Compounds, compositions, and methods for preventing and / or treating cancer

Abstract

The present invention includes compounds, compositions, and methods useful for preventing or treating melanoma or any other Myc-expressing cancer in a subject. In certain embodiments, the compounds comprise an estrogen (including an estrogen derivative or analog), a selective GPER agonist, and / or another small molecule G protein-coupled receptor (GPCR) agonist that increases differentiation of cancer cells.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 351,599, filed June 17, 2016, and U.S. Provisional Patent Application No. 62 / 367,174, filed July 27, 2016, all of which are incorporated herein by reference in their entirety.

[0003] Statement regarding federally funded research or development

[0004] This invention was made with government support under license number F31CA206325 granted by the National Cancer Institute. The government owns certain rights to this invention. Background Technology

[0005] Melanoma is the deadliest form of skin cancer and develops from pigmented cells called melanocytes. Melanoma typically occurs in the skin, but can also occur in the mouth, intestines, or eyes. The primary cause of melanoma is DNA damage caused by ultraviolet (UV) radiation exposure, especially in individuals with lighter skin and lower levels of skin pigment (melanin). Most melanomas develop from pre-existing nevi (moles). People with low baseline skin pigmentation, many nevi, a family history of melanoma, and weakened immune systems are at higher risk of developing melanoma. More than 80,000 new cases of melanoma are diagnosed in the United States each year. Current treatment options are limited, and only 30% of patients with metastatic melanoma survive for more than 5 years.

[0006] There is a need in the art for compounds, compositions, and methods that can be used to prevent and / or treat melanoma and other cancers in subjects. Such compounds, compositions, and methods should exhibit clinical efficacy comparable to and / or superior to current anticancer therapeutics, or optionally, when used in combination therapy, enhance the efficacy of other anticancer therapeutics. The present invention fulfills this need. Summary of the Invention

[0007] This invention provides a method for treating or preventing GPCR-expressing cancers in a subject. This invention further provides a method for treating or preventing Myc-expressing cancers in a subject. This invention further provides a composition comprising (i) an estrogen and / or a GPCR agonist and (ii) chemotherapy, engineered CAR T-cells, and / or an immune checkpoint inhibitor. This invention further provides a kit comprising (i) an estrogen and / or a GPCR agonist and (ii) chemotherapy, engineered CAR-cells, an immune checkpoint inhibitor, and / or radiotherapy, and instructional materials thereof for treating or preventing cancers in a subject.

[0008] In some embodiments, the method includes administering to a subject in need a therapeutically effective amount of estrogen and / or a G protein-coupled receptor (GPCR) agonist that increases cell differentiation in cancer. In other embodiments, the subject is further co-administered with at least one immunotherapeutic agent. In still other embodiments, the subject is further co-administered with at least one histone deacetylase inhibitor (HDAC).

[0009] In some implementations, the GPCR agonist comprises a selective G-protein-coupled estrogen receptor (GPER) agonist.

[0010] In some embodiments, the cancer expressing Myc- is selected from at least one of melanoma, Burkitt lymphoma, leukemia, sarcoma, lymphoma, multiple myeloma, brain cancer, neuroblastoma, medulloblastoma, astrocytoma, glioblastoma, ovarian cancer, cervical cancer, uterine cancer, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, thyroid cancer, liver cancer, prostate cancer, esophageal cancer, kidney cancer, bladder cancer, and gallbladder cancer. In other embodiments, the cancer expressing Myc- is selected from at least one of melanoma, pancreatic cancer, and lung cancer.

[0011] In some embodiments, the cancer expressing GPCRs is selected from at least one of melanoma, Burkitt lymphoma, leukemia, sarcoma, lymphoma, multiple myeloma, brain cancer, neuroblastoma, medulloblastoma, astrocytoma, glioblastoma, ovarian cancer, cervical cancer, uterine cancer, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, thyroid cancer, liver cancer, prostate cancer, esophageal cancer, kidney cancer, bladder cancer, and gallbladder cancer. In other embodiments, the cancer expressing GPCRs is selected from at least one of melanoma, pancreatic cancer, and lung cancer.

