WO2024091899A1 - Salts of galeterone and salts of next generation galeterone analogs, and uses thereof - Google Patents
Salts of galeterone and salts of next generation galeterone analogs, and uses thereof Download PDFInfo
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- WO2024091899A1 WO2024091899A1 PCT/US2023/077577 US2023077577W WO2024091899A1 WO 2024091899 A1 WO2024091899 A1 WO 2024091899A1 US 2023077577 W US2023077577 W US 2023077577W WO 2024091899 A1 WO2024091899 A1 WO 2024091899A1
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- 150000003839 salts Chemical class 0.000 title claims abstract description 130
- PAFKTGFSEFKSQG-PAASFTFBSA-N Galeterone Chemical class C1=NC2=CC=CC=C2N1C1=CC[C@H]2[C@H](CC=C3[C@@]4(CC[C@H](O)C3)C)[C@@H]4CC[C@@]21C PAFKTGFSEFKSQG-PAASFTFBSA-N 0.000 title description 29
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- 229940125904 compound 1 Drugs 0.000 claims abstract description 57
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 34
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- 101710139011 MAP kinase-interacting serine/threonine-protein kinase 1 Proteins 0.000 claims abstract description 19
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 14
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 13
- 239000010452 phosphate Substances 0.000 claims abstract description 13
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- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims abstract description 11
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims abstract description 9
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- A61P35/00—Antineoplastic agents
Definitions
- the disclosed embodiments further relate to methods of treatment of diseases, such as cancer in humans, using pharmaceutical compounds.
- BACKGROUND OF INVENTION Drugs and lead candidates in the drug discovery and development process are often formed as a weak acid or base, but this form is not always optimal for dissolution or absorption into the body [NPL-1, NPL-2]. Without absorption, a drug cannot have a therapeutic effect, so some forms require salinification, i.e., salt formation. Many medications need to be water- soluble as well. Therefore, drugs are often chemically made into their salt forms to enhance how the drug dissolves, boost its absorption into the body’s bloodstream, and increase its effectiveness.
- Galeterone 3 ⁇ -(hydroxy)-17-(1H-benzimidazole-1-yl)androsta-5,16-diene, (Gal or Compound 1) is a potent drug that simultaneously targets AR/AR-V7 and Mnk1/2 [NPL-5].
- Gal metastatic castration-resistance prostate cancer
- mCRPC metastatic castration-resistance prostate cancer
- NPL-6 metastatic castration-resistance prostate cancer
- VNPP433-3 ⁇ 3 ⁇ -(1H-imidazole-1-yl-17-(1H- benzimidazole-1-yl)androsta-5,16-diene (Compound 2) is an NGGA with high therapeutic index (TI), a lower minimum effective dose, and compelling therapeutic effects in castration-resistant drug-naive PC preclinical models in vitro and in vivo [NPL-8], and which inhibits prostate cancer stem cells [NPL-9].
- TI therapeutic index
- VNPP433-3 ⁇ effectively disrupts multiple cellular pathways that drive PC development, progression, and metastasis, it could possibly thwart therapy resistance, making it an excellent candidate for clinical development [NPL-10]. While such early results are promising, there continues to be a need to improve the efficacy of Gal, NGGAs (e.g., VNPP433- 3 ⁇ ) and salts thereof. BRIEF SUMMARY OF INVENTION [0006] With the aim of improving aqueous solubility, oral bioavailability and hence reducing the administered dosage of Gal and VNPP433-3 ⁇ , the inventors set about on the development and discovery of new salt forms of these two related compounds.
- the invention is directed to a salt of Galeterone (Compound 1), where the salt may be, but is not limited to, a hydrochloride, a dihydrochloride, a sodium, a tosylate, a mesylate, a succinate, a sulfate, a disulfate, a maleate, a fumarate, a phosphate, and a diphosphate salt.
- the salt may be, but is not limited to, a hydrochloride, a dihydrochloride, a sodium, a tosylate, a mesylate, a succinate, a sulfate, a disulfate, a maleate, a fumarate, a phosphate, and a diphosphate salt.
- the salt of Compound 1 is selected from the group consisting of: H + Cl- O H + N N S O - N O , , , O H + H+ S O - N N O- O- Compound 15, Compound 18, Compound 20, and Compound 22.
- the invention is directed to a salt of VNPP433-3 ⁇ (Compound 2), where the salt may be, but is not limited to, a hydrochloride, a dihydrochloride, a sodium, a tosylate, a mesylate, a succinate, a sulfate, a disulfate, a maleate, a fumarate, a phosphate, and a diphosphate salt.
- VNPP433-3 ⁇ (2) (Compound 2) [0010]
- the salt of Compound 2 is selected from the group consisting of: Compound 4, Compound 5, Compound 8, C Compound 16, Compound 17, Compound 23, and Compound 24.
- the invention is directed to Compound 3 Compound 3 .
- the invention is directed to Compound 4 Compound 4 .
- the invention is directed to Compound 5 Compound 5 .
- the invention is directed to a pharmaceutical composition, comprising any one of Compounds 1-24 and a pharmaceutically acceptable carrier, excipient and/or diluent.
- the invention is directed to a method of treating a disease, such as cancer, comprising administering a therapeutically effective amount of a salt of Compound 1 and/or a salt of Compound 2 to a subject in need thereof.
- the salt of Compound 1 is at least one of Compounds 3, 6, 7, 10, 13, 15, 18, 20 and 22.
- the salt of Compound 2 is at least one of Compounds 4, 5, 8, 9, 11, 12, 14, 16, 17, 19, 21, 23 and 24.
- the disease is at least one type of cancer, such as prostate cancer, bladder cancer, pancreatic cancer, breast cancer, a hematological cancer, and hepatocellular carcinoma, or benign prostatic hyperplasia, or Kennedy’s disease.
- the prostate cancer is castration-resistant prostate cancer.
- the invention is directed to a method of prolonging survival of a subject having cancer, comprising administering a therapeutically effective amount of a salt of Compound 1 and/or a salt of Compound 2 to the subject.
- the salt of Compound 1 is at least one of Compounds 3, 6, 7, 10, 13, 15, 18, 20 and 22.
- the salt of Compound 2 is at least one of Compounds 4, 5, 8, 9, 11, 12, 14, 16, 17, 19, 21, 23 and 24.
- the disease is a cancer, such as prostate cancer, bladder cancer, pancreatic cancer, breast cancer, a hematological cancer, and hepatocellular carcinoma, or benign prostatic hyperplasia, or Kennedy’s disease.
- the prostate cancer is castration-resistant prostate cancer.
- the invention is a method of inducing androgen receptor (AR) degradation in a subject, comprising administering a therapeutically effective amount of a salt of Compound 1 and/or a salt of Compound 2 to a subject in need thereof.
- AR androgen receptor
- the invention is a method of inducing Mnk1/2 transcriptional activity degradation in a subject, comprising administering a therapeutically effective amount of a salt of Compound 1 and/or a salt of Compound 2 to a subject in need thereof.
- FIG.1 Chemical structures of Galeterone (Compound 1) and VNPP433-3 ⁇ (Compound 2).
- FIG.2. Chemical structures of Galeterone (Compound 1) and VNPP433-3 ⁇ (Compound 2).
- FIG.3A and FIG.3B Effects of Compounds 1 and 3 (FIG.3A) and Compounds 2 and 4 (FIG.3B) compared to Enzalutamide (ENZ) on the expression of fAR, AR-V7, Mnk1, peIF4E, eIF4E, Bcl2, BAX and cyclin D1 in human CWR22Rv1 prostate cancer cells in vitro.
- FIG.4A, FIG.4B and FIG.4C are plasma pharmacokinetics profiles of Compounds 3, 4 and 5, respectively, after intravenous (IV) and oral (PO) administration to CD-1 male mice. Each point represents the mean ⁇ standard deviation of the mean (SDM) in three mice.
- FIGS.5A, 5B, 5C and 5D Male NRG mice bearing CWR22Rv1 tumors were treated with vehicle, Compounds 1, 2, 3 and 4 for 16 days, and tumor volumes were measured twice a week and the tumors were collected.
- FIG.5A shows the percent tumor growth inhibition (%TGI) values at the right of each growth curve, and the error bars are the SEM. Results are represented as means ⁇ SEM.
- FIG.5B shows the average tu * weights from each experimental group at day 16: p ⁇ 0.0256, ** p ⁇ 0.0104, *** p ⁇ 0.0041, **** p ⁇ 0.0069.
- FIG.5C is a photograph of excised tumors from each group at the end of the study.
- FIG.5D shows the mean body weights of mice determined during the study. [0028] FIGS.6A, 6B, 6C, 6D and 6E.
- a head-to-head comparison of the antitumor activity of Gal (Compound 1), Gal.HCl (Compound 3), Enzalutamide (ENZ), and Docetaxel (DTX) in human CRPC CWR22Rv1 tumor xenograft model is provided.
- Mice bearing CWR22Rv1 xenografts (5 mice per group) were treated with vehicle, Compound 1 (100 mg/kg/twice daily), Compound 3 (24, 48 or 96 mg/kg, once daily), ENZ (25 mg/kg, once daily) 5 days per week for 16 days, and DTX (5 mg/kg, IP, 3 times per week).
- FIG.6A tumor volumes were measured twice a week and the tumors were collected.
- %TGI Percent tumor growth inhibition
- FIG.6D provides the percent change in volume for each tumor (5 tumors per treatment group) after 16 days as shown as a waterfall plot.
- FIG.6E provides the mean body weights of mice determined during the study. [0029]
- ENZ Enzalutamide
- DTX Docetaxel
- mice bearing CWR22Rv1 xenografts (5 mice per group) were treated with vehicle, Compound 2 (20 mg/kg, once daily), Compound 4 (5 or 20 mg/kg, once daily), Compound 5 (5 or 20 mg/kg, once daily), ENZ (25 mg/kg, once daily) 5 days per week for 16 days, and DTX (5 mg/kg, IP, 3 times per week).
- FIG.7A tumor volumes were measured twice a week and the tumors were collected. Percent tumor growth inhibition (%TGI) values are indicated at the right of each growth curve, and the error bars are the SEM. Results are represented as means ⁇ SEM.
- FIG.7B provides the average tumor weights from each experimental group at day 16; *p ⁇ 0.0001 versus control for all the treated groups.
- FIG.7C provides the total percent change in mean tumor volumes of the initial volume after 16 days as shown as a waterfall plot. The p values are as in FIG.7A.
- FIG.7D is the percent change in volume for each tumor (5 tumors per treatment group) after 16 days as shown as a waterfall plot.
- FIG.7E shows the mean body weights of mice determined during the study.
- FIG.8A is a photograph of all the excised tumors after termination of the study.
- FIG. 8B is Western blot analyses of tumor samples from each experimental group after termination of the study.
- FIG.9A The quantification analysis of the proteins analyzed in FIG.3A on the expression of fAR, AR-V7, Mnk1, peIF4E and eIF4E.
- FIG.9B The quantification analysis of the proteins analyzed in FIG.3B on the expression of fAR, AR-V7, Mnk1, peIF4E and eIF4E.
- FIG.11 13 C-NMR of Compound 1 in CDCl 3 [0035] FIG.12. 1 H-NMR of Compound 3 in CDCl 3 [0036] FIG.13. 13 C-NMR of Compound 3 in CDCl 3 [0037] FIG.14. HRMS spectra of Compound 3 [0038] FIG.15. 1 H-NMR of Compound 2 in CDCl 3 [0039] FIG.16. 13 C-NMR of Compound 2 in CDCl 3 [0040] FIG.17. 1 H-NMR of Compound 4 in CDCl 3 [0041] FIG.18. 13 C-NMR of Compound 4 in CDCl 3 [0042] FIG.19.
- FIG.20 1 H-NMR of Compound 2 in DMSO-d 6 [0044]
- FIG.22. 1 H-NMR of Compound 5 in DMSO-d 6 [0046]
- FIG.23. 13 C-NMR of Compound 5 in DMSO-d 6 [0047]
- FIG.24. HRMS spectra of Compound 5 DETAILED DESCRIPTION OF THE INVENTION [0048] Unless otherwise noted, technical terms are used according to conventional usage.
- another may mean at least a second or more.
- singular terms include pluralities and plural terms include the singular.
- “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
- the term “about” generally refers to a range of numerical values (e.g., +/- 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.
- dose As used herein, the terms “dose”, “unit dose”, “dosage”, “effective dose”, “therapeutically effective dose”, and related terms refer to physically discrete units that contain a predetermined quantity of active ingredient or therapeutic drug (agent) calculated to produce a desired therapeutic effect. A single dose is thus a predetermined quantity of an agent of the invention that is administered to a subject.
- a “subject” is a human, a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.
- Disruption and/or perturbation of cap-dependent translation is essential for the development of cancers.
- eukaryotic translation initiation factor 4E eukaryotic translation initiation factor 4E
- eIF4E eukaryotic translation initiation factor 4E
- the oncogenic potential of eIF4E is dependent on serine 209 phosphorylation by MAPK-interacting kinases 1 and 2 (Mnk1/2).
- Mnk1/2-eIFE axis Given the implication of Mnk1/2-eIFE axis in the initiation and progressions of all types of solid tumors and hematologic cancers, successful agents for the prevention and treatment of all forms of breast and prostate cancers and other diseases will block the activity of Mnk 1/2.
- Androgen receptors are a well-established target for therapeutic intervention in certain diseases, including prostate cancer, bladder cancer, pancreatic cancer, breast cancer, a hematological cancer, hepatocellular carcinoma, benign prostatic hyperplasia (BPH) and Kennedy’s disease.
- Galeterone analogs are of substantial interest because of their multi-target anticancer activities, including AR and Mnk degrading activities.
- ARDAs potent androgen receptor degrading agents
- MNKDAs Mnk degrading agents
- PDAC pancreatic ductal adenocarcinoma
- the inventors developed salts of Galeterone and VNPP433-3 ⁇ in order to improve aqueous solubility and oral bioavailability of the compounds.
- the resulting changes to the compounds allow a reduced amount of the compounds to be administered to a subject, such during treatment of a medical condition.
- Exemplary salts of the compounds include hydrochloride, dihydrochloride, sodium, tosylate, mesylate, succinate, sulfate, disulfate, maleate, fumarate, phosphate, and diphosphate salts.
- the present invention is directed to the following specific compounds: salts of Galeterone including Gal-HCl (or Gal.HCl) (Compound 3), Gal-sodium (Compound 6), Gal-tosylate (Compound 7), Gal-mesylate (Compound 10), Gal-succinate (Compound 13), Gal- sulfate (Compound 15), Gal-Maleate (Compound 18), Gal-fumarate (Compound 20), and Gal- phosphate (Compound 22).
- Salts of Galeterone including Gal-HCl (or Gal.HCl) (Compound 3), Gal-sodium (Compound 6), Gal-tosylate (Compound 7), Gal-mesylate (Compound 10), Gal-succinate (Compound 13), Gal- sulfate (Compound 15), Gal-Maleate (Compound 18), Gal-fumarate (Compound 20), and Gal- phosphate (Compound 22).
