WO2020146845A1 - Compositions and methods for treating prostate cancer with enzalutamide and an inhibitor of monoamin oxidase a - Google Patents

Abstract

The invention provides novel therapeutically methods and pharmaceutical compositions for treating prostate cancer with increased survival rate and improved treatment outcome. Methods and compositions of the invention can be used to prevent, delay, and/or reduce drug resistance and to increase, restore and/or prolong the effective treatment of prostate cancer with anti -androgen compounds.

Description

COMPOSITIONS AND METHODS FOR TREATING PROSTATE CANCER WITH ENZALUTAMIDE AND AN INHIBITOR OF MONOAMIN OXIDASE A

Priority Claims and Related Applications

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62/791,474, filed January 11, 2019, the entire content of which is incorporated herein by reference for all purposes.

Technical Field of the Invention

[0002] The invention generally relates to novel therapeutic methods and pharmaceutical compositions for treating cancer. More particularly, the invention relates to a novel approach to addressing drug resistance in prostate cancer treatment.

Background of the Invention

[0003] Prostate cancer (PCa) is the most common form of cancer and the second leading cause of cancer death among men in the United States. (Centers for Disease Control and Prevention http://www.cdc.gov/cancer/dcpc/data/men.htm.) Approximately one in seven men will be diagnosed with prostate cancer during his lifetime, and about one in 38 men will die of the disease with an estimated 307,000 deaths worldwide in 2012. (Merseburger, et al. 2015 Ther Adv Urol 7, 9-21.)

[0004] The current standard treatment for advanced prostate cancer is androgen deprivation therapy (ADT), which may suppress prostate cancer progression by reducing androgen biosynthesis or by preventing androgen from binding to the androgen receptor. (Chang, et al. 1988 Science 240, 324-326; Heinlein, et al. 2004 Endocr Rev 25, 276-308; Chang, et al. 2014 Oncogene 33, 3225-3234; Niu, et al. 2010 Oncogene 29, 3593-3604.)

[0005] A newly developed anti-androgen compound, Enzalutamide (Enz), was shown to suppress castration resistant prostate cancer (CRPC) and could further extend patients overall survival by an average of 4.8 months. (Scher, et al. , 2012 N Engl JMed 367, 1187-1197.) Unfortunately, most ADT, including Enz, may fail after 1-2 years treatment due to development of castration resistance (EnzR). (Miyamoto, et al. 2005 Nature clinical practice. Oncology 2, 236-237; Miyamoto, et al. 2004 The Prostate 61, 332-353.) The detail mechanism, however, remains unclear.

[0006] Recent human clinical survey indicated that Enz might induce the expression of ARv7, an AR splicing variant that lacks androgen-binding-domain, to constitutively activate its downstream genes in the castration concentration of androgen (near 1-3 nM), which can then lead to the development of castration resistance. (Kaiser, J. 2009 Science 324, 165; Ryan, et al. 2013 The New England journal of medicine 368, 1458-1459.) The detailed mechanism why Enz- induced ARv7 may lead to the development of castration resistance, however, remains unclear.

[0007] Currently, there is not an effective way to delay, reduce or reverse such drug resistance, which has seriously limited the overall effectiveness and treatment outcome of anti androgen therapy. There remains an urgent need for novel and improved approaches that effectively address these issues.

Summary of the Invention

[0008] The invention is based in part on the unexpected discovery that inhibitors of MAO A can be used to prevent, delay, reduce and/or reverse EnzR resistance and to increase, restore and/or prolong the effective treatment of prostate cancer with anti-androgen compounds, such as Enz. As disclosed herein, MO AO inhibitors can delay the onset and development and/or reduce the magnitude of EnzR and suppress EnzR tumors.

[0009] As is disclosed herein for the first time, MAOA is highly expressed in various Enz resistant PCa cells. Targeting MAOA with its specific inhibitors, for example clorgyline or phenelzine, both FDA-proved for treating Parkinson and depression, can lead to re-sensitized Enz-R cells in response to Enz treatment to further suppress Enz-R cell growth. This result indicates that certain currently FDA-proved drugs may be immediately developed and used as new therapy to overcome the Enz-induced castration resistance.

[0010] Studies have shown that MAOA increase hypoxia signaling to promote the Enz resistance. Together, this newly identified mechanism showing Enz can function via altering the ARv7 to increase MAOA expression lays the foundation for the quick development of a new therapy for CRPC using current FDA-proved drugs ( e.g ., clorgyline or phenelzine) to further extend CRPC patients survival that already developed Enz-resistance. [0011] The present invention thus may fundamentally alter the treatment protocol for castration resistant prostate cancer patients. Therapeutic methods and pharmaceutical compositions of the invention may be used to treat castration resistant prostate cancer, or a related disease or condition, in ways that delay, reduce or reverse drug resistance while maximizing treatment outcome.

[0012] In one aspect, the invention generally relates to a pharmaceutical composition, which includes: a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, and a second compound which is an inhibitor of monoamine oxidase A, wherein each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in the treatment of prostate cancer, or a related disease or condition thereof, in a mammal, including a human, and a pharmaceutically acceptable carrier.

[0013] In another aspect, the invention generally relates to a unit dosage form comprising a pharmaceutical composition disclosed herein.

[0014] In yet another aspect, the invention generally relates to a method for treating castration resistant prostate cancer, or a related disease or condition thereof. The method includes administering to a subject in need thereof a pharmaceutical composition disclosed herein.

[0015] In yet another aspect, the invention generally relates to a method for treating castration resistant prostate cancer, or a related disease or condition thereof. The method includes administering to a subject in need thereof a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, and a second compound which is an inhibitor of monoamine oxidase A, in amounts effective in the treatment of castration resistant prostate cancer, or a related disease or condition thereof, in a mammal, including a human, wherein the subject has been detected of the development of drug resistance to the first compound.

[0016] In yet another aspect, the invention generally relates to a method for treating castration resistant prostate cancer, or a related disease or condition thereof. The method includes: administering to a subject in need thereof a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, in an amount effective in the treatment of castration resistant prostate cancer or a related disease or condition thereof in a mammal, including a human; monitoring the subject to detect a development of drug resistance to the first compound; upon the subject being detected of the development of drug resistance to the first compound, administering to the subject a second compound which is an inhibitor of monoamine oxidase A, in an amount effect to reduce or eliminate drug resistance to the first compound; and monitoring the subject to detect a level of drug resistance to the first compound.

[0017] In yet another aspect, the invention generally relates to a method for treating a drug resistance in connection with a cancer treatment. The method includes: administering to a subject in need thereof a second compound which is an inhibitor of monoamine oxidase A, in an amount effective to prevent, delay, reduce or reverse resistance to a treatment of prostate cancer, or a related disease or condition thereof in a mammal, including a human.

Brief Description of the Drawings

[0018] FIG. 1. MAOA expression is associated with the development of Enz-resistance.

(A) Heat map of most significantly changed genes in EnzR cells. (B) Statistical analysis of MAOA expression in published datasets. The expressions of MAOA in normal prostate tissues and PCa tissues were analyzed based on Singh (normal, n=50; cancer, n=52) and Vanaja (normal, n=8; cancer, n=32) datasets. The MAOA expressions in different Gleason score PCa samples were analyzed based on the Taylor dataset (Gleason score 6, n=79; Gleason score 7, n=50 and Gleason score 8, n=10; Gleason score 9, n=9).The MAOA expression in PCa samples with recurrence and no recurrence were analyzed based on Lapointe (no recurrence, n=14; recurrence, n=3) dataset. (C) The qPCR and Western blot analysis of MAOA levels in EnzRl- C4-2 and EnzSl-C4-2 cells. (D) MAOA activity analysis in EnzSl-C4-2 and EnzRl-C4-2 cells by using MAO-Glo assay. (E) MAOA and ARv7 mRNA levels by qPCR following treatment with DMSO or 10 mM Enz in EnzSl-C4-2 cells for 6 days. (F) MAOA activity analyzed in EnzSl-C4-2 cells treated with 10 mM Enz for 6 days. (G) Absolute copy numbers of MAOA mRNA normalized to copy number of RPL13A in CTCs isolations from 288 patients. Those samples were grouped by CTC and ARv7 status: CTC-, CTC+/ARv7-, and CTC+/ARv7+. (H) MAOA copy numbers normalized by RPL13A in patients whose ARv7 status remained negative after Enz treatment (N=30). (I) MAOA copy numbers normalized by RPL13A in patients whose ARv7 status changed from negative to positive after Enz treatment (N=13). (J-K) Relative MAOA copy number values at baseline (before Enz) and after Enz treatment (after Enz) in each patient whose ARv7 status changed from negative to positive after Enz treatment (N=13) (J) and in patients whose ARv7 status was positive at baseline (before Enz) and remained positive after Enz treatment (N=12) (K). Quantitation is mean ± SEM, P-value was determined by two-tailed paired t test. P-values are, **<0.005, ***<0.001.

[0019] FIG. 2. Targeting MAOA re-sensitizes EnzR cells to Enz and suppresses EnzR cell growth. (A) EnzSl-C4-2 cells were treated with/without (w/o) 10 mM Enz and 5 mM clorgyline and cell viability analyzed by MTT assay. (B-D) EnzRl-C4-2, EnzR2-C4-2, EnzR3- 22Rvl cells, respectively, were treated (w/o) 10 mM Enz and 5 mM clorgyline, and cell viability was analyzed by MTT assay. (E-G) EnzRl-C4-2, EnzR2-C4-2, EnzR3-22Rvl cells, respectively, were treated w/o 10 mM Enz and 5 mM phenelzine, and cell viability was analyzed. (H-J) The EnzRl-C4-2, EnzR2-C4-2, EnzR3-22Rvl cells with pLKO or shMAOA cells were treated w/o Enz and cell viability was analyzed. (K) MAO A was overexpressed (oeMAOA) in EnzSl-C4-2 cells and then the cells treated w/o Enz and cell viability was analyzed. (L) EnzSl-C4-2 cells were treated w/o 10 mM Enz and cell viability was analyzed. (M) EnzSl-C4-2 cells were first treated (w/o) 10 mM Enz for 1.5 month. And then the cells were seeding and the cell viability under 10 mM Enz treatment was analyzed by MTT assay. (N-O) EnzSl-C4-2 cells were treated w/o 10 mM Enz and 1 mM,2.5mM clorgyline (clg) for 1.5 month. And then the cell viability under 10 mM Enz treatment was analyzed. Quantitation is mean ± SEM, P-value was determined by two-tailed paired t test. P-values are, *<0.05, **<0.005.

