STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This disclosure was made in part with government support under Grant No.

R01CA 105304 awarded by U.S. National Institutes of Health. The United States Government has certain rights in this disclosure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Patent Application No. 62/465,081, file February 28, 2017, U.S. Patent Application No. 62/504,243, filed May 10, 2017, U.S.

Application No. 62/581,336, filed November 3, 2017, and U.S. Patent Application No.

62/581,477, filed November 3, 2017, each of which is incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to therapeutic compounds and compositions thereof, as well as methods for the treatment of various indications, including cancer. In particular, the disclosed compounds and compositions thereof find utility in therapy applications for treatment of prostate cancers including, but not limited to, primary/localized prostate cancer (newly diagnosed), locally advanced prostate cancer, recurrent prostate cancer, metastatic prostate cancer, metastatic castration-resistant prostate cancer (CRPC), and hormone-sensitive prostate cancer. Further, the compounds and compositions of the present disclosure can be useful in neoadjuvant and adjuvant therapies for various androgen-mediated or androgen-related diseases or conditions as well as in combination with different therapies, such as androgen ablation therapy.

BACKGROUND

Androgens mediate their effects through the androgen receptor (AR). Androgens play a role in a wide range of developmental and physiological responses and are involved in male sexual differentiation, maintenance of spermatogenesis, and male gonadotropin regulation (R. K. Ross et al. Eur Urol 35, 355-361 (1999); A. A. Thomson, Reproduction 121 , 187-195 (2001); N. Tanji, K. Aoki & M. Yokoyama, Arch Androl 47, 1-7 (2001)). Several lines of evidence show that androgens are associated with the development of prostate carcinogenesis. Firstly, androgens induce prostatic carcinogenesis in rodent models (R. L. Noble, Cancer Res 37, 1929— 1933 (1977); R. L. Noble, Oncology 34, 138-141 (1977)) and men receiving androgens in the form of anabolic steroids have a higher incidence of prostate cancer (J. T. Roberts & D. M. Essenhigh, Lancet 2, 742 (1986); J. A. Jackson et al. Arch Intern Med 149, 2365-2366 (1989); P. D. Guinan et al. Am J Surg 131, 599-600 (1976)). Secondly, prostate cancer docs not develop if humans or dogs are castrated before puberty (J Clin Endocrinol Metab 84, 4324-4331 (1999); G. Wilding, Cancer Surv 14, 113-130 (1992)). Castration of adult males causes involution of the prostate and apoptosis of prostatic epithelium while eliciting no effect on other male external genitalia (E. M. Bruckheimer & N. Kyprianou, Cell Tissue Res 301, 153-162 (2000); J. T. Isaacs, Prostate 5, 545-557 (1984)). This dependency on androgens provides the underlying rationale for treating prostate cancer with chemical or surgical castration (androgen ablation).

Androgens also play a role in female diseases such as polycystic ovary syndrome as well as cancers. One example is ovarian cancer where elevated levels of androgens arc associated with an increased risk of developing ovarian cancer (K. J. Helzlsoucr et al. JAMA 274, 1926- 1930 (1995); R. J. Edmondson et al. Br J Cancer 86, 879-885 (2002)). The AR has been detected in a majority of ovarian cancers (H. A. Risen, J Natl Cancer Inst 90, 1774-1786 (1998); B. R. Rao & B. J. Slotman, Endocr Rev 12, 14-26 (1991); G. M. Clinton & W. Hua, Crit Rev Oncol Hematol 25, 1-9 (1997)), whereas estrogen receptor-alpha (ERa) and the progesterone receptor are detected in less than 50% of ovarian tumors.

The only effective treatment available for advanced prostate cancer is the withdrawal of androgens which are essential for the survival of prostate luminal cells. Androgen ablation therapy causes a temporary reduction in tumor burden concomitant with a decrease in serum prostate-specific antigen (PSA). Unfortunately, prostate cancer can eventually grow again in the absence of testicular androgens (castration-resistant disease) (Huber et al. 1987 Scand J. Urol Nephrol. 104, 33-39). Castration-resistant prostate cancer that is still driven by AR is biochemically characterized by a rising titre of serum PSA (Miller et al. 1992 J. Urol. 147, 956- 961). Once the disease becomes castration-resistant most patients succumb to their disease within two years. The AR has distinct functional domains that include die carboxy-terminal ligand-binding domain (LBD), a DNA-binding domain (DBD) comprising two zinc finger motifs, and an N- termraiis domain (NTD) that contains two transcriptional activation units (taul and tauS) within activation function- 1 (AF-1). Binding of androgen (ligand) to the LBD of the AR results in its activation such that die receptor can effectively bind to its specific DNA consensus site, termed the androgen response element (ARE), on the promoter and enhancer regions of androgen regulated genes, such as PSA, to initiate transcription. The AR can be activated in the absence of androgen by stimulation of the cAMP-dcpcndcnt protein kinase (PKA) pathway, with interleukin-6 (1L-6) and by various growth factors (Culig et al. 1994 Cancer Res. 54, 5474-5478; Nazareth et al 1996 J. Biol. Chem. 271, 19900-19907; Sadar 1999 J. Biol. Chem. 21 A, 7777- 7783; Ueda et al 2002 A J. Biol. Chem. 211, 7076-7085; and Ueda et al 2002 B J. Biol. Chem. 277, 38087-38094).

Clinically available inhibitors of the AR include nonsteroidal antiandrogens such as bicalutamide (Casodex™), nilutamide, flutamide, and enzalutamidc. There is also a class of steroidal antiandrogens, such as cyproterone acetate and spironolactone. Both steroidal and nonsteroidal antiandrogens target the LBD of the AR and predominantly fail presumably due to poor affinity, mutations mat lead to activation of the AR by these same antiandrogens (Taplin, M.E. et al. Cancer Res., 59, 2511-2515 (1999)), and constitutively active AR splice variants lacking the AR ligand-binding domain. Abiraterone and antiandrogens have no effect on constitutively active AR splice variants that lack the ligand-binding domain (LBD), which are associated with castration-recurrent prostate cancer (Dehm SM et al. Cancer Res 68, 5469-77, 2008; Guo Z et al. Cancer Res. 69, 2305-13, 2009; Hu et al 2009 Cancer Res. 69, 16-22; Sun ct al 2010 J Clin Invest. 2010 120, 2715-30; Antonarakis et al. N Engl J Med. 2014, 371, 1028-38; Scher et al. JAMA Oncol. 2016).

AR antagonists other than the bisphenol ether derivatives previously reported that bind to full-length AR and/or truncated AR splice variants that are currently being developed include: AR degraders such as niclosamide (Liu G et al. 2014), galcteronc (Njar et al. 2015; Yu Z at al 2014), and ARV- 330/ Androgen receptor PROTAC (Ncklesa et al. 2016 J Clin Oncol 34 suppl 2S; abstr 267); AR DBD inhibitor VPC-14449 (Dalai K et al. 2014 J Biol Chem. 289(38):26417- 29; Li H et al. 2014 J Med Chem. 57(15):6458-67); antiandrogens apalutamidc (Cicgg NJ et al. 2012), ODM-201 (Moilanen AM et al. 2015), ODM-204 (Kallio et al. J Clin Oncol 2016 vol. 34 no. 2_suppl 230), TAS368I (Minamiguchi et al 2015 J Clin Oncol 33, suppl 7; abstr 266); and ARNTD inhibitors 3E10-AR441bsAb (Goicochea NL et al 201S), and sintokamide (Sadar et al 2008; Banuelos et al 2016).