- the present invention is also directed to the following specific compounds: salts of VNPP433-3 ⁇ , including VNPP433-3 ⁇ HCl (Compound 4), VNPP433-3 ⁇ dihydrochloride (Compound 5), VNPP433-3 ⁇ tosylate (Compound 8), VNPP433-3 ⁇ ditosylate (Compound 9), VNPP433-3 ⁇ mesylate (Compound 11), VNPP433-3 ⁇ (Compound 12), VNPP433-3 ⁇ succinate (Compound 14), VNPP433-3 ⁇ -sulfate (Compound 16), VNPP433-3 ⁇ -disulfate (Compound 17), VNPP433-3 ⁇ -maleate (Compound 19), VNPP433-3 ⁇ -fumarate (Compound 21), VNPP433-3 ⁇ - phosphate (Compound 23), and VNPP433-3 ⁇ -diphosphate (Compound 24).
- VNPP433-3 ⁇ HCl Compound 4
- hydrochloride salts of Gal Compound 1 and VNPP433-3 ⁇ (Compound 2) were synthesized and shown to have improved aqueous solubility and enhanced in vitro and in vivo anticancer activities.
- Three hydrochloride salts of two lead compounds were prepared and biologically evaluated (Compounds 3, 4 and 5), with the results provided below.
- Compounds 3, 4 and 5 exhibit drastically improved anticancer activities and are statistically significantly more potent than their parent compounds (Compounds 1 and 2).
- the HCl salts are significantly more potent than two FDA-approved prostate cancer drugs, Enzalutamide (ENZ) and Docetaxel (DTX), as shown in the experimental data provided herein.
- the disclosed compounds can be used in the treatment of a variety of diseases, including cancers, and in prolonging survival in subjects having a variety of diseases, including cancers. It will be apparent that particular combinations of Compounds may differ in effectiveness depending on the type of cancer, the stage and grade of a particular cancer, the physical location of the cancer within the subject, the molecular abnormalities in the cancer, and available means for administering the Compounds, among other factors.
- Exemplary diseases that may be treated via the methods of the invention include, but are not limited to, cancer, such as prostate cancer, bladder cancer, pancreatic cancer, breast cancer, a hematological cancer, and hepatocellular carcinoma, or benign prostatic hyperplasia (BPH) or Kennedy’s disease.
- compositions [0062]
- the compounds of the invention may be used themselves or as agents in various methods, including but not limited to methods of treating diseases as described below.
- the compounds used in the methods may be formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient and/or diluent.
- a suitable pharmaceutical composition may comprise a single compound or combinations comprising two or more compounds, and may include a single carrier, excipient and/or diluent, or a combination of carrier(s), excipient(s) and/or diluent(s).
- compositions may be formulated, for example, for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, pulmonary, topical or parenteral administration.
- Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (IM), intravenous (IV), intraperitoneal (IP), intra-arterial, intramedulary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Any known device useful for parenteral injection or infusion of formulations can be used to affect such administration.
- the pharmaceutical composition is administered to the subject as an intravenous (IV) formulation.
- IV intravenous
- Pharmaceutically acceptable carriers, excipients and diluents are those ingredients, solutions, substances or materials that can be used to produce formulations that are suitable to be administered to a subject, such as a human.
- carriers, excipients and diluents of the present invention are those useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and that may present pharmacologically favorable profiles, and includes carriers and diluents that are acceptable for veterinary use as well as human pharmaceutical use.
- Suitable pharmaceutically acceptable carriers, excipients and diluents are well known in art and can be determined by those of skill in the art as the clinical situation warrants.
- suitable carriers and diluents include dextrose, water, glycerol, ethanol, propylene glycol, polysorbate 80 (Tween-80TM), poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (e.g., Cremophor EL), poloxamer 407 and 188, a cyclodextrin or a cyclodextrin derivative (including HPCD ((2- hydroxypropyl)-cyclodextrin) and (2-hydroxyethyl)-cyclodextrin), hydrophilic and hydrophobic carriers, and combinations thereof.
- Hydrophobic carriers include, for example, fat emulsions, lipids, PEGylated phospholipids, polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes.
- the terms specifically exclude cell culture medium. More particularly: (1) 5% (w/v) dextrose, or (2) water (e.g., sterile water; Water-For-Injection), may be used as a pharmaceutically acceptable carrier.
- Pharmaceutically acceptable diluents also include tonicity agents that make the composition compatible with blood. Tonicity agents are particularly desirable in injectable formulations.
- Excipients included in a formulation have different purposes depending, for example on the nature of the agent, and the mode of administration.
- excipients include, without limitation: stabilizing agents, solubilizing agents and surfactants, buffers, antioxidants and preservatives, tonicity agents, bulking agents, lubricating agents, emulsifiers, suspending or viscosity agents, inert diluents, fillers, disintegrating agents, binding agents, wetting agents, lubricating agents, antibacterials, chelating agents, sweeteners, perfuming agents, flavoring agents, coloring agents, administration aids, and combinations thereof.
- the pharmaceutical compositions may contain common carriers and excipients, such as cornstarch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, alginic acid, croscarmellose sodium, and sodium starch glycolate.
- common carriers and excipients such as cornstarch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, alginic acid, croscarmellose sodium, and sodium starch glycolate.
- the particular carrier, diluent and excipient used will depend upon the means and purpose for which the active ingredient (i.e., Compounds 1-24) is being applied.
- Acceptable methods for preparing the pharmaceutical compositions according to the invention are known to those skilled in the art.
- compositions may be prepared following conventional techniques of the pharmaceutical chemist involving steps such as mixing, granulating, and compressing when necessary for tablet forms, or mixing, filling, and dissolving the ingredients as appropriate, to give the desired products for various routes of administration.
- Methods of Treatment [0069] As summarized discussed above, the present invention is also directed to methods of treating diseases, including cancer, or prolonging survival in a subject having diseases, including cancer, by administering therapeutically effective amounts of one or more of salts of Galeterone (Compound 1) and/or one or more salts of VNPP433-3 ⁇ (Compound 2), such as Compound 3, Compound 4 or Compound 5.
- the salts of Compound 1 include hydrochloride, dihydrochloride, sodium, tosylate, mesylate, succinate, sulfate, disulfate, maleate, fumarate, phosphate, and diphosphate salts.
- the salts of Compound 2 include hydrochloride, dihydrochloride, sodium, tosylate, mesylate, succinate, sulfate, disulfate, maleate, fumarate, phosphate, and diphosphate salts.
- the disclosed embodiments are directed to a method of treating cancer, particularly prostate cancer, in a subject comprising administering therapeutically effective amounts of a salt of Galeterone to a subject having the cancer, particularly prostate cancer.
- the salts of Galeterone include Gal-HCl (Compound 3), Gal-sodium (Compound 6), Gal-tosylate (Compound 7), Gal-mesylate (Compound 10), Gal-succinate (Compound 13), Gal- sulfate (Compound 15), Gal-Maleate (Compound 18), Gal-fumarate (Compound 20), and Gal- phosphate (Compound 22).
- the disclosed embodiments are also directed to a method of treating cancer, particularly prostate cancer, in a subject comprising administering therapeutically effective amounts of a salt of VNPP433-3 ⁇ to a subject having the cancer, particularly prostate cancer.
- VNPP433-3 ⁇ HCl Compound 4
- VNPP433-3 ⁇ dihydrochloride Compound 5
- VNPP433-3 ⁇ tosylate Compound 8
- VNPP433- 3 ⁇ ditosylate Compound 9
- VNPP433-3 ⁇ mesylate Compound 11
- VNPP433-3 ⁇ Compound 12
- VNPP433-3 ⁇ succinate Compound 14
- VNPP433-3 ⁇ -sulfate Compound 16
- VNPP433- 3 ⁇ -disulfate Compound 17
- VNPP433-3 ⁇ -maleate Compound 19
- VNPP433-3 ⁇ -fumarate Compound 21
- VNPP433-3 ⁇ -phosphate Compound 23
- VNPP433-3 ⁇ -diphosphate Compound 24
- Exemplary diseases that may be treated via the methods of the invention include, but are not limited to, cancers, such as prostate cancer, bladder cancer, pancreatic cancer, breast cancers, a hematological cancer, and hepatocellular carcinoma, or benign prostatic hyperplasia or Kennedy’s disease.
- the prostate cancer is castration-resistant prostate cancer.
- the terms “treating” cancer and “treatment” of cancer mean at least the mitigation of cancer, or a disease condition or symptom associated with cancer in a subject that is achieved by a reduction of growth, replication, and/or propagation, or death or destruction of cancer and/or cancer cells, on or in the subject.
- treating and “treatment” include curing, healing, inhibiting, relieving from, improving and/or alleviating, in whole or in part, the cancer or associated disease condition or symptom.
- the mitigation of cancer or associated disease condition or symptom may be about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% in the subject, versus a subject to which a compound of the invention (i.e., Compounds 1-24) has not been administered.
- treating means reducing the population of cancer cells causing the cancer in the subject to an undetectable level, where detection is by any conventional means, such as assay a blood sample in the laboratory.
- treating means complete healing of the cancer, shown by an absence of clinical symptoms associated with the cancer.
- treating means the mitigation of cancer or an associated disease condition or symptom by at least about 90% in the subject.
- treating means the mitigation of cancer or an associated disease condition or symptom by at least about 95% in the subject.
- the disclosed methods also include methods of prolonging survival of a subject having cancer comprising administering a therapeutically effective amount of a salt of Compound 1 to a subject having cancer.
- the salt of Compound 1 and the cancers are those defined herein.
- the cancer is prostate cancer, such as castration-resistant prostate cancer.
- the disclosed methods also include methods of prolonging survival of a subject having cancer comprising administering a therapeutically effective amount of a salt of Compound 2 to a subject having cancer.
- the salts of Compound 2 and the cancers are those defined herein.
- the cancer is prostate cancer, such as castration-resistant prostate cancer.
- prolonging survival means extending the life span of a subject having cancer by at least one day versus a subject having the same cancer that does not receive a compound of the invention. Prolonged survival includes increasing the life span of the subject by at least: 1, 2, 3, 4 or more weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months, or 1, 2, 3, 4, 5, or more years.
- the amount (dose) of the compound of the invention (Compounds 1-24) sufficient to have an effect on cancer (additive, synergistic or otherwise) in a subject will vary, for example, in view of the identity of the compound being used in the combination, the physical characteristics of the subject, the severity of the subject’s symptoms, the form of the cancer, the identity of the cancer, the formulations and means used to administer the compound, and the method being practiced.
- the specific dose for a given subject is usually set by the judgment of the attending physician.
- the daily dose of the salt of Compound 1 or the salt of Compound 2 may be in the range of 0.1 mg/kg to 1000 mg/kg per day, 1 mg/kg to 100 mg/kg per day, 1 mg/kg to 100 mg/kg, or 24 mg/kg to 96 mg/kg.
- the daily dose may be in the range of 1 mg/kg to 100 mg/kg or 5 mg/kg to 20 mg/kg.
- the daily dose may be in the range of 1 mg/kg to 100 mg/kg or 5 mg/kg to 20 mg/kg.
- the daily dose may be in the range of 1 mg/kg to 100 mg/kg or 5 mg/kg to 20 mg/kg.
- administration frequencies of the agents will generally include 4, 3, 2 or once daily, every other day, every third day, every fourth day, every fifth day, every sixth day, once weekly, every eight days, every nine days, every ten days, bi-weekly, monthly and bi-monthly, whether the drugs are administered alone or in combination, concurrently or sequentially.
- the concurrent or sequential administration is administration once daily.
- compositions and the compounds of the invention may be administered via means that include oral, enteral, sublingual, intranasal, intraocular, rectal, intravaginal, transdermal, mucosal, topical or parenteral administration.
- Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (IM), intravenous (IV), intraperitoneal (IP), intra-arterial, intramedullary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Any known device useful for parenteral injection or infusion of agents and formulations can be used to affect such administration.
- the compounds and pharmaceutical compositions are administered to the subject intravenously.
- the dose may be administered all at once, such as with an oral formulation in a capsule, or slowly over a period of time, such as with an intravenous (IV) administration.
- IV intravenous
- the administering period can be a matter of minutes, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or more minutes, or a period of hours, such as about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 or more hours.
- the administration of the dose may be interrupted, such as where the dose is administered via intravenous infusion and the dose is divided into two or more infusion bags.
- VNPP433- 3 ⁇ dihydrochloride Compound 5
- Compound 2 IPA/methanol followed by treatment with 3M HCl in methanol (Scheme 2). Both VNPP433-3 ⁇ salts were obtained in quantitative yields.
- C16 proton of steroid skeleton has chemical shift difference of 0.33 ppm, which makes its chemical shift slightly more than other protons of the benzimidazole ring.
- the resonance of ⁇ bond electrons between C16-C17 and benzimidazole may be the reason for the observed difference.
- the remaining protons in the steroid skeleton away from C17 do not have any chemical shift effect.
- the protons of imidazole ring at C3 in VNPP433-3 ⁇ mono-HCl (Compound 4) also experience significant chemical shifts.
- the proton at C-2” has highest difference in chemical shift of 1.13 ppm.
- Example 2 In Vitro Cellular Antiproliferative Activities Against Three Prostate Cancer Cell Lines
- the three salts (Compounds 3, 4 and 5) were evaluated over a range of concentrations to examine their antiproliferative activities against the following three different prostate cancer cell lines of (1) LNCaP (androgen-sensitive), (2) C42B (androgen-insensitive), and (3) CWR22Rv1 (castration-resistant), using the well-established 3-(4,5-dimethylthiazolyl-2)-2,5- diphenyl tetrazolium bromide (MTT) assay [NPL-12, NPL-13, NPL-14].
- MTT 3-(4,5-dimethylthiazolyl-2)-2,5- diphenyl tetrazolium bromide
- FIG.2 shows the antiproliferative effects of parent Compounds 1 and 2 compared to their corresponding salts (Compounds 3, 4 and 5), Enzalutamide (ENZ) and Abiraterone (ABI) against CWR22Rv1 human prostate cancer cells in vitro.
- the salts, Compound 3 and Compound 4 were further examined for their effects on the degradation of AR/ARV7 and Mnk1 and the other related downstream molecular targets in both CWR22Rv1 prostate cancer cells by Western blotting analysis (FIGS.3A and 3B).
- the parent compounds (Compounds 1 and 2) and the vehicle, DMSO, were used as positive and negative controls, respectively.
- the two salts significantly and dose-dependently reduced the expressions of Mnk1 and peIF4E, without noticeable effects on the expression of total eIF4E and the house-keeping protein, ⁇ -actin (FIGS.3A and 3B).
- FIGS.3A and 3B show the effects of Compounds 1 and 3 (FIG.3A) and Compounds 2 and 4 (FIG.3B) compared to Enzalutamide (ENZ) on the expression of fAR, AR-V7, Mnk1, peIF4E, eIF4E, Bcl2, BAX and cyclin D1 in human CWR22Rv1 prostate cancer cells in vitro.