[0020] FIG. 3. Mechanism dissection of how Enz increases the MAOA expression: via increasing ARv7. (A-B) PC3 cells were treated with/without (w/o) 10 mM Enz and 5 mM clorgyline or 10mM clorgyline. (The cell viability was analyzed by MTT assay. (C) EnzSl-C4-2 cells were treated with 10 mM Enz for different time points. The ARv7 and MAOA mRNA level were analyzed by qPCR (left) and western blot (right). (D) The mRNA levels of ARv7 and MAOA were analyzed in EnzRl-C4-2 PWPI and PWPI-ARv7 cells. (E) The expression of ARv7 and MAOA were analyzed in EnzSl_C4-2pWPI and pWPI-ARv7(oeARv7) cells by q-PCR (left) and western blot (right). (F-H) The mRNA and protein level of ARv7 and MAOA were analyzed in EnzRl_C4-2, EnzR2_C4-2 (no protein shown), EnzR3_CWR22Rvl pLKO and shARv7 cells. (I) EnzSl-C4-2 cells were infected with pLKO and shARv7 viruses. And then the cells were treated w/o Enz for 2 and 6 days, the ARv7 and MAOA levels were examined by qPCR. (J) EnzSl cells were infected by pWPI-ARv7, pLKO-shMAOA or both viruses. And then the cells were treated w/olO mM Enz and the cell viability analyzed by MTT assay. For C and F- I, quantitations are presented as mean±SEM, P-value was determined by two-tailed paired t test. P-values are **<0.005, ***<0.001.

[0021] FIG. 4. Enz up-regulates the MAOA transactivation in transcriptional level and protein level. (A) Schematic depiction of putative ARE on MAOA promoter region. The mutant ARE was marked by italic font. (B) ChIP assay was performed to identify that endogenous AR and Flag-ARv7 bind to the putative ARE on MAO A promoter in EnzRl-C4-2 and EnzSl -pWPI- flag-ARV7 cells. (C) EnzRl cells were infected by Flag-ARv7 or Flag-ARfl virus. The ChIP assay were performed to analyze Flag-AR and Flag-ARv7 binding on MAOA promoter region in EnzRl cells. (D) PC3 pWPI and pWPI-ARV7 cells were transfected by the PGL3-MAOA promoter plasmid, and the promoter activity was determined by luciferase assay. (E) EnzSl-C4-2 cells were treated with Enz at different time points and the MAOA promoter activity was identified by luciferase reporter assay. (F) MAOA promoter with mutated ARE was constructed into PGL3 plasmid, and then the MAOA promoter activity was analyzed in PC3 and PC3- oeARv7 cells. (G) ARv7 and flAR were transfected into EnzSl-C4-2 cells. And then the PGL3- MAOA promoter luciferase activity was assayed. (H) ARv7 and flAR were transfected into EnzSl-C4-2 cells. And then the MAOA expression was analyzed by WB. (I) EnzSl-C4-2 and EnzRl -C4-2 cells were treated with cycloheximide (CHX) for different time points. The MAOA protein level was analyzed by western blot. (J) EnzSl-C4-2 and EnzRl -C4-2 cells were treated with MG-132. The MAOA protein level was analyzed by western blot. (K) MAOA

phosphorylation is increased in EnzRl -C4-2 cells and the lysine phosphorylation of MAOA was detected by specific antibody. (L) The phosphorylation of p38 (p-p38) protein levels were detected by western blot in EnzSl-C4-2 and EnzRl -C4-2 cells. (M) EnzSl and EnzRl -C4-2 cells were treated with p38 inhibitor. The MAOA protein levels were analyzed by western blot. (N) EnzRl cells were treated with p38 inhibitors for 48hrs, and then the MAOA protein was pulled down, MAOA p-Ser level was examined by western blot. For C and D, Quantitation is mean ± SEM, P-value was determined by two-tailed paired t test. P value is <**0.005.

[0022] FIG. 5. MAOA activates Hypoxia signaling to promote the Enzalutamide- resistance. (A) The mRNA levels of Glutl, N-Cadherin, Timp and VEGF-A in EnzSl-C4-2 and EnzRl -C4-2 cells were analyzed by qPCR. (B) In EnzRl -C4-2 cells, MAOA was knocked down by shRNA. And the mRNA levels of MAOA, Slug, VEGF-A and HIF-Ia were analyzed by qPCR (left), as well asHIF-Ia and VEGF-A expression by western blot (right). (C) EnzRl -C4-2 cells were treated with/without 5mM clorgyline, and the mRNA and protein level of MAOA, HIF-Ia and VEGF-A were analyzed by qPCR (left) and western blot (right). (D) MAOA was overexpressed (oeMAOA) in EnzSl -C4-2 cells, and the mRNA and protein levels of MAOA, HIF-Ia and VEGF-A were analyzed by qPCR (left) and western blot (right). (E) HIF-lawas knocked down (shHIF-Ia) in EnzRl-C4-2 cells, and then the cells were treated with/without Enz. The cell viability was analyzed by MTT assay. Quantitation is presented as mean ± SEM, P-value was determined by two-tailed paired t test. P values are *<0.05, **<0.005, ***<0.001.

[0023] FIG. 6. Clorgyline and phenelzine, inhibitors of MAO A activity can overcome Enzalutamide-resistance. (A) The in vivo PDX-PCa mouse model data revealed that injection with Enz (30mg/kg/every other day) increased the MAOA, ARv7 and p-p38 level. (B, C) Mice implanted with EnzR3-22Rvl xenografts were treated with vehicle control, Enz (30 mg/kg), clorgyline (10 mg/kg), phenelzine (30 mg/kg), Enz+cl orgyline (30 mg/kg+10 mg/kg), or Enz+phenelzine (30 mg/kg+30 mg/kg). After sacrifice, tumors of the 6 groups were collected and weighed. (D, E) Mice implanted with EnzRl-C4-2 xenografts received the same treatments as EnzR3-22Rvl After sacrifice, tumors of the 6 groups were collected and weighed. (F) IHC staining of Ki-67 and VEGFA in EnzR3-22Rvl tumors were performed. (G) IHC staining of Ki- 67 and VEGF-A in EnzRl-C4-2 tumors were performed. For C, E, F and G, quantitation is mean ± SEM, P-value was determined by two-tailed paired t test. P value is ***<0.001.

[0024] FIG. 7. (A) Quantitative IHC analysis of MAO-A protein expression in normal prostate (n = 7), PCa clinical samples (gleason score 3) (n = 5), PCa clinical samples (gleason score 4) (n = 7), and PCa clinical samples (gleason score 5) (n = 8). Arrows indicate tumor areas. (B) Western blot analysis of MAO-A expression in EnzS4_C4-2B parental and Enz-R4_C4-2B EnzR cells. (C) VCaP cells were treated by Enz for 10 days and then the ARv7 and MAO-A protein levels were examined by WB. For A Quantitations are mean±SEM, P-value was determined by two-tailed paired t test. P values are **<0.005, ***<0.001.

[0025] FIG. 8. AR-FL copy numbers at baseline (before) and after Enzalutamide treatment (after) in patients whose ARv7 status (A) remained negative (N=30), (B) changed from negative to positive (N=13), and (C) remained positive (N=12) after Enzalutamide treatment. P-values were determined by two-tailed paired t-test.

[0026] FIG. 9. (A) MGEA11 and HSD3B1 expression EnzR and EnzS cells were analyzed by qPCR. (B) The EnzRl cells were treated w/o lOuM clorgyline for 24 hours. The MAO-A and ARv7 expression were analyzed by WB. [0027] FIG. 10. The ARfl and ARv7 binding on MAO-A promoter regions in presence of DHT (A) or in the absence of DHT (Etoh) (B) were analyzed based on ChIP-seq data (GEO: GSE106559). (C) The quantification of ARfl and ARv7 binding on the MAO-A promoter region.

[0028] FIG. 11. (A) Western blot analysis to identify the knocking efficiency of MAO-A in EnzRl_C4-2 and EnzR2 cells. (B) The qPCR analysis (left) to identify the mRNA level and Western blot analysis (right) of HIF-Ia and VEGF-A in EnzRl_C4-2 pLVTHM and shHIFla cells. For B Quantitations are mean±SEM, P-value was determined by two-tailed paired t test. P values are **<0.005, ***<0.001.

[0029] FIG. 12. (A) Mice implanted with EnzR3-22RVl-luc xenografts were treated with vehicle control, Enz (30 mg/kg), cl orgyline (10 mg/kg), phenelzine (30 mg/kg), Enz+cl orgyline (30 mg/kg+10 mg/kg), or Enz+phenelzine (30 mg/kg+30 mg/kg). The tumor sizes were monitored by In vivo imaging system (IVIS). (B-C) After sacrifice, tumors of the 6 groups were collected (B) and weighed (C). (D) IHC staining of Ki-67 and VEGF-A in EnzR3-22Rvl-luc tumors were performed. For D Quantitations are mean±SEM, P-value was determined by two- tailed paired t test. P values are **<0.005.

[0030] FIG. 13. Exemplary primer and plasmid sequences.

Definitions

[0031] Unless defined otherwise, 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. General principles of organic chemistry, as well as specific functional moieties and reactivity, are described in“Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 2006.

[0032] Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans- isomers, R- and ^-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.

Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention. [0033] Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90: 10, 95:5, 96:4, 97:3, 98:2, 99: 1, or 100:0 isomer ratios are contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.

[0034] If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic methods well known in the art, and subsequent recovery of the pure enantiomers.