[0012] In some implementations, the cancer is selected from melanoma, pancreatic cancer, and lung cancer.

[0013] In some embodiments, the immunotherapeutic agent comprises an immune checkpoint inhibitor. In other embodiments, the immune checkpoint inhibitor is selected from PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, LAG3 inhibitors, IDO(1 / 2) inhibitors, and B7-H3 inhibitors.

[0014] In some embodiments, at least one HDAC inhibitor is selected from valproic acid, vorinostat, romidepsin, trichostatin A, and panobinostat.

[0015] In some embodiments, estrogen and / or a GPCR agonist and at least one immune checkpoint inhibitor are co-administered to the subject. In other embodiments, estrogen and / or a GPCR agonist and at least one immune checkpoint inhibitor are co-formulated.

[0016] In some embodiments, estrogen and / or a GPCR agonist and at least one HDAC are administered to the subject together. In other embodiments, estrogen and / or a GPCR agonist and at least one HDAC are co-formulated.

[0017] In some embodiments, the estrogen and / or GPCR agonist is the only anticancer agent administered to the subject. In other embodiments, the estrogen and / or GPCR agonist is the only anticancer agent administered to the subject in an amount sufficient to treat or prevent cancer in the subject.

[0018] In some embodiments, the GPCR is selected from G-protein-coupled estrogen receptors (GPER), MC1R, CYSLTR2, F2R, HRH2, LPAR2 / 3 / 6, PTGER1, S1PR2, S1PR3, and TBXA2R. In other embodiments, the GPCR is selected from GPER, F2R, PTGER1, and TBXA2R.

[0019] In some embodiments, the estrogen includes a selection from estrone (E1), estradiol (E2), estriol (E3), estetrol (E4), 17β-estradiol, 27-hydroxycholesterol, dehydroepiandrosterone (DHEA), 7-oxo-DHEA, 7α-hydroxy-DHEA, 16α-hydroxy-DHEA, 7β-hydroxyepiandrosterone, Δ 4 -androstenedione, Δ 5 -Androstenediol, 3α-androstanediol, 3β-androstanediol, 2-hydroxyestradiol, 16-hydroxyestradiol, estradiol cyclopentylpropionate, estradiol valerate, estradiol acetate, estradiol benzoate, ethinylestradiol (EE), mesestrol, mokestrol, ethinylestradiol, diethylstilbestrol, phenylestrol, diethylstilbestrol acetate, diethylstilbestrol dipropionate, fosfestrol, hexanestilbestrol, mesestrol dipropionate, exogenous estrogen, phytoestrogens and mycoestrogens, or at least one of their salts, solvates, tautomers, enantiomers or diastereomers.

[0020] In some embodiments, the GPER agonist includes at least one selected from G-1, tamoxifen, fulvestrant, and raloxifene, or their salts, solvates, tautomers, enantiomers, or diastereomers.

[0021] In some embodiments, the GPER agonist comprises at least one selected from the following:

[0022] The numerator of formula (I): In (I):

[0023] R1 is selected from =O, =N-OH, =N-NHC(=O) (p-methoxyphenyl), =N-NHC(=O)CH(OMe)phenyl and =N-NH(5-iodopyridin-2-yl); R2 is selected from C1-C4 alkyl and C1-C4 haloalkyl; bond a is a single or double bond, such that: if bond a is a double bond, then R3 and R4 are H, and if bond a is a single bond, then R3 is selected from H, -OH, -OAc and halogen; R4 is selected from H, -OH, -OA c and -S (o-nitrophenyl); or R3 and R4 combined to form a bimole selected from -CH2-, -OCH2O-, -OCH(CH3)O- and -OC(CH3)2O-; R5 is selected from H, benzyl, C1-C4 alkyl and acetyl; R6 is selected from H, halogen, -NO2, C1-C4 alkyl, -C≡CH, -C≡C-Si(CH3)3 (or -C≡C-TMS), -O-benzyl, -OH, -OAc, C1-C4 alkoxy, -COOH and -COO (C1-C4 alkyl); R7 is selected from H, halogen, -NO2, C1-C4 alkyl, -OH, -OAc and C1-C4 alkoxy; R8 is selected from H, halogen, -NO2, C1-C4 alkyl, -O-benzyl, -N(R)(R), -SR, -COOH, -COO (C1-C4 alkyl), -OH, -OAc, C1-C4 alkoxy, 3-thionylbutadienyl-methoxy, - SO2 (morpholino) and -OCH2CH=CH2, wherein each R is independently selected from H and C1-C4 alkyl; R9 is selected from H, halogen, -NO2, C1-C4 alkyl, -OH, -OAc and C1-C4 alkoxy, or R8 and R9 combine to form a bimole selected from -OCH2O-, -OCH(CH3)O-, -OC(CH3)2O-, -O(CH2)2O-, -O-CH=CH- and -CH=CH-O-; R 10 Selected from H, C1-C4 alkyl, and halogen; wherein each occurrence of the benzyl group is independently and optionally substituted with at least one group selected from C1-C4 alkyl, -OH, C1-C4 alkoxy, halogen, and -NO2; and