- ENZ Enzalutamide
- Equal protein concentrations from prostate cancer cells treated with the compounds at different concentration (5, 10 and 20 ⁇ M) for 24 h were separated by SDS-PAGE and Western blots probed with the respective protein antibodies. Vehicle-treated cells were included as a control, and all blots were reprobed for GAPDH for loading control.
- the potencies of the two salts are enhanced compared to their parent compounds. The reason for this difference is unknown. One possibility could be due to enhanced diffusion of these salts across the cell membrane of this cell line resulting in enhanced intracellular concentrations and modulation of the cellular molecular target proteins.
- Example 4 Plasma Concentrations and Pharmacokinetic Parameters of HCl Salts (Compounds 3, 4 and 5) in male CD-1 Mice. [0098] The inventors previously reported the plasma pharmacokinetics of Compounds 1 and 2 in male CD-1 mice [NPL-8].
- the significant PK parameters are presented in Table 3.
- mice bearing CWR22Rv1 tumors were treated with vehicle, Compounds 1, 2, 3 and 4 for 16 days as described in the METHODS section below.
- Percent tumor growth inhibition (%TGI) values are indicated at the right of each growth curve, and the error bars are the SEM. Results are represented as means ⁇ SEM. All p vales are compared to vehicle control: * p ⁇ 0.0256, ** p ⁇ 0.0104, *** p ⁇ 0.0041. See FIG.5A. The average tumor weights from each experimental group at day 16: * p ⁇ 0.0256, ** p ⁇ 0.0104, *** p ⁇ 0.0041, **** p ⁇ 0.0069 (FIG.5B). Photographed excised tumors from each group at the end of the study are provided in FIG.5C. Mean body weights of mice determined during the study are shown in FIG.5D.
- the tumors in the vehicle-treated mice increased in size by ⁇ 12.5-fold (from ⁇ 100 mm 3 to ⁇ 1250 mm 3 , FIG.5A).
- Compound 3 (at 9.7-fold lower equimolar dose than Compound 1) caused tumor growth inhibition (TGI) of 76% versus control compared to Compound 1 (64% versus control).
- TGI tumor growth inhibition
- the use of Compound 3 allows 50% less-frequent dosing, i.e., from twice daily for Compound 1 to once daily for Compound 3.
- Example 6 Compound 1 and Compound 2 and their respective hydrochloride salts, Compounds 3, 4 and 5, are more efficacious than Enzalutamide (ENZ) and Docetaxel (DTX) in Castration-Resistant Prostate Cancer CWR22Rv1 Xenograft Model.
- ENZ Enzalutamide
- DTX Docetaxel
- FIGS.6A-6E CWR22Rv1 tumors were less sensitive to ENZ and DTX treatments with percent TGI values of 54.7% and 56.9%, respectively.
- TGI tumor regressions.
- Compound 3 (96 mg/kg; 147.5%, p ⁇ 0.0001 vs. vehicle) > Compound 3 (48 mg/kg; 136.8%, p ⁇ 0.0001 vs. vehicle) > Compound 3 (24 mg/kg; 80.3%, p ⁇ 0.0005 vs. vehicle). It is notable that Compound 3 (at 2.3-, 4.6-, and 9.1-fold lower molar doses) is more efficacious than parent Compound 1.
- Compound 3 has a once-a-day dosing regimen instead of twice-a-day, and permits smaller quantities to be dosed, which undoubtedly would mitigate some undesirable side effects. In general, no host toxicities were observed, as monitored by changes in animal body weight (FIG.6E) and visual observation of general health of the animals. [00110] Mice bearing CWR22Rv1 xenografts (5 mice per group) were treated with vehicle, Compound 1 (100 mg/kg/twice daily), Compound 3 (24, 48 or 96 mg/kg, once daily), ENZ (25 mg/kg, once daily) 5 days per week for 16 days, and DTX (5 mg/kg, IP, 3 times per week).
- FIG.6A provides the average tumor weights from each experimental group at day 16; *p ⁇ 0.0001 versus control for all the treated groups.
- FIG.6C provides the total percent change in mean tumor volumes of the initial volume after 16 days as shown as a waterfall plot.
- FIG.6D provides the percent change in volume for each tumor (5 tumors per treatment group) after 16 days as shown as a waterfall plot.
- FIG.6E provides the mean body weights of mice determined during the study.
- FIGS.7A-7E The results of the impact of parent Compound 2 and its two hydrochloride salts, Compounds 4 and 5, are presented in FIGS.7A-7E.
- the lower 5 mg/kg doses of the two salts (Compounds 4 and 5) were less potent than the precursor Compound 2 (20 mg/kg), but the higher doses of 20 mg/kg, each, caused significant tumor regressions.
- TGI The decreasing order of potency was as follows: Compound 5 (20 mg/kg; 152.9%, p ⁇ 0.0001 vs. vehicle) > Compound 4 (20 mg/kg; 139.6%, p ⁇ 0.0001 vs. vehicle) > Compound 2 (20 mg/kg; 83.8%, p ⁇ 0.0005 vs. vehicle) > Compound 5 (5 mg/kg; 79.2%, p ⁇ 0.0006 vs. vehicle) > Compound 4 (5 mg/kg; 74.2%, p ⁇ 0.0008 vs. vehicle).
- no host toxicities were observed, as monitored by changes in animal body weight (FIG.7E) and visual observation of general health of the animals.
- mice bearing CWR22Rv1 xenografts (5 mice per group) were treated with vehicle, Compound 2 (20 mg/kg, once daily), Compound 4 (5 or 20 mg/kg, once daily), Compound 5 (5 or 20 mg/kg, once daily), ENZ (25 mg/kg, once daily) 5 days per week for 16 days, and DTX (5 mg/kg, IP, 3 times per week).
- Tumor volumes were measured twice a week and the tumors were collected. Percent tumor growth inhibition (%TGI) values are indicated at the right of each growth curve, and the error bars are the SEM. Results are represented as means ⁇ SEM.
- FIG.7A provides the average tumor weights from each experimental group at day 16; *p ⁇ 0.0001 versus control for all the treated groups.
- FIG.7C provides the total percent change in mean tumor volumes of the initial volume after 16 days as shown as a waterfall plot. The p values are as in FIG.7A.
- FIG.7D is the percent change in volume for each tumor (5 tumors per treatment group) after 16 days as shown as a waterfall plot.
- FIG.7E shows the mean body weights of mice determined during the study.
- FIG.8A shows a photograph of all the excised tumors after termination of the study which corroborates the tumor volumes of FIG.6A through FIG.7E.
- FIG.8B shows the impact of the various treatments on key biomarker proteins.
- FIG. 8B is Western blot analyses of tumor samples from each experimental group on day 16. Equal protein concentrations from prostate cancer tumors treated with the compounds at the indicated doses for 16 days were separated by SDS-PAGE and Western blots probed with the respective protein antibodies. Vehicle-treated cells were included as a control, and all blots were reprobed for GAPDH for loading control.
- taxanes including DTX have been reported to cause decrease in expression of AR in in vitro and in vivo models of prostate cancer [NPL-32].
- Each of Compounds 3, 4 and 5 caused a decrease in the expression of the anti-apoptotic Bcl-2 and upregulation of pro-apoptotic BAX, but the impacts of Compounds 3, 4 and 5 were superior to the impacts of ENZ or DTX at the tested doses.
- Compounds 3, 4 and 5 inhibited tumor growth via inhibition of AR/AR-V7 and Mnk1-eIF4e signaling pathways and induction of apoptosis, like previous reports by the inventors [NPL-8, NPL-9, NPL-10, NPL- 33].
- the Compound 2 required for mono and di-salt preparation was synthesized from Compound 1 following our reported synthetic procedure [NPL-34].
- Anhydrous solvents isopropyl alcohol and methanol
- 3M methanolic-HCl solutions small ampules for controlled reaction in mono salt preparation and bottled where HCl in excess used for other two salts
- Melting points were recorded on Fisher-Johns melting point apparatus and are uncorrected.
- 1 H and 13 C NMR spectra were obtained on a Bruker Ascend 400 spectrometer, and chemical shifts ⁇ are expressed in ppm relative to TMS as internal standard.
- ESI is a soft-ionization technique that can form adducts in positive-ion mode (i.e., H+ or Na+).
- positive-ion mode i.e., H+ or Na+.
- negative-ion mode it is possible to observe an M + Cl- anion or the [M-H]- parent ion, but HCl will greatly inhibit ionization in the negative-ion mode. In either case, the ionization process sometimes strips the Cl-.
- BIOLOGICAL STUDIES Cell Culture and Western Blotting
- the human prostate cancer cell line CWR22Rv1 was procured from ATCC (Manassas, VA) and cultured in the recommended media supplemented with 10% heat-inactivated standard fetal bovine serum (FBS, GIBCO) and 1% penicillin-streptomycin (10,000 U/ml, Life Technologies) at 37 °C and 5% CO 2 .
- LNCaP and C4-2B cell lines were cultured in RPMI-1640 medium with 10% FBS and penicillin/streptomycin (100 U/ml in a humidified incubator with 5% CO2 at 37°C.
- Cell proliferation analysis was performed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) as previously described [NPL-37 and NPL-10]. Briefly, 2500 cells/well (CWR22Rv1, LNCaP, and C4-2B) were seeded in 96 well plate for 24 h and treated with the indicated concentrations of test compounds for 2nd and 5th day in a span of eight days. Growth inhibitory concentration (GI 50 ) was calculated based on a non-linear regression curve fit using GraphPad prism 5.0 software (La Jolla, CA).
- mice Male CD-1 mice (20 – 35 g) were dosed with the test salts intravenously (IV) or oral gavage (PO) at 1 mg/kg and 10 mg/kg, respectively. The compounds were dissolved in 20% ⁇ - cyclodextrin in saline. Blood samples (0.1 mL were obtained via lateral saphenous vein at 0.083, 0.25, 1, 2, 4, 8, and 24 h time points and collected in heparinized tubes. After collection of blood samples at each time point, the blood samples were stored on ice, prior to centrifugation. Blood samples were centrifuged within 0.25 h of collection to separate plasma. Centrifugation was conducted at 10640rcf, 5 min, 4°C.
- the plasma was separated and transferred to pre-labelled matrix tubes and promptly frozen at –20 ⁇ 10°C. After 8 h time-point, all the plasma samples were transferred to bioanalytical group on dry ice and stored at -80°C until bioanalysis. Samples were identified by test item, group, animal number and collection time point. Test samples concentrations were determined by LC-MS/MS, and the PK parameters were calculated by non- compartmental analysis module in Phoenix WinNonlin® (Version 8.1) (Pharsight, Mountain View, trapezoidal rule) as previously reported by the inventors [NPL-16].
- Absolute Bioavailability (%F) AUC PO /AUC IV ⁇ Dose IV /Dose PO ⁇ 100.
- the mice (age 5–6 weeks) were procured from the Veterinary Resources, University of Maryland School of Medicine (Baltimore, MD, USA), housed under sterile conditions and fed with sterile pellets and water ad libitum. After a week of acclimatization, 3x10 6 cells were subcutaneously injected into the left flank of mice.
- mice After three weeks of inoculation and upon reaching the tumor volume of ⁇ 100 mm 3 , the mice were randomized into 12 groups of five animals, each.
- the control groups of mice were orally administered with vehicle (20% ⁇ -cyclodextrin in saline, PO).
- Docetaxel was administered as IP injection. All other compounds were orally administered as doses as indicated for five days a week for 16 days.
- the animals were monitored daily for general health and body weight recorded three times a week.
- the tumor size was measured thrice a week using digital calipers and tumor volume calculated using the formula length ⁇ width 2 ⁇ 0.5 (mm 3 ).
- mice Upon reaching the tumor length close to 20 mm in control groups (16th day), the experiment was terminated, the animals were euthanized, and tumors were excised for further analyses. All in vivo studies in mice were performed in accordance with the humane use of experimental animals following review and approval by the Institutional Animal Care and Use Committee (IACUC), University of Maryland School of Medicine, Baltimore, MD, USA, per IACUC No. # 0421007 dated 01/31/2022. [00127] Statistical analysis All experiments were carried out in at least triplicates and are expressed as mean ⁇ S.E. where applicable. Treatments were compared to controls using the student’s t-test with either GraphPad Prism or Sigma Plot. Differences between groups were considered statistically significant at P ⁇ 0.05.
- IACUC Institutional Animal Care and Use Committee
- NPL-8 A.K. Kwegyir-Afful, S. Ramalingam, V.P. Ramamurthy, P. Purushottamachar, F.N. Murigi, T.S. Vasaitis, W. Huang, M.A. Kane, Y. Zhang, N. Ambulos, S. Tiwari, P. Srivastava, I.P. Nnane, A. Hussain, Y. Qiu, D.J. Weber, V.C.O.
- VNPP433- 3beta the lead next-generation galeterone analog inhibits prostate cancer stem cells by downregulating epithelial-mesenchymal transition and stem cell markers, Mol Carcinog 61 (2022) 643-654.
- NPL-10 E. Thomas, R.S. Thankan, P. Purushottamachar, W. Huang, M.A. Kane, Y. Zhang, N.P. Ambulos, D.J. Weber, V.C.O.
- NPL-12 H.W. Mbatia, S. Ramalingam, V.P. Ramamurthy, M.S. Martin, A.K. Kwegyir- Afful, V.C.
- Novel C-4 heteroaryl 13-cis-retinamide Mnk/AR degrading agents inhibit cell proliferation and migration and induce apoptosis in human breast and prostate cancer cells and suppress growth of MDA-MB-231 human breast and CWR22Rv1 human prostate tumor xenografts in mice, J Med Chem, 58 (2015) 1900-1914.
- NPL-13 S. Ramalingam, L. Gediya, A.K. Kwegyir-Afful, V.P. Ramamurthy, P. Purushottamachar, H. Mbatia, V.C.
- NPL-14 S. Ramalingam, V.P. Ramamurthy, L.K. Gediya, F.N. Murigi, P. Purushottamachar, W. Huang, E.Y. Choi, Y. Zhang, T.S. Vasaitis, M.A. Kane, R.G. Lapidus, V.C.O.
- Patilea-Vrana G. Gampa, H.K. Seneviratne, J.D. Unadkat, J.R. Kenny, K. Nagapudi, L. Di, L. Zhou, M. Zak, M.R. Wright, N.N. Bumpus, R. Zang, X. Liu, Y. Lai, S.C. Khojasteh, Drug Concentration Asymmetry in Tissues and Plasma for Small Molecule-Related Therapeutic Modalities, Drug Metab Dispos, 47 (2019) 1122-1135. [NPL-20] S. Ramalingam, V.P. Ramamurthy, L.K. Gediya, F.N. Murigi, P.
- NPL-29 C. Liu, W. Lou, Y. Zhu, N. Nadiminty, C.T. Schwartz, C.P. Evans, A.C. Gao, Niclosamide inhibits androgen receptor variants expression and overcomes enzalutamide resistance in castration-resistant prostate cancer, Clin Cancer Res, 20 (2014) 3198-3210.
