Treatment of cancer with drug combinations

Abstract

Disclosed herein are methods of treating androgen-dependent cancers, including androgen dependent prostate cancer.

Description

TREATMENT OF CANCER WITH DRUG COMBI NATIONSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application 63 / 601,425, filed November 21, 2023, the contents of which are hereby incorporated in its entirety.FIELD OF THE INVENTION

[0002] The invention relates to methods of treating androgen-dependent cancers, including androgen dependent prostate cancer.BACKGROUND

[0003] Cancer is an extremely complex disease that involves initiating mutations epigenetic changes in the DNA and promoting agents that stimulate cell division. The prominent risk factors for prostate cancer are family history, race, age and diet. According to the American Cancer Society having a father or brother with prostate cancer more than doubles a man's risk of developing this disease. In the USA, Prostate cancer is the second leading cause of cancer-related death affecting one of every nine men. The prostate is a walnut-shaped gland that produces the seminal fluid to nourish and transport sperm in males.

[0004] Cancer is an extremely complex disease that involves initiating mutations epigenetic changes in the DNA and promoting agents that stimulate cell division. The prominent risk factors for prostate cancer are family history, race, age and diet. According to the American Cancer Society, having a father or brother with prostate cancer more than doubles a man's risk of developing this disease. In the USA, Prostate cancer is the second leading cause of cancer-related death affecting one of every nine men. The prostate is a walnut-shaped gland that produces the seminal fluid to nourish and transport sperm in males.

[0005] There remains a need for improved systems and methods for treating cancer. There remains a need for improved systems and methods for treating androgen-dependent cancer. There remains a need for improved systems and methods for treating androgendependent prostate cancer.BRIEF DESCRIPTION OF THE FIGURES

[0006] Figure 1 depicts the cytotoxic effects of KBU2046 and calcitriol on the androgen dependent human prostate cancer cell line LNCaP.

[0007] Figure 2 depicts how genistein at micromolar concentrations decreases the viability of androgen dependent human prostate cancer cells after a 24 hour exposure.

[0008] Figure 3 depicts how genistein at micromolar concentrations decrease the viability of androgen dependent human prostate cancer cells at both 24 hour and 48 hour exposure times.

[0009] Figure 4 depicts how KBU2046 is noncytotoxic to androgen dependent prostate cancer cells (LNCaP) after a 24 hour exposure.

[0010] Figure 5 depicts how KBU2046 is noncytotoxic to androgen dependent prostate cancer cells (LNCaP) after 24 hour and 48 hour exposure.

[0011] Figure 6 depicts calcitriol Does Not Decrease the Viability of Human Prostate Cancer Cells (LNCaP) When Used Alone.

[0012] Figure 7 depicts the effect of ellagic acid on LNCaP.

[0013] Figure 8 depicts that ellagic acid decreases the viability of prostate cancer cells after exposure for 48 hr.

[0014] Figure 9 depicts that curcumin decreases the viability of androgen dependent prostate cancer cells following exposure for 24 hours.

[0015] Figure 10 depicts prolonged exposure for 48 hours increases the cytotoxicity of curcumin toward androgen dependent prostate cancer cells.

[0016] Figure 11 depicts how the active form of vitamin D3, calcitriol, does not decrease the viability of androgen dependent prostate cancer cells.

[0017] Figures 12A, 12B, 12C, 12D, 12E, and 12F depict the in vitro effects of various organic compound combinations on the viability of androgen dependent human prostate cancer cells.

[0018] Figure 13 depicts the combination of KBU2046 and calcitriol decrease the viability of androgen dependent prostate cancer cells.

[0019] Figure 14A, 14B, and 14C depicts KBU2046 and calcitriol inhibit the viability of LNCaP tumor spheroids after exposure for 72 hours.

[0020] Figure 15 depicts the time dependent treatment of PC3 cells androgen independent PC3 cell line initially responsive to KBU and calcitriol at 48 hours becomes resistant to the inhibitory effects at 72 hours.

[0021] Figure 16 depicts the combination of KBU2046 and calcitriol are not cytotoxic toward human peripheral blood mononuclear cells.

[0022] Figure 17 depicts 22-oxacalcitriol and KBU2046 in combination are not cytotoxic androgen dependent prostate cancer LNCaP cell line.

[0023] Figure 18 depicts higher concentration 22-oxacalcitriol and KBU2046 in combination are not cytotoxic androgen dependent prostate cancer LNCaP cell line.

[0024] Figure 19 depicts paricalcitol and KBU2046 in Combination are not cytotoxic on androgen dependent prostate cancer LNCaP cell line.

[0025] Figure 20 depicts higher concentrations of paricalcitol and KBU2046 in combination are not cytotoxic to the LNCaP cell line of androgen-dependent prostate cancer (<5 uM).

[0026] Figure 21 depicts a combination of KBU2046 and calcitriol inhibit proliferation of human androgen dependent prostate cancer cells over a 12 day period.

[0027] Figure 22 depicts the combination of KBU2046 and calcitriol inhibits wound healing in vitro on the androgen-dependent prostate cell line LNCaP.

[0028] Figure 23A, 23B, and 23C depict the combination of KBU2046 and calcitriol inhibits migration in vitro on the androgen-dependent prostate cell line LNCaP. The anti-migratory effects of (6.4 D) KBU2046 alone, (6.4 E) calcitriol (D3), and (6.4 F) KBU2046.DETAILED DESCRIPTION

[0029] Before the present methods and systems are disclosed and described, it is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0030] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes-, from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0031] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0032] Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers or steps. "Exemplary" means "an example of" and is not intended to convey anindication of a preferred or ideal embodiment. "Such as" is not used in a restrictive sense, but for explanatory purposes.

[0033] Disclosed are components that can be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0034] Compounds disclosed herein may be provided in the form of acceptable salts. Examples of such salts are acid addition salts formed with inorganic acids, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids and the like; salts formed with organic acids such as acetic, oxalic, tartaric, succinic, maleic, fumaric, gluconic, citric, malic, methanesulfonic, p-toluenesulfonic, napthalenesulfonic, and polygalacturonic acids, and the like; salts formed from elemental anions such as chloride, bromide, and iodide; salts formed from metal hydroxides, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, and magnesium hydroxide; salts formed from metal carbonates, for example, sodium carbonate, potassium carbonate, calcium carbonate, and magnesium carbonate; salts formed from metal bicarbonates, for example, sodium bicarbonate and potassium bicarbonate; salts formed from metal sulfates, for example, sodium sulfate and potassium sulfate; and salts formed from metal nitrates, for example, sodium nitrate and potassium nitrate.

[0035] Disclosed herein are methods for processing biomass including the step of administering to a subject in need thereof the compound of Formula (I):and administering to the subject a vitamin D analog. As used herein, the compound of Formula (1) may also be designated KBU2046.