[0035] Enzalutamide ( ak.a ., MDV3100 and Xtandi, CAS No. 915087-33-1) is a synthetic, non-steroidal pure antiandrogen that was developed for the treatment of metastatic castration- resistant prostate cancer. The IUPAC name of the compound is 4-(3-(4-Cyano-3- (trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-l-yl)-2-fluoro-N- methylbenzamide. The structure of Enzalutamide is depicted by formula (I).

[0036] As used herein, the term“effective amount” of an active agent refers to an amount sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound of the invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the patient.

[0037] As used herein, the terms“treatment” or“treating” a disease or disorder refers to a method of reducing, delaying or ameliorating such a condition before or after it has occurred. Treatment may be directed at one or more effects or symptoms of a disease and/or the underlying pathology. The treatment can be any reduction and can be, but is not limited to, the complete ablation of the disease or the symptoms of the disease. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique. [0038] As used herein, the terms“prevent”,“preventing”, or“prevention” refer to a method for precluding, delaying, averting, or stopping the onset, incidence, severity, or recurrence of a disease or condition. For example, a method is considered to be a prevention if there is a reduction or delay in onset, incidence, severity, or recurrence of a disease or condition or one or more symptoms thereof in a subject susceptible to the disease or condition as compared to a subject not receiving the method. The disclosed method is also considered to be a prevention if there is a reduction or delay in onset, incidence, severity, or recurrence of osteoporosis or one or more symptoms of a disease or condition in a subject susceptible to the disease or condition after receiving the method as compared to the subject's progression prior to receiving treatment. Thus, the reduction or delay in onset, incidence, severity, or recurrence of osteoporosis can be about a 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between.

[0039] As used herein, the term“pharmaceutically acceptable” excipient, carrier, or diluent refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymer as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. [0040] As used herein, the terms“isolated” or“purified” refer to a material that is substantially or essentially free from components that normally accompany it in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

[0041] As used herein, the term“subject” refers to any animal ( e.g ., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms“subject” and“patient” are used interchangeably herein in reference to a human subject.

[0042] As used herein, the term“low dosage” refers to at least 5% less (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) than the lowest standard recommended dosage of a particular compound formulated for a given route of administration for treatment of any human disease or condition. For example, a low dosage of an agent that is formulated for administration by inhalation will differ from a low dosage of the same agent formulated for oral administration.

[0043] As used herein, the term“high dosage” is meant at least 5% (e.g., at least 10%, 20%, 50%, 100%, 200%, or even 300%) more than the highest standard recommended dosage of a particular compound for treatment of any human disease or condition.

[0044] As used herein, the term“prodrug” (or“pro-drug”) refers to a pharmacological derivative of a parent drug molecule that requires biotransformation, either spontaneous or enzymatic, within the organism to release the active drug. Such prodrugs are pharmaceutically active in vivo, when they undergo solvolysis under physiological conditions or undergo enzymatic degradation. Prodrug compounds herein may be called single, double, triple, etc., depending on the number of biotransformation steps required to release the active drug within the organism, and the number of functionalities present in a precursor-type form.

[0045] Prodrug forms often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism. (See, Bundgard, Design of Prodrugs, pp. 7-9,21-24,

Elsevier, Amsterdam 1985 and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp. 352-401, Academic Press, San Diego, Calif., 1992). Prodrugs commonly known in the art include well-known acid derivatives, such as, for example, esters prepared by reaction of the parent acids with a suitable alcohol, amides prepared by reaction of the parent acid compound with an amine, basic groups reacted to form an acylated base derivative, etc. Of course, other prodrug derivatives may be combined with other features disclosed herein to enhance bioavailability. As such, those of skill in the art will appreciate that certain of the presently disclosed compounds having free amino, arnido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds having an amino acid residue, or a polypeptide chain of two or more ( e.g ., two, three or four) amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy or carboxylic acid groups of the presently disclosed compounds. The amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma- aminobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine sulfone. Prodrugs also include compounds having a carbonate, carbamate, amide or alkyl ester moiety covalently bonded to any of the above substiruents disclosed herein.

[0046] Isotopically-labeled compounds are also within the scope of the present disclosure.

As used herein, an "isotopically-labeled compound" refers to a presently disclosed compound including pharmaceutical salts and prodrugs thereof, each as described herein, in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds presently disclosed include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸0, ¹⁷0, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶C1, respectively.

[0047] By isotopically-labeling the presently disclosed compounds, the compounds may be useful in drug and/or substrate tissue distribution assays. Tritiated (³H) and carbon-14 (¹⁴C) labeled compounds are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (²H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds presently disclosed, including pharmaceutical salts, esters, and prodrugs thereof, can be prepared by any means known in the art.

[0048] Further, substitution of normally abundant hydrogen (¾) with heavier isotopes such as deuterium can afford certain therapeutic advantages, e.g., resulting from improved absorption, distribution, metabolism and/or excretion (ADME) properties, creating drugs with improved efficacy, safety, and/or tolerability. Benefits may also be obtained from replacement of normally abundant ¹²C with ¹³C. (See, WO 2007/005643, WO 2007/005644, WO 2007/016361, and WO 2007/016431.)

[0049] Stereoisomers ( e.g ., cis and trans isomers) and all optical isomers of a presently disclosed compound (e.g., R and S enantiomers), as well as racemic, diastereomeric and other mixtures of such isomers are within the scope of the present disclosure.

[0050] Compounds of the present invention are, subsequent to their preparation, preferably isolated and purified to obtain a composition containing an amount by weight equal to or greater than 95% (“substantially pure”), which is then used or formulated as described herein. In certain embodiments, the compounds of the present invention are more than 99% pure.

[0051] Solvates and polymorphs of the compounds of the invention are also contemplated herein. Solvates of the compounds of the present invention include, for example, hydrates.

[0052] Any appropriate route of administration can be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intraventricular, intracorporeal, intraperitoneal, rectal, or oral administration. Most suitable means of administration for a particular patient will depend on the nature and severity of the disease or condition being treated or the nature of the therapy being used and on the nature of the active compound.

[0053] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof are admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (i) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (ii) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (iii) humectants, as for example, glycerol, (iv) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (v) solution retarders, as for example, paraffin, (vi) absorption accelerators, as for example, quaternary ammonium

compounds, (vii) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (viii) adsorbents, as for example, kaolin and bentonite, and (ix) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art.

[0054] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, such as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3- butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like. Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.

[0055] Materials, compositions, and components disclosed herein can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. It is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

Detailed Description of the Invention

[0056] The invention provides a unique approach to treatment of prostate cancer, particularly drug resistance prostate cancer. The novel therapeutic methods and compositions provided herein can benefit prostate cancer patients in terms of increased survival rate and improved treatment outcome. Methods and compositions of the invention can be used to prevent, delay, reduce and/or reverse drug resistance and to increase, restore and/or prolong the effective treatment of prostate cancer with anti-androgen compounds.

[0057] Enzalutamide, an anti-androgen agent, has been successfully used as the last line therapy to extend lives of CRPC patients. After prostate cancer patients develop CRPC, the current standard therapy to further suppress CRPC involves either docetaxel (Doc)- chemotherapy or ADT with either using Enz to prevent androgens binding to AR. or using abiraterone (ABI) to further suppress the androgen synthesis in renal. Unfortunately, many patients still develop Enz resistance after an average of 4.8 months response to Enz. (Scher, et al. 2012 N Engl J Med 367 , 1187-1197; Dhingra, et al. 2013 Mini Rev Med Chem 13, 1475-1486.)

Enzalutamide (Enz)

[0058] The mechanism by which CRPC patients received Enz develop EnzR remains not fully understood. Certain studies indicated the possible involvement of multiple mechanisms. For instance, Enz or its derivative ARN-509 ( a.k.a ., JNJ-56021927) may induce an AR. point mutation at AR.876 (a missense mutation of phenylalanine 876 to leucine in the Ligand-Binding- Domain (LBD) of AR, named AR-F876L) that is no longer sensitive to Enz treatment. (Korpal, et al. 2013 Cancer Discovery 3, 1030-1043; Joseph et al. 2013 Cancer Discovery 3, 1020-1029.)

ARN-509

[0059] Other studies showed increased glucocorticoid receptor (GR) signals by Enz in a subset of prostate cancer cells due to relief of AR-mediated feedback repression of GR expression. GR and AR are closely related members of the nuclear receptor superfamily with similar DNA-binding-domains. It is possible that the GR could replace some AR roles during development of EnzR. Studies also found that the GR agonist dexamethasone was sufficient to confer EnzR, whereas a GR antagonist could partially restore sensitivity. (Arora, et al. 2013 Cell 155, 1309-1322; Yemelyanov, et al. 2012 Cell Cycle 11, 395-406; Sharifi 2014 New England Journal of Medicine 370, 970-971.)

[0060] Development of AR splicing variant ARv7 is also linked to the development of EnzR. Recent clinical sample survey reported that CRPC patients received ADT-Enz had increased ARv7, and ARv7 expression was higher in patients developed Enz resistance. (Antonarakis, et al. 2014 The New England journal of medicine 371, 1028-1038.) For example, clinical studies from CRPC patients demonstrated that 39% of metastatic CRPC patients treated with Enz had detectable ARv7 in their circulating tumor cells. These ARv7 positive patients had lower Prostate-Specific Antigen (PSA) response rates than ARv7 negative patients with shorter PSA progression-free survival (median, 1.4 months vs. 6.0 months), indicating that CRPC patients with ARv7 might have poor response to Enz treatment and that Enz treatment might enhance ARv7 expression. (Antonarakis et al. 2014 New England Journal of Medicine 371, 1028-1038.) [0061] Instead of directly targeting ARv7, however, compounds identified herein target MAOA, a key gene downstream of ARv7, to mediate the ARv7-indiced Enz resistance. The advantages of using MAOA inhibitors to re-sensitize Enz resistance include improved efficacy to increase the Enz sensitivity in Enz resistant cells (FIG. 2B-D) as well as delaying the development of Enz resistance during combination therapy with Enz to treat CRPC cells (FIG.