- NPL-30 S. Qu, K. Wang, H. Xue, Y. Wang, R. Wu, C. Liu, A.C. Gao, P.W. Gout, C.C. Collins, Y.
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Abstract
A salt of 3β-(hydroxy)-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (Compound 1) or a salt of 3β-(1H-imidazole-1-yl-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (Compound 2), wherein the salt is at least one selected from the group consisting of a hydrochloride, a dihydrochloride, a sodium, a tosylate, a mesylate, a succinate, a sulfate, a disulfate, a maleate, a fumarate, a phosphate, and a diphosphate salt; a pharmaceutical composition, comprising the salt; a method of treating cancer, benign prostatic hyperplasia (BPH), and/or Kennedy's disease in a subject, comprising administering the salt to the subject; a method of prolonging survival of a subject having cancer, comprising administering the salt to the subject; a method of inducing androgen receptor (AR) degradation in a subject comprising administering the salt to the subject; and a method of inducing Mnk1/2 transcriptional activity degradation in a subject, comprising administering the salt to the subject.
Description
SALTS OF GALETERONE AND SALTS OF NEXT GENERATION GALETERONE ANALOGS, AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Application No.63/419,139, filed October 25, 2022, which is incorporated herein by reference in its entirety. STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] This invention was made with government support under Grant No. CA224696 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD [0003] The disclosed embodiments relate to salts of pharmaceutical compounds for use in the treatment of diseases. The disclosed embodiments further relate to methods of treatment of diseases, such as cancer in humans, using pharmaceutical compounds. BACKGROUND OF INVENTION [0004] Drugs and lead candidates in the drug discovery and development process are often formed as a weak acid or base, but this form is not always optimal for dissolution or absorption into the body [NPL-1, NPL-2]. Without absorption, a drug cannot have a therapeutic effect, so some forms require salinification, i.e., salt formation. Many medications need to be water- soluble as well. Therefore, drugs are often chemically made into their salt forms to enhance how the drug dissolves, boost its absorption into the body’s bloodstream, and increase its effectiveness. Drugs with poor aqueous solubility are liable to have low and variable oral bioavailability [NPL-3, NPL-4]. As a result, it is important to identify solubility enhancement strategies for drugs early in the drug discovery and development process. [0005] Galeterone, 3β-(hydroxy)-17-(1H-benzimidazole-1-yl)androsta-5,16-diene, (Gal or Compound 1) is a potent drug that simultaneously targets AR/AR-V7 and Mnk1/2 [NPL-5]. Although Gal successfully advanced through Phase II clinical trials in treating metastatic castration-resistance prostate cancer (mCRPC), it faced setbacks in the pivotal Phase III clinical
trials due to the limited number of patients studied, and the need to administer relatively higher clinical doses [NPL-6]. Consequently, a series of Gal analogs were designed and synthesized. These are next generation galeterone analogs (NGGAs), and they were evaluated for their potential development to treat all stages of prostate cancer (PC) focusing on enhanced efficacy, safety, and minimum effective dose [NPL-7] . VNPP433-3β, 3β-(1H-imidazole-1-yl-17-(1H- benzimidazole-1-yl)androsta-5,16-diene (Compound 2) is an NGGA with high therapeutic index (TI), a lower minimum effective dose, and compelling therapeutic effects in castration-resistant drug-naive PC preclinical models in vitro and in vivo [NPL-8], and which inhibits prostate cancer stem cells [NPL-9]. The molecular mechanisms of action and efficacy VNPP433-3β in AR-overexpressing castration resistant prostate cancer in vitro and in vivo models were reported [NPL-10]. As VNPP433-3β effectively disrupts multiple cellular pathways that drive PC development, progression, and metastasis, it could possibly thwart therapy resistance, making it an excellent candidate for clinical development [NPL-10]. While such early results are promising, there continues to be a need to improve the efficacy of Gal, NGGAs (e.g., VNPP433- 3β) and salts thereof. BRIEF SUMMARY OF INVENTION [0006] With the aim of improving aqueous solubility, oral bioavailability and hence reducing the administered dosage of Gal and VNPP433-3β, the inventors set about on the development and discovery of new salt forms of these two related compounds. As described herein, and after concerted efforts, new salt forms of Gal and VNPP433-3β were developed. For example, hydrochloride, dihydrochloride, sodium, tosylate, mesylate, succinate, sulfate, disulfate, maleate, fumarate, phosphate, and diphosphate salts were developed. The present invention is directed to these compounds and to methods of using these compounds in treatment of diseases, such as cancer. [0007] In a first embodiment, the invention is directed to a salt of Galeterone (Compound 1), where the salt may be, but is not limited to, a hydrochloride, a dihydrochloride, a sodium, a tosylate, a mesylate, a succinate, a sulfate, a disulfate, a maleate, a fumarate, a phosphate, and a diphosphate salt.
(Compound 1) [0008] In particular aspects of this embodiment, the salt of Compound 1 is selected from the group consisting of: H+ Cl- O H + N N S O- N O
, , , O H + H+ S O- N N O- O-
Compound 15, Compound 18,
Compound 20, and Compound 22. [0009] In a second embodiment, the invention is directed to a salt of VNPP433-3β (Compound 2), where the salt may be, but is not limited to, a hydrochloride, a dihydrochloride, a sodium, a tosylate, a mesylate, a succinate, a sulfate, a disulfate, a maleate, a fumarate, a phosphate, and a diphosphate salt.
VNPP433-3β (2) (Compound 2) [0010] In particular aspects of this second embodiment, the salt of Compound 2 is selected from the group consisting of:
Compound 4, Compound 5, Compound 8,
C
Compound 16, Compound 17,
Compound 23, and Compound 24. [0011] In a third embodiment, the invention is directed to Compound 3
Compound 3 . [0012] In a fourth embodiment, the invention is directed to Compound 4
Compound 4 . [0013] In a fifth embodiment, the invention is directed to Compound 5
Compound 5 . [0014] In a sixth embodiment, the invention is directed to a pharmaceutical composition, comprising any one of Compounds 1-24 and a pharmaceutically acceptable carrier, excipient and/or diluent. [0015] In a seventh embodiment, the invention is directed to a method of treating a disease, such as cancer, comprising administering a therapeutically effective amount of a salt of Compound 1 and/or a salt of Compound 2 to a subject in need thereof. [0016] In one aspect of the seventh embodiment, the salt of Compound 1 is at least one of Compounds 3, 6, 7, 10, 13, 15, 18, 20 and 22. In another aspect of this embodiment, the salt of Compound 2 is at least one of Compounds 4, 5, 8, 9, 11, 12, 14, 16, 17, 19, 21, 23 and 24. [0017] In a further aspect of the seventh embodiment, the disease is at least one type of cancer, such as prostate cancer, bladder cancer, pancreatic cancer, breast cancer, a hematological cancer, and hepatocellular carcinoma, or benign prostatic hyperplasia, or Kennedy’s disease. In certain aspects, the prostate cancer is castration-resistant prostate cancer. [0018] In an eighth embodiment, the invention is directed to a method of prolonging survival of a subject having cancer, comprising administering a therapeutically effective amount of a salt of Compound 1 and/or a salt of Compound 2 to the subject. [0019] In one aspect of this eighth embodiment, the salt of Compound 1 is at least one of Compounds 3, 6, 7, 10, 13, 15, 18, 20 and 22. In another aspect of this embodiment, the salt of Compound 2 is at least one of Compounds 4, 5, 8, 9, 11, 12, 14, 16, 17, 19, 21, 23 and 24. [0020] In a further aspect of this eighth embodiment, the disease is a cancer, such as prostate cancer, bladder cancer, pancreatic cancer, breast cancer, a hematological cancer, and hepatocellular carcinoma, or benign prostatic hyperplasia, or Kennedy’s disease. In certain aspects, the prostate cancer is castration-resistant prostate cancer. [0021] In a ninth embodiment, the invention is a method of inducing androgen receptor (AR) degradation in a subject, comprising administering a therapeutically effective amount of a salt of Compound 1 and/or a salt of Compound 2 to a subject in need thereof. [0022] In a tenth embodiment, the invention is a method of inducing Mnk1/2 transcriptional activity degradation in a subject, comprising administering a therapeutically effective amount of a salt of Compound 1 and/or a salt of Compound 2 to a subject in need thereof.
BRIEF DESCRIPTION OF THE DRAWINGS [0023] FIG.1. Chemical structures of Galeterone (Compound 1) and VNPP433-3β (Compound 2). [0024] FIG.2. Antiproliferative effects of Galeterone (Compound 1) and VNPP433-3β (Compound 2) compared to their corresponding salts (Compound 3, Compound 4 and Compound 5), Enzalutamide (ENZ) and Abiraterone (ABI) against CWR22Rv1 human prostate cancer cells in vitro. [0025] FIG.3A and FIG.3B. Effects of Compounds 1 and 3 (FIG.3A) and Compounds 2 and 4 (FIG.3B) compared to Enzalutamide (ENZ) on the expression of fAR, AR-V7, Mnk1, peIF4E, eIF4E, Bcl2, BAX and cyclin D1 in human CWR22Rv1 prostate cancer cells in vitro. [0026] FIG.4A, FIG.4B and FIG.4C are plasma pharmacokinetics profiles of Compounds 3, 4 and 5, respectively, after intravenous (IV) and oral (PO) administration to CD-1 male mice. Each point represents the mean ± standard deviation of the mean (SDM) in three mice. [0027] FIGS.5A, 5B, 5C and 5D. Male NRG mice bearing CWR22Rv1 tumors were treated with vehicle, Compounds 1, 2, 3 and 4 for 16 days, and tumor volumes were measured twice a week and the tumors were collected. FIG.5A shows the percent tumor growth inhibition (%TGI) values at the right of each growth curve, and the error bars are the SEM. Results are represented as means ± SEM. All p vales are compared to vehicle control: *p < 0.0256, **p < 0.0104, ***p < 0.0041. FIG.5B shows the average tu *
weights from each experimental group at day 16: p < 0.0256, **p < 0.0104, ***p < 0.0041, ****p < 0.0069. FIG.5C is a photograph of excised tumors from each group at the end of the study. FIG.5D shows the mean body weights of mice determined during the study. [0028] FIGS.6A, 6B, 6C, 6D and 6E. A head-to-head comparison of the antitumor activity of Gal (Compound 1), Gal.HCl (Compound 3), Enzalutamide (ENZ), and Docetaxel (DTX) in human CRPC CWR22Rv1 tumor xenograft model is provided. Mice bearing CWR22Rv1 xenografts (5 mice per group) were treated with vehicle, Compound 1 (100 mg/kg/twice daily), Compound 3 (24, 48 or 96 mg/kg, once daily), ENZ (25 mg/kg, once daily) 5 days per week for 16 days, and DTX (5 mg/kg, IP, 3 times per week). In FIG.6A, tumor volumes were measured twice a week and the tumors were collected. Percent tumor growth inhibition (%TGI) values are indicated at the right of each growth curve, and the error bars are the SEM. Results are
represented as means ± SEM. All p vales are compared to vehicle control: 1p < 0.0480, 2p < 0.0409, 3p < 0.0013, 4p < 0.0005, 5p < 0.0001 and 6p < 0.0001. FIG.6B provides the average tumor weights from each experimental group at day 16; *p < 0.0001 versus control for all the treated groups. FIG.6C provides the total percent change in mean tumor volumes of the initial volume after 16 days as shown as a waterfall plot. FIG.6D provides the percent change in volume for each tumor (5 tumors per treatment group) after 16 days as shown as a waterfall plot. FIG.6E provides the mean body weights of mice determined during the study. [0029] FIGS.7A, 7B, 7C, 7D and 7E. A head-to-head comparison of the antitumor activity of Compounds 2, 4 and 5, Enzalutamide (ENZ), and Docetaxel (DTX) in human CRPC CWR22Rv1 tumor xenograft model is provided. Mice bearing CWR22Rv1 xenografts (5 mice per group) were treated with vehicle, Compound 2 (20 mg/kg, once daily), Compound 4 (5 or 20 mg/kg, once daily), Compound 5 (5 or 20 mg/kg, once daily), ENZ (25 mg/kg, once daily) 5 days per week for 16 days, and DTX (5 mg/kg, IP, 3 times per week). In FIG.7A, tumor volumes were measured twice a week and the tumors were collected. Percent tumor growth inhibition (%TGI) values are indicated at the right of each growth curve, and the error bars are the SEM. Results are represented as means ± SEM. All p vales are compared to vehicle control: 1p < 0.0360, 2p < 0.0303, 3p < 0.0001, 4p < 0.0008, 5p < 0.0006, 6p < 0.0001 and 7p < 0.0001. FIG.7B provides the average tumor weights from each experimental group at day 16; *p < 0.0001 versus control for all the treated groups. FIG.7C provides the total percent change in mean tumor volumes of the initial volume after 16 days as shown as a waterfall plot. The p values are as in FIG.7A. FIG.7D is the percent change in volume for each tumor (5 tumors per treatment group) after 16 days as shown as a waterfall plot. FIG.7E shows the mean body weights of mice determined during the study. [0030] FIG.8A is a photograph of all the excised tumors after termination of the study. FIG. 8B is Western blot analyses of tumor samples from each experimental group after termination of the study. [0031] FIG.9A. The quantification analysis of the proteins analyzed in FIG.3A on the expression of fAR, AR-V7, Mnk1, peIF4E and eIF4E. [0032] FIG.9B. The quantification analysis of the proteins analyzed in FIG.3B on the expression of fAR, AR-V7, Mnk1, peIF4E and eIF4E.