[0036] In some implementations, the compound of Formula (1) can be administered in combination with a vitamin D analog such as calcitriol, calcipotriol (22-ene-26,27-dehydro- la,25(OH)2D3), paricalcitol (19-nor-la,25(OH)2D2), 22-oxacalcitriol (22-oxa-l,25(OH)2D3), alfacalcidol (la(OH)D3), hectorol (la(0H )DZ), fa leca Icitriol (1,25-(OH )Z-26,27F6 D3), tacalicitol (la,24( R)(O H)2D3), eldecalcidol (2p-(3-hydroxypropoxy)-la,25(OH)2D3), seocalcitol (22,24 diene-24,26,27-trishomo-la,25(OH)2D3), 20-epi-la,25(OH)2D3, lexicalcitol (20-epi-22-oxa- 24,26,27-trishomo-la,25(OH)2D3), 17-methyl-19-nor-21-nor-23-yne-26,27-F6-la,25(OH)2D3, inecalcitol (19-nor-14-epi-23-yne-la,25(OH)2D3), 19-nor-14,20-bisepi-23-yne-la,25(OH)2D3,2-methylene-19-nor-(20S)-la25(OH)2D3, seco-C-9,11 bisnor-17-methyl-20-epi-26,27-F6- la,25(OH)2D3, ((5E,7E)-22-ene-26,27-dehydro-la,25(OH)2D3, 16-ene-23-yne-la,25(OH)2D3,2-amino-3-deoxy-19-nor-22-ene-26-dihomo-27-dihomo-25(OH)D3, (lR,3S,5Z)-5-((8E)-2-((3R)-3-((2R,3S)-3-(5-cyclopropyl-3H-l,2-dioxol-3-yl)-2-ethyl-3-methylcyclohexylidene)ethylidene-4-methylenecyclohexane-l,3-diol), ((24E)-(lS)-25-dehydro-24a-homo-la,25(OH)2D3, 20R-(4- hydroxy-4-methylpentyl)-23-yne-26,27-hexafluoro-19-nor-la,25(OH)2D3, 20R-21(3-hydroxy- 3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-la,25(OH)2D3, 20R-21(3- trideuteromethyl-3-hydroxy-4,4,4-trideteurobutyl)-23-yne-26,27-hexafluoro-19-nor- la,25(OH)2D3, 19-nor-2a-(3-hydroxypropyl)-la,25(OH)2D3, la,25(OH)2D3-3-bromoacetate, 16,23Z-diene-26,27-F6-19-nor-la,25(OH)2D3, or a combination thereof. In certain implementations the vitamin D analog is calcitriol.

[0037] The compound of Formula (1) may be administered at a variety of dosage levels. In certain implementations, the subject may receive a total daily dose of the compound of Formula (1) from 0.1-5,000 mg / day, from 0.1-10 mg / day, from 5-100 mg / day, from 50-250 mg / day, from 100-500 mg / day, from 250-750 mg / day, from 500-1,000 mg / day, from 500- 1,500 mg / day, from 1,000-2,000 mg / day, from 1,000-2,500 mg / day, or from 2,000-5,000 mg / day.

[0038] The vitamin D analog (for example calcitriol) may be administered at a variety of dosage levels. In some implementations, the subject may receive a total daily dose of the vitamin D analog from 0.01-1,000 pg / day, from 0.01-500 pg / day, from 0.01-100 pg / day, from 0.01-50 pg / day, from 0.01-25 pg / day, from 0.01-15 pg / day, from 0.01-10 pg / day, from 0.01-5 pg / day, from 0.01-1 pg / day, from 0.05-1 pg / day, from 0.1-1 pg / day, from 0.1-0.5 pg / day, from 0.5-1 pg / day, or from 0.5-1.5 pg / day.

[0039] In some implementations, the subject can receive the compound of Formula (1) in combination with a single vitamin D analog. In some implementations, the subject can receive the compound of Formula (1) and calcitriol, with no other vitamin D analogs beingadministered. In other implementations, the subject can receive the compound of Formula (1) in combination with two or more vitamin D analogs. For example, the subject can receive the compound of Formula (1), calcitriol, and one or more additional vitamin D analogs. In some implementations, the subject receives daily dose of all vitamin D analogs from 0.01- 1,000 pg / day, from 0.01-500 pg / day, from 0.01-100 pg / day, from 0.01-50 pg / day, from 0.01- 25 pg / day, from 0.01-15 pg / day, from 0.01-10 pg / day, from 0.01-5 pg / day, from 0.01-1 pg / day, from 0.05-1 pg / day, from 0.1-1 pg / day, from 0.1-0.5 pg / day, from 0.5-1 pg / day, or from 0.5-1.5 pg / day. In some implementations, the subject receives calcitriol in an amount from 0.01-1,000 pg / day, from 0.01-500 pg / day, from 0.01-100 pg / day, from 0.01-50 pg / day, from 0.01-25 pg / day, from 0.01-15 pg / day, from 0.01-10 pg / day, from 0.01-5 pg / day, from 0.01-1 pg / day, from 0.05-1 pg / day, from 0.1-1 pg / day, from 0.1-0.5 pg / day, from 0.5-1 pg / day, or from 0.5-1.5 pg / day, and a second vitamin D analog in an amount from 0.01- 1,000 pg / day, from 0.01-500 pg / day, from 0.01-100 pg / day, from 0.01-50 pg / day, from 0.01- 25 pg / day, from 0.01-15 pg / day, from 0.01-10 pg / day, from 0.01-5 pg / day, from 0.01-1 pg / day, from 0.05-1 pg / day, from 0.1-1 pg / day, from 0.1-0.5 pg / day, from 0.5-1 pg / day, or from 0.5-1.5 pg / day.

[0040] The compound of Formula (1) and vitamin D analog(s) may be administered according to a variety of dosing schedules. The compound of Formula (1) can be administered over a first treatment period, and the vitamin D analog(s) can be administered over a second treatment period.

[0041] In some implementations the first treatment period is consecutive with the second treatment period, for example, for each day the subject receives the compound of Formula (1), the subject also receives the vitamin D analog(s). In some implementations, the compound of Formula (1) and vitamin D analog(s) can be administered at approximately the same time. In such embodiments, the compounds can be administered in a single unitary composition, or the compounds can be administered in separate compositions at roughly the same time. In other implementations, the subject can receive the compound of Formula (1) and vitamin D at separate times during the same day, for example at an interval of at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, or at least 12 hours.

[0042] In some implementations, the first treatment period occurs prior to the second treatment period, for example, the subject receives a course of the compound of Formula (1), administration of the compound of Formula (1) is halted, and the subject then receivesthe vitamin D analog(s). In other implementations, the first treatment period occurs subsequent to the second treatment period.

[0043] In certain implementations, the first treatment period partially overlaps the second treatment period. For example, the compound of Formula (1) may be administered to the subject for one or more days (a loading phase), and then the subject receives both the compound of Formula (1) and the vitamin D analog(s) (a coadministration phase). In certain implementations, the dosage level of the compound of Formula (1) during the loading phase is the same as the dosage level of the compound of Formula (1) during the coadministration phase. In other implementations, the dosage level of the compound of Formula (1) during the loading phase is greater than the dosage level of the compound of Formula (1) during the coadministration phase. In other implementations, the dosage level of the compound of Formula (1) during the loading phase is lower than the dosage level of the compound of Formula (1) during the coadministration phase.

[0044] In some implementations, the vitamin D analog(s) may be administered first in a loading phase, and then administered in combination with the compound of Formula (1). In certain implementations, the dosage level of the vitamin D analog(s) during the loading phase is the same as the dosage level of the vitamin D analog(s) during the coadministration phase. In other implementations, the dosage level of the vitamin D analog(s) during the loading phase is greater than the dosage level of the vitamin D analog(s) during the coadministration phase. In other implementations, the dosage level of the vitamin D analog(s) during the loading phase is lower than the dosage level of the vitamin D analog(s) during the coadministration phase.

[0045] The combinations disclosed herein may be used to treat subjects with a variety of androgen-related cancers. In some implementations, the subject can be diagnosed with androgen-dependent prostate cancer. In some implementations, the subject can be diagnosed with a predisposition to androgen-dependent prostate cancer or precancerous prostate disease. In certain implementations the subject can be diagnosed with prostate intraepithelial neoplasia. In some implementations the subject can be diagnosed with proliferative inflammatory atrophy. In some implementations the subject may be diagnosed with Atypical small acinar proliferation

[0046] In some implementations, the subject can be diagnosed with prostate cancer, for example can be adenocarcinoma of the prostrate, transitional cell carcinoma of the prostrate, prostrate sarcoma, squamous cell carcinoma of the prostrate, neuroendocrine tumors of the prostrate, small cell prostate cancer, or a combination thereof.

[0047] In some implementations, the prostate cancer can be acinar adenocarcinoma, prostatic ductal adenocarcinoma, or a combination thereof.

[0048] In some implementations, the prostate cancer can be recurrent prostate cancer.

[0049] In some implementations, the prostate cancer can be metastatic prostate cancer.

[0050] In some implementations, in addition to the compound of Formula (1) and vitamin D compound, the subject further receives one or more additional therapies, for example chemotherapy, hormonal therapy, immunotherapy, gene therapy, radical prostatectomy, orchiectomy, radiation beam therapy, focal therapy, or a combination thereof.