2J).

[0062] MAOA is a key enzyme in catalyzing the deamination of amines, and play key roles in inducing some neurotransmitters including norepinephrine, dopamine and serotonin.

(Dorfman, et al. 2014 Current topics in behavioral neurosciences 17, 297-313.) Interestingly, recent studies also reported that higher MAOA might be linked to the PCa progression via inducing the hypoxia and EMT signals. (Flamand, et al. 2010 Journal of cancer research and clinical oncology 136, 1761-1771; Wu, et al. 2014 The Journal of clinical investigation 124, 2891-2908.)

[0063] It has now been unexpectedly discovered, as disclosed herein, that inhibitors of MO AO, such as clorgyline or phenelzine, can be used to effectively prevent, delay, reduce and/or reverse EnzR resistance and to increase, restore and/or prolong the effective treatment of prostate cancer with anti-androgen compounds, such as Enzalutamide and its derivative ARN-

509.

Phenelzine

[0064] Clorgyline is a selective and irreversible inhibitor of MAOA. Phenelzine is a non- selective and irreversible MAOA inhibitor of the hydrazine class. Phenelzine is approved and used as an antidepressant and anxiolytic. [0065] Potential side effects of treatment with MAOA inhibitors such as phenelzine include dizziness, drowsiness, tiredness, weakness, problems sleeping, constipation, and dry mouth. To CRPC patients that have already developed EnzR, the potential benefit of MAOA inhibitors in restoration of Enz sensitivity would in many cases outweigh the risk of such side effects. In addition, where side effects are severe, dosage adjustment may offer patient therapeutic benefit with manageable risk exposure.

[0066] Significantly, certain MAOA inhibitors have been clinically studied and or approved and may be immediately applied to treating Enz resistant CRPC patients. For example, cl orgyline or phenelzine has been used to treat Parkinson's disease and depression since 1960s. (Riederer, et al. 2011 Experimental neurobiology 20, 1-17.) It’s worth noting that the suggested dose of clorgyine for depression is near 30 mg/day, whereas doses used in in vitro cell line studies disclosed herein is at 1.5 mg/mL and in vivo mice is 1 mg/Kg/day, which is lower than the human dose. This indicates that the dose used to treat the human depression can be also used to effectively re-sensitize the Enz resistance.

[0067] Thus, the present invention enables a novel and ready therapy to suppress the Enz- resistant CRPC progression and to extend survival of CRPC patients. In certain embodiments of the invention, as discussed in details herein, a combination of a MO AO inhibitor and Enz may be used at the beginning of therapy. In certain other embodiments, sequential treatment with Enz therapy at the beginning may be followed by addition of a MO AO inhibitor at or after confirmation of EnzR ( e.g ., when decreased PSA start to rise again).

[0068] In one aspect, the invention generally relates to a pharmaceutical composition, which includes: a first compound of Formula (I)

in an amount such that the pharmaceutical composition is effective in the treatment of prostate cancer, or a related disease or condition thereof, in a mammal, including a human, and a pharmaceutically acceptable carrier.

[0069] In certain embodiments, each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in delaying the onset of or delaying the development of drug resistance. In certain embodiments, each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in delaying the onset of drug resistance. In certain embodiments, each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in delaying the development of drug resistance.

[0070] In certain embodiments, each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in reducing or reversing drug resistance. In certain embodiments, each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in reducing drug resistance. In certain embodiments, each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in reversing drug resistance.

[0071] The first and second compounds may be present in the pharmaceutical composition at any suitable ratios. For example, the weight ratio of the first compound to the second compound is from about 10 : 1 to about 1 : 10 (e.g., from about 7 : 1 to about 1 : 7, from about 5 : 1 to about 1 : 5, from about 3 : 1 to about 1 : 3, from about 2 : 1 to about 1 : 2, about 1 : 1).

[0072] The first compound may be present in the pharmaceutical composition in any suitable amount, for example in a unit dosage of, in the range of about 0.1 mg to about 1,000 mg, about 1 mg to about 500 mg, about 1 mg to about 10 mg, about 10 mg to about 250 mg, about 10 mg to about 125 mg, about 10 mg to about 100 mg, about 10 mg to about 75 mg, about 10 mg to about 50 mg, about 10 mg to about 25 mg.

[0073] The second compound may be present in the pharmaceutical composition in any suitable amount, for example in a unit dosage of, in the range of about 0.1 mg to about 1,000 mg, about 1 mg to about 500 mg, about 1 mg to about 10 mg, about 10 mg to about 250 mg, about 10 mg to about 125 mg, about 10 mg to about 100 mg, about 10 mg to about 75 mg, about 10 mg to about 50 mg, about 10 mg to about 25 mg. [0074] In certain embodiments, the pharmaceutical composition is suitable for oral administration. In certain embodiments, the pharmaceutical composition is suitable for intravenous, intramuscular, or subcutaneous administration.

[0075] Any suitable inhibitor(s) of monoamine oxidase may be used in preparing the pharmaceutical composition of the invention. In certain embodiments, the pharmaceutical composition includes a single inhibitor of monoamine oxidase ( e.g ., MAOA). In certain embodiments, the pharmaceutical composition includes two or more inhibitors of monoamine oxidase (e.g., MAOA) in a combination.

[0076] Exemplary inhibitors of MAOA include cl orgyline and phenelzine.

[0077] In certain embodiments, the second compound is clorgyline. In certain embodiments, the weight ratio of the first compound to clorgyline is from about 7 : 1 to about 1 : 7, from about 5 : 1 to about 1 : 5, from about 3 : 1 to about 1 : 3, from about 2 : 1 to about 1 : 2, about 1 : 1 [0078] In certain embodiments, the second compound is phenelzine. In certain

embodiments, the weight ratio of the first compound to phenelzine is from about 7 : 1 to about 1 : 7, from about 5 : 1 to about 1 : 5, from about 3 : 1 to about 1 : 3, from about 2 : 1 to about 1 : 2, about 1 : 1.

[0079] In another aspect, the invention generally relates to a unit dosage form comprising a pharmaceutical composition disclosed herein.

[0080] In certain embodiments, the unit dosage form is in the form of a tablet or capsule suitable for oral administration.

[0081] In certain embodiments, the unit dosage form is in the form of a liquid solution or suspension suitable for intravenous, intramuscular, or subcutaneous administration.

[0082] In yet another aspect, the invention generally relates to a method for treating prostate cancer, or a related disease or condition thereof. The method includes administering to a subject in need thereof a pharmaceutical composition disclosed herein.

[0083] In certain embodiments, the method further include: administering to the subject one or more other anti-cancer agents. The one or more other anti-cancer agents may be any suitable agent, for example, a chemotherapeutic agent.

[0084] Exemplary chemotherapeutic agents include Erlotinib (TARCEVA®, Genentech/OSI Pharm.), Bortezomib (VELCADE®, Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate (GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib

(TYKERB®, GSK572016, Glaxo Smith Kline), Lonafarnib (SCH 66336), Sorafenib (BAY43- 9006, Bayer Labs), and Gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271;

Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. , calicheamicin, especially calicheamicin gammall and calicheamicin omegall (Angew Chem. Inti. Ed. Engl. (1994) 33: 183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,

dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, ADRIAMYCIN^® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esonibicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamniprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate,

epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;

demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;

losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK^® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;

tenuazonic acid; triaziquone; 2,2' ,2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine;

mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. , TAXOL^® (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE^® (doxetaxel; Rhone- Poulenc Rorer, Antony, France); chloranmbucil; GEMZAR^® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE^® (vinorelbine);

novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA^®);

ibandronate; CPT-11 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0085] In yet another aspect, the invention generally relates to a method for treating castration resistant prostate cancer, or a related disease or condition thereof. The method includes administering to a subject in need thereof a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, and a second compound which is an inhibitor of monoamine oxidase A, in amounts effective in the treatment of prostate cancer, or a related disease or condition thereof, in a mammal, including a human, wherein the subject has been detected of the development of drug resistance to the first compound.

[0086] In certain embodiments, the second compound is administered simultaneously with the first compound, or subsequently after the administration of the first compound.

[0087] In certain embodiments, the subject has been detected of the development of drug resistance to the first compound and the method is applied.

[0088] In certain embodiments, the subject has not been detected of the development of drug resistance to the first compound and the method is applied.

[0089] In certain embodiments, the drug resistance results from or is related to Arv7 and/or AR mutant.

[0090] In certain embodiments, the first and/or the second compound is administered orally.

[0091] In certain embodiments, the first and/or the second compound is administered intravenously, intramuscularly, or subcutaneously.

[0092] In certain embodiments, the second compound is clorgyline.

[0093] In certain embodiments, the second compound is phenelzine.

[0094] In certain embodiments, the method further includes administering to the subject one or more other anti-cancer agents (e.g., a chemotherapeutic agent).

[0095] In yet another aspect, the invention generally relates to a method for treating prostate cancer, or a related disease or condition thereof. The method includes: administering to a subject in need thereof a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, in an amount effective in the treatment of prostate cancer or a related disease or condition thereof in a mammal, including a human; monitoring the subject to detect a development of drug resistance to the first compound; upon the subject being detected of the development of drug resistance to the first compound, administering to the subject a second compound which is an inhibitor of monoamine oxidase A, in an amount effect to reduce or eliminate drug resistance to the first compound; and monitoring the subject to detect a level of drug resistance to the first compound.

[0096] In certain embodiments, the method further includes: upon the subject being detected of the development of drug resistance to the first compound, continuing to administer the subject the first compound.

[0097] In certain embodiments, the method further includes: upon the subject being detected of the development of drug resistance to the first compound, halting the administration of the first compound, and upon the subject being detected of a substantial reduction or disappearance of drug resistance, re-starting the administration of the first compound.

[0098] In certain embodiments, the drug resistance results from or is related to Arv7 and/or AR mutant.

[0099] In certain embodiments, the first and/or the second compound is administered orally.