[0033] FIG.10. 1H-NMR of Compound 1 in CDCl3 [0034] FIG.11. 13C-NMR of Compound 1 in CDCl3 [0035] FIG.12. 1H-NMR of Compound 3 in CDCl3 [0036] FIG.13. 13C-NMR of Compound 3 in CDCl3 [0037] FIG.14. HRMS spectra of Compound 3 [0038] FIG.15. 1H-NMR of Compound 2 in CDCl3 [0039] FIG.16. 13C-NMR of Compound 2 in CDCl3 [0040] FIG.17. 1H-NMR of Compound 4 in CDCl3 [0041] FIG.18. 13C-NMR of Compound 4 in CDCl3 [0042] FIG.19. HRMS spectra of Compound 4 [0043] FIG.20. 1H-NMR of Compound 2 in DMSO-d6 [0044] FIG.21.13C-NMR of Compound 2 in DMSO-d6 [0045] FIG.22. 1H-NMR of Compound 5 in DMSO-d6 [0046] FIG.23.13C-NMR of Compound 5 in DMSO-d6 [0047] FIG.24. HRMS spectra of Compound 5 DETAILED DESCRIPTION OF THE INVENTION [0048] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technical references. [0049] As used herein, “a” or “an” may mean one or more. As used herein when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular. [0050] As used herein, “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about”
generally refers to a range of numerical values (e.g., +/- 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure. [0051] As used herein, the terms “dose”, “unit dose”, “dosage”, “effective dose”, “therapeutically effective dose”, and related terms refer to physically discrete units that contain a predetermined quantity of active ingredient or therapeutic drug (agent) calculated to produce a desired therapeutic effect. A single dose is thus a predetermined quantity of an agent of the invention that is administered to a subject. [0052] As used herein, a “subject” is a human, a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal. [0053] Disruption and/or perturbation of cap-dependent translation is essential for the development of cancers. Hyper-activation of eukaryotic translation initiation factor 4E (eIF4E), the mRNA 5’ cap-binding protein of cap-dependent translation promotes exquisite transcript- specific translation of key mRNAs that are indispensable in cancer initiation, progression and metastases. The oncogenic potential of eIF4E is dependent on serine 209 phosphorylation by MAPK-interacting kinases 1 and 2 (Mnk1/2). Given the implication of Mnk1/2-eIFE axis in the initiation and progressions of all types of solid tumors and hematologic cancers, successful agents for the prevention and treatment of all forms of breast and prostate cancers and other diseases will block the activity of Mnk 1/2. [0054] Androgen receptors (AR) are a well-established target for therapeutic intervention in certain diseases, including prostate cancer, bladder cancer, pancreatic cancer, breast cancer, a hematological cancer, hepatocellular carcinoma, benign prostatic hyperplasia (BPH) and Kennedy’s disease. [0055] Galeterone analogs are of substantial interest because of their multi-target anticancer activities, including AR and Mnk degrading activities. In the course of studies to develop potent androgen receptor degrading agents (ARDAs), novel ARDAs were discovered that also effectively target oncogenic eukaryotic protein translation, via modulation of Mnk-eIF4E axis, so as to function as Mnk degrading agents (MNKDAs) that suppress oncogenic peIF4E via degradation of Mnk1 and 2. These targets have been implicated in the development, progression,
metastasis and drug resistance of a variety of cancers, including prostate and pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC), as well as breast cancer and hematological cancers. [0056] As noted above, the inventors developed salts of Galeterone and VNPP433-3β in order to improve aqueous solubility and oral bioavailability of the compounds. The resulting changes to the compounds allow a reduced amount of the compounds to be administered to a subject, such during treatment of a medical condition. Exemplary salts of the compounds include hydrochloride, dihydrochloride, sodium, tosylate, mesylate, succinate, sulfate, disulfate, maleate, fumarate, phosphate, and diphosphate salts. [0057] In particular, the present invention is directed to the following specific compounds: salts of Galeterone including Gal-HCl (or Gal.HCl) (Compound 3), Gal-sodium (Compound 6), Gal-tosylate (Compound 7), Gal-mesylate (Compound 10), Gal-succinate (Compound 13), Gal- sulfate (Compound 15), Gal-Maleate (Compound 18), Gal-fumarate (Compound 20), and Gal- phosphate (Compound 22). [0058] The present invention is also directed to the following specific compounds: salts of VNPP433-3β, including VNPP433-3β HCl (Compound 4), VNPP433-3β dihydrochloride (Compound 5), VNPP433-3β tosylate (Compound 8), VNPP433-3β ditosylate (Compound 9), VNPP433-3β mesylate (Compound 11), VNPP433-3β (Compound 12), VNPP433-3β succinate (Compound 14), VNPP433-3β-sulfate (Compound 16), VNPP433-3β-disulfate (Compound 17), VNPP433-3β-maleate (Compound 19), VNPP433-3β-fumarate (Compound 21), VNPP433-3β- phosphate (Compound 23), and VNPP433-3β-diphosphate (Compound 24). [0059] In particular, the hydrochloride salts of Gal (Compound 1) and VNPP433-3β (Compound 2) were synthesized and shown to have improved aqueous solubility and enhanced in vitro and in vivo anticancer activities. Three hydrochloride salts of two lead compounds were prepared and biologically evaluated (Compounds 3, 4 and 5), with the results provided below. Compounds 3, 4 and 5 exhibit drastically improved anticancer activities and are statistically significantly more potent than their parent compounds (Compounds 1 and 2). Further, the HCl salts are significantly more potent than two FDA-approved prostate cancer drugs, Enzalutamide (ENZ) and Docetaxel (DTX), as shown in the experimental data provided herein. [0060] The disclosed compounds can be used in the treatment of a variety of diseases, including cancers, and in prolonging survival in subjects having a variety of diseases, including
cancers. It will be apparent that particular combinations of Compounds may differ in effectiveness depending on the type of cancer, the stage and grade of a particular cancer, the physical location of the cancer within the subject, the molecular abnormalities in the cancer, and available means for administering the Compounds, among other factors. [0061] Exemplary diseases that may be treated via the methods of the invention include, but are not limited to, cancer, such as prostate cancer, bladder cancer, pancreatic cancer, breast cancer, a hematological cancer, and hepatocellular carcinoma, or benign prostatic hyperplasia (BPH) or Kennedy’s disease. Pharmaceutical Compositions [0062] The compounds of the invention (i.e., Compounds 1-24) may be used themselves or as agents in various methods, including but not limited to methods of treating diseases as described below. The compounds used in the methods may be formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient and/or diluent. It will be apparent that depending on the identity of the compound being used, a suitable pharmaceutical composition may comprise a single compound or combinations comprising two or more compounds, and may include a single carrier, excipient and/or diluent, or a combination of carrier(s), excipient(s) and/or diluent(s). [0063] The pharmaceutical compositions may be formulated, for example, for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, pulmonary, topical or parenteral administration. Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (IM), intravenous (IV), intraperitoneal (IP), intra-arterial, intramedulary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Any known device useful for parenteral injection or infusion of formulations can be used to affect such administration. In preferred aspects of each of the embodiments on the invention, the pharmaceutical composition is administered to the subject as an intravenous (IV) formulation. [0064] Pharmaceutically acceptable carriers, excipients and diluents are those ingredients, solutions, substances or materials that can be used to produce formulations that are suitable to be administered to a subject, such as a human. In particular, carriers, excipients and diluents of the
present invention are those useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and that may present pharmacologically favorable profiles, and includes carriers and diluents that are acceptable for veterinary use as well as human pharmaceutical use. Suitable pharmaceutically acceptable carriers, excipients and diluents are well known in art and can be determined by those of skill in the art as the clinical situation warrants. Examples of suitable carriers and diluents include dextrose, water, glycerol, ethanol, propylene glycol, polysorbate 80 (Tween-80™), poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (e.g., Cremophor EL), poloxamer 407 and 188, a cyclodextrin or a cyclodextrin derivative (including HPCD ((2- hydroxypropyl)-cyclodextrin) and (2-hydroxyethyl)-cyclodextrin), hydrophilic and hydrophobic carriers, and combinations thereof. Hydrophobic carriers include, for example, fat emulsions, lipids, PEGylated phospholipids, polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes. The terms specifically exclude cell culture medium. More particularly: (1) 5% (w/v) dextrose, or (2) water (e.g., sterile water; Water-For-Injection), may be used as a pharmaceutically acceptable carrier. Pharmaceutically acceptable diluents also include tonicity agents that make the composition compatible with blood. Tonicity agents are particularly desirable in injectable formulations. [0065] Excipients included in a formulation have different purposes depending, for example on the nature of the agent, and the mode of administration. Examples of generally used excipients include, without limitation: stabilizing agents, solubilizing agents and surfactants, buffers, antioxidants and preservatives, tonicity agents, bulking agents, lubricating agents, emulsifiers, suspending or viscosity agents, inert diluents, fillers, disintegrating agents, binding agents, wetting agents, lubricating agents, antibacterials, chelating agents, sweeteners, perfuming agents, flavoring agents, coloring agents, administration aids, and combinations thereof. [0066] The pharmaceutical compositions may contain common carriers and excipients, such as cornstarch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, alginic acid, croscarmellose sodium, and sodium starch glycolate. [0067] The particular carrier, diluent and excipient used will depend upon the means and purpose for which the active ingredient (i.e., Compounds 1-24) is being applied.
[0068] Acceptable methods for preparing the pharmaceutical compositions according to the invention are known to those skilled in the art. For example, pharmaceutical compositions may be prepared following conventional techniques of the pharmaceutical chemist involving steps such as mixing, granulating, and compressing when necessary for tablet forms, or mixing, filling, and dissolving the ingredients as appropriate, to give the desired products for various routes of administration. Methods of Treatment [0069] As summarized discussed above, the present invention is also directed to methods of treating diseases, including cancer, or prolonging survival in a subject having diseases, including cancer, by administering therapeutically effective amounts of one or more of salts of Galeterone (Compound 1) and/or one or more salts of VNPP433-3β (Compound 2), such as Compound 3, Compound 4 or Compound 5. [0070] The salts of Compound 1 include hydrochloride, dihydrochloride, sodium, tosylate, mesylate, succinate, sulfate, disulfate, maleate, fumarate, phosphate, and diphosphate salts. [0071] The salts of Compound 2 include hydrochloride, dihydrochloride, sodium, tosylate, mesylate, succinate, sulfate, disulfate, maleate, fumarate, phosphate, and diphosphate salts. [0072] In a particular aspect, the disclosed embodiments are directed to a method of treating cancer, particularly prostate cancer, in a subject comprising administering therapeutically effective amounts of a salt of Galeterone to a subject having the cancer, particularly prostate cancer. The salts of Galeterone include Gal-HCl (Compound 3), Gal-sodium (Compound 6), Gal-tosylate (Compound 7), Gal-mesylate (Compound 10), Gal-succinate (Compound 13), Gal- sulfate (Compound 15), Gal-Maleate (Compound 18), Gal-fumarate (Compound 20), and Gal- phosphate (Compound 22). [0073] In a particular aspect, the disclosed embodiments are also directed to a method of treating cancer, particularly prostate cancer, in a subject comprising administering therapeutically effective amounts of a salt of VNPP433-3β to a subject having the cancer, particularly prostate cancer. The salts of VNPP433-3β include VNPP433-3β HCl (Compound 4), VNPP433-3β dihydrochloride (Compound 5), VNPP433-3β tosylate (Compound 8), VNPP433- 3β ditosylate (Compound 9), VNPP433-3β mesylate (Compound 11), VNPP433-3β (Compound
12), VNPP433-3β succinate (Compound 14), VNPP433-3β-sulfate (Compound 16), VNPP433- 3β-disulfate (Compound 17), VNPP433-3β-maleate (Compound 19), VNPP433-3β-fumarate (Compound 21), VNPP433-3β-phosphate (Compound 23), and VNPP433-3β-diphosphate (Compound 24). [0074] Exemplary diseases that may be treated via the methods of the invention include, but are not limited to, cancers, such as prostate cancer, bladder cancer, pancreatic cancer, breast cancers, a hematological cancer, and hepatocellular carcinoma, or benign prostatic hyperplasia or Kennedy’s disease. In a particular aspect, the prostate cancer is castration-resistant prostate cancer. [0075] The terms “treating” cancer and “treatment” of cancer mean at least the mitigation of cancer, or a disease condition or symptom associated with cancer in a subject that is achieved by a reduction of growth, replication, and/or propagation, or death or destruction of cancer and/or cancer cells, on or in the subject. The terms “treating” and “treatment” include curing, healing, inhibiting, relieving from, improving and/or alleviating, in whole or in part, the cancer or associated disease condition or symptom. The mitigation of cancer or associated disease condition or symptom may be about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% in the subject, versus a subject to which a compound of the invention (i.e., Compounds 1-24) has not been administered. In one aspect, treating means reducing the population of cancer cells causing the cancer in the subject to an undetectable level, where detection is by any conventional means, such as assay a blood sample in the laboratory. In another aspect, treating means complete healing of the cancer, shown by an absence of clinical symptoms associated with the cancer. In a further aspect of the invention, treating means the mitigation of cancer or an associated disease condition or symptom by at least about 90% in the subject. In an additional aspect, treating means the mitigation of cancer or an associated disease condition or symptom by at least about 95% in the subject. [0076] The disclosed methods also include methods of prolonging survival of a subject having cancer comprising administering a therapeutically effective amount of a salt of Compound 1 to a subject having cancer. The salt of Compound 1 and the cancers are those defined herein. In one aspect, the cancer is prostate cancer, such as castration-resistant prostate cancer.
[0077] The disclosed methods also include methods of prolonging survival of a subject having cancer comprising administering a therapeutically effective amount of a salt of Compound 2 to a subject having cancer. The salts of Compound 2 and the cancers are those defined herein. In one aspect, the cancer is prostate cancer, such as castration-resistant prostate cancer. [0078] The term “prolonging survival” means extending the life span of a subject having cancer by at least one day versus a subject having the same cancer that does not receive a compound of the invention. Prolonged survival includes increasing the life span of the subject by at least: 1, 2, 3, 4 or more weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months, or 1, 2, 3, 4, 5, or more years. [0079] The amount (dose) of the compound of the invention (Compounds 1-24) sufficient to have an effect on cancer (additive, synergistic or otherwise) in a subject will vary, for example, in view of the identity of the compound being used in the combination, the physical characteristics of the subject, the severity of the subject’s symptoms, the form of the cancer, the identity of the cancer, the formulations and means used to administer the compound, and the method being practiced. The specific dose for a given subject is usually set by the judgment of the attending physician. [0080] The daily dose of the salt of Compound 1 or the salt of Compound 2 may be in the range of 0.1 mg/kg to 1000 mg/kg per day, 1 mg/kg to 100 mg/kg per day, 1 mg/kg to 100 mg/kg, or 24 mg/kg to 96 mg/kg. In the case of Compound 3, the daily dose may be in the range of 1 mg/kg to 100 mg/kg or 5 mg/kg to 20 mg/kg. In the case of Compound 4, the daily dose may be in the range of 1 mg/kg to 100 mg/kg or 5 mg/kg to 20 mg/kg. In the case of Compound 5, the daily dose may be in the range of 1 mg/kg to 100 mg/kg or 5 mg/kg to 20 mg/kg. [0081] The timing of administration used in the methods of the invention will vary depending on a number of factors, including whether there is concurrent or sequential administration, the identity of the compound of the invention, the identity of the cancer, the physical characteristics of the subject, the severity of the subject’s symptoms, and the formulation and the means used to administer the compounds, among other factors. However, administration frequencies of the agents will generally include 4, 3, 2 or once daily, every other day, every third day, every fourth day, every fifth day, every sixth day, once weekly, every eight
days, every nine days, every ten days, bi-weekly, monthly and bi-monthly, whether the drugs are administered alone or in combination, concurrently or sequentially. In certain aspects, the concurrent or sequential administration is administration once daily. The duration of treatment will be based on the cancer being treated and will be best determined by the attending physician. Under some conditions, treatment will be continued for a number of days, weeks, or months. Under other conditions, complete treatment will be achieved through administering one, two or three doses of the combinations over the entire course of treatment. [0082] The pharmaceutical compositions and the compounds of the invention may be administered via means that include oral, enteral, sublingual, intranasal, intraocular, rectal, intravaginal, transdermal, mucosal, topical or parenteral administration. Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (IM), intravenous (IV), intraperitoneal (IP), intra-arterial, intramedullary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Any known device useful for parenteral injection or infusion of agents and formulations can be used to affect such administration. In certain aspects of each of the embodiments of the invention, the compounds and pharmaceutical compositions are administered to the subject intravenously. [0083] Depending on the means of administration, the dose may be administered all at once, such as with an oral formulation in a capsule, or slowly over a period of time, such as with an intravenous (IV) administration. For slower means of administration, the administering period can be a matter of minutes, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or more minutes, or a period of hours, such as about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 or more hours. The administration of the dose may be interrupted, such as where the dose is administered via intravenous infusion and the dose is divided into two or more infusion bags. Under such circumstances, the administration of the dose may be interrupted while the infusion bags are changed.