The AR-NTD is also a target for drug development (e.g. WO 2000/001813), since die NTD contains Activation-Function- 1 (AF-1) which is the essential region required for AR transcriptional activity (Jenster et al 1991. Mol Endocrinol. 5, 1396-404). The AR-NTD importandy plays a role in activation of the AR in the absence of androgens (Sadar, M.D. 1999 J. Biol. Chcm. 21 A, 7777-7783; Sadar MD et al. 1999 Endocr Rclat Cancer. 6, 487-502; Ucda et al. 2002 J. BioL Chem. 277, 7076-7085; Ueda 2002 J. Biol. Chem. 277, 38087-38094;

Blaszczyk et al. 2004 Clin Cancer Res. 10, 1860-69; Dehm et al 2006 J Biol Chem. 28, 27882- 93; Gregory et al. 2004 J Biol Chem. 219, 7119-30). The AR-NTD is important in hormonal progression of prostate cancer as shown by application of decoy molecules (Quayle et al. 2007, Proc Natl Acad Sci USA. 104,1331-1336).

While the crystal structure has been resolved for the AR C-terminal LBD, this has not been the case for the NTD due to its high flexibility and intrinsic disorder in solution (Reid et al. 2002 J. Biol. Chem. 277, 20079-20086) thereby hampering virtual docking drug discovery approaches.

SUMMARY

This disclosure is based in part on the fortuitous discovery that certain compositions described herein modulate androgen receptor (AR) activity. Specifically, the pharmaceutical compositions identified herein, show inhibition of AR N-Terminal Domain (NTD)

transactivation, which can be useful for blocking in vivo tumor growth in the presence and absence of androgens. The compositions described herein can be used for in vivo or in vitro research uses {i.e., non-clinical) to investigate the mechanisms of orphan and nuclear receptors (including steroid receptors such as the androgen receptor). Further, these compositions can be used individually or as part of a kit for in vivo or in vitro research to investigate signal transduction pathways and/or the activation of orphan and nuclear receptors using recombinant proteins, cells maintained in culture, and/or animal models. Alternatively, the compositions described herein can be combined with commercial packaging and/or instructions for use.

This disclosure is also based in part on the surprising discovery mat the compositions described herein can also be used to modulate the androgen receptor activity either in vivo or in vitro for both research and therapeutic uses. The pharmaceutical compositions can be used in an effective amount so that androgen receptor activity can be modulated. The androgen receptor can be mammalian. The androgen receptor can be human. In particular, the pharmaceutical compositions can be used to inhibit transactivau ^' on of the AR N-tcrmmal domain (NTD). The compounds' modulatory activity can be used in either an in vivo or an in vitro model for the study of at least one of the following indications: prostate cancer, breast cancer, ovarian cancer, endometrial cancer, hair loss, acne, hirsutism, ovarian cysts, polycystic ovary disease, precocious puberty, and agc-rclatcd macular degeneration. Furthermore, the compositions' modulatory activity can be used for the treatment of at least one of the following indications: prostate cancer, breast cancer, ovarian cancer, endometrial cancer, hair loss, acne, hirsutism, ovarian cysts, polycystic ovary disease, precocious puberty (tcstoxicosis), and age-related macular

degeneration.

In one aspect, described herein is a composition comprising a compound having the structure of one or more of Formulae (I), (II), (ΙΠ), (TV), (V), (VI), (VII), (VIII), (IX), or (X):

or a pharmaceutically acceptable salt thereof; and a second therapeutic agent. In certain embodiments, the compositions provided herein are pharmaceutical compositions.

In accordance with some embodiments, provided here are uses a composition as set out herein for modulating androgen receptor (AR) activity. Alternatively, the use can be for the preparation of a medicament for modulating androgen receptor (AR). Alternatively, the use can be for the treatment of or for the preparation of a medicament for the treatment of at least one indication selected from the group consisting of: prostate cancer, breast cancer, ovarian cancer, endometrial cancer, hair loss, acne, hirsutism, ovarian cysts, polycystic ovary disease, precocious puberty, and age-related macular degeneration. The indication can be prostate cancer. The prostate cancer can be androgen- independent prostate cancer. The prostate cancer can be androgen-dependent prostate cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

5 FIGS. 1 A-F show Pinl depletion impairs transcription mediated by AR.

FIGS. 2A-F show inhibition of Pinl by compounds blocks AR transcriptional activity. FIGS. 3A-I show Pinl interacts with the AR NTD and regulates transactivation.

FIGS. 4A-F show ATRA and other Pinl inhibitors suppress prostate cancer cell proliferation.

0 FIGS. 5A-G show ATRA synergizes with AR NTD antagonism by EPI-002.

FIGS.6A and 6B summarize select Pinl inhibitors.

FIGS. 7A and 7B are bar graphs showing synergy between juglone and EPI-002 on inhibiting transactivation of GaW-ARN by (FIG. 7A) IL-6 and (FIG. 7B) FSK.

FIGS. 8A-8D show the effects of sequential EPI and ATRA combinations on the cellS cycle of LN95 cells.

FIGS. 9A and 9B show Pinl siRNA inhibit AR transcriptional activity.

FIGS. iOA and 10B show Pinl inhibitors effectively block AR transcriptional activity.

FIGS. 11A and 1 IB show Pin 1 inhibition blocks transactivation of the AR N-tcrmmal domain by the IL-6/STAT3 pathway.

0 FIGS. 12A-C show Pinl predominately interacts with the region containing the amino acids 234 -391 of the AR N-Terminal Domain.

FIGS. I3A-C show Pinl inhibitors arc effective in blocking the growth of LNCaP95 cells, which exhibit androgen-independent growth.

FIGS. 14A and 14B show ATRA synergizes with EPI-002 to induce Gl arrest.5 FIG. IS shows EPI-002 and Wecl inhibitor combination therapy inhibits cell growth of

LN95 cells in vitro.

FIGS. 16A and I6B shows EPI-002 and Wee I inhibitor combination therapy induces DNA damage and cell cycle arrest.

FIG. 17 shows EPI-002 is an inhibitor of the transcriptional activity of the AR.0 FIG. 18A-D show inhibition of AR transcriptional activity by EPI-002 is enhanced by N- acetylcysteine (NAC). FIG. 19 shows NAC optimizes EPI-002 inhibition of androgen-induced PSA protein levels.

FIG. 20 shows NAC alters protein levels of NRF2.

DETAILED DESCRIPTION

This disclosure relates to therapeutics, their uses and methods for the treatment of various indications, including various cancers. In particular, the disclosure relates to therapies and methods of treatment for cancers, such as prostate cancer, including all stages and androgen dependent, andro gcn-sensitivc and androgen-indcpcndent (also referred to as hormone refractory, castration resistant, androgen deprivation resistant, androgen ablation resistant, androgen depletion-independent, castration-recurrent, and anti-androgen-recurrent) prostate cancer.

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.

The methods and techniques of die present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g., "Principles of Neural Science", McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, "taturtive Biostatistics", Oxford University Press, Inc. (1995); Lodish et al., "Molecular Cell Biology, 4th ed", W. H. Freeman & Co., New York (2000); Griffiths et al., "Introduction to Genetic Analysis, 7th ed", W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., "Developmental Biology, 6th ed.", Sinauer Associates, Inc., Sunderland, MA (2000). Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by "The McGraw-Hill Dictionary of Chemical Terms", Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

AH of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

A "patient," "subject," or "individual" are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bo vines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

"Treating" a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. As used herein, and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment

The term "preventing" is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount

"Administering" or "administration of a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutancously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdcrmally (by absoφtion _t e.g., through a skin duct). A composition can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the composition. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended -periods.

Appropriate methods of administering a substance, in particular a composition of the disclosure as disclosed herein, to a subject will also depend, for example, on the age and/or the physical condition of the subject and the chemical and biological properties of the composition (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a substance, in particular a composition of the disclosure as disclosed herein, is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

As used herein, the phrase "conjoint administration" refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which can include synergistic effects of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

The term "modulate" as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.

The phrase "pharmaceutically acceptable" is art-rccognized. Tn certain embodiments, the term includes compositions, excipicnts, adjuvants, polymers and other materials and/or dosage forms which arc, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. "Pharmaceutically acceptable salt" or "salt" is used herein to refer to an acid addition salt or a basic addition salt that is suitable for or compatible with the treatment of patients.