[00100] In certain embodiments, the first and/or the second compound is administered intravenously, intramuscularly, or subcutaneously.

[00101] In certain embodiments, the second compound is clorgyline.

[00102] In certain embodiments, the second compound is phenelzine.

[00103] In certain embodiments, the method further includes administering to the subject one or more other anti-cancer agents ( e.g ., a chemotherapeutic agent). [00104] In yet another aspect, the invention generally relates to a method for treating a drug resistance in connection with a cancer treatment. The method includes: administering to a subject in need thereof a second compound which is an inhibitor of monoamine oxidase A, in an amount effective to prevent, delay, reduce or reverse resistance to a treatment of prostate cancer, or a related disease or condition thereof in a mammal, including a human.

[00105] In certain embodiments, the method is effective in delaying the onset of or delaying the development of drug resistance.

[00106] In certain embodiments, the method is effective in reducing or reversing drug resistance.

[00107] In certain embodiments, the resistance is to a compound having Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof.

[00108] The following examples are meant to be illustrative of the practice of the invention, and not limiting in any way.

Examples

[00109] It was unexpectedly found that the expression of MAO A was significantly increased in several EnzR CRPC cells, and higher MAOA expression was associated with positive ARv7 detection in CRPC patients following Enz treatment. Targeting MAOA with specific inhibitors, including clorgyline or phenelzine re-sensitized the EnzR cells to Enz treatment and further suppressed EnzR cell growth in the multiple in vitro EnzR CRPC cells and in vivo mouse models. Mechanism dissection indicates that Enz via increasing ARv7 expression can

transcriptionally regulate the MAOA expression via binding to ARE on its 5’ promoter region, and increased MAOA can then increase Enz-resi stance via altering the hypoxia HIF-Ia signals. Together, results disclosed herein showed that targeting MAOA with anti-depression phenelzine or clorgyline can restore Enz-sensitivity to further suppress EnzR cell growth via altering the Enz/ARv7/MAOA signaling.

Increased MAO A expression after the development ofEnz resistance

[00110] The antiandrogen Enz is clinically effective in treating CRPC patients. However, development of Enz-resi stance is inevitable, emphasizing the need to further dissect the mechanisms of Enz-resi stance in basic preclinical studies using in vitro cell lines and in vivo mouse models. (Antonarakis, et al. 2014 The New England journal of medicine 371, 1028-1038.)

[00111] We first searched for potential altered genes after the development of Enz-resi stance in EnzR CRPC cells generated after chronic culture of CRPC C4-2 cells in media containing increasing Enz concentrations from 10 to 30 mM for 1 year (named as EnzRl-C4-2), or after continuously culturing the C4-2 cells in media containing fixed 10 mM Enz for 6 months (named as EnzR2-C4-2), We also used the naturally Enz-resistant cell line, CWR22Rvl, and named them as EnzR3-CWR22Rvl. Finally, we also obtained the EnzR C4-2B cells from Dr. Allen Gao and named as EnzR4-C4-2B in these studies.

[00112] We then applied RNAseq assay to compare the expression profiles in EnzRl-C4-2 cells and the C4-2 parental Enz-sensitive (EnzSl-C4-2) cells. Among many genes that were highly differentially expressed in EnzRl-C4-2 cells, MAO A expression was increased significantly in the EnzRl-C4-2 cells by more than 6 folds (FIG. 1A). We analyzed MAOA expression in 4 different human PCa datasets. The results revealed that higher expression of MAOA in human PCa tissues compared to normal prostate tissues (FIG. IB), and higher MAOA expression in higher Gleason score tumors and recurrent PCa (FIG. IB, FIG. 7A).

[00113] We further validated the RNAseq data, and found MAOA (mRNA and protein) expression were significantly increased in the EnzRl-C4-2 cells as compared to the EnzSl-C4-2 cells (FIG. 1C). In addition, MAOA activity was higher in EnzRl-C4-2 cells than EnzSl-C4-2 cells by MAO-GLO assay (FIG. ID). Similar results were also obtained when we replaced another paired EnzR vs EnzS cell line (EnzR4-C4-2B vs EnzS4-C4-2B cells) (FIG. 7B).

[00114] Importantly, we found that treating the EnzSl-C4-2 cells with 10 mM Enz for 6 days could increase both MAOA and ARv7 mRNA expressions (FIG. IE), suggesting that Enz may increase MAOA expression by activating key transcription factors, such as ARv7. Similar results were obtained when we treated EnzS5-VCaP cells with lOuM for 10 days (FIG. 7C). Results from MAO-GLO assay also confirmed that adding 10 mM Enz led to increased MAOA activity in EnzSl-C4-2 cells (FIG. IF).

[00115] Therefore, similar to an increase of ARv7 expression in cells treated with Enz, an increase in MAOA expression in Enz-treated cells may also play a role in mediating the Enz- resistance.

Elevated MAOA expression detected in circulating tumor cells (CTCs) from mCRPC patients treated with Enz

[00116] To further corroborate the in vitro cell lines data, we evaluated MAOA expression in a total of 288 blood-CTCssamples collected from men with mCRPC undergoing treatment with standard-of-care systemic therapies. These 288 blood samples were processed for CTCs and then cDNA andfmally were divided into three biomarker groups: 1. CTC negative (CTC-) (n=90); 2.

CTC positive (CTC+)/ARV7- (n=127); and 3. CTC+/ARv7+ (n=71). We found significantly increased MAOAmRNAin ARv7+ CTCs compared to ARv7- CTCs (FIG. 1G), suggesting a positive correlation between ARv7and MAOA expression.

[00117] Subseqeunt analysis focused on patients treated with Enz with paired samples collected at treatment baseline and at disease progression. Among the 288 samples, there were 30 pairs of samples (n=60) from those that were negative for ARv7 at baseline and remained negative at the time of progression on Enz (ARv7- to ARv7- group), 13 pairs of samples (n=26) from those with a conversion from ARv7- status to ARv7+ status (ARv7- to ARv7+ group), and 12 pairs of samples (n=24) from those who were positive for ARv7 at baseline and remained positive at the time of progression (ARv7+ to ARv7+ group). In those CTCs that remained

ARv7- after Enz treatment, MAOA expression showed little difference (FIG. 1H). In contrast, in those CRPC patients whose ARv7 expression changed from negative to positive after Enz treatment, the MAOA expression levels increased dramatically, suggesting that MAOA

expression may only increase in those ARv7+ patients (FIG. 1I-J). In CRPC patients with

ARv7+ at baseline and remained positive after Enz treatment, there was no further increase of MAOA expression after Enz treatment, MAOA expression trended higher after treatment with Enz, but the difference was not significant (FIG. IK). This is consistent with lack of further ARv7 increase after Enz treatment if ARv7 was detected at the baseline. (Antonarakis, et al.

2014 The New England journal of medicine 371, 1028-1038.) We did not find statistically significant differences for the full-length AR (AR-FL) comparing pre- and post-treatment samples in any of the paired groups (FIG. 8A-C), suggesting that elevated MAOA expression following treatment with Enz may be mediated by the ARv7.

[00118] Together, results from human CRPC patients’ CTC analysis (FIG. 1G-K)

demonstrate that MAOA expression is positively associated with ARv7 expression in CRPC patients treated with Enz. Together with our in vitro cell lines data (FIG. 1C-D), these human clinical findings suggest that Enz can increase both ARv7 and MAOA expression, and Enz- induced MAOA expression may depend on the ARv7 status.

Clorgyline or Phenelzine, the selective inhibitor of MAOA, can restore Enz-sensitivity to further suppress EnzR cell growth

[00119] To further link the increased MAOA expression to the development of Enz-resi stance in CRPC cells, we treated the CRPC cells with a selective MAOA inhibitor, Clorgyline, used as anti-depression drug. Results from MTT proliferation assay revealed that while Clorgyline treatment alone failed to suppress EnzSl-C4-2cell proliferation, the combination of Clorgyline and Enz treatment significantly inhibited (85%) EnzSl-C4-2cell growth at day 6 (FIG. 2A).

Importantly, EnzRl-C4-2 cell proliferation was also suppressed by adding Clorgyline to Enz treatment (49% suppression), suggesting restoration of Enz sensitivity (FIG. 2B). Similar results were obtained in EnzR2-C4-2 cells (56% suppression) (FIG. 2C) or EnzR3-CWR22RVl(43% suppression) cells (FIG. 2D).

[00120] We repeated the same set of experiments with the FDA-approved MAOA inhibitor, Phenelzine (lOuM) used to treat depression¹⁹, and results also revealed that suppressing the

MAOA activity could re-sensitize EnzRl-C4-2 (51% suppression), EnzR2-C4-2

(46%suppression) and EnzR3-CWR22RVl cells (48%suppression) to Enz treatment (FIG. 2E- G).

[00121] We then applied the gene expression perturbation approach using MAOA-shRNA to suppress MAOA, and results revealed that targeting MAOA with MAOA-shRNA also

significantly increased Enz sensitivity and suppressed EnzR cells proliferation (FIG. 2H-J), with suppression rates of 42% in EnzRl-C4-2, 35% in EnzR2-C4-2, and 28% in EnzR3- CWR22Rvlcells. In contrast, overexpressing MAOA in EnzSl-C4-2 cells resulted in Enz- resistance (FIG. 2K).

[00122] Together, results from FIG. 2A-K using multiple EnzR CRPC cells with various inhibitors/blockers to either suppress MAOA activity or suppress MAOA expression all demonstrate that Enz-induced MAOA expression contributed to the development of Enz- resistance, and suppressing the MAOA activity with anti-depression drugs Clorgyline or Phenelzine (or MAOA-shRNA to suppress its expression) all lead to restore Enz-sensitivity to further suppress EnzR cell proliferation.