[0024] The molecule of formula (II): In (II):

[0025] R1 is selected from =O and =N-OH; R2 is a C1-C4 alkyl group; R5 is selected from H, benzyl and C1-C4 alkyl groups; R8 and R9 are independently selected from H and C1-C4 alkoxy groups, or R8 and R9 are combined to form a dimer selected from -OCH2O-, -OCH(CH3)O- and -OC(CH3)2O-, or salts, solvates, tautomers, enantiomers or diastereomers, or any mixture thereof.

[0026] In some embodiments, the GPER agonist comprises at least one selected from the following:

[0027] The molecule of formula (I-1): In (I-1):

[0028] R1 is selected from =O and =N-OH; R2 is a C1-C4 alkyl group; bond a is a single or double bond such that: if bond a is a double bond, then R3 and R4 are H, and if bond a is a single bond, then R3 and R4 are independently selected from H and -OH, or R3 and R4 combine to form a bimolecular group selected from -OCH2O-, -OCH(CH3)O-, and -OC(CH3)2O-; R5 is selected from H, benzyl, and C1-C4 alkyl; R6 is selected from H and halogen; R8 and R9 are independently selected from H and C1-C4 alkoxy groups, or R8 and R9 combine to form a bimolecular group selected from -OCH2O-, -OCH(CH3)O-, and -OC(CH3)2O-; and

[0029] The molecule of formula (II): In (II):

[0030] R1 is selected from =O and =N-OH; R2 is a C1-C4 alkyl group; R5 is selected from H, benzyl and C1-C4 alkyl groups; R7 and R8 are independently selected from H and C1-C4 alkoxy groups, or R7 and R8 are combined to form a dimer selected from -OCH2O-, -OCH(CH3)O- and -OC(CH3)2O-, or salts, solvates, tautomers, enantiomers or diastereomers, or any mixture thereof.