The term "pharmaceutically acceptable acid addition salt" as used herein means any nontoxic organic or inorganic salt of any base compounds disclosed herein. Illustrative inorganic acids that form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycohc, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, malek, benzoic, phenylacetic, cmnamic and salicylic acids, as well as sulfonic acids such as p-toluenc sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts can exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds disclosed herein are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, eg., oxalates, can be used, for example, in the isolation of compounds of the disclosure for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

The term "pharmaceutically acceptable basic addition salt" as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds of the disclosure, or any of their intermediates. Illustrative inorganic bases that form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as mcthylamine, trimethylamme and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.

Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center can be present in an R or an S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stcreoisomcric forms such as enantiomeric and diastereoisomeric forms of die compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g. , WO 01 /062726. Furthermore, certain compounds which contain alkenyl groups can exist as Z

(zusammen) or E (entgegen) isomers. In each instance, the disclosure includes both mixtures and separate individual isomers.

Some of the compounds can also exist in tautomeric forms. Such forms, although not explicitly indicated in the formulae described herein, are intended to be included within the scope of the present disclosure.

"Prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized, for example hydrolyzcd or oxidized, in the host after administration to form the compound of the present disclosure (e.g., compounds of Formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof, the second therapeutic agent). Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, animated, dcaminatcd, hydroxylated, dehydroxylated, hydrolyzcd, dehydroh/zed, alkylated, dealkylatcd, acylated, deacylated, phosphorylatcd, or dephosphorylated to produce the active compound. Examples of prodrugs using ester or phosphoramidate as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of that are incorporated herein by reference. The prodrugs of this disclosure are metabolized to produce a compound, or a pharmaceutically acceptable salt thereof. The present disclosure includes within its scope, prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs" Ed. H. Bundgaard, Elsevier, 1985.

The phrase "pharmaceutically acceptable carrier" as used herein means a

pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.

Compositions of the Disclosure

The compounds of the present disclosure can be conjointly administered either simultaneously with, or before or after, one or more other therapeutic agents. The compositions of the present disclosure can be administered separately, by the same or different route of administration, or together in the same composition as the other agents. Specifically, provided herein are compositions comprising a compound having the structure of one or more of Formulae

or a pharmaceutically acceptable salt thereof; and a second therapeutic agent.

In some embodiments the second therapeutic agent is an inhibitor of Pin 1. In some such embodiments, the inhibitor of Pin 1 is selected from juglone; plumbagin; PiB, epigallocatechin gallatc (EGCG); all-trans retinoic acid (ATRA), dipentamethylene thiauram monosulfide, and TME-00I, or a pharmaceutically acceptable salt thereof. In some embodiments, the inhibitor of Pin I is an analogue of retinoic acid. In yet other embodiments, the inhibitor of Pin I is selected from:

or a pharmaceutically acceptable salt thereof. Tn yet other embodiments, the inhibitor of Pin 1 is a peptide. In some such embodiments, the peptide is selected from: CRYPEVE1C, wherein the cysteine residues of said peptide are cyclized by a disulfide bond (SEQ ID NO: 1); Ac-Lys(N- biotinoyl>Ah-AU-Bth-I>Thr(P03H2)-Pip-Nal-Gln-NH2 (SEQ ID NO: 2); Ac-Phe-D- Thr(P03H2>Pip-Nal-Gln-NH2 (SEQ ID NO: 3); and Ac-Phe-Phe-pSer-T[(Z)CHdC-Pro-Arg- N¾ (SEQ ID NO: 4).

In other embodiments, the second therapeutic agent is an antiandrogen. In some such embodiments, the antiandrogen is selected from bicahitamidc, fhitamidc, nilutamidc, apahitamide, darolutamide, enzalutamide, proxahitamide, topilutamide, cimetidine,

chlormadinone acetate, cyproterone acetate, megcstrol acetate, osaterone acetate, nomegcstrol acetate, dienogest, oxcndolone, drospirenone, spironolactone, and medrogestone, or a pharmaceutically acceptable salt thereof.

In other embodiments, the second therapeutic agent is a PARP inhibitor. In some such embodiments, the PARP inhibitor is selected from olaparib, rucaparib, niraparib, iniparib, talazoparib, veliparib, CEP9722, E7016, BGB-290, and 3-aminobenzamide, or a

pharmaceutically acceptable salt thereof.

In yet other embodiments, the second therapeutic agent is a Wcc-1 inhibitor. In some such embodiments, the Wee-I inhibitor is MK1775, or a pharmaceutically acceptable salt thereof.

In still other embodiments, the second therapeutic agent is N-acetyl cysteine (NAC).

In other embodiments, the second therapeutic agent is a derivative of N-acetyl cysteine (see, e.g., Lhi et al. Med Chem Comm, 2017, 8, 2238-2247; Hickman ct al. Aus. J. Chem. 1985, 38 (6), 899-904; U.S. Patent No. 5,296,500).

According to some embodiments, prodrugs of the compounds as described herein are also provided. Those of ordinary skill in the art will appreciate that prodrugs are compounds which are converted to the compounds as described herein or salts thereof under specified conditions. Specified conditions can include, for example, and without limitation, in vivo enzymatic or non- enzymatic means. Conversion of the prodrug can occur, for example, and without limitation, spontaneously, or it can be catalyzed, induced by another agent, or a change in a physical parameter or environmental parameter, for example, an enzyme, light, acid, temperature or pH. In some embodiments, the prodrug can have little or no pharmacological activity themselves, and then when converted into the compounds as described herein have the desired activity. Prodrugs can be prepared, for example, and without limitation, by converting appropriate functional groups (for example, a carboxylic acid functional group -COOH, an alcohol functional group - OH, or primary or secondary amine functional group) in the compounds as described herein with suitable moieties. Suitable moieties would be understood to and can be determined by those of ordinary skill in the art. For example, and without limitation, a prodrug can be formed by converting a primary or secondary amino functionality to an amide functionality. For example, and without limitation, a prodrug can be formed by converting a carboxylic acid functionality to an ester functionality, or converting an alcohol functionality to an ether functionality. A prodrug moiety can be, for example, and without limitation, a protecting group that acts to mask a functional group, a group that acts as a substrate for one or more active or passive transport mechanism, or a group that acts to impart or enhance a property of the compound, for example, solubility, bioavailability or localization. In some embodiments, the compounds as described herein or salts thereof can themselves be prodrugs of other compounds as described herein.

Compounds as described herein can be in the free form or in the form of a salt thereof. In some embodiments, compounds as described herein can be in the form of a pharmaceutically acceptable salt, which are known in the art (Bergc et al., J. Pharm. Sci. 1977, 66, 1).

Pharmaceutically acceptable salt as used herein includes, for example, salts that have the desired pharmacological activity of the parent compound (salts which retain the biological effectiveness and/or properties of the parent compound and which are not biologically and/or otherwise undesirable). Compounds as described herein having one or more functional groups capable of forming a salt can be, for example, formed as a pharmaceutically acceptable salt. Compounds containing one or more basic functional groups can be capable of forming a pharmaceutically acceptable salt with, for example, a pharmaceutically acceptable organic or inorganic acid. Pharmaceutically acceptable salts can be derived from, for example, and without limitation, acetic acid, adipic acid, alginic acid, aspartic acid, ascorbic acid, benzoic acid, benzenesulfonic acid, butyric acid, cinnamic acid, citric acid, camphoric acid, camphorsulfbnic acid, cyclopentancpropionic acid, dicthylacctic acid, digluconic acid, dodccylsulfonic acid, ethanesulfonic acid, formic acid, fiimaric acid, glucoheptanoic acid, gluconic acid,