Clorgyline delays the development of Enz resistance in CRPC cells

[00123] In addition to suppressing the EnzR cell growth, we are interested to see if Clorgyline can also delay the development of Enz-resi stance in CRPC cells. We first treated EnzSl-C4-2 cells withlO mM Enz alone or combined 10 mM Enz with 1 mM or 2.5 mM Clorgyline for 1.5 months. We then treated the cells with Enz and evaluated cell proliferation by MTT assays on days 0, 2, 4, and 6. The results revealed that whilel.5 months of Enz treatment alone could induce the development of Enz-resi stance by reducing Enz sensitivity from 54% to 24%. (FIG. 2L vs FIG. 2M), the combination of Enz and Clorgyline delayed the development of Enz- resistance by increasing Enz sensitivity from 54%®24% to 54%®33% (at ImM Clorgyline, FIG. 2M vs FIG. 2N) or from 54%®24% to 54%®43% (at 2.5mM Clorgyline, FIG. 2M vs FIG. 20)

[00124] Together, these results (FIG. 2L-0) suggest that targeting MAOA with the inhibitor Clorgyline can also delay the development of Enz-resistance.

Mechanism dissection of how Enz can induce MAOA expression: via increasing ARv7 expression

[00125] To dissect the molecular mechanism underlying Enz-induced MAOA expression, we focused on the ARv7. We first examined the effect of MAOA inhibition in AR-negative PC-3 cells, and found that adding Clorgyline alone or Clorgyline and Enz resulted in little effects on PC-3 cell proliferation (FIG. 3A) at 5 mM Clorgyline (FIG. 3B) and 10 mM Clorgyline, suggesting that Enz-increased MAOA expression and effect of MAOA inhibition is rather specific to AR-positive cells and may depend on AR (or ARv7) signals. As expected, adding Enz to EnzSl-C4-2 cells led to increase the expression of ARv7 and MAOA at both mRNA and protein levels (FIG. 3C), In addition, adding ARv7-cDNA (OE-ARv7) also increased MAOA mRNA expression in EnzRl-C4-2 cells (FIG. 3D, at mRNA level) and EnzSl-C4-2 cells (FIG. 3E, at both mRNA and protein levels), while suppressing ARv7 via adding ARv7-shRNA led to decrease MAOA mRNA level in multiple EnzR cells, including EnzRl-C4-2, EnzR2-C4-2 and EnzR3-CWR22Rvl cells (FIG. 3F-H, respectively), as well as a decrease in MAOA protein expression in the EnzRl-C4-2 and EnzR3-CWR22Rvl cells (FIG. 3F-H). To explore whether ARv7 induction by Enz is essential for the MAOA increase, we treated EnzSl-C4-2-pLKO and EnzSl-C4-2-shARv7 cells w/o Enz and then detected the ARv7 and MAOA level. As shown in FIG. 31, only in EnzSl-C4-2-pLKO cells, the ARv7 and MAOA level can be induced by Enz treatment, however, the ARv7 and MAOA expression cannot be induced by Enz in EnzSl-C4-2- shARv7 cells. To identify whether MAOA is the key downstream gene of ARv7 to confer EnzR, we manipulated ARv7 and MAOA expression in the EnzSl-C4-2 cells. As shown in the FIG.

3J, OE-ARv7 can decrease the Enz sensitivity, however, knockdown MAOA can reverse the ARv7 effects on Enz sensitivity, suggesting that ARv7 decrease Enz sensitivity dependent on MAOA.

[00126] Finally, to test whether cl orgyline can also suppress the expression of MAOA (and ARv7), in addition to suppress the MAOA activity, we found that adding clorgyline failed to impact significantly the expression of MAOA (and ARv7) in the EnzRl cells during 3 days treatment (FIG. 9B), suggesting that clorgyline’ s effect on the altering Enz sensitivity could be mainly from suppressing the MAOA activity.

[00127] Together, results from FIG. 3A-J suggest that Enz may function via increasing ARv7 to increase MAOA expression during the development of Enz-resistance.

Mechanism dissection of how Enz-increased ARv7 can increase the MAOA expression: via transcriptional resulation

[00128] Next, to dissect the mechanism of how Enz-increased ARv7 can increase the MAOA expression at the molecular level, we searched for the ARv7 response elements (AREs) on the 3kb MAOA promoter region, and found a putative ARE on the 5' promoter region (FIG. 4A). We first checked ARv7 binding on this ARE and ChIP assay showed strong binding signal of Flag-ARv7, but not endogenous ARfl (FIG. 4B). To better compare the binding ability of ARv7 and ARfl, we then overexpressed Flag-flAR and Flag-ARv7 in EnzRl-C4-2 cells (which were maintained in lOuM Enz) and used Flag antibody to pull-down flAR and ARv7, suggesting that ARv7 has much stronger activity to bind in vivo to this ARE on the MAOA promoter region as compared to flAR (FIG. 4C). Such result is consistent with the previous reports that in EnzR cells, flAR activity is suppressed significantly.

[00129] To confirm the results of ChIP assay, we checked AR and ARv7 binding on 5kb MAOA promoter region through ChIP-seq database (GEO: GSE106559). The results showed that in the presence of DHT, AR and ARv7 can bind to MAOA promoter in different areas (FIG. 5A). However, without DHT, AR lost its ability to bind to MAOA promoter, yet ARv7 still showed very strong binding signal (FIG. 11B). Interestingly, according to ChIP-seq data, although the strongest signal of ARv7 binding is on 4.5kb upstream of MAOA coding region, the ARv7 can still bind to the predicted ARE area in the absence of DHT. This data is consistent with our FIG. 4B-C showing ARv7 has stronger activity to bind to MAOA promoter.

[00130] Last, we also quantitated the enrichment of AR or ARv7 binding on MAOA promoter region. As shown in FIG. 11C, ARv7 binding in both conditions (w/o DHT) is stronger than AR.

[00131] Together, results from multiple approaches all confirmed that ARv7 has stronger binding ability than AR.

[00132] Results from luciferase assay via constructing the 3Kb MAOA promoter containing this ARE (or mutant ARE) into PGL3 reporter plasmid also confirmed that treating with Enz to increase ARv7 expression or direct adding ARv7-cDNA (OE-ARv7) could increase the MAOA expression at the transcriptional level with wild-type ARE in both EnzSl-C4-2 and PC3 cells (FIG. 4D-E), and not with mutant ARE (see sequences in FIG. 4 A) in the PC3 cells (FIG. 4F).

[00133] To further confirm ARv7, and not flAR, has better effects to increase MAOA expression, we overexpressed flAR and ARv7 in the EnzSl-C4-2 cells (cultured in 10%FBS RPMI which has 3nM DHT), and then assayed the MAOA promoter activity. As shown in FIG. 4G, only ARv7, and not flAR, could enhance MAOA promoter activity, suggesting that ARv7 had stronger ability to promote the MAOA transcription. [00134] To further confirm this data, we also assayed the MAOA expression in EnzSl-AR and EnzSl-ARv7 cells. As shown in FIG. 4H, both flAR and ARv7 can increase MAOA expression, however, ARv7 showed much better effect compared to flAR.

[00135] Together, results from multiple assays including qPCR, western blot, ChIP-on in vivo binding assay, and luciferase reporter assay (FIG. 4A-H) all demonstrate that Enz-increased ARv7 expression may lead to increase MAOA expression by transcriptional regulation via direct binding to the ARE on the MAOA 5' promoter region.

Mechanism dissection of how Enz-increased ARv7 expression can lead to increase the MAOA expression: via protein stability resulation

[00136] In addition to the transcriptional regulation, we also investigated whether Enz- increased ARv7 can lead to increase the MAOA expression via protein stability. By treating Enz- S1-C4-2 and Enz-Rl-C4-2 cells with the protein synthesis inhibitor cycloheximide (CHX), we found the MAOA protein degradation rate is much lower in EnzRl-C4-2 cells than that in the EnzSl-C4-2 cells (FIG. 41), suggesting that MAOA protein is more stable in EnzRl-C4-2 cells than in EnzSl-C4-2 cells. In contrast, we found that the MAOA protein expression in EnzSl-C4- 2 and EnzRl-C4-2 cells was similar after adding the proteasome inhibitor, MG132, suggesting that the degradation of MAOA protein by the proteasome system is suppressed in EnzRl-C4-2 cells (FIG. 4J).

[00137] To further dissect the mechanism why the MAOA protein stability is higher in Enz- R1-C4-2 cells, we compared the phosphorylation of MAOA in EnzSl-C4-2 and EnzRl-C4-2 cells, since early reports suggested that the phosphorylation could influence protein stability^{30 31}.

The results from immunoprecipitation of MAOA from EnzSl-C4-2 and EnzRl-C4-2 cells revealed that serine phosphorylation (p-Ser) significantly increased in EnzRl-C4-2 cells (FIG.

4K).

[00138] We also assayed the p38 effects since it might also phosphorylate MAOA to alter the MAOA protein stability, and results revealed that the phosphorylation of p38 (p-p38)

significantly increased in EnzRl-C4-2 cells (FIG. 4L), and adding the p38 inhibitor could only reduce MAOA expression and p-MAOA level in EnzRl-C4-2 cells but not in EnzSl-C4-2 cells and (FIG. 4M&N), suggesting that p38 activity was enhanced in EnzRl-C4-2 cells and indicating that MAO A was phosphorylated by higher p38 activity to increase the protein stability.

[00139] Taken together, results from FIG. 4I-N suggest that in addition to increase MAOA expression via transcriptional regulation, Enz can also increase MAOA expression via increasing its protein stability.

Mechanism dissection of how targeting the Enz/ ARv7 /MAOA signaling can increase the Enz- sensitivity to further suppress the EnzR cell growth

[00140] Next, to dissect the mechanisms of how targeting the Enz/ARv7/MAOA signaling can overcome the Enz-resi stance in multiple EnzR cells, we focused on hypoxia signals since recent studies indicated MAOA might exert its biological functions via altering the key hypoxia signals²⁴. We first examined the MAOA effects on the hypoxia signals with the HIF-1 and its downstream target genes including the Glutl, N-Cadherin, Timp and VEGF-A. The results revealed higher expression of these hypoxia downstream genes in EnzRl-C4-2 cells than those in EnzSl-C4-2 cells (FIG. 5A). Suppressing MAOA with MAOA-shRNA (FIG. 10A) also led to reduce the expression of HIF-1 and its target genes expression at mRNA (FIG. 5B, left) and the protein levels of HIF-1 and VEGF-A (FIG. 5B, right) in both EnzSl-C4-2 cells and

EnzRl-C4-2 cells. Suppressing HIF-Ia with HIF-1 -shRNA also reduced its target gene

VEGF-A expression at mRNA and protein levels in EnzRl-C4-2cells. (FIG. 4B).