[0031] In some embodiments, the GPER agonist comprises at least one selected from the following: G-1; CMPD1 (rel-1-((3aS,4R,9bR)-4-(benzo[d][1,3]m-dioxacyclopenten-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopentadieno[c]quinolin-8-yl) aceto-1-one); CMPD2 (rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]m-dioxacyclopenten-5-yl)-5-methyl-3a,4,5,9b-tetrahydro-3H-cyclopentadieno[c]quinolin-8-yl) aceto-1-one); CMPD3 ( rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]m-dioxacyclopenten-5-yl)-2,3,3a,4,5,9b-hexahydro-1H-cyclopentadien[c]quinoline-8-yl) ethyl-1-one); CMPD4(rel-1-((3aS,4R,9bR)-5-benzyl-4-(6-bromobenzo[d][1,3]m-dioxacyclopenten-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopentadien[c]quinoline-8-yl) ethyl-1-one); CMPD5(rel-1-((3aS,4R,9bR)-4-(2-bromophenyl)-3a ,4,5,9b-tetrahydro-3H-cyclopentadieno[c]quinoline-8-yl) ethyl-1-one); CMPD6(rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]m-dioxacyclopenten-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopentadieno[c]quinoline-8-yl) ethyl-1-one oxime); CMPD7(rel-1-((3aS,4R,9bR)-4-(2-bromo-4,5-dimethoxyphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopentadieno[c]quinoline-8-yl) ethyl-1-one); CMPD8(rel-1-( (3aS,4R,9bR)-4-(6-chlorobenzo[d][1,3]m-dioxacyclopenten-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopentadieno[c]quinolin-8-yl) ethyl-1-one); CMPD9(rel-1-((6R,6aS,7aS,10aR,10bR)-6-(6-bromobenzo[d][1,3]m-dioxacyclopenten-5-yl)-9,9-dimethyl-6,6a,7,7a,10a,10b-hexahydro-5H-[1,3]m-dioxacyclopenten[4',5':3,4]cyclopentadieno[1,2-c]quinolin-2-yl) ethyl-1-one);CMPD10(rel-1-((1R,2S,3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]m-dioxacyclopenten-5-yl)-1,2-dihydroxy-2,3,3a,4,5,9b-hexahydro-1H-cyclopentadien[c]quinolin-8-yl) ethyl-1-one); CMPD11(rel-1-((3aS,4R,9bR)-4-(2-bromo-4,5-dimethoxyphenyl)-2,3,3a,4,5,9b) -hexahydro-1H-cyclopentadieno[c]quinolin-8-yl)ethyl-1-one; and CMPD12(rel-1-((4S,5aS,6R,11aR)-4,5,5a,6,11,11a-hexahydro-4,6-bridged methylene[1,3]m-dioxacyclopenteno[4',5':5,6]benzo[1,2-c]acrid-8-yl)ethyl-1-one), or salts, solvates, tautomers, enantiomers or diastereomers, or any mixture thereof.

[0032] In some embodiments, the GPCR agonist is selected from the following: afamelanotide (N-acetyl-L-seryl-L-tyrosyl-L-seryl-L-leucyl-L-α-glutamyl-L-histyl-D-phenylalanyl-L-arginyl-L-tryptophanyl-glycyl-L-lysyl-L-prolyl-L-valamid), N-methylLTC4 (N-methyl-5S-hydroxy-6R-(S-glutathione)-7E,9E,11Z,14Z-eicosatetraenoic acid), TFLLR-NH2 (Thr-Phe-Leu-Leu-Arg-NH2), and impromide (2-[3-(1H-imidazol-5-yl)propyl]-1-[2-[(5-methyl-1-yl)-1-yl]-[2-[(5-methyl-1-yl)-1-yl]-[2-[(5-methyl-1-yl)-[2-yl] ... H-imidazol-4-yl)methylmercapto]ethyl]guanidine), carbonylcholine (2-[(aminocarbonyl)oxy]–N,N,N-trimethylethylammonium chloride), thioprostone ((Z)-7-[(1R,3R)-3-hydroxy-2-[(E,3R)-3-hydroxy-4-phenoxybut-1-enyl]-5-oxocyclopentyl]-N-methylsulfonylhept-5-enamide), FTY720 (2-amino-2-[2-(4-octyl-phenyl)-ethyl]-propane-1,3-diol hydrochloride) and U46619 ((E)-7-((1R,4R,5S,6R)-6-((S,Z)-3-hydroxyoct-1-en-1-yl)-2-oxabicyclo[2.2.1]hept-5-yl)hept-5-enic acid).

[0033] In some embodiments, an estrogen or GPCR agonist is administered to the subject as a pharmaceutical composition, the pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier. In other embodiments, the subject is further administered at least one additional anticancer treatment. In still other embodiments, at least one additional anticancer treatment includes chemotherapy, engineered chimeric antigen receptor (CAR) T-cell therapy, immune checkpoint inhibitors, and / or radiation therapy. In still other embodiments, chemotherapy is selected from histone deacetylase inhibitors (HDAC), temozolomide, dacarbazine (DTIC), vemurafenib, dabrafenib, and trametinib. In still other embodiments, the immune checkpoint inhibitor is selected from PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG3 inhibitors, IDO(1 / 2) inhibitors, TIGIT inhibitors, and B7-H3 inhibitors. In other embodiments, the estrogen or GPCR agonist is administered to the subject via at least one route of administration selected from the following: inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, intrathecal, intracranial, and intravenous. In some embodiments, the subject is a mammal. In other embodiments, the mammal is a human.