glycerophosphoric acid, glycolic acid, hcmisulfonic acid, heptanoic acid, hcxanoic acid, hydrochloric acid, hydrobromic acid, hydriodic acid, 2-hydroxyethanesulfonic acid, isonicotinic acid, lactic acid, malic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid, 2- naphthalcncsulfonic acid, naphthalcncdisulphonic acid, p-tolucnesulfonic acid, nicotinic acid, nitric acid, oxalic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, phosphoric acid, picric acid, pimelic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, succinic acid, sulfuric acid, sulfamic acid, tartaric acid, thiocyanic acid or undecanoic acid. Compounds containing one or more acidic functional groups can be capable of forming pharmaceutically acceptable salts with a pharmaceutically acceptable base, for example, and without limitation, inorganic bases based on alkaline metals or alkaline earth metals or organic bases such as primary amine compounds, secondary amine compounds, tertiary amine compounds, quaternary amine compounds, substituted amines, naturally occurring substituted amines, cyclic amines or basic ion-exchange resins. Pharmaceutically acceptable salts can be derived from, for example, and without limitation, a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation such as ammonium, sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese or aluminum, ammonia, benzathine, meglumine, mcthylamine,

dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, ^propylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2- diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidinc, caffeine, hydrabamine, choline, betaine, cmylcmechaminc, glucosamine, ghicamine, memylgmcaminc, theobromine, purines, piperazine, pipcridmc, procaine, N-ethylpipcridinc, theobromine, tctramethylammonium compounds, tetraethyl ammonium compounds, pyridine, N,N-dimethylaniIine, N- methylpiperidme, morpholine, N- methylmorpholine, N-ethylmorpholine, dicyclohexylamine, dibenzylamine, N5N- dibenzylphenethylamine, 1-ephenamine, N.hT-dibenzylcthylenediamine or poryamine resins. In some embodiments, compounds as described herein can contain both acidic and basic groups and can be in the form of inner salts or zwhterions, for example, and without limitation, betames. Salts as described herein can be prepared by conventional processes known to a person skilled in the art, for example, and without limitation, by reacting the free form with an organic acid, an inorganic acid, an organic base or an inorganic base, or by anion exchange or cation exchange from other salts. Those skilled in the art will appreciate that preparation of salts can occur in situ during isolation and/or purification of the compounds or preparation of salts can occur by separately reacting an isolated and/or purified compound. In some embodiments, compounds and all different forms thereof (.e.g., free forms, salts, polymorphs, isomeric forms) as described herein can be in the solvent addition form, for example, solvates. Solvates contain either stoichiometric or non-stoichiomctric amounts of a solvent in physical association with a compound or salt thereof. The solvent can be, for example, and without limitation, a pharmaceutically acceptable solvent. For example, hydrates are formed when the solvent is water or alcoholates are formed when the solvent is an alcohol.

In some embodiments, compounds and all different forms thereof (e.g., free forms, salts, solvates, isomeric forms) as described herein can include crystalline and/or amorphous forms, for example, polymorphs, pseudopolymorphs, conformational polymorphs, amorphous forms, or a combination thereof. Polymorphs include different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability and/or solubility. Those skilled m the art will appreciate that various factors including recrystallization solvent, rate of crystallization and storage temperature can cause a single crystal form to dominate.

In some embodiments, compounds and all different forms thereof (e.g., free forms, salts, solvates, polymorphs) as described herein include isomers such as geometrical isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, individual cnantiomcrs, individual diastereomers, raccmates, diastereomeric mixtures and combinations thereof, and arc not limited by the description of the formula illustrated for the sake of convenience.

Pharmaceutical Compositions and Administration Thereof

The compositions and methods disclosed herein can be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non- human mammal. When administered to an animal, such as a human, the composition is preferably administered as a pharmaceutical composition comprising, for example, at least one of a compound of Formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically salt thereof; a second therapeutic agent; and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In certain embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for rcconstitution, powder, solution, syrup, suppository, injection, or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as an ointment or cream.

A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the disclosure. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation of pharmaceutical composition can be a self-emulsifying drug delivery system or a sclf- microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the disclosure. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that arc relatively simple to make and administer.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a

pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. 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: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflowcr oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (1 1) polyols, such as glycerin, sorbitol, maimitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (IS) alginic acid; (16) pyrogen-free water, (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue);

absorption through the oral mucosa (e.g., sublingually); anally, rcctalry or vaginally (for example, as a pessary, cream or foam); parentcrally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally;

intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The compositions can also be formulated for inhalation. In certain embodiments, the composition can be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6, 1 10,973,

5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the disclosure, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations arc prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the disclosure suitable for oral administration can be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water- in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient

Compositions or compounds can also be administered as a bolus, electuary, or paste.

To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stcarate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type can also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent

The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They can also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They can be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents and can be of a composition that they release the active ingredients) only, or

preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylenc glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tctrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfiiming and preservative agents.

Suspensions, in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostcaryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonitc, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions for rectal, vaginal, or urethral administration can be presented as a suppository, which can be prepared by mixing one or more active compounds with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations of the pharmaceutical compositions for administration to the mouth can be presented as a mouthwash, or an oral spray, or an oral ointment

Alternatively or additionally, compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices can be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellents that can be required. The ointments, pastes, creams and gels can contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and poh/amidc powder, or mixtures of these substances. Sprays can additionally contain customary propellents, such as chlorofluorohydrocarbons and volatile unsubstitutcd hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present disclosure to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the composition across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the composition in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this disclosure. Exemplary ophthalmic formulations are described in U.S. Publication Nos. 2005/0080056, 2005/0059744, 2005/0031697, and 2005/004074; and U.S. Patent No. 6,583,124, the contents of which arc incorporated herein by reference. If desired, liquid ophthalmic formulations have properties similar to that of lacrimal fluids, aqueous humor or vitreous humor or are compatible with such fluids.

The phrases "parenteral administration" and "administered parenteralfy" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which can be reconstituted into sterile injectable solutions or dispersions just prior to use, which can contain antioxidants, buffets, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that can be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug men depends upon its rate of dissolution, which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microcmulsions that are compatible with body tissue.

For use in the methods of this disclosure, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1% to about 99.5% (more preferably, about 0.5% to about 90.0%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Kits

In some embodiments, the disclosure provides a kit comprising one or more separate compositions, preferably pharmaceutical composition, at least one of which contains a compound of Formulae or a

pharmaceutically acceptable salt thereof; and a second therapeutic agent. In some such embodiments, the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules, and the like.

The kit of the disclosure can be used for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit of the disclosure typically comprises directions for administration. In the combination therapies of the disclosure, the compound of Formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof, and a second therapeutic agent can be manufactured and/or formulated by the same or different manufacturers.

Moreover, the compound of Formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X) and the second therapeutic agent can be brought together into the composition of the disclosure via a combination therapy: (i) prior to release of the combination product to physicians (e.g., in the case of a kit comprising the compound of Formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X) and the second therapeutic agent); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (in) in the patient themselves, e.g., during sequential administration of the compound of Formulae (0, (Π), (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), and the second therapeutic agent.

Methods of Use

The compositions of the disclosure can be used for treatment of at least one indication selected from die group consisting of: prostate cancer, breast cancer, ovarian cancer, endometrial cancer, hair loss, acne, hirsutism, ovarian cysts, polycystic ovary disease, precocious puberty and age-related macular degeneration. In certain embodiments, the compositions of the disclosure can be used for treatment of prostate cancer. In some such embodiments, the prostate cancer is selected from primary/localized prostate cancer, locally advanced prostate cancer, recurrent prostate cancer, metastatic prostate cancer, advanced prostate cancer, castration-resistant prostate cancer (CRPC), and hormone-sensitive prostate cancer. In certain embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC), in particular metastatic CRPC.