[00141] In contrast, treating with MAOA inhibitor Clorgyline decreased these hypoxia-related genes at mRNA and protein levels in EnzRl-C4-2 cells (FIG. 5C).

[00142] Using an opposite approach via increasing MAOA via adding with MAOA-cDNA in EnzSl-C4-2 cells also resulted in increasing the target genes HIF-1 and VEGF-A expression at both mRNA and protein levels (FIG. 5D). More importantly, our results showed that when knocking down HIF-1 a led to increase the Enz-sensitivity in EnzRl-C4-2 cells (FIG. 5E), which suggested that MAOA can promote EnzR via activating HIFla signaling.

[00143] Together, results from FIG. 5A-E suggest that Enz/ARv7/MAOA axis can overcome the Enz-resi stance via altering the hypoxia signals in EnzRl-C4-2 and EnzSl-C4-2cells. Preclinical studies using in vivo mouse models to prove that targeting Enz/ARv7 /MAO A signaling with Clorgyline and Phenelzine led to further suppress EnzR tumor growth.

[00144] To establish a preclinical proof-of-principle in the in vivo mouse model, we first established the in vivo PDX-PCa mouse model in 6 mice, and results revealed that mice received Enz (30 mg/kg/every other day) had an increase in MAOA, ARv7 and p-p38 level in the

xenografted PDX tumors (FIG. 6A).

[00145] We then established the 2^nd in vivo mouse model by implanted EnzR3-22Rvl cells in the nude mice for the purpose of examining the effects of Clorgyline and Phenelzine on EnzR tumors. To make sure all data are repeatable and reach the statistical significance, we performed two sets of experiments, with implanted EnzR3-22RVl-Luc cells (cells which express luciferase plasmid) in the l^st-set and implanted EnzR3-22RVl cells in the 2^nd-set into nude mice. Once the tumor formation was detectable (after 4 weeks), the mice were treated in groups as follows, 1) vehicle, 2) Enz(10mg/kg), 3) clorgyline (lOmg/kg), 4) phenelzine(30mg/kg), 5) Enz + clorgyline, and 6) Enz + phenelzine, and i.p. injected every two days for 4 weeks. For EnzR3-22RV14uc cells, we used in vivo imaging system (IVIS) to monitor the tumor sizes weekly. After 4 weeks injections, we sacrificed the mice (4 mice/group in the l^st-set and 10 mice/group in the 2^nd-set) 2 days after the final treatment, and then measured the tumor weights.

[00146] The results revealed that adding Enz alone has little effect on the EnzR3-22Rvl (FIG.

6B) or EnzR3-22Rvl4uc (FIG. 12A-B) growth. In contrast, combining Enz (lOmg/kg every two days for 4 weeks) with Clorgyline (lOmg/kg every two days for 4 weeks) or Phenelzine

(30mg/kg every two days for 4 weeks) significantly suppressed EnzR3-22Rvl(FIG. 6B) or

EnzR3-22Rvl4uc (FIG. 12A-B) tumors growth. Tumor volumes and weight after sacrifice also confirmed that combining Enz and Clorgyline or Phenelzine can suppress EnzR3-22Rvl and EnzR3-22Rvl4uc tumors progression in mice compared with Enz alone and control vehicle group (FIG. 6C & FIG. 12C)

[00147] Finally, to further confirm the above two in vivo mouse models (PDX and EnzR3- 22Rvl), we also established the 3^rd in vivo mouse model with implanted another EnzR cells

(EnzRl-C4-2) cells into B-NDG mice and repeated the treatments on EnzR3-22Rvl tumors. As expected, we obtained the similar results showing Clorgyline and Phenelzine can also reverse the EnzRl-C4-2 resistance. (FIG. 6D-E) [00148] We also perform the IHC staining on EnzR3-22Rvl, EnzR3-22Rvl-luc and EnzRl- C4-2 tumors to assay the proliferation markers Ki-67 and VEGF-A, and results showed that in all three EnzR tumors, the expression of these proliferation proteins were significantly inhibited by the combination treatment (FIG. 6F-G & FIG. 12D).

[00149] Together, results from FIG. 6A-G & FIG. 12A-D using three preclinical in vivo mouse models all conclude that combining Enz with the MAOA inhibitors Clorgyline or

Phenelzine can overcome Enz-resi stance to further suppress the growth of EnzR tumors in the well-established EnzR in vivo models.

Experimental

Cell culture and reagents

[00150] CWR-22RV1, VCaP and PC-3 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 with 10% FBS.

HEK293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM with 10% FBS. The EnzSl-C4-2 cell line was a gift from Dr.

Leland W.K Chung from Cedars-Cinai. EnzS4-C4-2B and EnzR4-C4-2B cell lines were gifts from Dr Allen Gao from UC Davis. C4-2 EnzR cell lines were generated via chronic culture of CRPC C4-2 cells in media containing increasing Enz (from 10 mM to 30 mM), with the increased concentration added when cells were no longer sensitive (EnzRl_C4-2) or continuous culture with 10 mM Enz for 6 months (EnzR2_C4-2). All cells were maintained in a humidified 5% C0₂ environment at 37°C.A11 cell lines have been authenticated by ATCC and periodically re- authenticated by PCR and determined to be mycoplasma and bacteria free following ATCC's instructions. The cycloheximide, p38 map kinase inhibitor IV and clorgyine were purchased from Sigma-Aldrich (St. Louis, MO). Enzalutamide was purchased from

MedChemExpress(South Brunswick, New Jersey).

Cell Proliferation Assays

[00151] We plated lxl 0⁴ C4-2, PC3 and C4-2 EnzR cells into each well of 24-wellplates for MTT assays on days 0, 2, 4, 6 for the PCa cells and on days 0, 1,2,3 for the EnzSl_C4-2 and EnzRl_C4-2. After the various time points, the MTT assay was performed by adding 100 pi of 5 mg/ml MTT to each well. We included one set of wells with MTT, but no cells (control), then incubated for 3 hrs at 37 °C, removed media and added 150 mΐ DMSO, covered the plates with foil, agitated the cells on an orbital shaker for 15 min, and then read the absorbance at 570 nm.

Lentivirus packaging and cell transfection

[00152] The sh-ARv7 was constructed into the pLKO.1 lentiviral vector as reported previously. The pLKO. l shMAOA, together with package and envelope plasmids, psPAX2 and pMD2G, were co-transfected into 293T cells for 48hrs to produce the MAOA shRNA lentivirus particle soup, which was then collected and frozen at -80°C for later use in transduction of PCa cells.

RNA extraction and quantitative real-time PCR (qRT-PCR) analysis

[00153] Total RNAs were isolated using Trizol reagent (Invitrogen, Grand Island, NY). One pg of total RNA was subjected to reverse transcription using Superscript III transcriptase (Invitrogen). RT-PCR was conducted using a Bio-Rad CFX96 system with SYBR green to determine the mRNA expression level of a gene of interest. Expression levels were normalized to GAPDH level.

RNAseq

[00154] The EnzS_C4-2 and EnzRl_C4-2 cells were prepared for RNA-seq. For cDNA library construction and sequencing, mRNA was first isolated from total RNA treated with DNase I using Magnetic Oligo (dT) Beads and was fragmented. Then, the double-stranded cDNA was synthesized with random hexamer primers and was further subjected to end-repair and adapter ligation using T4 DNA ligase. The products of ligation reaction were purified on 2% agarose gel and cDNA fragments (about 200 bp) were recovered. PCR was carried out to enrich the purified cDNA template. Finally, the cDNA library was constructed. After validating on Quit and Bioanalyzer, the library was sequenced using Illumina HiSeq 2500 according to the manufacturer's instruction. Western blot analysis

[00155] Cells were lysed in RIPA buffer and proteins (20-40 pg) were separated on 8-10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking membranes, they were incubated with primary antibodies, then HRP-conjugated secondary antibodies, and visualized using ECL system (Thermo Fisher Scientific, Rochester, NY). The MAOA, GAPDH, tubulin, VEGF-A, Ki67and HIF-1 antibodies were from Santa Cruz Biotechnology, Inc (Santa Cruz). The ARv7 antibody was purchased from Precision Antibody (Columbia, MD). P-P38 antibody was purchased from Cell signaling Technology (Danvers, MA).

MAOA activity assay

[00156] MAO-Glo assay systems were purchased from Promega (Madison, WI). The cells were lysed by luciferase lysis buffer, and the cell lysates were applied to analyze the MAOA activity by the MAO-Glo kit.

Chromatin Immunoprecipitation Assay ( ChIP )

[00157] Cells were cross-linked with 4% formaldehyde for 10 minutes followed by cell collection and sonication with a predetermined power to yield genomic DNA fragments of 300- 1000 bp long. Lysates were precleared sequentially with normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and protein A-agarose. Anti-ARv7 antibody (2.0 pg) was added to the cell lysates and incubated at 4°C overnight. For the negative control, IgG was used in the reaction. Specific primer sets were designed to amplify a target sequence within human MAOA promoter and PCR products were analyzed by agarose gel electrophoresis.

Luciferase assay

[00158] PC3-Pwpi and PC3-oeARv7 cells were plated in 24-well plates and co-transfected with PGL3-MAOA-promotercontaining the WT-ARE or mutant ARE and pRL-TK, which is used as internal control using Lipofectamine (Invitrogen). After 48hrs transfection, . Luciferase activity was measured by Dual-Luciferase Assay (Promega, Madison, WI) according to the manufacturer’s manual. The EnzSl cells were transfected with PGL3-MAOA-promoter containing the WT-ARE and pRL-TK using Lipofectamine. After 12 hrs of transfection, the cells were treated with lOuM Enz for 1, 2 and 4 days and then the luciferase activity was measured.