[0051] In certain implementations, the subject may receive abiraterone acetate, estrogens, luteinizing hormone-releasing hormone agonists for example, leuprolide, goserelin, and buserelin, antiandrogens for example flutamide, bicalutamide, enzalutamide, apalutamide, nilutamide, and darolutamide, ketoconazole, aminoglutethimide, hydrocortisone, progesterone, or a combination thereof.

[0052] In certain implementations, the subject may also receive a surgical treatment, cryosurgical treatment, radiation therapy, ultrasound therapy, proton beam therapy, photodynamic therapy, hormone therapy, chemotherapy, targeted therapy, immunotherapy, or a combination thereof.

[0053] In certain implementations, the subject also receives one or more chemotherapeutic agents. In some implementations the subject receives abiraterone acetate, Akeega (niraparib tosylate and abiraterone acetate), apalutamide, bicalutamide, cabazitaxel, darolutamide, degarelix, docetaxel, leuprolide acetate, enzalutamide, firmagon, flutamide, feuprolide acetate, Lutetium Lu 177 fipivotide tetraxetan, mitoxantrone hydrochloride, nilutamide, olaparib, vipivotide tetraxetan), relugolix, rucaparib camsylate, Sipuleucel-T, talazoparib tosylate, , radium 223 dichloride, goserelin acetate, or a combination thereof.

[0054] In certain implementations, the subject receives cabazitaxel or docetaxel.

[0055] Also disclosed herein are pharmaceutical compositions, comprising the compound of Formula (1) and a vitamin D analog.

[0056] In certain implementations, the pharmaceutical composition containing the compound of Formula (1) may include one or more vitamin D analog(s) such as calcitriol, calcipotriol (22-ene-26,27-dehydro-la,25(OH)2D3), paricalcitol (19-nor-la,25(OH)2D2), 22- oxacalcitriol (22-oxa-l,25(OH)2D3), alfacalcidol (la(OH) D3), hectorol (la(OH)D2), fa leca Icitriol (1,25-(OH)2-26,27F6 D3), tacalicitol ( la,24(R)(OH )2D3), eldecalcidol (2|3-(3-hydroxypropoxy)- la,25(OH)2D3), seocalcitol (22,24 diene-24, 26, 27-trishomo-la,25(OH)2D3), 20-epi- la,25(OH)2D3, lexicalcitol (20-epi-22-oxa-24,26,27-trishomo-la,25(OH)2D3), 17-methyl-19-nor-21-nor-23-yne-26,27-F6-la,25(OH)2D3, inecalcitol (19-nor-14-epi-23-yne-la,25(OH)2D3), 19-nor-14,20-bisepi-23-yne-la,25(OH)2D3, 2-methylene-19-nor-(20S)-la25(OH)2D3, seco-C- 9,11 bisnor-17-methyl-20-epi-26,27-F6-la,25(OH)2D3, ((5E,7E)-22-ene-26,27-dehydro- la,25(OH)2D3, 16-ene-23-yne-la,25(OH)2D3, 2-amino-3-deoxy-19-nor-22-ene-26-dihomo-27- dihomo-25(OH)D3, (lR,3S,5Z)-5-((8E)-2-((3R)-3-((2R,3S)-3-(5-cyclopropyl-3H-l,2-dioxol-3-yl)- 2-ethyl-3-methylcyclohexylidene)ethylidene-4-methylenecyclohexane-l,3-diol), ((24E)-(1S)- 25-dehydro-24a-homo-la,25(OH)2D3, 20R-(4-hydroxy-4-methylpentyl)-23-yne-26,27- hexafluoro-19-nor-la,25(OH)2D3, 20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)- 23-yne-26,27-hexafluoro-la,25(OH)2D3, 20R-21(3-trideuteromethyl-3-hydroxy-4,4,4- trideteurobutyl)-23-yne-26,27-hexafluoro-19-nor-la,25(OH)2D3, 19-nor-2a-(3- hydroxypropyl)-la,25(OH)2D3, la,25(OH)2D3-3-bromoacetate, 16,23Z-diene-26,27-F6-19- nor-la,25(OH)2D3, or a combination thereof.

[0057] In certain implementations, the composition includes the compound of Formula (1) and calcitriol.

[0058] The composition is formulated for oral administration or parenteral administration. Solid dosage forms for oral administration include, but are not limited to, tablets, soft or hard gelatin or non-gelatin capsules, and caplets. However, liquid dosage forms, such as solutions, syrups, suspension, shakes, etc. can also be utilized. The compositions can include a pharmaceutically acceptable carrier composed of materials that are considered safe and effective and can be administered to an individual without causing undesirable biological side effects or unwanted interactions. As generally used herein "carrier" includes, but is not limited to, diluents, binders, lubricants, disintegrators, fillers, pH modifying agents, preservatives, antioxidants, solubility enhancers, and coating compositions.EXAMPLES

[0059] The following examples are for the purpose of illustration of the invention only and are not intended to limit the scope of the present invention in any manner whatsoever.

[0060] Example 1: Experimental Design

[0061] Cell culture was performed as follows: In RPMI-1640 media (Sigma-Aldrich, St. Louis, MO) with 10% FBS, 1% penicillin / streptomycin, and L-glutamine added, LNCaP cells (ATCC) were cultured. To reach 60-80% confluence, the cells were maintained for 24-48 hours at 37°C and 5% CO2. After 24-48 hours of incubation, the media and removed and replaced with either media alone as a negative control, or media containing different concentrations of each compound alone and / or in combination.

[0062] A resazurin, or alamar blue, assay was used to determine the cytotoxicity of genistein (Sigma-Aldrich, St. Louis, MO), ellagic acid (Cayman Chemical, Ann Arbor, Ml), curcumin (Sigma-Aldrich, St. Louis, MO), KBU2046 (MedChemExpress, Monmouth Junction, NJ), and calcitriol (Sigma-Aldrich, St. Louis, MO) on LNCaP cells. The resazurin assay procedure is based on the mitochondrial respiratory chain converting oxidized non- fluorescent blue resazurin to a red fluorescent dye (resorufin) in live cells. The amount of resorufin produced is proportional to the number of live cells in the organism. Resorufin has an Ex / Em wavelength of 530-560 / 590 nm and absorbance of 570 nm. Prostate cancer cells LNCaP were cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin, and L-glutamine. To reach 60-80 percent confluence, cells were cultured for 24-48 hours at 37°C and 5% CO2. In a 96-well plate, cells were plated at a density of 5 x 103cells per well and incubated for 24 hours to attain confluence. The medium was removed after incubation and replaced with either media alone as a negative control or media containing differing concentrations of each given compound and incubated for 24-72 hours. The ICso was investigated for each compound as a baseline for efficacy. ICso is the concentration of a drug required to block a biological process or reaction by 50%. In whole cell tests, ICso is often employed as a measure of efficacy.

[0063] After the designated treatment time, alamar blue reagent (20 pl) was added to each well of the 96-well plate. After 4 hr. incubation (379C), the absorbance was read on a microtiter plate reader (VersaMax Tunable) at 570 nm / 590nm and the results recorded. The absorbance of the treated cells were compared to the non-treated controls at corresponding dose levels; and a graph of % cell viability compared to dosage was made.

[0064] The dye trypan blue is used to count the number of viable cells in a cell suspension. Live cells have intact cell membranes that keep specific dyes out, such as trypan blue, eosin, or propidium, but dead cells do not. In this test, the cell suspension is combined with dye and then visually analyzed to see if the cells absorb or exclude the dye. A healthy cell will have a clear cytoplasm, but a nonviable cell will have a blue cytoplasm. Trypan blue assay is used to visually determine the percentage of dead cells.