EXAMPLES Example 1: Design, synthesis, and characterization of Gal and VNPP433-3β salts [0084] Hydrochloride salts of Compound 1 and Compound 2 were synthesized and tested. The synthesis of Gal.HCl (Compound 3) is presented in Scheme 1. Compound 1, dissolved in isopropyl alcohol (IPA) was treated with anhydrous 3M methanolic-HCl (i.e., 3 times excess molar ratio) followed by stirring at room temperature for 12 hours to provide the desired product in quantitative yield. [0085] N + - N HCl N N i HO HO Galeterone (1) 3 Scheme 1: Synthesis of Galeterone HCl (Compound 3) Reagents and conditions: i) IPA, 3M HCl in MeOH, room temperature (rt.) [0086] The synthesis of VNPP433-3β hydrochloride (Compound 4) (Scheme 2) was achieved by treating a mole of Compound 2 in methanol with half-molar ratio of anhydrous 3M methanolic-HCl at room temperature. The salt formation is preferential with imidazole moiety over benzimidazole ring due to its stronger nucleophilic nature. For the synthesis of VNPP433- 3β dihydrochloride (Compound 5), Compound 2 IPA/methanol followed by treatment with 3M HCl in methanol (Scheme 2). Both VNPP433-3β salts were obtained in quantitative yields. [0087]
VNPP433-3β (2) Scheme 2: Synthesis of Compound 4 and Compound 5 Reagents and conditions: i) MeOH, 3M HCl in MeOH, rt.; ii) IPA/MeOH, 3M HCl in MeOH, rt. [0088] The three salts (Compounds 3, 4 and 5) were characterized by analysis of their 1H- NMR compared to the respective parent compounds. The position of protonation is evident from NMR chemical shift of corresponding ring hydrogens and to some extent hydrogens of steroid skeleton which are closer to protonated nitrogen. The protonated nitrogen atom causes downfield shift effect on protons of adjacent carbons. The difference in chemical shifts observed among important protons in both free base and HCl salts of all compounds are shown in Table 1. There are significant chemical shift differences between Compound 1 (Gal free base) and Compound 3 (Gal.HCl salt) for the protons in the vicinity of salt forming nitrogen atom. The chemical shift of protons at C-2’ in Gal and its HCl salt are δ 7.96 and 9.26, respectively, with difference of 1.3 ppm. As the C-2’ proton is close to salt forming nitrogen, its chemical shift difference is the
highest among protons of benzimidazole ring. Surprisingly, C16 proton of steroid skeleton, has chemical shift difference of 0.33 ppm, which makes its chemical shift slightly more than other protons of the benzimidazole ring. The resonance of π bond electrons between C16-C17 and benzimidazole may be the reason for the observed difference. The remaining protons in the steroid skeleton away from C17 do not have any chemical shift effect. [0089] Similarly, the protons of imidazole ring at C3 in VNPP433-3β mono-HCl (Compound 4) also experience significant chemical shifts. The proton at C-2” has highest difference in chemical shift of 1.13 ppm. Modest downfield shifts were observed for the other protons of the imidazole moiety (0.21 ppm between free base and salt). Unlike C16 proton chemical shift in Compound 3 (Gal-HCl), the C3 proton in steroid skeleton of VNPP433-3β mono-HCl (Compound 4) has minimum effect on chemical shift (0.23 ppm), which might be due to absence of π bond between imidazole ring and C2-C3 or C3-C4 bonds. The fact that chemical shifts were observed in the imidazole protons with no difference in chemical shift in the benzimidazole moiety is a strong piece of evidence for the mono-HCl formation, specifically on imidazole ring. The shift in the aromatic protons of both imidazole at C3 and benzimidazole at C17 including C16 and C3 in the case of VNPP433-3β-dihydrochloride (Compound 5) indicates successful formation of the dihydrochloride salt. Chemical shift differences were also observed in the 13C- NMR spectrum between free bases and salts. Unlike the 1H-NMR, the chemical shifts are shifted up field, in agreement with literature precedence [NPL-11]. The 1H, 13C spectral data is provided in the METHODS section below and FIGS.12-24.
Proton Compound 1 Compound 3 Compound 2 Compound 4 *Compound 5 number (Mono-HCl of (Mono-HCl of (Di-HCl of Compound 1) Compound 2) Compound 2)
Deuterium solvents used and chemical shift in δ ppm
Table 1: 1H-NMR chemical shifts of Compound 1 and Compound 2 compared to their respective hydrochloride salts, Compound 3, Compound 4 and Compound 5. *Di-HCl salt of Compound 2 (Compound 5) is insoluble in CDCl3, therefore, the chemical shifts data for Compound 5 in DMSO-d6 solvent is provided. Important protons where chemical shifts were observed upon salination are proton numbers 16, 2’, 2”, 4” and 5”. Bold-face indicates important protons where chemical shifts were observed in CDCl3 solvent or DMSO-d6 solvent.
Example 2: In Vitro Cellular Antiproliferative Activities Against Three Prostate Cancer Cell Lines [0090] The three salts (Compounds 3, 4 and 5) were evaluated over a range of concentrations to examine their antiproliferative activities against the following three different prostate cancer cell lines of (1) LNCaP (androgen-sensitive), (2) C42B (androgen-insensitive), and (3) CWR22Rv1 (castration-resistant), using the well-established 3-(4,5-dimethylthiazolyl-2)-2,5- diphenyl tetrazolium bromide (MTT) assay [NPL-12, NPL-13, NPL-14]. Compound 1 (Gal)was used as the positive control and two FDA approved prostate cancer drugs, Abiraterone (ABI) and Enzalutamide (ENZ), were included in the assays for the sake of comparison with Compound 1, Compound 2, and their salts (Compounds 3, 4 and 5). Typical dose-response curves are presented in FIG.2, and the GI50 values from all compounds against the three PC cell lines are presented in Table 2. [0091] FIG.2 shows the antiproliferative effects of parent Compounds 1 and 2 compared to their corresponding salts (Compounds 3, 4 and 5), Enzalutamide (ENZ) and Abiraterone (ABI) against CWR22Rv1 human prostate cancer cells in vitro. The dose-response curves were generated from MTT assays after 6-day exposure of different concentrations of the compounds. Each point is an average (mean) of replicates from three independent experiments. The GI50 values were determined from the dose-response curves by nonlinear regression analysis using GraphPad Prism. [0092]
Table 2: Head-to-head comparison of the antiproliferative activities of Compounds 1 to 5 versus FDA-approved prostate cancer drugs, Abiraterone (ABI) and Enzalutamide (ENZ).
[0093] When compared to ABI and ENZ, Compound 1 was found to have more potent antiproliferative activity against the three PC cell lines (GI50 mean = 2.81 vs 11.72 and 14.78 µM, respectively, for ABI and ENZ). Compound 2 and the three salts (Compounds 3, 4, and 5), with GI50 values in the mid-nanomolar range, possess potent antiproliferative activities against all three PC cell lines. Unlike Compound 3 (Gal.HCl), which was found to be 7.4-fold more potent than its parent (Compound 1) with lower GI50 mean values for the three cell lines (GI50 mean = 0.38 vs 2.81 µM), the two salts of parent Compound 2, i.e., Compounds 4 and 5, were found to be equipotent to Compound 2 with the same GI50 mean values for the three cell lines (GI50 mean = 0.27 vs 0.27 µM). The reason for the significantly enhanced antiproliferative activity of Compound 3 compared to Compound 1 is unknown at this time. One possibility could be due to enhanced diffusion of Compound 3 molecules across the cell membrane of these cell lines resulting in enhanced intracellular concentrations and inhibition of its proliferation. It is also reasonable to suggest that the lack of difference in the antiproliferative activities of Compound 2 and its two salts, Compounds 4 and 5, could be due to equivalent cellular diffusions into the PC cells. Example 3. Effects of Gal and VNPP433-3β salts on Mnk1, eIF4E, peIF4E, cyclin D1, Bcl2 and Bax [0094] The inventors previously reported that the antiproliferative effects of Compound 1 and Compound 2 in prostate cancer cells was due to degradation of AR/AR-V7 and Mnk1/2 with consequent inhibition of AR signaling and depletion of peIF4E, respectively, and modulation of the downstream molecular targets [NPL-5, NPL-8, NPL-9, NPL-10]. [0095] The salts, Compound 3 and Compound 4, were further examined for their effects on the degradation of AR/ARV7 and Mnk1 and the other related downstream molecular targets in both CWR22Rv1 prostate cancer cells by Western blotting analysis (FIGS.3A and 3B). The parent compounds (Compounds 1 and 2) and the vehicle, DMSO, were used as positive and negative controls, respectively. The two salts significantly and dose-dependently reduced the expressions of Mnk1 and peIF4E, without noticeable effects on the expression of total eIF4E and the house-keeping protein, β-actin (FIGS.3A and 3B). Compounds 3 and 4 also cause significant
depletion of the downstream target, cyclin D1, and induction of apoptosis via significant downregulation of antiapoptotic Bcl-2 and upregulation of proapoptotic Bax (FIGS.3A and 3B). [0096] FIGS.3A and 3B show the effects of Compounds 1 and 3 (FIG.3A) and Compounds 2 and 4 (FIG.3B) compared to Enzalutamide (ENZ) on the expression of fAR, AR-V7, Mnk1, peIF4E, eIF4E, Bcl2, BAX and cyclin D1 in human CWR22Rv1 prostate cancer cells in vitro. Equal protein concentrations from prostate cancer cells treated with the compounds at different concentration (5, 10 and 20 µM) for 24 h were separated by SDS-PAGE and Western blots probed with the respective protein antibodies. Vehicle-treated cells were included as a control, and all blots were reprobed for GAPDH for loading control. [0097] The potencies of the two salts are enhanced compared to their parent compounds. The reason for this difference is unknown. One possibility could be due to enhanced diffusion of these salts across the cell membrane of this cell line resulting in enhanced intracellular concentrations and modulation of the cellular molecular target proteins. This phenomenon may be similar to observations with cabazitaxel and docetaxel, molecules that share a high degree of structural similarity, where cabazitaxel was taken up into MCF7 human breast cancer cells significantly faster and better intracellular retention than docetaxel and exhibited significantly more potent antiproliferative activity than docetaxel [NPL-15]. Example 4: Plasma Concentrations and Pharmacokinetic Parameters of HCl Salts (Compounds 3, 4 and 5) in male CD-1 Mice. [0098] The inventors previously reported the plasma pharmacokinetics of Compounds 1 and 2 in male CD-1 mice [NPL-8]. In this Example 4, the inventors focused of the plasma pharmacokinetics of the three salts (Compounds 3, 4 and 5) after intravenous (IV) and oral (PO) administration to CD-1 male mice. FIGS.4A-4C depict plasma concentrations versus time profiles, which show that Compound 3 is rapidly cleared from the systemic circulation in mice (T½ = 1.02 and 5.16 h). The significant PK parameters are presented in Table 3.
[0099]
Table 3: Plasma pharmacokinetic parameters of HCl salts, Compounds 3, 4 and 5, after intravenous (IV) and oral (PO) administration to CD-1 male mice Notes: C0: extrapolated plasma concentration at time 0; Cmax: maximum observed plasma concentration; Tmax: time to maximum concentration; T1/2: elimination half-life; AUClast: area under the concentration-time curve from dosing (time 0) to the time of the last measured concentration. Absolute Bioavailability (%F) = AUC PO/AUC IV × Dose IV/Dose PO × 100. [00100] Except from the increased half-life (T½) which was extended by 3.9 h (5.16 h for Compound 3 versus 1.26 h for Compound 1), the oral plasma PK profile of Compound 3 was rather surprising, as it is significantly inferior to the oral plasma PK profile of parent base (Compound 1) reported previously [NPL-8]. Overall, Compound 3 exhibited high clearance, high volume of distribution and low oral bioavailability. The reason(s) for this unexpected result is unknown at this time and would require additional PK studies to also investigate the tissue distribution of Compound 3 following oral administration. The data shows in FIG.5A through FIG.8B that Compound 3 at significantly lower oral doses, and requiring once daily oral dosing, unlike parent Compound 1 that requires high twice daily oral dosing, is exceptionally more efficacious than Compound 1 against aggressive difficult-to-treat CWR22Rv1 human CRPC xenograft tumors. This suggests significant accumulation of Compound 3 in the tumors. There are reports of small molecules that accumulate at higher concentrations in animal tissues versus plasma [NPL-16, NPL-17, NPL-18 and NPL-19], which is very likely the case for Compound 3. An excellent example is the finding that cabazitaxel concentrations were 40-fold higher in
tumors versus plasma observed in a plasma pharmacokinetics and drug distribution in tumors study [NPL-18]. [00101] In contrast to Compound 3, the two HCl salts of parent Compound 2, Compounds 4 and 5, exhibited improved oral PK profiles compared to Compound 2, as the mean maximum concentration (Cmax) and oral bioavailability (%F) were each enhanced by 2-fold (mean Cmax = 1394.2 ng/mL for Compound 4 and Compound 5 versus 706.27 ng/mL for Compound 2) and 3.1-fold (mean %F = 153.08 for Compound 4 and Compound 5 versus 49.5 for Compound 2), respectively. As shown in Table 3, the absolute bioavailability of Compounds 4 and 5 are greater than 100%, indicating complete absorption of the Compounds upon oral administration. These data are in accordance with several of the inventors’ previous reports [NPL-8, NPL-20, NPL-21] as well as others’ reports [NPL-36, NPL-37, NPL-38, NPL-39 and NPL-40] of drugs that have absolute oral bioavailability of more than 100%. This phenomenon is attributed to possible implication of transporter interactions in the systemic disposition of small molecules and drugs [NPL-24, NPL-25 and NPL-26]. [00102] The half-lives (T½) of the two salts, Compound 4 (17.09 h) and Compound 5 (17.51 h), following IV injection were comparable and were extended by ~3 h compared to the parent base, Compound 2. However, it was not possible to calculate the half-lives (0.693/elimination rate constant) of Compounds 4 and 5 following oral administration, because it was not possible to calculate the elimination rate constant (k) which requires a minimum of 3 concentrations, each, in the elimination phase. It is assumed that the relatively high half-lives are likely due to long absorption phases that overlap with the elimination phases. For a similar reason, it was recently reported that reliable values of the half-life of Compound 2 was not determined following oral administration of Compound 2 to male and female rats [NPL-27]. Overall, both Compounds 4 and 5 exhibited low clearance, high volume of distribution and high oral bioavailability. For the sake of clarity, it is important that the PK study in CD-1 mice of the salts (Compounds 3, 4 and 5) was conducted after the CWR22Rv1 xenograft antitumor efficacy studies described above.