[0034] In some embodiments, the subject has cancer or has been diagnosed with cancer. In other embodiments, the subject does not have cancer or has not been diagnosed with cancer. In still other embodiments, estrogen and / or a GPCR agonist are administered to the subject over a period of 3 weeks or less. In still other embodiments, estrogen or a GPCR agonist is administered to the subject over a period of 2 weeks or less. In still other embodiments, estrogen or a GPCR agonist is administered to the subject over a period of 1 week or less. Attached Figure Description

[0035] The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. Some embodiments are depicted in the drawings for the purpose of illustrating the invention. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0036] Figure 1 It is a non-restrictive schematic model of how sex hormones affect the normal pigment production and / or differentiation process in melanocytes.

[0037] Figure 2 The diagram illustrates the unrestricted balance of differentiation and proliferation under normal homeostatic conditions, where more differentiated cells are generally considered to have lower tumorigenicity.

[0038] Figure 3 This is a set of figures and images illustrating the findings that exposure to continuous pregnancy-related physiological levels of estrogen increases melanocyte differentiation status—as indicated by increased melanin production. Brief exposure to the same concentration of estrogen for only 4 days induces the same changes in melanocyte differentiation that are durable and persist indefinitely after estrogen withdrawal.

[0039] Figures 4A-4F The diagram illustrates the discovery that GPER signaling slows proliferation and drives differentiation in mouse and human melanomas. Figure 4A B16F10 and WM46 (BRAF) treated with estrogen (E2) V600E WM51 (BRAF) V600E ) and WM3702 (NRAS Q61L Cell proliferation was measured over 5 days. * indicates significance obtained through a two-tailed t-test. n=3 per group. Figure 4B E2-processed B16F10, WM46 (BRAF) V600E WM51 (BRAF) V600E ) and WM3702 (NRAS Q61L Melanin levels in cells were measured over 5 days. * indicates significance obtained through a two-tailed t-test. n = 3 per group. Figure 4C B16F10 and WM46 (BRAF) treated with the GPER agonist (G-1) V600E WM51 (BRAF) V600E ) and WM3702 (NRAS Q61L Cell proliferation was measured over 5 days. * indicates significance obtained through a two-tailed t-test. n = 3 per group. Figure 4D B16F10 and WM46 (BRAF) treated with G-1 V600E WM51 (BRAF) V600E ) and WM3702 (NRAS Q61L Melanin levels in cells were measured over 5 days. * indicates significance obtained through a two-tailed t-test. n = 3 per group. Figure 4E : 3-day proliferation assay of B16F10 cells treated with dose-responsive G-1. * indicates significance of one-way ANOVA and Tukey's multiple comparison test. n=5 for each group. Figure 4F Western blot of B16F10 cells treated with saturated dose-response G-1 for 16 hours. All error bars are equal to the standard deviation of the sample.

[0040] Figure 5 This is a set of diagrams and images illustrating the discovery that estrogen pretreatment inhibits the in vivo growth of melanoma cell lines.

[0041] Figure 6This is a diagram illustrating the discovery that in vivo treatment with G-1 inhibited the growth of the mouse melanoma cell line B16F10.

[0042] Figures 7A-7C The diagram illustrates the discovery that multiple pregnancies suppress melanoma development. Figure 7A Western blot validation was performed using doxycycline-induced BRAF (V600E), dominant-inactivated p53 (R248W), active CDK4 (R24C), and hTERT transduction in normal human melanocytes. (Figure) Figure 7B Representative photograph of SCID mice with human-designed melanoma xenografts. Figure 7C : MITF immunohistochemistry of all non-breeding and breeding mice, * indicates Figure 1 The repeat shown is at a scale bar of 100 μM.