[0065] LNCaP was cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin, and L-glutamine. After 60-80 percent confluence, cells were plated at lxlO4cells per well in 3 ml medium in six well plates and incubated for 24 hours to adhere. The medium was removed after incubation and replaced with either media alone as a negative control, media containing vehicles (DMSO / ethanol, final concentration 0.1%), or KBU2046 / calcitriol at the indicated concentrations. Every three days, the medium holdingthe vehicle and / or drugs was replaced. To determine if the growth inhibition caused by KBU2046 and / or calcitriol is long-lasting, LNCaP cells treated with these compounds were examined to see if the inhibition was reversed by removing the compounds. Cells were plated at low confluence and treated for six days. Each compound was removed after day six and the vehicle and cells proceeded to grow for an additional six days. Cells were counted every three days for twelve days total. Every three days a given population's live (unstained) and dead (blue) cells were directly identified and counted using the trypan blue staining technique. Trypan blue was added to a cell suspension following each given time trial, and the outcome was visually assessed to determine the amount of cell growth. Following treatment, each well's conditioned media was removed and placed into an Eppendorf tube. 2 ml of PBS was dispensed into each well with 0.5 pL of Tryple Express and incubated for 5- 10 minutes for cell detachment. Individual plastic pipettes per treatment were used to blast each well to dislodge cells. Cells were removed and placed into a second set of Eppendorf tubes. Cells were centrifuged in the second set of Eppendorf tubes for 2,500 rpm for 5 minutes. The supernatant was decanted off each tube and the cell pellet was resuspended in 400 pL of the initial media. Cells were counted via hemocytometer and recorded as viable cell count.

[0066] The wound healing assay is a common in vitro approach for studying two- dimensional collective cell migration and quantifies the pace at which cells move in a cell monolayer to fill a cell-free gap. In this assay, a cell-free region in a confluent monolayer is formed by mechanical exclusion or by eliminating the cells from the area through physical, thermal, or chemical damage. Sheet migration is a form of collective cell movement investigated by the wound healing experiment. This migration is characterized by the movement of epithelial and endothelial monolayers in two dimensions while retaining their intercellular connections. Sheet migration is seen in a variety of processes, including cancer metastasis, embryonic morphogenesis, and tissue damage. One of the primary reasons for mortality and unsuccessful therapy in prostate cancer is metastasis. The most frequent method for simulating wounding in vitro is to scratch a confluent monolayer with a pipette tip, needle, or other sharp object to produce a gap. Once the gap is created, optical microscopy is applied to monitor cells migrating into the wound area over time points (24-72 hr.). After acquiring photographs of the gap closing time points, the change in wound area is determined as a percentage of wound closure and compared to the original as percent wound healing inhibition.

[0067] LNCaP was cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin, and L-glutamine. Cells were plated at 2x10scells per well in a 24 well plate and incubated for 24 hours to adhere. The monolayer of each well was scraped directly down the center with a 100 pL pipette tip. The medium was removed following the "scratch" and each well washed with PBS with Ca2+and Mg2+. The medium was replaced with either serum free media alone as a negative control or serum free media containing differing concentrations of each compound in triplicate for 24-72 hours. The "scratch" was analyzed using Fiji ImageJ programming software to quantify the area / percentage of the wound comparably before and after treatment.

[0068] Example 2: Experimental results

[0069] Figure 1 depicts: (A) Parental LNCaP cells at very low density displaying epithelial morphology. (B) LNCaP cells at 70-80% confluence, the morphology of the cells are presented as fibroblastic, elongated, and aggregated indicating healthy cells pre-treatment. (C) LNCaP cells post-treatment with KBU2046 and calcitriol, the cells had a decline in viability as noted by a decrease in cell number with visible shrinkage of cell size and formation of apoptotic bodies.

[0070] Cells were plated at a density of 5 x 103cells per well and incubated for 24 hours for adherence. After incubation, the cells were treated with varying concentrations of genistein for 24 / 48 hours. Prior to usage, genistein was dissolved in DMSO as a stock solution and diluted in culture media to final concentrations (0-100 pM). The cytotoxicity of genistein on LNCaP cells was assessed using the Alamar Blue (resazurin) Assay. The findings indicate that genistein can induce inhibition in cancer cells, which is consistent from prior studies. As the genistein concentration increased, the assay revealed a steady decrease in viability with 24 hour treatment. The p-value indicated that the data was statistically significant with concentrations of 20 pM to 45 pM (p 0.05), 50 pM to 70 pM (p 0.01), and 80 pM (p 0.001). The 48 hour treatment followed a trend similar to the 24 hour treatment with concentrations 30 pM to 60 pM (p 0.05) and 70 pM to 80 pM (p 0.01). The cells' ability to decrease viability increased significantly above 55 pM (p 0.05). Between 0 and 55 pM, there was a statistically significant decrease in the viability of cells (p 0.05). The IC50of genistein on LNCaP cells was calculated and found to be between 40 and 50 pM.

[0071] Cells were plated at a density of 5 x 103cells per well and incubated for 24 hours. After incubation, the cells were treated with varying concentrations of KBU2046 for 24 and 48 hours. Prior to usage, KBU2046 was dissolved in DMSO as a stock solution and diluted in culture media to final concentrations (0-10 pM). Alamar blue assay indicated KBU2046 doesnot significantly induce inhibition in cancer cells at less than 10 pM. As the concentration of KBU2046 was increased, no significant change in cell viability occurred following 24 hour treatment. Treatment for 48 hours followed a similar trend. After 48 hours of treatment there was no significant change in cell viability. With >5 pM concentration there was an increase in viability, indicating KBU2046 is noncytotoxic to LNCaP cells for the given treatments / time points.

[0072] Cells were plated at a density of 5 x 103cells per well and incubated for 24 hours. After incubation, the cells were treated with varying concentrations based on prior studies of ellagic acid for 24 / 48 hours. Prior to usage, ellagic acid was dissolved in DMSO as a stock solution and diluted in culture media to final concentrations (0-32.5 pM). The cytotoxic effect of Ellagic acid on LNCaP cells was assessed using the Alamar Blue (resazurin) Assay. The findings indicate that ellagic acid does not significantly decrease viability in cancer cells (<32.5 pM). As the ellagic acid concentration increased, the assay revealed after 24 hour treatment there were no significant effects on cell viability except that 8.125 pM showcased an (p- value = <0.05) increase in viability. 48 hour treatment followed a trend similar. After 48 hour treatment there was no significant change in cell viability until treatment with 32.5 pM (p- value = <0.05) indicated a decrease in 50% viability (ICso), indicating ellagic acid is cytotoxic to LNCaP cells for >48 hour treatment in a time and dose dependent manner.

[0073] Cells were plated at a density of 5 x 103cells per well and incubated for 24 hours. After incubation, the cells were treated with varying concentrations based on prior studies of curcumin for 24 / 48 hours. Prior to usage, curcumin was dissolved in ethanol as a stock solution and diluted in culture media to final concentrations (0-40 pM). The cytotoxicity of curcumin on LNCaP cells was assessed using the Alamar Blue (resazurin) Assay. The findings indicate that curcumin can decrease viability in cancer cells. As curcumin concentration increased, the assay revealed a steady decrease in viability with 24 hour treatment. At 40 pM concentration there was a statistically significant (p- value = <0.05) decrease in cell viability. 48 hour treatment also followed a trend similar to the 24 hour treatment.Cytotoxicity increased significantly after 48 hours between 20 to 40 pM (p 0.05). The ICsoof curcumin in LNCaP cells was calculated to be 40 pM.

[0074] Cells were plated at a density of 5 x 103cells per well and incubated for 24 hours for adherence. After adherence, the cells were treated with varying concentrations of calcitriol in a serial dilution due to lack of prior studies on LNCaP cytotoxicity to establish a baseline for the ICso. Calcitriol was dissolved in ethanol as a stock solution and diluted in culture media to final concentrations (0-2.5 pM). The cytotoxicity of calcitriol on LNCaP cells wasassessed using the Alamar Blue (resazurin) Assay. The findings indicated that calcitriol does not decrease viability in prostate cancer cell line LNCaP at < 2.5 pM. As the calcitriol concentration increased, the assay revealed a slight increase in viability with 24 hour treatment. There were no significant differences after 24 hour treatment (p- value = <0.05). 48 hour treatment followed the inhibition trend similar. There is a decrease in cell viability for 48 hour treatment, as concentration increases, however, no significant differences were observed.