In some embodiments, the compositions of the disclosure can be used for preparation of a medicament lor treatment of at least one indication selected from the group consisting of:

prostate cancer, breast cancer, ovarian cancer, endometrial cancer, hair loss, acne, hirsutism, ovarian cysts, polycystic ovary disease, precocious puberty and age-related macular

degeneration. In certain embodiments, the compositions of the disclosure can be used for the preparation of a medicament for treatment of prostate cancer. In some such embodiments, the prostate cancer is selected from primary/localized prostate cancer, locally advanced prostate cancer, recurrent prostate cancer, metastatic prostate cancer, advanced prostate cancer, castration-resistant prostate cancer (CRPC), and hormone-sensitive prostate cancer. In certain embodiments, the prostate cancer is castration-resistant prostate cancer (CRPC), in particular metastatic CRPC.

In some embodiments, the compositions of the disclosure can also be used, in assays and for research purposes. Ligand-independent activation of the AR refers to transactivation of the AR m the absence of androgen (ligand) by, for example, stimulation of the cAMP-dependent protein kinase (PKA) pathway with forskolin (FSK). Some compounds and compositions of this disclosure can inhibit both FSK and androgen (e.g., R1881) induction of ARE-luciferasc (ARE- hic). Such compounds can block a mechanism that is common to both ligand-dependent and ligand-independent activation of the AR. This could involve any step in activation of the AR including dissociation of heat shock proteins, essential posttranslational modifications (e.g., acetylation, phosphorylation), nuclear translocation, protein-protein interactions, formation of the transcriptional complex, release of co-rcprcssors, and/or increased degradation. Some compounds and compositions of this disclosure can inhibit Rl 881 only and can interfere with a mechanism specific to ligand-dependent activation (e.g., accessibility of the ligand binding domain (LBD) to androgen). Numerous disorders in addition to prostate cancer involve the androgen axis (e.g., acne, hirsutism, alopecia, benign prostatic hyperplasia) and compositions interfering with this mechanism can be used to treat such conditions. Some compositions of this disclosure can only inhibit FSK induction and can be specific inhibitors to ligand-tndependent activation of the AR. Such compositions can interfere with the cascade of events that normally occur with FSK and/or PKA activity or any downstream effects that can play a role on the AR (e.g., FSK increases MAPK activity that has a potent effect on AR activity). Examples can include an inhibitor of cAMP and or PKA or other kinases. Compositions of this disclosure can induce basal levels of activity of the AR (no androgen or stimulation of the PKA pathway). Some compositions of this disclosure can increase induction by R 1881 or FSK. Such compounds and compositions can stimulate transcription or transactivation of the AR. Some compounds and compositions of this disclosure can inhibit activity of the androgen receptor N-terminal domain (AR-NTD).

Intcrleukin-6 (1L-6) also causes ligand-independent activation of the AR in LNCaP cells and can be used in addition to FSK. In some embodiments, compositions of this disclosure can interact with the AR-NTD or with another protein required for transactivation of the AR-NTD.

In some embodiments, the compositions of the disclosure can be used, for example, and without limitation, in combination with other treatment methods for at least one indication selected from the group consisting of: prostate cancer, breast cancer, ovarian cancer, endometrial cancer, hair loss, acne, hirsutism, ovarian cysts, polycystic ovary disease, precocious puberty and age-related macular degeneration. For example, compounds and all their different forms as described herein can be used as neoadjuvant (prior), adjunctive (during), and/or adjuvant (after) therapy with surgery, radiation (brachythcrapy or external beam), or other therapies (e.g., HIFU).

In general, compounds of the disclosure should be used without causing substantial toxicity. Toxicity of the compositions of the disclosure can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LDso (the dose lethal to 50% of the population) and the LDioo (the dose lethal to 100% of the population). In some circumstances, however, such as in severe disease conditions, it can be necessary to administer substantial excesses of the compositions. Some compounds of mis disclosure can be toxic at some concentrations. Titration studies can be used to determine toxic and non-toxic concentrations. Toxicity can be evaluated by examining a particular compound's or composition's specificity across cell lines using PC3 cells as a negative control that do not express AR.

Animal studies can be used to provide an indication if the composition has any effects on other tissues. Systemic therapy that targets the AR will not likely cause major problems to other tissues since an ti androgens and androgen in sensitivity syndrome are not fatal.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. EXAMPLES

General Methods

Cell culture. LNCaP cells were maintained in RPMI-1640 supplemented with 5% FBS. LN95 cells were provided by the University of Washington, and maintained in RPMI-1640 media containing 10% charcoal-stripped serum (CSS). PC-3 and Dill 45 cells were cultured in DMEM (Invitrogen) supplemented with 5% and 10% FBS respectively, and 2 mM L-glutamine, and 1 mM sodium pyruvate. VCaP cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% FBS, 2 mM L-glutammc, and 1 mM sodium pyruvate. All cell lines were cultured at 37 °C in a humidified incubator containing 5% CO2.

Compounds and reagents. ATRA, 13cRA, Juglone, and EGCG were purchased from Sigma-Aldrich. EPI-002 was provided by NAEJA. Enzalutamide was purchased from OmegaChcm, and apalutamide was from Selleck Chemicals. R1881 was purchased from Perkin- Elmer, IL-6 from R&D Systems, and forskolin from EMD Milfipore. All mutations were carried out by site-directed mutagenesis using the QuikChange II kit from Agilent. Antibodies against various proteins were from the following sources, mouse monoclonal antibodies: Pin I (8C10), Cyclin Dl (DCS-6) from Santa Cruz Biotechnology; STAT3 (124H6), Cyclin A2 (BF683) from Cell Signaling Technology; Lamin A/C (612162) from BD Biosciences; GAPDH (6CS) from Irrvttrogen; β-actin (A5441) from Sigma-Aldrich; rabbit monoclonal: Cyclin Bl (D5C10), p21 Wafl/Cipl (12D1), p27 Kipl (D69C12) from Cell Signaling Technology; rabbit polyclonal: Androgen Receptor (N-20) from Santa Cruz Biotechnology; phospho-Androgen Receptor (Ser81) from EMD Millipore; phospho-STAT3 (Tyr705; D3A7), p44/42 MAPK (9102), phospho-p44/42 MAPK (Thr202/Tyr204) from Cell Signaling Technology; goat polyclonal: PSA (C-19) from Santa Cruz Biotechnology.

Transfections and luciferase assay. Plasmids and transfection procedures have been previously described by Ocd&et al. J Biol Chem 277, 7076-7083 (2002); Andersen et al Cancer Cell 17, 53S-S46 (2010). Transfections with siRNA were carried out using lipofectamine RNAiMAX in Opti-MEM media and 10 nM of Pin 1 -targeting (si 0544, si 0546) or scramble control siRNAs (Invitrogcn). Transfections for reporter gene assays were performed in serum- free media using Lipofectin (invitrogcn) and FugeneHD (Promega) reagents. Luciferase activities were measured by Dual Luciferase assay system (Promega) and then normalized to total protein concentrations determined by the Bradford method.

Extraction of nuclear proteins. After incubation with compounds, cells were washed in PBS before being resuspensed in a hypotonic lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM KC1, 3 mM MgCL¾ 0.5% NP-40), with freshly added protease and phosphatase inhibitors (Roche), and then incubated on ice for 15 min. The crude nuclei were isolated by centrifugation at 425g for 10 min, washed once, and resuspended in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS) with protease and phosphatase inhibitors. Nuclear extracts were homogenized by sheering with an insulin syringe and cleared by centrifugation. Protein concentrations were quantified by BCA assay and 20 μg of total protein per sample were analyzed by SDS-PAGE.