[0043] Figures 8A-8E The diagram illustrates the findings that multiple pregnancies suppress melanoma development and drive differentiation. Figure 8A Experimental timeline of genetically defined human xenograft melanoma in SCID mice, n=5 per group. Figure 8B Histological characterization of representative anteroposterior skin and resulting tumors, including hematoxylin and eosin (H / E), melanocytes and proliferation markers MITF, Ki67 / MART and Fontana Masson (melanin), scale bar = 100 μM. Figure 8C-8E : Epidermal MITF staining ( Figure 8C Ki67 proliferation index ( Figure 8D ) and melanin staining in epidermal keratinocytes ( Figure 8E The quantitative value of ) is indicated by *, which represents the significance of passing the Mann-Whitney test.

[0044] Figure 9 It is a set of bar charts illustrating the findings that MSH (the endogenous equivalent of afanotide) and afanotide (also known as melanotide II or NDP-α-MSH) increase pigment production (top) and differentiation (bottom) in human melanocytes.

[0045] Figure 10A-10D It is a set of bar charts that illustrate the discovery that agonists of certain inflammations associated with melanocyte GPCRs (CCR10, F2R, PTGER1, and TBXA2R) regulate melanin synthesis in human melanocytes. Figure 10A It showed reduced differentiation, while Figure 10B-10D Each exhibits increased differentiation.

[0046] Figure 11A-11E The diagram illustrates the findings of GPER signaling driving stable differentiation in normal human melanocytes and melanoma. Figure 11A Long-term melanin assay, in which normal human melanocytes were briefly treated with progesterone (P4) or estrogen (E2). A subset of these groups (red) were treated with another brief pulse of P4 on day 27. Error bars equal the standard deviation of the samples. Figure 11B Western blot of melanocyte differentiation markers after a brief 4-day treatment with a carrier or estrogen followed by an 8-day withdrawal period. Figure 11C Experimental timelines of mouse and human melanoma cells pretreated with estrogen or GPER agonist (G-1), n=5 per group. Figure 11D : Relative tumor weight of mouse and human melanoma pretreated with estrogen. * indicates significance by the Mann-Whitney test. Figure 11E : Relative tumor weight of mouse and human melanoma pretreated with G-1, * indicates significance by the Mann-Whitney test.

[0047] Figure 12 The images illustrate the finding that GPER signaling leads to c-Myc loss in melanoma. Images AC: Western blots of heMel (Image A), WM46 (Image B), and B16F10 (Image C) melanoma cells treated briefly with E2 for 3 days followed by a 4-day treatment period. Image D: Western blot of WM46 cells treated with a specific GPER agonist (G-1) for 16 hours. Image E: Western blot of WM46 cells transduced with luciferase or c-Myc after 16 hours of G-1 treatment. Image F: Western blot of WM46 cells treated with G-1 throughout the time period. Image G: Western blot of WM46 cells treated with G-1, 100 μM PKA inhibitor Rp-8-Br-cAMPS (PKAi), or both for 1 hour. Image H: Western blot of WM46 cells treated with G-1, 2.5 μM proteasome inhibitor (MG132), or both for 1 hour. Image I: Western blots of WM46 cells treated with 10 μg / ml cyclohexylamide (CHX) with and without G-1.

[0048] Figure 13A-13G The diagram illustrates the discovery that transient GPER activation inhibits proliferation and enhances the response to melanoma immunotherapy. Figures 13A-13C B16F10 was briefly treated with pregnancy-associated concentrations of E2 (25 nM) or optimized concentrations of G-1 (500 nM). Figure 13A WM46 Figure 13B ) and YUMM1.7 ( Figure 13C Protein imprints of melanoma cells. Figure 13D Experimental timelines: B16F10 cells pretreated with vector or G-1 and then treated with αPD-1 antibody or allotype antibody control (2A3), n=5 per group. Figure 13ETumor volume in the treatment group on day 14, * indicates significance using one-way ANOVA and Tukey's multiple comparison test. Figure 13F Survival curves of mice with tumors pretreated with a vector or G-1 followed by an allotype antibody control (2A3) or αPD-1 antibody. Significance between groups as measured by Log-Rank (Mantel-Cox) is listed in the table below. Figure 13G Western blot of WM46 cells transduced with luciferase or c-Myc after treatment with G-1 for 16 hours.