[0075] LNCaP cells were plated at a density of 5 x 103cells per well and incubated for 24 hours. After adherence, the cells were treated with varying concentrations of compounds based on the prior viability studies above; results are presented in the Figures. The cells were treated with various combinations of KBU2046, ellagic acid, curcumin, and calcitriol. Genistein was not studied in the combination studies because KBU2046 is a noncytotoxic analogue to normal cells. The cells were treated with % of each compound's determined ICso and followed in a 1 / 8 serial dilution and incubated for 24 and 48 hours with each drug to determine if the compounds were able to exert a significant difference at lower concentrations. The cytotoxicity of the drugs in combination on LNCaP cells was assessed using the Alamar Blue (resazurin) Assay.

[0076] Combination of ellagic acid with curcumin revealed a trend of decreased viability after treatment for 24 hrs. The 48 hour treatment also followed a similar trend. However, no significant differences were observed, P-value <0.05 = *, <0.01=**, and <0.001 = ***. Thus, indicating curcumin and ellagic acid do not exhibit significant effects in combination.

[0077] Curcumin and KBU2046 were studied in combination and there was a trend in decreased cell viability for both 24 and 48 hours of exposure. As the concentration increases however, no statistically significant differences were observed, P-value <0.05 = *, <0.01=**, and <0.001 = ***. 48 hour treatment followed a similar trend. The data suggests that in combination curcumin and KBU2046 do not exhibit significant inhibitory effects.

[0078] Curcumin and calcitriol were studied in combination. As the concentration of curcumin and calcitriol increased, a decrease in cell viability with 24 hour treatment at % the original IC5o occurred (40 pM curcumin + 2.5 pM calcitriol). Treatment for 48 hours followed a similar trend with a decrease in viability exhibited at the % concentration (20 pM curcumin + 1.25 pM calcitriol). Based on the information gathered the data suggests that in combination curcumin and calcitriol may have minor effect on viability in prostate cancer cells. However, there was no statistically significant difference in the ICso whether the drugs were used in combination or alone.

[0079] KBU2046 and ellagic acid were studied in combination, and as the KBU2046 and ellagic acid concentration increased no significant change in viability occurred with 24 hour treatment. Treatment for 48 hour followed a similar trend. There was no significant change in viability after 48 hours of treatment until 5 pM KBU2046 +32.5 pM ellagic acid. The result is significantly different from the untreated cells (P-value <0.05). While the combination exhibited a lower IC50 value compared to cells treated with KBU2046 alone, the IC50is not significantly different from the IC50for ellagic acid when used alone. Thus, it was concluded the decrease of cell viability of the combination was not significant between KBU2046 and ellagic acid.

[0080] For the calcitriol and ellagic acid combination study, as the calcitriol and ellagic acid concentration increased, the assay revealed no significant difference in viability with 24 hour treatment. The 48 hour treatment followed similar results. The data suggests that in combination calcitriol and ellagic acid did not induce a significant effect in LNCaP cells, (P- value <0.05) in the comparison of the IC50 value in combination compared to the drugs alone. Thus indicating calcitriol and ellagic acid do not exhibit a significant effect in combination.

[0081] KBU2046 and calcitriol were studied in combination as shown. As the KBU2046 and calcitriol concentration increased, a significant decrease in viability with 24 hour treatment occurred. Treatment for 48 hours followed a similar trend. A significant decrease in viability occurred. When KBU2046 and calcitriol are used in combination they are significantly cytotoxic in LNCaP cancer cells at half the original IC5o dosage (5 pM KBU2046 +2.5 pM calcitriol). There is a significant difference (P-value <0.05) in the IC5o in combination compared to the drugs alone. The active form of Vitamin D3, Calcitriol, or KBU2046 alone did not significantly decrease the viability of LNCaP cells.

[0082] KBU2046 and calcitriol treatment significantly decreased LNCaP cell viability when cells were exposed for 24 and 48 hours in the above combination study. In combination, KBU2046 and calcitriol can induce viability inhibition in LNCaP cancer cells at half the original IC50 dosage (5 pM KBU2046 +2.5 pM calcitriol). There is a significant difference (P-value <0.05) in the IC50in combination compared to the drugs alone. The experiment was conducted with the same concentrations as previous for 72 hours. A significant decrease in viability after 72 hour treatment was observed with 5 pM KBU2046 +2.5 pM calcitriol. In combination, KBU2046 and calcitriol can induce viability inhibition in androgen dependent cancer cells at half the original ICso (5 pM KBU2046 +2.5 pM calcitriol) in a dose and time dependent manner. There is a significant difference (P-value <0.05) in the IC5o incombination with the same 7z concentration (5 pM KBU2046 +2.5 pM) previously used for the 24 / 48 hour treatment compared to the effects with either drugs alone when additive. Thus, indicating KBU2046 and Calcitriol exhibit a synergistic effect on prostate cancer cell line LNCaP.

[0083] As a complex transitional model between 2D monolayer models and in vivo solid tumors, the multicellular tumor spheroids (MCTs) model is emerging as a key tool in the study of cancer. MCTs share many characteristics with in vivo solid tumors, including diverse architecture, internal gradients of signaling substances, nutrition, and oxygenation. MCTs have growth dynamics that are comparable to those of in vivo tumors, and the interactions between the cells in a spheroid match those of the tumors' physical structures, such as cell- to-cell and cell-to-extracellular matrix connections. These similarities offer significant prospects for understanding the basic characteristics of tumors and a useful framework for testing new drugs and assessing the efficacy of treatments.

[0084] LNCaP cells were seeded at 4 x 104cells per well in a round bottom 96 well plate and centrifuged for 10 minutes at 2,500 rpm and 1010 g-force to form spheroids. Spheroids were incubated for 72 hours prior to treatment. After incubation, media was removed and replaced with new media, KBU2046, and Calcitriol in combination and / or alone. Spheroids were treated for 48 and 72 hours. The cytotoxicity of KBU2046 and calcitriol on LNCaP cells was graphed as % cell viability, using the alamar blue assay. LNCaP tumor spheroids treated with KBU2046 alone had no significant difference in % cell viability. LNCaP tumor spheroids treated with calcitriol alone decreased spheroid % cell viability to ~75% for all treatments had no significant differences (P-value <0.05 = *, <0.01=**, and <0.001 = ***). LNCaP tumor spheroids treated KBU2046 and Calcitriol limited the viability of LNCaP Tumor Spheroids significantly.

[0085] LNCaP tumor spheroids treated with KBU2046 or calcitriol alone for 72 hours showed a similar effect as the 48 hour treatment. Spheroids treated 72 hours with KBU2046 and calcitriol in combination followed a similar trend as 48 hour treatment, except at half the individual IC5o values (5 pM KBU2046 +2.5 pM calcitriol) the combination caused a significant difference in % cell viability (P-value <0.05). Significant difference in cell viability between the organic compounds alone or in combination further suggests that KBU2046 and calcitriol in combination have a significant impact on LNCaP cell viability.

[0086] Prostate cancer cell line PC3 (obtained from ATCC, Manassas, Virginia) was tested with KBU2046 and calcitriol alone and in combination to investigate if the above concentration were ubiquitous to Androgen-independent cancer cell lines. PC3 cells lack theexpression of AR and PSA, and their proliferation is androgen independent. PC3 were cultured in RPMI-1640 and treated with KBU2046 and / or ca Icitriol. Utilizing an Alamar blue exclusion test, cytotoxicity was assessed 48 and 72 hours after treatment. Alamar blue reagent (20 pl) was added to each well of the 96-well plate after 48 and 72 hours of treatment. The absorbance was measured at 570 and 590 nm using a plate reader after the plates had been incubated at 37°C and 5% CO2 for 4 hours. The cytotoxicity of KBU2046 and calcitriol on PC3 cells was graphed as % cell viability. There was significant difference in PC3 % cell viability of calcitriol alone, KBU2046 alone, and in combination after 48 hour treatment. The % cell viability increased exponentially after 72 hours of treatment, concluding treatment is time dependent for PC3. KBU2046 and Calcitriol alone / and in combination do not exhibit viability inhibition on PC3 at the concentrations used for LNCaP. Androgen independent PC3 cell line was initially responsive to KBU and Calcitriol at 48 hours but becomes resistant to the inhibitory effects at 72 hours.