Immunoprecipitation and imrmmoblotting. Irnmunoprecipftation for STAT3 complexes have been previously described by Banuelos et al. "Sintokamide A Is a Novel Antagonist of Androgen Receptor That Uniquely Binds Activation Function- 1 in Its Amino- tcrminal Domain." J Biol Chcm 291, 22231-22243 (2016). For his-tag pull-down assay, LNCaP cells were transfected with 1 μg of Pinl plasmid and 8-11 μg of expression plasmid encoding his-taggcd ARH(1-558), AR-(l-233), AR-(234-391), or AR-(392-558). The following day, cells were treated with compounds and stimulated with IL-6 (SO ng/ml) for 6 h. When harvesting, cell pellets were washed in PBS, flash-frozen in liquid nitrogen, and stored at -80°C. To extract proteins, thawed cell pellets were resuspended in 1 ml of lysis buffer (50 mM HEPES, 150 mM NaCL 10 mM imidazole, and 0.5% Triton X-100). The lysatcs were passed several times through an insulin syringe and then cleared by centrifugation. Lysates were incubated with 200 μΙ of Ni- NTA agarose beads for 1 h at 4°C, washed twice with HEPES/NaCl buffer (pH 8.0) containing 60 mM imidazole, and chxted with HEPES/NaCl buffer (pH 8.0) containing 300 mM imidazole. Eluted samples were resolved on 12.5% SDS-PAGE along with input samples (10% of starting cell lysate material) and analyzed by immunoblotting for Pin 1 (8C10) and polyhisndine (HIS-1). For immunoblotting, antibodies were diluted in 5% non-fat milk (w/v) in Tris-buffered saline (pH 7.4) containing 0.05% Tween-20 (TBS-T). Membranes were incubated overnight with primary antibodies (1:500-5,000), washed, and incubated with secondary antibody conjugated to HRP (1:10,000) for 1 h. Chcmilumincscencc was detected b y ECL Prime Reagent (Amersham) and images were captured using the ChemiDoc MP Imaging System (Bio-Rad). Densitometric analyses were performed using ImageJ software.

Fluorescence microscopy. LNCaP cells grown on covcrslips were transfected with 2 ug of expression vector encoding for YFP-AR fusion protein. On the following day, cells were pre- treated with the indicated compounds for 1 h, and then stimulated with R1881 (1 nM) or vehicle (ethanol) for 3 hours. After incubations, the cells were fixed with 4% paraformaldehyde, washed, and then mounted and counterstained with DAPI. The slides were examined on a Zeiss

Axioplan-2 Fluorescence Microscope. Quantification was performed by ImageJ software to determine the ratio of cell fluorescence after background correction. For each treatment, at least 9 images containing 6 or more cells in field were used for quantification (>50 cells total from 3 independent experiments).

BrdU cell proliferation assay. Cells were seeded in 96-well plates (LNCaP, 5,000/well; LN95, 8,000/well) in reduced serum media (0.5-1%). The following day, cells were pre-treatcd with compounds for 1 h, and then stimulated with 0.1 nM of R 1881 as needed. After the indicated incubation times, BrdU labeling for 2 hours was performed by BrdU ELISA kit (Roche) and measured using a VersaMax Microplate Reader (Molecular Devices).

Cell cycle analysis by flow cytometry. Cells were grown in 10 cm dishes in 10% CSS for 3 days before the media was changed to 1.5% CSS and ATRA and/or EP1-O02 were added at the indicated concentrations. After treatments, cells were labeled with BrdU (10 uM) for 2 hours, and then trypsinized, washed, and fixed in 70% EtOH/PBS at -20 °C. Fixed cells were labeled with fluorescein (FITC)-conjugated anti-BrdU antibody (B44; BD Biosciences) and DNA was stained with 7-AAD (Sigma). Data was collected with a BD FACSCalibur flow cytomctcr (BD Biosciences). FITC and 7-AAD fluorescence were detected in the FL1 and FL3 channels respectively, using CellQuest Pro software and analyzed by Flow Jo V10 software.

Colony formation assay. Cells were treated with indicated concentrations of ATRA and/or EP1-002 for 24 hours in the presence of limited serum (1.5% CSS). After incubations, the cells were trypsinized, filtered, and counted using a hemocytometer, and then seeded in 6-well plates (500 cells/well) with RPMI containing 10% CSS. Fourteen days later, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. Colonies containing >50 cells were counted using ImageJ software, and the surviving fraction was determined by nonnalizing the plating efficiencies (number of colonies/cells seeded) to the control group.

Statistical analysis. Statistic differences were determined by GraphPad Prism by analysis of variance (ANOVA) using Dunnett's multiple comparison's test, where *P < 0.05, **P < 0.01, ***P < 0.001 , ****/> < 0.0001

Example 1: Pin J Depletion Impairs Transcription Mediated bvAR.

FIG. 1A is a schematic diagram depicting the locations of putative Pinl motifs on AR indicated by proline residues, with prediction of regions of intrinsic disorder (>0.5, RONN plot; lower panel). FIG. IB is an immunoblot of Pinl protein in human prostate cancer cell lines. FIG. 1C is a bar graph showing the activities of AR-rcsponsive reporter genes (PSA- and PB- luciferase) in LNCaP cells treated with siRNA targeting Pinl, and then incubated with synthetic androgen (Rl 881 , 1 nM) for 48 h. FIG. ID is a representative immunoblot demonstrating the effectiveness of the siRNAs targeting Pinl. Below, graph shows quantification of Pinl protein levels normalized to beta-actin. FIG. E is a bar graph showing the depiction of Pinl did not inhibit the activities of CMV and AP-1 reporters, which are not AR-driven. FIG. IF is a plot showing cellular localization of Pin 1 and AR in LNCaP cells treated with R1881 (10 nM) for up to 48h. Results shown are the mean ± s.e.m. from 3 independent experiments. *P < 0.05, ****p < 0.0001, as determined by Dunnett's test

Example 2: Inhibition of Pinl by Compounds Blocks AR Transcriptional Activity.

FIG. 2A are bar graphs showing activities of PSA- and PB-hiciferase gene reporters in

LNCaP cells treated with Pinl inhibitors: vehicle (DMSO), juglone (JUG, 20 μΜ), EGCG (20 μΜ), or ATRA (10 μΜ); in the presence and absence of androgen (Rl 881 , 1 nM) for 24 h. FIG. 2B are bar graphs showing no inhibitory effects were observed for CMV- and AP-l-luciferase reporters, which arc not regulated by AR. 13cRA is an active conformcr of ATRA which does not inhibit Pinl . FIG.2C shows immunoblotting analysis of PSA protein from LNCaP cells treated with Pinl inhibitors and R1881 (left), with graph showing quantification of the data (right). FIG. 2D shows an immunoblot of purified AR, showing the levels of phosphorylation of AR serine 81 (P-AR Ser81) from cells treated with indicated concentrations of juglone and R1881 (left), and graph showing the average band intensity quantified from 5 separate experiments (right). FIG. 2E shows subcellular localization of YFP-AR in LNCaP cells following treatment with anti-androgens (bicalutamide, BIC; and enzalutamide, ENZA) or Pinl inhibitors, and vehicle (EtOH) or Rl 881 (1 nM) for 2 h. FIG. 2F is a bar graph showing the ratio of nuclear to cytoplasmic AR-YFP from at least 50 cells. Scale bar represents 20 pm. Results are representative of 3 separate experiments unless stated otherwise. Error bars represent the mean ± s.c.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001, as determined by Dunnett's test.

Example 3: Pinl Interacts with the AR NTD and Regulates Transactivation.