[0049] Figure 14A-14F The figure illustrates the finding that treatment with G-1 and αPD-1 immunotherapy significantly prolonged the survival of mice carrying melanoma. Figure 14A Experimental timelines of mice carrying B16F10 treated with the vector or G-1 and αPD-1 antibody or isotype antibody control (2A3), n=10 per group. Figure 14B Tumor volume in the treatment group on day 14, * indicates significance using one-way ANOVA and Tukey's multiple comparison test. Figure 14C Survival curves of mice treated with the vector or G-1 and the isotype antibody control (2A3) or αPD-1 antibody. The significance between groups as measured by the Log-Rank (Mantel-Cox) test is listed in the table below. Figure 14D Summary of experiments using mice carrying YUMM1.7 treated with the vector or G-1 and an isotype antibody control (2A3) or αPD-1 antibody. Treatment began on day 14 after the tumor diameter reached 4-5 mm, with n=5 per group. Figure 14E Tumor volume in the treatment group over time. Figure 14F Survival curves of mice treated with the vector or G-1 and αPD-1 antibody or allotype antibody control (2A3). Significance between groups as determined by Log-Rank (Mantel-Cox) is listed below. Figure 14F In the table.

[0050] Figures 15A-15C The diagram illustrates the discovery that G-1 treatment alters tumor-infiltrating immune cells in vivo. Figure 15A : Experimental timeline of mice carrying YUMM1.7 melanoma or treated with G-1. Figure 15B This study summarizes heatmaps of immune profiles across biological replicas, with n=5 per group. * indicates that each immune population is assumed to be an independent measure of immune activation, and the significance is determined by two-way ANOVA. Figure 15C From Figure 15B The quantitative analysis of individual immune populations was performed, with n=5 for each group.

[0051] Figures 16A-16CThe diagram illustrates the discovery that G-1 treatment drives histone acetylation and synergizes with HDAC inhibitors in melanoma. Figure 16A Mass spectra of normal human melanocytes treated with estrogen for a short period of time. Red data points (*) indicate significant upregulation of histone acetylation regulated by CBP / p300. Figure 16B Western blots of p-RB, c-Myc, and H3K56ac in melanoma cells after treatment with G-1, HDACi, or a combination thereof. Figure 16C Proliferation assay of B16F10 melanoma cells after treatment with G-1, HDACi, or a combination thereof.

[0052] Figures 17A-17D The diagram illustrates the findings that GPER signaling reduces the proliferation of pancreatic ductal adenocarcinoma (PDAC) cells in vitro and in vivo. Figure 17A Western blot of c-Myc in several PDAC cell lines treated with 500 nM G-1 for 1 hour. Figure 17B Proliferation assays of several PDAC cell lines treated with 500 nM G-1 for 5 days. Figure 17C : Experimental timeline of mice carrying PDAC tumors treated with vectors or G-1. Figure 17D Tumor weight in mice carrying PDAC tumors treated with the vector or G-1.

[0053] Figure 18A-18G The diagram illustrates the discovery that GPER signaling reduces NSCLC cell proliferation in vitro and in vivo and has a combination effect with αPD-1 immunotherapy. Figure 18A Western blot of pCREB from LLC1 cells treated with 500 nM G-1 for 30 minutes. Figure 18B Proliferation assay of LLC1 cells treated with 500 nM G-1 for 6 days. Figure 18C Western blot of c-Myc protein from LLC1 cells treated with 500 nM G-1 for 6 days. Figure 18D Western blot of c-Myc protein from LLC1 cells treated with 500 nM G-1 over a period of time. Figure 18E Western blots of pCREB and c-Myc in TC-1 cells treated with 500 nM G-1 for 1 hour. Figure 18F Tumor volume changes over time in mice carrying LLC1 tumors treated with vector or G-1 and αPD-1 antibody or isotype antibody control (2A3). Figure 18G Survival curves of mice treated with the vector or G-1 and αPD-1 antibody or allotype antibody control (2A3). The significance between groups as measured by Log-Rank (Mantel-Cox) is listed in the table below.