Example 5: Antitumor Activity in CWR22Rv1 Tumor Xenograft (Pilot Study). [00103] Because of the promising antitumor efficacies of Compounds 1 and 2 against CRPC CWR22Rv1 xenografts that the inventors previously reported [NPL-8, NPL-10], and the significantly enhanced (7-fold) antiproliferative activity of Compound 3 compared to Compound 1 against the three prostate cancer cell lines, in vitro, a pilot head-to-head comparison study of Compounds 1 and 2 versus their corresponding mono-HCl salts, Compounds 3 and 4, respectively, was conducted in CWR22Rv1 xenografts in vivo. [00104] Male NRG mice bearing CWR22Rv1 tumors were treated with vehicle, Compounds 1, 2, 3 and 4 for 16 days as described in the METHODS section below. Mice bearing CWR22Rv1 xenografts (4 mice per group) were treated with vehicle, Compound 1 (100 mg/kg = 0.515 mmol/kg, twice daily, 5 days/week), Compound 2 (20 mg/kg = 0.0456 mmol/kg, once daily, 5 days per/week), Compound 3 (22.6 mg/kg = 0.0532 mmol/kg, once daily, 5 days per/week), and Compound 4 (5 mg/kg = 0.0105 mmol/kg, once daily, 5 days per/week). Tumor volumes were measured twice a week and the tumors were collected. Percent tumor growth inhibition (%TGI) values are indicated at the right of each growth curve, and the error bars are the SEM. Results are represented as means ± SEM. All p vales are compared to vehicle control: *p < 0.0256, **p < 0.0104, ***p < 0.0041. See FIG.5A. The average tumor weights from each experimental group at day 16: *p < 0.0256, **p < 0.0104, ***p < 0.0041, ****p < 0.0069 (FIG.5B). Photographed excised tumors from each group at the end of the study are provided in FIG.5C. Mean body weights of mice determined during the study are shown in FIG.5D. [00105] After 16 days of treatment, the tumors in the vehicle-treated mice increased in size by ~12.5-fold (from ~100 mm3 to ~1250 mm3, FIG.5A). As shown in FIGS.5A, 5B and 5C, the parent compounds exhibited statistically significant robust inhibition of CWR22Rv1 tumor growth t=at the tested doses. Remarkably, Compound 3 (at 9.7-fold lower equimolar dose than Compound 1) caused tumor growth inhibition (TGI) of 76% versus control compared to Compound 1 (64% versus control). In addition, the use of Compound 3 allows 50% less-frequent dosing, i.e., from twice daily for Compound 1 to once daily for Compound 3. With respect to Compound 2, a 4.3-fold lower equimolar dose of its corresponding HCl salt (Compound 4) exhibited comparable efficacy (TGIs = 82% for each compound) as the parent compound. No
host toxicities were observed, as monitored by changes in animal body weight FIG.5D and visual observation of general health of the animals. [00106] Based on these promising data of the pilot study, the inventors proceeded to conduct a more rigorous antitumor efficacy study, that included head-to-head comparison of Compounds 1, 2, 3, 4 and 5 and two FDA-approved prostate cancer drugs, Enzalutamide (ENZ) and docetaxel (DTX). Example 6: Compound 1 and Compound 2 and their respective hydrochloride salts, Compounds 3, 4 and 5, are more efficacious than Enzalutamide (ENZ) and Docetaxel (DTX) in Castration-Resistant Prostate Cancer CWR22Rv1 Xenograft Model. [00107] Following the promising in vitro data, the therapeutic activities of Compound 1 and Compound 2 and their respective hydrochloride salts, Compounds 3, 4 and 5, were assessed in vivo using difficult-to-treat castration-resistant prostate cancer (CRPC) CWR22Rv1 xenografts. In addition, the efficacy of FDA-approved prostate cancer drugs, Enzalutamide [NPL-28, NPL- 29] and Docetaxel [NPL-30] were assessed as comparisons using their respective reported efficacious doses in animal models of CRPC. [00108] Male NRG mice bearing CWR22Rv1 tumors were treated with vehicle, Compound 3 (3 doses), Compounds 2 and 4 (2 doses), Compound 5 (2 doses), Enzalutamide (ENZ) or Docetaxel (DTX) for 16 days as described in the METHODS section below. ENZ and DTX were administered at their optimal preclinical dosing regimens [NPL-30 and NPL-31]. Importantly, higher doses of DXT have been shown to be toxic to mice. After 16 days of treatment, the tumors in the vehicle-treated mice increased in size by ~20-fold (from ~100 mm3 to ~2000 mm3, FIGS.6A and 7A). The results are presented in two figures (FIGS.6A-6E and FIGS.7A-7D) for the sake of clarity, because there were two groups of mice in this study. [00109] As shown in FIGS.6A-6D, CWR22Rv1 tumors were less sensitive to ENZ and DTX treatments with percent TGI values of 54.7% and 56.9%, respectively. Compound 1 (200 mg/kg) was more efficacious than ENZ and DTX, causing a significant TGI of 76.2% (p = 0.0013 vs. vehicle). Gratifyingly, Compound 3 caused a dose-dependent inhibition of tumor growth, with the two larger doses causing tumor regressions. The decreasing order of potency (TGI) was as follows: Compound 3 (96 mg/kg; 147.5%, p < 0.0001 vs. vehicle) > Compound 3 (48 mg/kg;
136.8%, p < 0.0001 vs. vehicle) > Compound 3 (24 mg/kg; 80.3%, p < 0.0005 vs. vehicle). It is notable that Compound 3 (at 2.3-, 4.6-, and 9.1-fold lower molar doses) is more efficacious than parent Compound 1. Remarkably, Compound 3 has a once-a-day dosing regimen instead of twice-a-day, and permits smaller quantities to be dosed, which undoubtedly would mitigate some undesirable side effects. In general, no host toxicities were observed, as monitored by changes in animal body weight (FIG.6E) and visual observation of general health of the animals. [00110] Mice bearing CWR22Rv1 xenografts (5 mice per group) were treated with vehicle, Compound 1 (100 mg/kg/twice daily), Compound 3 (24, 48 or 96 mg/kg, once daily), ENZ (25 mg/kg, once daily) 5 days per week for 16 days, and DTX (5 mg/kg, IP, 3 times per week). Tumor volumes were measured twice a week and the tumors were collected. Percent tumor growth inhibition (%TGI) values are indicated at the right of each growth curve, and the error bars are the SEM. Results are represented as means ± SEM. All p vales are compared to vehicle control: 1p < 0.0480, 2p < 0.0409, 3p < 0.0013, 4p < 0.0005, 5p < 0.0001 and 6p < 0.0001. See FIG.6A. FIG.6B provides the average tumor weights from each experimental group at day 16; *p < 0.0001 versus control for all the treated groups. FIG.6C provides the total percent change in mean tumor volumes of the initial volume after 16 days as shown as a waterfall plot. The p values are the same as in FIG.6A. FIG.6D provides the percent change in volume for each tumor (5 tumors per treatment group) after 16 days as shown as a waterfall plot. FIG.6E provides the mean body weights of mice determined during the study. [00111] The results of the impact of parent Compound 2 and its two hydrochloride salts, Compounds 4 and 5, are presented in FIGS.7A-7E. Here, the lower 5 mg/kg doses of the two salts (Compounds 4 and 5) were less potent than the precursor Compound 2 (20 mg/kg), but the higher doses of 20 mg/kg, each, caused significant tumor regressions. The decreasing order of potency (TGI) was as follows: Compound 5 (20 mg/kg; 152.9%, p < 0.0001 vs. vehicle) > Compound 4 (20 mg/kg; 139.6%, p < 0.0001 vs. vehicle) > Compound 2 (20 mg/kg; 83.8%, p < 0.0005 vs. vehicle) > Compound 5 (5 mg/kg; 79.2%, p < 0.0006 vs. vehicle) > Compound 4 (5 mg/kg; 74.2%, p < 0.0008 vs. vehicle). In general, no host toxicities were observed, as monitored by changes in animal body weight (FIG.7E) and visual observation of general health of the animals.
[00112] Mice bearing CWR22Rv1 xenografts (5 mice per group) were treated with vehicle, Compound 2 (20 mg/kg, once daily), Compound 4 (5 or 20 mg/kg, once daily), Compound 5 (5 or 20 mg/kg, once daily), ENZ (25 mg/kg, once daily) 5 days per week for 16 days, and DTX (5 mg/kg, IP, 3 times per week). Tumor volumes were measured twice a week and the tumors were collected. Percent tumor growth inhibition (%TGI) values are indicated at the right of each growth curve, and the error bars are the SEM. Results are represented as means ± SEM. All p vales are compared to vehicle control: 1p < 0.0360, 2p < 0.0303, 3p < 0.0001, 4p < 0.0008, 5p < 0.0006, 6p < 0.0001 and 7p < 0.0001. See FIG.7A. FIG.7B provides the average tumor weights from each experimental group at day 16; *p < 0.0001 versus control for all the treated groups. FIG.7C provides the total percent change in mean tumor volumes of the initial volume after 16 days as shown as a waterfall plot. The p values are as in FIG.7A. FIG.7D is the percent change in volume for each tumor (5 tumors per treatment group) after 16 days as shown as a waterfall plot. FIG.7E shows the mean body weights of mice determined during the study. [00113] FIG.8A shows a photograph of all the excised tumors after termination of the study which corroborates the tumor volumes of FIG.6A through FIG.7E. [00114] FIG.8B shows the impact of the various treatments on key biomarker proteins. FIG. 8B is Western blot analyses of tumor samples from each experimental group on day 16. Equal protein concentrations from prostate cancer tumors treated with the compounds at the indicated doses for 16 days were separated by SDS-PAGE and Western blots probed with the respective protein antibodies. Vehicle-treated cells were included as a control, and all blots were reprobed for GAPDH for loading control. [00115] To further validate the molecular mechanisms underlying the activities of the Compounds 3, 4 and 5 compared to ENZ and DTX, the inventors evaluated the expression levels of f-AR, AR-V7, Mnk1 and its downstream targets, eIF4E and cyclin D1 and markers of apoptosis, Bcl-2 and BAX by western blot analyses (FIG.8B). As expected, the levels of f-AR and AR-V7 were significantly reduced by Compound 1, Compound 2, and DTX. ENZ had no impact on the levels of AR and AR-V7 (FIG.8B). It should be noted that taxanes, including DTX, have been reported to cause decrease in expression of AR in in vitro and in vivo models of prostate cancer [NPL-32]. Each of Compounds 3, 4 and 5 caused a decrease in the expression of the anti-apoptotic Bcl-2 and upregulation of pro-apoptotic BAX, but the impacts of Compounds
3, 4 and 5 were superior to the impacts of ENZ or DTX at the tested doses. Thus, Compounds 3, 4 and 5 inhibited tumor growth via inhibition of AR/AR-V7 and Mnk1-eIF4e signaling pathways and induction of apoptosis, like previous reports by the inventors [NPL-8, NPL-9, NPL-10, NPL- 33]. Considering the superior efficacy of Compounds 3, 4 and 5 over ENZ and DTX, these results provide a strong rationale for clinical studies comparing Compounds 3, 4 and 5 with DTX or ENZ in men with mCRPC with disease progression on first line ENX or DTX. [00116] These results demonstrate for the first time that the hydrochloride salts, Compounds 3, 4 and 5, are orally bioavailable, safe and are more efficacious against difficult-to-treat CWR22Rv1 cells and tumor xenografts and are superior to their parent compounds (Compounds 1 and 2). Further Compounds 3, 4 and 5 are more highly efficacious than two blockbuster FDA- approved prostate cancer drugs Enzalutamide (Xtandi) and Docetaxel (DTX, Taxotere). Collectively, these results classify Compounds 2, 3, 4 and 5 as valuable lead candidates for further evaluation in prostate cancer clinical Phase I studies. Because Compound 1 has been shown to be safe in humans in the clinic and was successful in Phases I and II clinical trials in men with prostate cancer, the enhanced efficacy of Compound 3 compared to Compound 1 would undoubtedly enable its accelerated Phase II clinical assessment in men with prostate cancer. METHODS General Chemical Synthesis methods [00117] Compound 1 required for the synthesis of salts was gift from Tokai Pharmaceuticals, Inc. The Compound 2 required for mono and di-salt preparation was synthesized from Compound 1 following our reported synthetic procedure [NPL-34]. Anhydrous solvents (isopropyl alcohol and methanol), 3M methanolic-HCl solutions (small ampules for controlled reaction in mono salt preparation and bottled where HCl in excess used for other two salts) were purchased from Aldrich. Melting points were recorded on Fisher-Johns melting point apparatus and are uncorrected.1H and 13C NMR spectra were obtained on a Bruker Ascend 400 spectrometer, and chemical shifts δ are expressed in ppm relative to TMS as internal standard. All 1H NMR data analyzed, and reports were generated by using ACD/NMR Processor Academic Edition, where 13C data were first processed in freely available SpinWorks-4.2, saved