FIG. 3A is a schematic diagram depicting the intact AR NTD and fragments, with putative Pinl motifs indicated by black bars. FIG. 3B shows the results of a pull-down assay of his-tagged AR NTD fragments from LNCaP cells incubated with IL-6 (50 ng/ml) for 6 h, which demonstrated the interactions between Pinl and specific fragments of the AR NTD. FIGS. 3C and 3D are bar graphs showing the effects of juglone (20 μΜ), EPI-002 (25 jiM), or a combination of the two compounds, on transactivation of AR NTD fused to a Gal4-DNA binding domain (Gal4-ARN) in LNCaP cells stimulated with (FIG. 3C) IL-6 (50 ng/ml) or (FIG. 3D) forskolin (FSK, 25 uM) for 24 h. FIG. 3E is a bar graph showing the results of transactivation assays, which demonstrated mat the 234-391 region of the AR NTD is necessary for most of the inhibitory effects of juglone (20 uM). FIG. 3F is a bar graph showing the effect of proline to glycine mutations of putative Pinl motifs on the transactivation of AR 234-391 induced by IL-6 and its sensitivity to juglone (20 μΜ). FIG. 3G shows the results of an immunoprecipitation assay demonstrating that EPI-002 (35 μΜ) and juglone (30 μΜ) prevent interactions between endogenous AR and STAT3 after treatment with IL-6 for 6h, and quantification of the ratio of AR to STAT3 immunoprecipitatcd (below). FIGS. 3H and 31 are representative immunoblots showing the levels of phosphorylated STAT3 (FIG. 3H) and MAPK (FIG. 31) in LNCaP cells following treatment with juglone (20 μΜ) and then stimulation with IL-6 or FSK for 15 min. Results shown are the mean ± s.ejn from 3-5 independent experiments. *P < 0.05, **P< 0.01, ***P < 0.001 , ****P < 0.0001 as determined by Dunnett's test.

Example 4: A TRA and Other Pinl Inhibitors Suppress Prostate Cancer Cell Proliferation.

FIGS. 4A-D are bar graphs showing the results of a BrdU incorporation assays comparing the effects of AR inhibitors (ENZA; APA, apalutamide; EPI) and Pinl inhibitors (JUG, EGCG, ATRA) on proliferation of (FIG. 4A) androgen-sensitive LNCaP cells for 72 hours, (FIG. 4B) androgen-independent LN95 cells for 48 hours, in reduced serum media. FIGS. 4C AND 4 D show potential synergy of ATRA with EPI, but not APA, on AR-driven proliferation regardless of ligand. Results shown are the mean ± s.e.m. from 3-5 independent experiments. FIG. 4e shows immunoblotting of indicated cell cycle proteins in LN95 treated with increasing concentrations of ATRA or 13cRA for 48 hours in limited serum media. FIG. 4F shows quantification of the data after normalizing to beta-actin. Graphs show the average band intensity from 3-4 independent experiments. Error bars represent the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, as determined by Dunnett's test. Example 5: ATRA svnerpizes with AR NTD antagonism by EPI-002.

FIG. 5A shows cell cycle analyses comparing BrdU incorporation and DNA content from LN95 cells incubated with EPI-002 (25 uM), ATRA (5 or 10 μΜ), or combinations for 24 or 48h in limited serum media (1.5% CSS). Bivariate plots shown are representative of 3 independent experiments. FIG. 5B provides a summary of the cell cycle distribution as the percentage of total cells. FIG. 5C is a representative image and FIG. 5D is a bar graph showing quantification of the colonies formed from LN95 cells treated with the indicated concentrations of EPI-002, ATRA, or combinations, from 3 independent experiments. Error bars show the mean ± s.e.m. *P < 0.05, **P < 0.01, ****P < 0.0001, as determined by Dunnett's test FIG. 5E is a bar graph showing the results of a PSA-luciferasc activity in LNCaP cells treated with different doses of EPI-002, in the presence and absence of ATRA (5 μΜ). FIG. 5F is a graph showing addition of ATRA (5 uM) shifting the dose-rcsponse of EPI-002 to the left, thus lowering the IC» value; and FIG. 5G is a graph showing surpassing the additive effect predicted by the Bliss independence model, which implies synergy. Graphs represent mean ± s.e.m. from 4-6 independent experiments. Example 6: Summary of Select Pinl Inhibitors

FIG. 6A provides a brief description of inhibitory mechanisms and chemical structures of juglone, EGCG, and ATRA. FIG. 6B are graphs showing the ability to inhibit Pinl PPlase activity in a fluorescence-based kinetic assay. Uncatalyzed value indicate the basal conversion rate without Pinl in the reaction. Graphs represent mean ± s.e.m. from 4 independent experiments.*P< 0.05, **P < 0.01, ***P < 0.001. ****P < 0.0001, as determined by Dunnett's test.

Example 7: Further Svnerev Experiments and Analysis

FIGS. 7A and 7B are bar graphs showing synergy between juglone and EPI-002 on inhibiting transactivation of Gal4-ARN by (FIG. 7A) IL-6 and (FIG. 7B) FSK. Graphs show the fractional responses of combination treatment compared to treatment in isolation. The combination index (CI) was calculated according to the Bliss independent model, and the dotted lines indicates the predicted additive effect. Bars show the mean fractional response ± s.e.m. from FIGS. 3C and 3D.

Example 8: Effects of Treatment on the Cell Cvcle.

FIGS. 8A-C are bivariate plots showing flow cytometric data of cell cycle distribution of LN95 cells incubated for 48 hours with (FIG. 8A) control, (FIG. 8B) EPI-002 (25 μΜ), then ATRA, (FIG. 8C) ATRA, then EPI-002 (25 jiM). The second treatment was added 24 hours after the first treatment, without changing the media. FIG. 8D show bar graphs comparing the average from 3 separate experiments.

Example 9: Inhibition of Proline Isomerase Pin 1 interrupts the Function of the Androgen

Receptor N-Terminal Domain and Suppresses Androeen-Independent Growth of Prostate

Cancer Cells

Summary

Several known inhibitors of Pin I were tested in cell-based assays that measure proliferation or transcription mediated by AR. The results demonstrated that inhibition of Pin 1 interrupted the function of the AR NTD. The Pin I inhibitor juglpne effectively and specifically blocked transcription mediated by AR induced by androgen, as well as transactivauon of the AR NTD in the presence of IL-6. It was found that Pin 1 predominantly interacted with a specific region of the AR NTD containing two Pin 1 binding sites, and by inhibiting Pin 1 the interactions between endogenous AR and STAT3 became attenuated. Furthermore, Pin 1 inhibitors were more effective than second-generation antiandrogens In blocking androgen-independent proliferation of LNCaP95 cells driven by AR variants. Without being bound by any one particular theory, Pin 1 seems to be a critical factor for transcription mediated by AR, regardless of ligand, by regulating the AR NTD.

Effect of siRNA Targeting Pfai 1 The activities of AR-responsive reporter genes (PSA- and PB-luciferase) in LNCaP cells incubated with siRNA targeting Pin 1 and androgen (R1881, 1 nM) for 48 hours are shown in FIG. 9A. FIG. 9B shows Pin I knockdown did not inhibit activities of reporters for CMV and AP-1, which are not regulated by AR.

Effect of Pin 1 Inhibitors

FIG. 10 A shows the actrvites of PSA- and PB-luciferase reporters in LNCaP cells treated with various Pin 1 inhibitors, including juglonc (JUG, 20 uM), EGCG (20 μΜ), or ATRA (10 μΜ), in die presence of R1881 (1 nM) for 48 h. FIG. 10B shows no inhibitory effects were observed by the inhibitors on CMV- and AP-luciferase reporter constructs.

Effect on Transactziivation of the AR N-Terminal Domain

FIG. 1 1A shows the effect of juglonc (20 μΜ), AR NTD antagonist EPI-002 (25 μΜ), or a combination of the two compounds on trans activation of human AR NTD (1-588) fused to a Gal4-DNA binding domain (ARN-Gal4DBD) in LNCaP cells stimulated with IL-6 (50 ng/mL) or forskolin (FSK- 25 μΜ) for 24 hours. FIG. 11 B are graphs showing the fractional response of combination treatment compared to treatment in isolation.

Interaction with the AR-N-Terminal Domain

FIG. 12A shows His-tagged AR NTD fragments purified from LNCaP cells treated with

IL-6, demonstrating interaction between Pin 1 and specific regions of AR NTD. FIG. 12B shows trans activation assays suggest that the 234-491 region of the AR NTD is necessary for the majority of inhibitory effects observed with juglonc (20 μΜ). FIG. 12C shows proline to glycine mutation of the putative Pin 1 motifs within this region indicates mat both these Pin 1 motifs arc required.