In vivo mouse model PDX implantation and different compound treatments

[00159] The PCa-133 PDX samples are the gifts from Dr. Sankar N. Maity from MD

Andersen Cancer center, who generated it from CRPC patients as described before⁵⁰. The PCa- 133 samples were implanted into 6 SCID mice subcutaneously as described previously⁵¹. After average tumor’s volumes reached 200mm3, we i.p injected two mice with DMSO and 5 mice with Enz (30mg/kg) every other day. After 10 injections, the mice were sacrificed and the tumors were collected for WB. All the mice were purchased from NCI. All experiments were conducted after approval from University of Rochester medical center and follow the regulations of University Committee on Animal Resources (UCAR).

In vivo tumorigenesis assay

[00160] EnzR3-22Rvl, EnzR3-22Rvl-luc and EnzRl-C4-2 (lxlO⁶) were mixed with Matrigel (1 : 1) and injected into the prostates of 6- to 7-week old male nude mice (EnzR3-22Rvl-luc and EnzR3-22Rvl) or B-NDG mice (EnzRl-C4-2). Tumor-bearing mice were randomized into four groups and treated by i.p. every other day for 4 weeks as follows: 1) vehicle, 2) Enz (10 mg/kg), 3) clorgyline (10 mg/kg), 4) phenelzine (30 mg/kg), 5) Enz+clorgyline, and 6) Enz+phenelzine. For EnzR3-22Rvl-luc tumors, IVIS was used weekly to monitor tumor growth. We imaged the mice a final time 2 days after the final treatment, sacrificed the mice, and monitored tumor growth with the IVIS (EnzR3-22Rvl-luc) as well as tumor sizes and tumor weights.

Analysis of circulating tumor cells (CTC)

[00161] CTC samples used for MAOA expression analysis were excess“left-over” cDNA samples from an ongoing prospective blood-based CTC ARv7 study in men with metastatic CRPC. Patient enrollment, blood collection, processing, CTC isolation, cDNA prepartion, and ARv7 detection procedures were described previosuly (Antonarakis ES, Lu C, Luber B, et al. Clinical Significance of Androgen Receptor Splice Variant-7 mRNA Detection in Circulating Tumor Cells of Men With Metastatic Castration-Resistant Prostate Cancer Treated With First- and Second-Line Abiraterone and Enzalutamide. This study was approved by the Johns Hopkins University institutional review board, and patients provided written informed consent. Selection for left-over CTC samples for MAOA expression analysis involved a retrospective review of available cDNA samples prepared from CTCs and stored at -80°C. A total of 288 frozen cDNA samples were selected on the basis of availability of adequate cDNA for analysis, and processed for quantitative PCR analysis on CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). PCR cycles at 95°C x 5min, 40 cycles of 95°C x 10s, 58°C x 30s, and 72°C x 30s were followed by melting curve analysis. Primer sequences used for MAOA were 5'- AATTCAGCGGCTTCCAATGG-3'(forward) and 5'-CAAGTCGATCAGCTTTCCGG- 3'(reverse); Primer sequences used for RPL13A were 5'- CCTGGAGGAGAAGAGGAAAGAGA-3 ' (forward) and 5'-

TTGAGGACCTCTGTGTATTTGTCAA-3' (reverse) resistance. Patient treatment status and sample collection timepoints were unblinded after laboratory data was generated. Among the 288 cDNA samples, 90 were CTC negative (CTC-), 127 were CTC positive (CTC+) but ARv7 negative (ARv7-), and 71 were CTC+ and ARv7 positive (ARv7+). MAOA expression data was normalized to the control gene (RPL13A), and normalized data presnted for each biomaker group according to CTC and ARv7 status, as well as each pre- and post-treatment pairs.

Difference of MAOA copy numbers according to CTC and ARv7 status were compared using two-tailed unpaired t test. Changes of MAOA expression before and after Enz treatment were compared by two-tailed paired t test. P-values of 0.05 or less were considered statistically significant. All statistical tests were performed by GraphPad Prism version 7.02 (GraphPad software, San Diego, CA).

[00162] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference, unless the context clearly dictates otherwise.

[00163] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Methods recited herein may be carried out in any order that is logically possible, in addition to a particular order disclosed.

Incorporation by Reference

[00164] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure.

All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.

Equivalents

[00165] The representative examples disclosed herein are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. The examples herein contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

What is claimed is: CLAIMS

1. A pharmaceutical composition comprising:

a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, and

a second compound which is an inhibitor of monoamine oxidase A, wherein each of the first and second compounds is present in an amount such that the pharmaceutical composition is effective in the treatment of prostate cancer, or a related disease or condition thereof, in a mammal, including a human, and a pharmaceutically acceptable carrier.

2 The pharmaceutical composition of claim 1, wherein each of the first and second

compounds is present in an amount such that the pharmaceutical composition is effective in delaying the onset of drug resistance.

3 The pharmaceutical composition of claim 1, wherein each of the first and second

compounds is present in an amount such that the pharmaceutical composition is effective in delaying the development of drug resistance.

4 The pharmaceutical composition of claim 1, wherein each of the first and second

compounds is present in an amount such that the pharmaceutical composition is effective in reducing drug resistance.

5 The pharmaceutical composition of claim 1, wherein each of the first and second

compounds is present in an amount such that the pharmaceutical composition is effective in reversing drug resistance.

6 The pharmaceutical composition of any of claims 1-5, wherein the weight ratio of the first compound to the second compound is from about 10 : 1 to about 1 : 10.

7. The pharmaceutical composition of any of claims 1-6, being suitable for one or more of oral administration, intravenous, intramuscular, and subcutaneous administration.

8 The pharmaceutical composition of any of claims 1-7, wherein the second compound is clorgyline.

9. The pharmaceutical composition of any of claims 1-7, wherein the second compound is phenelzine.

10 A unit dosage form comprising the pharmaceutical composition of any of claims 1-9.

11 The unit dosage form of claim 10, being in the form of a tablet or capsule suitable for oral administration, or in the form of a liquid solution or suspension suitable for intravenous, intramuscular, or subcutaneous administration.

12 A method for treating prostate cancer, or a related disease or condition thereof,

comprising administering to a subject in need thereof the pharmaceutical composition of any of claims 1-11.

13. The method of claim 12, further comprising administering to the subject one or more other anti -cancer agents.

14. The method of claim 13, wherein the one or more other anti-cancer agents comprise a chemotherapeutic agent.

15. The method of any of claims 12-14, wherein the prostate cancer is castration resistant prostate cancer.

16. A method for treating prostate cancer, or a related disease or condition thereof,

comprising administering to a subject in need thereof a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, and a second compound which is an inhibitor of monoamine oxidase A, in amounts effective in the treatment of prostate cancer, or a related disease or condition thereof, in a mammal, including a human, wherein the subject has been detected of the development of drug resistance to the first compound.

17. The method of claim 16, wherein the second compound is administered simultaneously with the first compound.

18. The method of claim 16, wherein the second compound is administered after the

administration of the first compound.

19. The method of any of claims 16-18, wherein the first and/or the second compound is administered orally, intravenously, intramuscularly, or subcutaneously.

20 The method of any of claims 16-19, wherein the weight ratio of the first compound to the second compound is from about 10 : 1 to about 1 : 10.

21 The method of any of claims 16-20, wherein the second compound is cl orgyline.

22 The method of any of claims 16-20, wherein the second compound is phenelzine.

23. The method of any of claims 16-22, further comprising administering to the subject one or more other anti-cancer agents.

24. The method of claim 23, wherein the one or more other anti-cancer agents comprise a chemotherapeutic agent.

25. The method of any of claims 16-24, wherein the prostate cancer is castration resistant prostate cancer.

26. A method for treating prostate cancer, or a related disease or condition thereof,

comprising:

administering to a subject in need thereof a first compound of Formula (I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof, in an amount effective in the treatment of prostate cancer or a related disease or condition thereof in a mammal, including a human; monitoring the subject to detect a development of drug resistance to the first compound;

upon the subject being detected of the development of drug resistance to the first compound, administering to the subject a second compound which is an inhibitor of monoamine oxidase A, in an amount effect to reduce or eliminate drug resistance to the first compound; and

monitoring the subject to detect a level of drug resistance to the first compound.

27. The method of claim 26, further comprising:

upon the subject being detected of the development of drug resistance to the first compound, continuing to administer the subject the first compound.

28. The method of claim 26, further comprising:

upon the subject being detected of the development of drug resistance to the first compound, halting the administration of the first compound, and

upon the subject being detected of a substantial reduction or disappearance of drug resistance, re-starting the administration of the first compound.

29. The method of any of claims 26-28, wherein the first and/or the second compound is administered orally, intravenously, intramuscularly, or subcutaneously.

30. The method of any of claims 26-29, wherein the drug resistance is related to Arv7 and/or AR mutant and detecting a development of drug resistance to the first compound is by measuring ARv7.

31. The method of any of claims 26-30, wherein the prostate cancer is castration resistant prostate cancer.

32. The method of any of claims 26-31, wherein the second compound is cl orgyline or

phenelzine.

33. A method for treating a drug resistance in connection with a prostate cancer treatment, comprising administering to a subject in need thereof a compound which is an inhibitor of monoamine oxidase A, in an amount effective to prevent, delay, reduce or reverse resistance to a treatment of prostate cancer, or a related disease or condition thereof in a mammal, including a human.

34. The method of claim 33, wherein the drug resistance is related to Arv7 and/or AR

mutant.

35. The method of claim 33 or 34, wherein the resistance is to a compound having Formula

(I)

or a pharmaceutically acceptable salt, ester or pro-drug thereof.

36. The method of any of claims 33-35, wherein the inhibitor of monoamine oxidase A is clorgyline.

37. The method of any of claims 33-35, wherein the inhibitor of monoamine oxidase A is phenelzine.