[0087] To investigate if KBU2046 and calcitriol are selective for cancer cells, Peripheral blood mononuclear cells (PBMC) were cultured in RPMI-1640 and treated with KBU2046 and / or calcitriol with phytohemagglutinin (1 pL / mL) to stimulate lymphocyte proliferation. Utilizing a trypan blue exclusion test, cytotoxicity was assessed 48 hours after treatment. Alamar blue reagent (20 pl) was added to each well of the 96-well plate after 48 hours of treatment. The absorbance was measured at 570 and 590 nm using a plate reader after the plates were incubated at 37°C and 5% CO2 for 4 hours. The cytotoxicity of KBU2046 and calcitriol on PBMC cells was graphed as % cell viability. There was no significant difference in % cell viability of treated PBMCs compared to untreated. Calcitriol and KBU2046 are noncytotoxic to PBMCs following 48 hours treatment at the exhibited concentrations. Compared to androgen dependent LNCaP cells, PBMCs treated with calcitriol and KBU2046 exhibited no difference in % cell viability, indicating selectivity of the combination.

[0088] With the strong evidence that calcitriol and KBU2046 are able to act effectively in combination, 22-oxa-calcitriol was investigated in combination with KBU2046. 22-oxa- calcitriol (OCT) is a novel vitamin D3 analog that has been shown by previous studies to reduce proliferation and promote differentiation without raising calcium levels to a hypercalcemic state, like the negative aspect of calcitriol. 22-oxa-calcitriol stock was dissolved in ethanol and diluted in culture media to final concentrations (0-2 pM). LNCaP were plated at 5 x 103cells per well and incubated for 24 hours. After incubation, cells were treated with concentrations based on prior studies with calcitriol for 48 hours. Alamar blue reagent (20 pl) was added to each well of the 96-well plate after 48 hours of treatment. Theabsorbance was measured at 570 and 590 nm using a plate reader after the plates had been incubated at 37°C and 5% CO2 for 4 hours. The cytotoxicity of 22-oxa-calcitriol treated LNCaP cells was graphed as % cell viability. Treatment with 22-oxacalcitriol is noncytotoxic to LNCaP cells after 48 hours. There was no statistically significant change in % cell viability (p- value = <0.05) as drug concentration increased. The experiment was repeated with a higher concentration of 22-oxacalcitriol compared to ca Icitriol, there was no significant difference (P-value <0.05) observed in cell viability. As 22-oxacalcitriol dose increased, there is no significant effect on LNCaP cell viability.

[0089] With significant evidence that calcitriol and KBU2046 can decrease cell viability in combination, vitamin D analog, paricalcitol was explored in combination with KBU2046. Previous research has demonstrated that paricalcitol treatment reduces proliferation and promotes differentiation without elevating calcium levels to a hypercalcemic state, which are the negative aspects of calcitriol treatment. Studies have shown paricalcitol treatment produced dose-dependent inhibition in primary prostatic cultures and cell lines from prostate cancer. Prior to usage, paricalcitol stock was dissolved in ethanol as a stock solution and diluted in culture media to final concentrations (0-5 pM). LNCaP cells were plated at a density of 5 x 103cells per well and incubated for 24 hours. After incubation, the cells were treated with concentrations based on prior studies of paricalcitol for 48 hours. Alamar blue reagent (20 pl) was added to each well of the 96-well plate after the 48 hours of incubation. The absorbance was measured at 570 and 590 nm using a plate reader after the plates had been incubated at 37°C and 5% CO2for 4 hours. The cytotoxicity of paricalcitol treatment on LNCaP cells was graphed as % cell viability. The findings indicate that paricalcitol treatment is noncytotoxic to LNCaP cells after 48 hours at low dose concentrations. There was no statistically significant change in % cell viability (p- value = <0.05) as paricalcitol concentration increased. The experiment was repeated with higher concentrations of paricalcitol (5 pM) compared to the previous calcitriol concentration used (2.5 pM).Concentration of paricalcitol was doubled compared to calcitriol, there was no significant difference (P-value <0.05) in % cell viability as concentration of paricalcitol increased.

[0090] To determine if viability inhibition caused by KBU2046 and / or calcitriol is long- lasting, LNCaP cells treated with these compounds were examined to see if the inhibition was reversed by removing the compounds. LNCaP was cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin, and L-glutamine. After 60-80 percent confluence, cells were plated at lxl04cells per well in 3 ml medium in 6 well plates and incubated for 24 hours to adhere. The medium was removed after incubation andreplaced with either media alone as a negative control, media containing vehicles (DMSO / ethanol, final concentration 0.1%), or KBU2046 / calcitriol at the indicated concentrations. Every three days, the medium holding the vehicle and / or drugs was replaced. Cells were removed from the plate by incubating them with 0.5 ml of 0.05% Trypsin-EDTA, stopping the reaction with an equivalent amount of media containing serum, and washing them with PBS without calcium or magnesium at the given time points.

[0091] Cells were plated at and treated for six days. Each compound was removed after day six and cells proceeded to grow for an additional six days. Cells were counted every three days for twelve days total. Trypan blue was added to a cell suspension following each given time trial, and the outcome was visually assessed to determine the amount of cell growth inhibition. Results show that cells that were continuously treated with KBU2046 and calcitriol in combination had significant inhibition of LNCaP growth. No significant growth was observed in populations that had been treated with calcitriol and KBU2046 for 6 days and then left to develop in the absence of the combination for 6 days. On day nine, there was a minor rise in growth. On day 12, the combination was significantly low compared to untreated cells.

[0092] The "scratch" was analyzed using Fiji Image programming software (Rasband, W.S., Imaged, U. 5. National Institutes of Health, Bethesda, Maryland, USA) to quantify the area / percentage of the wound comparably before and after treatment. The wound healing assay confirmed KBU2046 treatment alone decreased migration, as noted by previous studies. Calcitriol treatment followed a similar pattern. When combined (at half the original IC50 concentration (5 pM KBU2046 +2.5 pM calcitriol) there was a significant decrease in migration. When the data is expressed as % open wound it showed a significant increase compared to the compounds treated alone. A significant increase in percent of open wound for 24, 48, and 72 hour treatment was exhibited as KBU2046 and calcitriol treatment concentration increased. There were significant differences observed as concentration and time increased for each time point P-value <0.05 = *, <0.01=**, and <0.001 = ***. KBU2046 and calcitriol treatment in combination are thus migratory inhibitors.

[0093] A combination of KBU2046 and calcitriol treatment significantly decreased both viability and migration of an androgen dependent prostate cancer cell line. None of the compounds investigated in this study had this dual effect when tested individually. Numerous functions support cancer growth and migration at the systemic level.

[0094] The combination of calcitriol and KBU2046 treatment was likely effective due to the negative effects of calcitriol on cell cyclins and the negative effects of KBU2046 on kinasesrequired for migration. My data suggests that the combination of KBU2046 and calcitriol may be an effective therapy for prostate cancer; however, one must take into consideration negative affects toward normal bodily function, particularly the immune system. It has been reported that KBU2046 alone was nontoxic to normal cells. However, the "normal cells" were not truly normal because they were a cell line obtained by immortalizing an epithelial cell line. Thus, I chose to study adverse effects on human lymphocytes. Remarkably the duel inhibitory effects of this combination had no adverse effects on PBMC viability, indicating that the combination is selective for cancer cells.