Effect on Androgen Independent Prostate Cancer Growth

FIG. 13A shows BrdU incorporation of LNCP95 cells measured after 48 h incubation with AR or Pin 1 inhibitors (ENZA, enzalutamide; APA, apalutamide). FIG. 13B shows BrdU incorporation after 24 h of incubation with ATRA with APA or EPI-002 suggest a possible synergistic interaction between ATRA and EPI-002 (AR NTD antagonist). FIG. 13C demonstrates the dose-dependent effect of ATRA in LNCaP95 ceils, downregulating Pin 1 and CyclinDl protein levels compared to the inactive conformer (13-cis-RA).

Effect on Cell Cycle Progression

FIGS. 14A and 14B shows BrdU incorporation and DNA content from LNCaP95 cells incubated in 1.5% CSS with EPI-002 (25 μΜ), ATRA (5-10 μΜ), or a combination for 24 or 48 h. Bivariate plots show cell cycle distribution of a representative experiment.

Example 10: Combination Therapy with EPI-002 and PARP Inhibitor for Castration-Resistant Prostate Cancer

Introduction and Objective

Following the results of the TOPARP-A Phase II trial, Olaparib, an oral PARP inhibitor was recently recognized as a FDA breakthrough therapy for metastatic castration recurrent prostate cancer (mCRPC) patients who have germline mutations in DNA repair genes. Although these results are noteworthy, the problem remains of how to treat the remaining mCRPC patients who do not have detectable germline mutations of DNA repair genes. Androgen receptor (AR) signaling regulates DNA repair in prostate cancer and AR modulating drugs induce DNA damage. Thus a combination approach using AR modulating drugs with a PARP inhibitor could be a promising option for the treatment of mCRPC. All currently approved AR modulating drugs, such as cnzalutamidc and abiratcronc, cither directly or indirectly target the AR C- terminus ligand-binding domain (LBD). Such drugs arc often unsuccessful due to the emergence of AR splice variants (ARV-7, ARv567es) that are constitutively active and lack a LBD. EPI-002 is a first-in-class AR antagonist that targets both full-length AR and AR splice variants. Herein, data is presented to support that a combination of Olaparib, a PARP inhibitor, and EPI-002 have beneficial effects in vitro.

Methods

Combination therapy using EPI-002 and Olaparib were evaluated in vitro using human prostate cancer cells, LNCaP (androgen sensitive and expresses full-length AR) and LNCaP95, an androgen-independent cell line that expresses full-length AR and AR-V7 and is resistant to enzahitamide. The effects of monotherapy and combination therapy on cell cycle and DNA damage were analysed using FACS and Western blot Results

Unexpectedly, EPI-002 caused an enormous decrease of Chkl protein levels in LNCaP and LNCaP95 cells. Whereas, enzalutamide also decreased the expression of Chkl in LNCaP, it had no effect on Chkl levels in LNCaP95 cells. Consistent with these data, AR knockdown in LNCaP cells also decreased Chkl levels. The PARP inhibitor, Olaparib, induced

phosphorylation of Chkl. EPI-002 induced Gl cell cycle arrest whereas Olaparib induced G2/M cell cycle arrest. FACS analysis of γΗ2ΑΧ showed increased ΌΝΑ damage with combination therapy compared to monotherapies.

Conclusions

EPI-002 decreased the expression of Chkl in prostate cancer cells that expressed both full-length AR and AR-V7. Combination therapy of EPI-002 plus Olaparib may provide a therapeutic approach for prostate cancer that expresses AR-V7.

Example 11: EPI-002 and Weel Inhibitor Combination Therapy Showed Accumulation ofDNA Damage and Cell Cycle Arrest

FIG. IS shows a combination therapy with EPI-002 and MK-1775 significantly decreased proliferation in vitro compared with vehicle and monotherapy-treated group. LNCaP95 cells were treated with DMSO, EPI-002 (25 uM), MK 1775 (100 nM or 200 nM), or a combination tor 48 hours. Proliferation was measured by BrU incorporation.

FIGS. I6A and 16B shows EPI-002 and Wee-1 inhibitor combination therapy induces DNA damage accumulation and cell cycle arrest. LNCaP95 cells were treated with DMSO, MK 1775 (200 nM), EPI-002 (25 μΜ), enzalutamide (5 μΜ), or a combination for 48h and then whole cell lysates were prepared and subjected to western blot (FIG. 16 A). LNCaP95 cells were treated with DMSO, MK 1775 (200 nM), EPI-002 (25 uM), or a combination for 24h or 28h and then subjected to cell cycle analysis using flow cytometry.

Example 12: Enhancing EPI-002 Inhibition of Androgen Receptorin LNCaP Cells by Co- Administration ofN-acetvlcvsteine (NAC).

Summary

Preliminary findings from luciferase reporter assays for AR transcriptional activity show that combination treatment yielded enhanced inhibition of AR transcriptional activity, compared to EPI-002 monotherapy.

Combination of NAC with EPI-002 resulted in a statistically significant decrease of the

ECJO for EPI-002.

Thus, inhibition of AR transcriptional activity by EPI-002 was enhanced with NAC. This modulation demonstrates the complexity of cellular mechanisms which influence the efficacy of targeted therapeutics.

Discussion FIG. 17 shows androgen (R1881)-induced PSA luciferase activity is inhibited by EPI- 002. LNCaP human prostate cancer cells were transiently transfected with the PSA(6.1kb)- luciferase reporter gene construct Bars represent the mean of 3 independent experiments each with 4 technical replicates per condition per experiment. The mean relative luciferase units/mg protein/minute was normalized to the control to determine fold-induction.

FIG. 18A shows NAC (10 mM) enhances inhibition of R1881-induced PSA-lucifcrasc by EPI-002. LNCaP human prostate cancer cells were transiently transfected with the PSA(6. Ikb)- Iucifcrasc reporter gene construct. Bars represent the mean of 3 independent experiments each with 4 technical replicates per condition per experiment The mean relative luciferase units/mg protein/minute was normalized to the control to determine fold-induction.

FIG. 18B shows the dose-response of EPI-002 inhibition of androgen (R1881)-induced PSA-hiciferase activity in the presence of NAC (10 mM). LNCap human prostate cancer cells were transiently transfected with the PSA(6.1kb)-luciferase reporter gene construct Bars represent the mean of 3 independent experiments each with 4 technical replicates per condition per experiment. The mean relative luciferase units/mg protein/minute was normalized to the control to determine fold-induction.

FIG. 18C shows NAC (10 mM) decreases the IC» of EPI-002 to inhibit R1881-induccd PSA-luciferasc activity. LNCaP human prostate cancer cells were transiently transfected with the PSA(6. lkb)-lucifcrase reporter gene construct Bars represent the mean of 3 independent experiments each with 4 technical replicates per condition per experiment. The luciferase activity with Rl 881 in the absence of EPI-002 was set to 100%.

FIG. 18D shows NAC enhances the inhibition of AR transcriptional activity by EPI-002. Bars represented the mean of 6 independent experiments each with 4 technical replicates per experiment. Application of two-tailed Student's unpaired t-test comparing EPI-002 monotherapy with EPI-002 and NAC combination therapy revealed significant difference between means.

FIG. 19 shows the effects of EPI-002 and NAC on the protein levels of prostate specific antigen (PSA) in LNCaP cells. Cells were pre-treated with EPI-002 (35 μΜ) alone or in combination with NAC (10 mM) for lh before the addition of androgen (R1881, 1 nM). After 24 hours, cells were harvested and fractionated into crude cytoplasmic and nuclear extracts.

Immunoblot shows levels of PSA protein and beta-actin as a control FIG. 20 shows the effect of EPI-002 and NAC on the levels of NRF2 protein in LNCaP prostate cancer cells. LNCaP ceils were pre-treated with EPI-002 (35 μΜ) alone or in combination with NAC (10 mM) for Ih before the addition of androgen (Rf 881, 1 nM) for 24 hours.