid comprises blood, plasma, urine, or spinal cord fluid.

22. The method of claim 16, wherein the evaluating comprises mass spectroscopy.

23. The method of claim 22, wherein the mass spectroscopy is LC-MS or GC-MS.

24. A method of evaluating a subject having an advanced solid tumor, the method comprising: determining 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 neo activity product, in the subject, that has been treated with Compound 1, to thereby evaluate the subject.

25. A method of evaluating a subject having an advanced solid tumor, 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 determining the level of Compound 1 or the level of an alpha hydroxy neoactivity product, in the subject, to thereby evaluate the subject.

26. The method of claim 24 or 25, wherein the alpha hydroxy neoactivity product is 2-hydroxyglutarate (2HG).

27. The method of claim 24 or 25, wherein the subject has been administered Compound 1 at a dose of about 50 mg to about 800 mg.

28. The method of claim 27, wherein Compound 1 is administered orally.

29. The method of claim 28, wherein Compound 1 is administered once or twice daily.

30. The method of claim 28, wherein Compound 1 is administered twice daily.

31. The method of claim 24 or 25, wherein the advanced solid tumor is selected from glioma, intrahepatic cholangiocarcinomas (IHCC), chondrosarcoma, prostate cancer, colon cancer, melanoma, and non- small cell lung cancer (NSCLC).

32. The method of claim 24 or 25, wherein the advanced solid tumor is characterized by the presence of a mutant allele of IDH1.

33. The method of claim 32, wherein the mutant allele of IDH1 has an R132X mutation.

34. The method of claim 33, wherein the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G.

35. The method of claim 33, wherein the R132X mutation is R132H or R132C.

36. The method of claim 33, wherein the R132X mutation is R132H.