[0095] Docetaxel (Taxotere), is one of the most prominent chemotherapy agents for prostate cancer, it is generally administered in conjunction with prednisone, a steroid medication. Docetaxel toxicity can limit the dose that may be delivered to patients as there are many adverse side effects. Another widely used drug for therapy of prostate cancer is Lupron. It works by preventing the production of testosterone; however; prostate cancer becomes androgen independent within a few years. Due to the many routes that contribute to the development of cancer, monotherapy has a limited ability to prevent cancer and is often unsuccessful in treating it. Perhaps a combination of the findings presented in this thesis with the use of Lupron may improve the life expectancy of patients with this disease.

[0096] The outcomes of this study promote additional research into the therapeutic application of KBU2046 and calcitriol combination in the prevention and remission of human prostate cancer.ADDITIONAL EMBODIMENTS1. A method of treating prostate cancer, comprising administering to a subject in need thereof the compound of Formula (I):[Formula (1)], and administering to the subject a vitamin D analog.2. The method according to a preceding embodiment, wherein the vitamin D analog comprises calcitriol, calcipotriol (22-ene-26,27-dehydro-la,25(OH)2D3), paricalcitol (19-nor- la,25(OH)2D2), 22-oxacalcitriol (22-oxa-l,25(OH)2D3), alfacalcidol (lajOHjDs), hectorol (la(OH)D2), fa leca Icitriol (1,25-(OH)2-26,27F6 D3), tacalicitol (la,24(R)(OH)2D3), eldecalcidol (2P-(3-hydroxypropoxy)-la,25(OH)2D3), seocalcitol (22,24 diene-24,26,27-trishomo- la,25(OH)2D3), 20-epi-la,25(OH)2D3, lexicalcitol (20-epi-22-oxa-24,26,27-trishomo-la,25(OH)2D3), 17-methyl-19-nor-21-nor-23-yne-26,27-F6-la,25(OH)2D3, inecalcitol (19-nor- 14-epi-23-yne-la,25(OH)2D3), 19-nor-14,20-bisepi-23-yne-la,25(OH)2D3, 2-methylene-19- nor-(20S)-la25(OH)2D3, seco-C-9,11 bisnor-17-methyl-20-epi-26,27-F6-la,25(OH)2D3, ((5E,7E)-22-ene-26,27-dehydro-la,25(OH)2D3, 16-ene-23-yne-la,25(OH)2D3, 2-amino-3- deoxy-19-nor-22-ene-26-dihomo-27-dihomo-25(OH)D3, (lR,3S,5Z)-5-((8E)-2-((3R)-3-((2R,3S)- 3-(5-cyclopropyl-3H-l,2-dioxol-3-yl)-2-ethyl-3-methylcyclohexylidene)ethylidene-4- methylenecyclohexane-l,3-diol), ((24E)-(lS)-25-dehydro-24a-homo-la,25(OH)2D3, 20R-(4- hydroxy-4-methylpentyl)-23-yne-26,27-hexafluoro-19-nor-la,25(OH)2D3, 20R-21(3-hydroxy- 3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-la,25(OH)2D3, 20R-21(3- trideuteromethyl-3-hydroxy-4,4,4-trideteurobutyl)-23-yne-26,27-hexafluoro-19-nor- la,25(OH)2D3, 19-nor-2a-(3-hydroxypropyl)-la,25(OH)2D3, la,25(OH)2D3-3-bromoacetate, 16,23Z-diene-26,27-F6-19-nor-la,25(OH)2D3, or a combination thereof. The method of any preceding embodiment, wherein the vitamin D analog comprises calcitriol. The method of any preceding embodiment, wherein the prostate cancer comprises adenocarcinoma of the prostrate, transitional cell carcinoma of the prostrate, prostrate sarcoma, squamous cell carcinoma of the prostrate, neuroendocrine tumors of the prostrate, small cell prostate cancer, or a combination thereof. The method of any preceding embodiment, wherein the prostate cancer comprises acinar adenocarcinoma, prostatic ductal adenocarcinoma, or a combination thereof. The method of any preceding embodiment, wherein the prostate cancer comprises recurrent prostate cancer. The method of any preceding embodiment, wherein the prostate cancer comprises metastatic prostate cancer. The method of any preceding embodiment, wherein the subject further receives chemotherapy, hormonal therapy, immunotherapy, gene therapy, radical prostatectomy, radiation beam therapy, focal therapy, or a combination thereof. The method of any preceding embodiment, wherein the compound of Formula (1) is administered during a first treatment period, the vitamin D analog is administered during a second treatment period, and the first treatment period is consecutive with the second treatment period; the first treatment period occurs prior to the second treatment period; or the first treatment period occurs subsequent to the second treatment period.The method according to a preceding embodiment, wherein the subject receives one or more additional prostate cancer treatments. The method according to a preceding embodiment, wherein the subject further receives chemotherapy, hormonal therapy, immunotherapy, gene therapy, radical prostatectomy, radiation beam therapy, focal therapy, or a combination thereof. The method according to a preceding embodiment, wherein the subject further receives abiraterone acetate, Akeega (niraparib tosylate and abiraterone acetate), apalutamide, bicalutamide, cabazitaxel, darolutamide, degarelix, docetaxel, leuprolide acetate, enzalutamide, firmagon, flutamide, feuprolide acetate, Lutetium Lu 177 fipivotide tetraxetan, mitoxantrone hydrochloride, nilutamide, olaparib, vipivotide tetraxetan), relugolix, rucaparib camsylate, Sipuleucel-T, talazoparib tosylate, , radium 223 dichloride, goserelin acetate, or a combination thereof. A pharmaceutical composition, comprising the compound of Formula (1) and a vitamin D analog. The pharmaceutical composition according to a preceding embodiment, wherein the vitamin D analog comprises calcitriol, calcipotriol (22-ene-26,27-dehydro-la,25(OH)2D3), paricalcitol (19-nor-la,25(OH)2D2), 22-oxacalcitriol (22-oxa-l,25(OH)2D3), alfacalcidol (la(0H)D3), hectorol (la(0H)D2), falecalcitriol (1,25-(OH)2-26,27F6 D3), tacalicitol (la,24(R)(OH)2D3), eldecalcidol (2p-(3-hydroxypropoxy)-la,25(OH)2D3), seocalcitol (22,24 diene-24, 26,27- trishomo-la,25(OH)2D3), 20-epi-la,25(OH)2D3, lexica Icitol (20-epi-22-oxa-24,26,27-trishomo- la,25(OH)2D3), 17-methyl-19-nor-21-nor-23-yne-26,27-F6-la,25(OH)2D3, inecalcitol (19-nor- 14-epi-23-yne-la, 25(OH )2D3), 19-nor-14,20-bisepi-23-yne-la,25(OH)2D3, 2-methylene-19- nor-(20S)-la25(OH)2D3, seco-C-9,11 bisnor-17-methyl-20-epi-26,27-F6-la,25(OH)2D3, ((5E,7E)-22-ene-26,27-dehydro-la,25(OH)2D3, 16-ene-23-yne-la,25(OH)2D3, 2-amino-3- deoxy-19-nor-22-ene-26-dihomo-27-dihomo-25(OH)D3, (lR,3S,5Z)-5-((8E)-2-((3R)-3-((2R,3S)- 3-(5-cyclopropyl-3H-l,2-dioxol-3-yl)-2-ethyl-3-methylcyclohexylidene)ethylidene-4- methylenecyclohexane-l,3-diol), ((24E)-(lS)-25-dehydro-24a-homo-la,25(OH)2D3, 20R-(4- hydroxy-4-methylpentyl)-23-yne-26,27-hexafluoro-19-nor-la,25(OH)2D3, 20R-21(3-hydroxy- 3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-la,25(OH)2D3, 20R-21(3- trideuteromethyl-3-hydroxy-4,4,4-trideteurobutyl)-23-yne-26,27-hexafluoro-19-nor- la,25(OH)2D3, 19-nor-2a-(3-hydroxypropyl)-la,25(OH)2D3, la,25(OH)2D3-3-bromoacetate, 16,23Z-diene-26,27-F6-19-nor-la,25(OH)2D3, or a combination thereof. The pharmaceutical composition of any preceding embodiment, wherein the vitamin D analog comprises calcitriol.16. The pharmaceutical composition of any preceding embodiment, wherein the composition is formulated for oral administration or parenteral administration.