dx format and reports were generated ACD/NMR Processor. High-resolution mass spectrometry was on Bruker 12T APEX-Qe FTICR-MS instrument by positive ion ESI mode by Isaiah Ruhl, Facility Director, College of Sciences Major instrument cluster, Old Dominion University, Norfolk, VA. Because electrospray ionization (ESI) was used in positive-ion mode to acquire the HRMS data, the Cl- is cleaved to give the M + H+. ESI is a soft-ionization technique that can form adducts in positive-ion mode (i.e., H+ or Na+). In negative-ion mode, it is possible to observe an M + Cl- anion or the [M-H]- parent ion, but HCl will greatly inhibit ionization in the negative-ion mode. In either case, the ionization process sometimes strips the Cl-. [00118] 3β-Hydroxide-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene hydrochloride (Compound 3) 0.5g of Compound 1 (1.28 mmol) was dissolved in IPA (30 mL) to get a clear solution, prior to the addition of 1.29 mL of fresh solution of 3M HCl in methanol (3.86 mmol) in two parts over a period of 15 min. A dense white precipitate obtained was continued to stir for 2-3 hours then evaporated under vacuum and dried under high vacuum at 40°C to obtain 0.54g (quantitative yield) of Compound 3; m.p.183-184°C, 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.06 (s, 6 H), 3.50 - 3.63 (m, 1 H), 5.41 (d, J=4.89 Hz, 1 H), 6.31 (br. s., 1 H), 7.53 - 7.68 (m, 3 H), 8.12 (d, J=7.34 Hz, 1 H), 9.26 (br. s., 1 H); 13C NMR (101 MHz, CHLOROFORM-d) δ ppm 16.07, 19.32, 20.49, 30.29, 30.73, 30.93, 31.53, 34.34, 36.74, 37.10, 42.18, 47.54, 50.28, 55.98, 71.53, 112.70, 116.55, 120.62, 127.25, 130.91, 131.00, 132.02, 137.88, 141.36, 144.65; 1H NMR (400 MHz, DMSO-d6) δ ppm 0.99 (s, 3 H), 1.00 (s, 3 H), 3.24 - 3.33 (m, 1 H), 5.34 (d, J=4.65 Hz, 1 H), 6.33 (br. s., 1 H), 7.50 - 7.59 (m, 2 H), 7.75 - 7.84 (m, 1 H), 7.85 - 7.91 (m, 1 H), 9.34 (br. s., 1 H); 13C NMR (101 MHz, DMSO-d6) δ ppm 15.86, 19.53, 20.58, 30.33, 30.54, 30.95, 31.85, 33.80, 36.78, 37.29, 42.69, 47.28, 50.44, 55.70, 70.41, 113.45, 116.78, 120.47, 125.86, 126.21, 129.53, 133.08, 141.55, 142.13, 145.07. HRMS calcd 389.2587 (C26H32N2O H+) found 389.2585. [00119] 3β-(1H-Imidazol-1-yl)-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene hydrochloride (Compound 4) Compound 2 (0.05g, 0.114 mmol) was dissolved in methanol (2 mL) at room temperature (cloudy solution). To which, 0.038 ml of ice-cold solution of 3M HCl in methanol (0.0042g, 0.0114 mmol) was added and mixture stirred at room for 12 hours. The solvent was evaporated
under reduced pressure to obtain 0.054g (quantitative yield) of Compound 4, m.p.188-189°C; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.04 (s, 3 H) 1.16 (s, 3 H) 2.74 (t, J=12.84 Hz, 1 H) 4.06 - 4.25 (m, 1 H) 5.54 (d, J=4.89 Hz, 1 H) 6.01 (br. s., 1 H) 7.15 (br. s., 1 H) 7.28 - 7.37 (m, 3 H) 7.46 - 7.58 (m, 1 H) 7.75 - 7.86 (m, 1 H) 7.98 (s, 1 H) 8.80 (br. s., 1 H); 13C NMR (101 MHz, CHLOROFORM-d) δ ppm 16.03, 19.32, 20.59, 29.65, 30.24, 30.26, 31.02, 34.76, 36.84, 37.56, 39.95, 47.22, 50.35, 55.74, 59.19, 111.12, 120.20, 122.54, 123.28, 123.46, 124.13, 134.54, 138.97, 141.59, 143.20, 147.08; 1H NMR (400 MHz, DMSO-d6) δ ppm 0.99 (s, 3 H), 1.14 (s, 4 H), 2.64 - 2.81 (m, 2 H), 4.09 - 4.23 (m, 1 H), 5.50 (d, J=4.40 Hz, 1 H), 6.08 (br. s., 1 H), 7.20 - 7.36 (m, 2 H), 7.43 (br. s., 1 H), 7.58 (d, J=7.82 Hz, 1 H), 7.71 (d, J=7.34 Hz, 1 H), 7.76 (br. s., 1 H), 8.28 (s, 1 H), 8.71 (br. s., 1 H); 13C NMR (101 MHz, DMSO-d6) δ ppm 16.15, 19.44, 20.64, 29.30, 30.26, 30.31, 30.96, 34.35, 36.88, 37.69, 47.10, 50.30, 55.67, 58.58, 111.80, 119.99, 122.37, 122.63, 123.70, 124.34, 134.69, 140.39, 142.76, 143.32, 146.88. HRMS calcd 439.2856 (C29H34N4 H+) found 439.2852. [00120] 3β-(1H-Imidazol-1-yl)-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene dihydrochloride (Compound 5) To a suspension of 0.2 g of Compound 2 (0.46 mmol) in IPA (4 mL) was added methanol (3 mL) to get a cloudy solution. To which, 0.91 mL of fresh solution of 3M HCl in methanol (2.73 mmol) was added and stirred at room temperature for 20 minutes to get clear solution. The mixture was stirred for 2-3 hours then evaporated under vacuum to obtain white oily solid. The white solid was dried under high vacuum to obtain brittle white solid which further kept under high vacuum at 80°C to remove traces of IPA to give 230 mg (quantitative yield) of Compound 5, m.p.191-192°C, 1H NMR (400 MHz, DMSO-d6) δ ppm 1.01 (s, 3 H), 1.14 (s, 3 H), 4.27 (t, J=12.10 Hz, 1 H), 5.51 (d, J=4.89 Hz, 1 H), 6.30 (br. s., 1 H), 7.46 - 7.56 (m, 2 H), 7.73 (t, 1 H), 7.74 - 7.80 (m, 1 H), 7.81 - 7.88 (m, 1 H), 8.00 (s, 1 H), 9.16 (br. s., 1 H), 9.31 (s, 1 H); 13C NMR (101 MHz, DMSO-d6) δ ppm 15.92, 19.40, 20.55, 25.95, 28.93, 30.23, 30.49, 30.91, 33.84, 36.88, 37.56, 38.91, 47.26, 50.23, 55.60, 59.46, 113.14 117.37, 120.13,120.91, 122.62, 125.29, 125.75, 128.67, 133.39, 134.47, 140.10, 141.77, 145.39. HRMS calcd 439.2856 (C29H34N4 H+) found 439.2849.
BIOLOGICAL STUDIES [00121] Cell Culture and Western Blotting The human prostate cancer cell line CWR22Rv1 was procured from ATCC (Manassas, VA) and cultured in the recommended media supplemented with 10% heat-inactivated standard fetal bovine serum (FBS, GIBCO) and 1% penicillin-streptomycin (10,000 U/ml, Life Technologies) at 37 °C and 5% CO2. LNCaP and C4-2B cell lines were cultured in RPMI-1640 medium with 10% FBS and penicillin/streptomycin (100 U/ml in a humidified incubator with 5% CO2 at 37°C. Primary antibodies against Mnk1, eIF4E, p-eIF4E, Cyclin D1, Bcl-2, BAX, β- actin, and secondary HRP-conjugated anti-rabbit used in the study were procured from Cell Signaling Technology, USA. Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer supplemented with 1X protease inhibitors (Roche, Indianapolis, IN, USA), phosphatase inhibitors (Thermo Scientific, Waltham, MA, USA), 1 mmol/L EDTA and 1 mmol/L PMSF (Sigma) and immunoblotting analyses were performed as previously described [NPL-9, NPL-35 and NPL-36]. [00122] Cell proliferation analysis Cell proliferation assay was performed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) as previously described [NPL-37 and NPL-10]. Briefly, 2500 cells/well (CWR22Rv1, LNCaP, and C4-2B) were seeded in 96 well plate for 24 h and treated with the indicated concentrations of test compounds for 2nd and 5th day in a span of eight days. Growth inhibitory concentration (GI50) was calculated based on a non-linear regression curve fit using GraphPad prism 5.0 software (La Jolla, CA). [00123] Determination of Pharmacokinetic Parameters in mice [00124] The PK study was performed by the Certified Research Organization (CRO), Aragen Life Sciences Private Limited (Aragen), Hyderabad, India (www.aragen.com). All procedures of the present study were in accordance with the guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) as published in The Gazette of India, January 7, 2010. CPCSEA renewal number for institute is 1125/PO/Rc/S/CPCSEA on February 13, 2014. Institutional Animal Ethics Committee (IAEC) approval has been taken to initiate the study (IAEC Approval Number: B-011A).
[00125] Male CD-1 mice (20 – 35 g) were dosed with the test salts intravenously (IV) or oral gavage (PO) at 1 mg/kg and 10 mg/kg, respectively. The compounds were dissolved in 20% β- cyclodextrin in saline. Blood samples (0.1 mL were obtained via lateral saphenous vein at 0.083, 0.25, 1, 2, 4, 8, and 24 h time points and collected in heparinized tubes. After collection of blood samples at each time point, the blood samples were stored on ice, prior to centrifugation. Blood samples were centrifuged within 0.25 h of collection to separate plasma. Centrifugation was conducted at 10640rcf, 5 min, 4°C. The plasma was separated and transferred to pre-labelled matrix tubes and promptly frozen at –20 ± 10°C. After 8 h time-point, all the plasma samples were transferred to bioanalytical group on dry ice and stored at -80°C until bioanalysis. Samples were identified by test item, group, animal number and collection time point. Test samples concentrations were determined by LC-MS/MS, and the PK parameters were calculated by non- compartmental analysis module in Phoenix WinNonlin® (Version 8.1) (Pharsight, Mountain View, trapezoidal rule) as previously reported by the inventors [NPL-16]. The equation used to calculate absolute bioavailability is: Absolute Bioavailability (%F) = AUCPO/AUCIV × DoseIV/DosePO × 100. [00126] In vivo tumor xenograft studies The castration resistant prostate cancer (CRPC) cell line CWR22Rv1 was used to induce tumor xenografts in immunodeficient male NRG mice. The mice (age 5–6 weeks) were procured from the Veterinary Resources, University of Maryland School of Medicine (Baltimore, MD, USA), housed under sterile conditions and fed with sterile pellets and water ad libitum. After a week of acclimatization, 3x106 cells were subcutaneously injected into the left flank of mice. After three weeks of inoculation and upon reaching the tumor volume of ~100 mm3, the mice were randomized into 12 groups of five animals, each. The control groups of mice were orally administered with vehicle (20% β-cyclodextrin in saline, PO). Docetaxel was administered as IP injection. All other compounds were orally administered as doses as indicated for five days a week for 16 days. The animals were monitored daily for general health and body weight recorded three times a week. The tumor size was measured thrice a week using digital calipers and tumor volume calculated using the formula length × width2 × 0.5 (mm3). Upon reaching the tumor length close to 20 mm in control groups (16th day), the experiment was terminated, the animals were euthanized, and tumors were excised for further analyses. All in vivo studies in
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V.C. Njar, VN/14-1 induces ER stress and autophagy in HP-LTLC human breast cancer cells and has excellent oral pharmacokinetic profile in female Sprague Dawley rats, Eur J Pharmacol, 734 (2014) 98-104. [NPL-22] C. Gardelli, E. Nizi, E. Muraglia, B. Crescenzi, M. Ferrara, F. Orvieto, P. Pace, G. Pescatore, M. Poma, R. Ferreira Mdel, R. Scarpelli, C.F. Homnick, N. Ikemoto, A. Alfieri, M. Verdirame, F. Bonelli, O.G. Paz, M. Taliani, E. Monteagudo, S. Pesci, R. Laufer, P. Felock, K.A. Stillmock, D. Hazuda, M. Rowley, V. Summa, Discovery and synthesis of HIV integrase inhibitors: development of potent and orally bioavailable N-methyl pyrimidones, J Med Chem, 50 (2007) 4953-4975. [NPL-23] S.H. Hwang, H.J. Tsai, J.Y. Liu, C. Morisseau, B.D. Hammock, Orally bioavailable potent soluble epoxide hydrolase inhibitors, J Med Chem, 50 (2007) 3825-3840. [NPL-24] M.V. Varma, C.M. Ambler, M. Ullah, C.J. Rotter, H. Sun, J. Litchfield, K.S. Fenner, A.F. El-Kattan, Targeting intestinal transporters for optimizing oral drug absorption, Curr Drug Metab, 11 (2010) 730-742. [NPL-25] K.W. Ward, L.M. Azzarano, C.A. Evans, B.R. Smith, Apparent absolute oral bioavailability in excess of 100% for a vitronectin receptor antagonist (SB-265123) in rat. I. Investigation of potential experimental and mechanistic explanations, Xenobiotica, 34 (2004) 353-366. [NPL-26] K.W. Ward, L.B. Hardy, J.R. Kehler, L.M. Azzarano, B.R. Smith, Apparent absolute oral bioavailability in excess of 100% for a vitronectin receptor antagonist (SB-265123) in rat. II. Studies implicating transporter-mediated intestinal secretion, Xenobiotica, 34 (2004) 367-377. [NPL-27] E. Thomas, R.S. Thankan, P. Purushottamachar, J. Guo, R.A. Parise, J.H. Beumer, V.C.O. Njar, Murine Toxicology and Pharmacokinetics of Lead Next Generation Galeterone Analog, VNPP433-3beta, Steroids, (2023) 109184. [NPL-28] C. Liu, C. Armstrong, Y. Zhu, W. Lou, A.C. Gao, Niclosamide enhances abiraterone treatment via inhibition of androgen receptor variants in castration resistant prostate cancer, Oncotarget, 7 (2016) 32210-32220.
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antitumor activity in the LAPC4 human prostate cancer xenograft model, J Med Chem, 48 (2005) 2972-2984. [00129] While the invention has been described with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the specific embodiments described.
Claims
3. The salt according to claim 1, wherein the salt of Compound 1 is selected from the group consisting of:
H , C N
, Compound 6 , C
, Compound 10
8. A pharmaceutical composition, comprising the salt of any one of claims 1-7, and a pharmaceutically acceptable carrier, excipient and/or diluent.
9. A method of treating cancer, comprising: administering a therapeutically effective amount of the salt of claim 1 or claim 2 to a subject in need thereof.
10. The method according to claim 9, wherein the salt is selected from the group consisting of:
H , C N
, Compound 6
15. The method according to any one of claims 9-14, wherein the cancer is prostate cancer, bladder cancer, pancreatic cancer, breast cancer, a hematological cancer, or hepatocellular carcinoma.
16. The method of claim 15, wherein the cancer is prostate cancer.
17. The method of claim 16, wherein the prostate cancer is castration-resistant prostate cancer.
18. A method of prolonging survival of a subject having cancer, comprising: administering a therapeutically effective amount of the salt of claim 1 or claim 2 to the subject in need thereof.
20. The method according to claim 18, wherein the salt is selected from the group consisting of: , C
, Compound 5
24. The method according to any one of claims 18-24, wherein the cancer is prostate cancer, bladder cancer, pancreatic cancer, breast cancer, a hematological cancer, or hepatocellular carcinoma.
25. The method according to claim 24, wherein the cancer is prostate cancer.
26. The method according to claim 25, wherein the prostate cancer is castration-resistant prostate cancer.
27. A method of treating benign prostatic hyperplasia (BPH) or Kennedy’s disease, comprising: administering a therapeutically effective amount of the salt according to claim 1 or claim 2 to a subject in need thereof.
29. The method according to claim 27, wherein the salt is selected from the group consisting of: , C
, Compound 5
33. A method of inducing androgen receptor (AR) degradation in a subject, comprising: administering a therapeutically effective amount of the salt according to claim 1 or claim 2 to a subject in need thereof.
34. The method according to claim 33, wherein the salt is selected from the group consisting of: , C
, Compound 6
35. The method according to claim 33, wherein the salt is selected from the group consisting of: ,
, Compound 5 7
39. A method of inducing Mnk1/2 transcriptional activity degradation in a subject, comprising: administering a therapeutically effective amount of the salt according to claim 1 or claim 2 to the subject in need thereof.
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