[0097] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The term "comprising" and variations thereof as used herein is used synonymously with the term "including" and variations thereof and are open, nonlimiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" can be used in place of "comprising" and "including" to provide for more specific embodiments of the invention and are also disclosed. Other than in the examples, or where otherwise noted, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches

Claims

CLAIMSWhat is claimed is:

1. A method of treating prostate cancer, comprising administering to a subject in need thereof the compound of Formula (I):and a vitamin D analog.

2. The method according to claim 1, wherein the vitamin D analog comprises calcitriol, calcipotriol (22-ene-26,27-dehydro-la,25(OH)2D3), paricalcitol (19-nor-la,25(OH)2D2), 22-oxacalcitriol (22-oxa- 1,25(OH)2D3), alfacalcidol (la(OH)D3), hectorol (la(OH)D2), falecalcitriol (1,25-(OH)2-26,27F6 D3), tacalicitol (la,24( R)(OH)2D3), eldecalcidol (2P-(3-hydroxypropoxy)-la,25(OH)2D3), seocalcitol (22,24 diene-24,26,27-trishomo-la,25(OH)2D3), 20-epi-la,25(OH)2D3, lexica Icitol (20-epi-22-oxa-24, 26,27- trishomo-la,25(OH)2D3), 17-methyl-19-nor-21-nor-23-yne-26,27-F6-la,25(OH)2D3, inecalcitol (19- nor-14-epi-23-yne-la,25(OH)2D3), 19-nor-14,20-bisepi-23-yne-la,25(OH)2D3, 2-methylene-19-nor- (20S)-la25(OH)2D3, seco-C-9,11 bisnor-17-methyl-20-epi-26,27-F6-la,25(OH)2D3, ((5E,7E)-22-ene-26.27-dehydro-la,25(OH)2D3, 16-ene-23-yne-la,25(OH)2D3, 2-amino-3-deoxy-19-nor-22-ene-26- dihomo-27-dihomo-25(OH)D3, (1 R,3S,5Z)-5-((8E)-2-((3R)-3-((2R,3S)-3-(5-cyclopropyl-3 H-l,2-dioxol- 3-yl)-2-ethyl-3-methylcyclohexylidene)ethylidene-4-methylenecyclohexane-l,3-diol), ((24E)-(lS)-25- dehyd ro-24a-homo-la, 25(01-1 )2D3, 20R-(4-hydroxy-4-methylpentyl)-23-yne-26,27-hexafluoro-19- nor-la,25(OH)2D3, 20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27- hexafluoro-la,25(OH)2D3, 20 -21(3-trideuteromethyl-3-hydroxy-4,4,4-trideteurobutyl)-23-yne-26.27-hexafluoro-19-nor-la,25(OH)2D3, 19-nor-2a-(3-hydroxypropyl)-la,25(OH)2D3, la,25(OH)2D3- 3-bromoacetate, 16,23Z-diene-26,27-F6-19-nor-la,25(OH)2D3, or a combination thereof.

3. The method of claim 1, wherein the vitamin D analog comprises calcitriol.

4. The method of any of claims 1-3, wherein the prostate cancer comprises adenocarcinoma of the prostrate, transitional cell carcinoma of the prostrate, prostrate sarcoma, squamous cell carcinoma of the prostrate, neuroendocrine tumors of the prostrate, small cell prostate cancer, or a combination thereof.

5. The method of any of claims 1-3, wherein the prostate cancer comprises acinar adenocarcinoma, prostatic ductal adenocarcinoma, or a combination thereof.

6. The method of any of claims 1-3, wherein the prostate cancer comprises recurrent prostate cancer.

7. The method of any of claims 1-3, wherein the prostate cancer comprises metastatic prostate cancer.

8. The method of any of claims 1-3, wherein the subject further receives chemotherapy, hormonal therapy, immunotherapy, gene therapy, radical prostatectomy, radiation beam therapy, focal therapy, or a combination thereof.

9. The method of any of claims 1-3, wherein the compound of Formula (1) is administered during a first treatment period, the vitamin D analog is administered during a second treatment period, and• the first treatment period is consecutive with the second treatment period;• the first treatment period occurs prior to the second treatment period; or• the first treatment period occurs subsequent to the second treatment period.

10. The method of any of claims 1-3, wherein the subject receives one or more additional prostate cancer treatments.

11. The method of any of claims 1-3, wherein the subject further receives chemotherapy, hormonal therapy, immunotherapy, gene therapy, radical prostatectomy, radiation beam therapy, focal therapy, or a combination thereof.

12. The method of claim 11, wherein the subject further receives abiraterone acetate, Akeega (niraparib tosylate and abiraterone acetate), apalutamide, bicalutamide, cabazitaxel, darolutamide, degarelix, docetaxel, leuprolide acetate, enzalutamide, firmagon, flutamide, feuprolide acetate, Lutetium Lu 177 fipivotide tetraxetan, mitoxantrone hydrochloride, nilutamide, olaparib, vipivotide tetraxetan), relugolix, rucaparib camsylate, Sipuleucel-T, talazoparib tosylate, , radium 223 dichloride, goserelin acetate, or a combination thereof.

13. A pharmaceutical composition, comprising the compound of Formula (1) and a vitamin D analog.

14. The pharmaceutical composition according to claim 13, wherein the vitamin D analog comprises calcitriol, calcipotriol (22-ene-26,27-dehydro-la,25(OH)2D3), paricalcitol (19-nor-la,25(OH)2D2), 22- oxacalcitriol (22-oxa-l,25(OH)2D3), alfacalcidol (la(0H ) D3), hectorol (la(OH)D2), faleca Icitriol (1,25- (OH)2-26,27F6 D3), tacalicitol (la,24( R)(OH)2D3), eldecalcidol (2p-(3-hydroxypropoxy)-la,25(OH)2D3), seocalcitol (22,24 diene-24,26,27-trishomo-la,25(OH)2D3), 20-epi-la,25(OH)2D3, lexicalcitol (20-epi- 22-oxa-24,26,27-trishomo-la,25(OH)2D3), 17-methyl-19-nor-21-nor-23-yne-26,27-F6-la,25(OH)2D3, inecalcitol (19-nor-14-epi-23-yne-la,25(OH)2D3), 19-nor-14,20-bisepi-23-yne-la,25(OH)2D3, 2- methylene-19-nor-(20S)-la25(OH)2D3, seco-C-9,11 bisnor-17-methyl-20-epi-26,27-F6-la,25(OH)2D3, ((5E,7E)-22-ene-26,27-dehydro-la,25(OH)2D3, 16-ene-23-yne-la,25(OH)2D3, 2-amino-3-deoxy-19- nor-22-ene-26-dihomo-27-dihomo-25(OH)D3, ( 1 R,3S,5Z)-5-((8E)-2-((3 R)-3-((2 R,3S)-3-(5-cyclopropyl- 3H-l,2-dioxol-3-yl)-2-ethyl-3-methylcyclohexylidene)ethylidene-4-methylenecyclohexane-l,3-diol), ((24E)-(lS)-25-dehydro-24a-homo-la,25(OH)2D3, 20R-(4-hydroxy-4-methylpentyl)-23-yne-26,27-hexafluoro-19-nor-la,25(OH)2D3, 20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23- yne-26,27-hexafluoro-la,25(OH)2D3, 20R-21(3-trideuteromethyl-3-hydroxy-4,4,4-trideteurobutyl)- 23-yne-26,27-hexafluoro-19-nor-la,25(OH)2D3, 19-nor-2a-(3-hydroxypropyl)-la,25(OH)2D3, la,25(OH)2D3-3-bromoacetate, 16,23Z-diene-26,27-F6-19-nor-la,25(OH)2D3, or a combination thereof.

15. The pharmaceutical composition of claim 13 or 14, wherein the vitamin D analog comprises calcitriol.

16. The pharmaceutical composition of claim 13 or 14, wherein the composition is formulated for oral administration or parenteral administration.

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