[0054] Figure 19These are Western blot images derived from WM46 melanoma cells treated with increased doses of G-1. The data show that in response to increased concentrations of the GPER agonist G-1, pRB and c-myc levels decreased, while GPER expression increased. Detailed Implementation

[0055] This invention relates to the unexpected discovery that estrogen and / or small molecule G protein-coupled receptor (GPCR) agonists increase melanocyte differentiation and can be used to prevent and / or treat cancers in subjects that express GPCRs (e.g., GPER-expression) and / or Myc-expression, such as, but not limited to, melanoma, pancreatic cancer, and / or non-small cell lung cancer. In some embodiments, the GPCR is a G-protein-coupled estrogen receptor (GPER), melanocortin receptor (MC1R), or other GPCRs that activate similar downstream signaling events.

[0056] Myc protein is a transcription factor that activates the expression of several genes by binding to enhancer cassette sequences and / or recruiting histone acetyltransferases. Myc can also act as a transcriptional repressor, inhibiting the expression of certain target genes. Myc plays a direct role in controlling DNA replication, cell proliferation, differentiation, cancer invasion, angiogenesis, cell survival, and cancer cell evasion of immune surveillance. Myc is expressed in many cancer types and is functionally important. Therefore, it is a biologically attractive therapeutic target. However, targeting Myc with pharmacological compounds is very difficult, and no cancer drugs are currently approved to directly target Myc.

[0057] Myc is activated by various mitotic signals, such as serum stimulation, or by Wnt, Shh, and EGF (via the MAPK / ERK pathway). Myc is a potent oncogene protein, frequently upregulated in many types of cancer, including melanoma, Burkitt lymphoma, leukemia, sarcoma, lymphoma, multiple myeloma, brain cancer, neuroblastoma, medulloblastoma, astrocytoma, glioblastoma, ovarian cancer, cervical cancer, uterine cancer, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, stomach cancer, thyroid cancer, liver cancer, prostate cancer, esophageal cancer, kidney cancer, bladder cancer, and / or gallbladder cancer.

[0058] Melanocytes are the cells of origin for melanoma, while many other human cell types express non-canonical steroid hormone receptor G-protein-coupled estrogen receptor 1 (GPER1). As demonstrated in this paper, activation of GPER in melanocytes by estrogen or, exemplarily non-restrictive, selective GPER-specific agonists (G-1) has been found to increase the differentiation state of human melanocytes. In doing so, GPER agonists inhibit the ability of melanocytes to proliferate and increase their production of melanin and melanocyte differentiation antigen proteins (G-1). Figure 1This reduces their ability to form cancer. This reflects the fact that differentiation and proliferation are generally considered to be in balance under normal conditions, with more differentiated melanocytes having lower tumorigenicity. Figure 2 ).

[0059] In one aspect, the present invention relates to the unexpected discovery that estrogen and / or GPCR agonists (e.g., selective GPER agonists, such as G-1) can be used not only to treat and / or prevent cancers that are classically responsive to estrogen and other sex hormones—including (e.g., cancers that develop in reproductive tissues, such as, but not limited to, breast cancer, ovarian cancer, prostate cancer, and / or endometrial cancer)—but also to treat and / or prevent any cancer in which cancer cells express GPER or any other GPCR, such as, but not limited to, Myc-expressing cancers. It is not intended to limit one to any theoretical notion that estrogen and / or GPCR agonists bind to GPCRs, leading to the downregulation of Myc in cancer cells. Figure 19 As shown, treatment of GPER-expressing cells with estrogen or GPCR agonists (e.g., G-1 as a selective agonist of GPER) resulted in significant depletion of Myc protein and an unexpected increase in GPER protein. This increase in GPER itself further sensitized cancer cells to the effects of G-1 and / or other GPCR agonists.

[0060] As shown in this article, mouse and human melanoma cells respond similarly to estrogen, which slows tumor cell proliferation. Human and mouse melanoma cells treated with estrogen or the specific synthetic GPER agonist G-1 grew more slowly in mice and formed significantly smaller tumors. Figure 5-6 A brief exposure to estrogen or G-1 is sufficient to observe antitumor effects. Without being bound by any theoretical limitations, brief estrogen exposure is sufficient to induce epigenetic memory, leading to a more differentiated cellular state. The data presented in this paper show that melanocytes briefly treated with estrogen maintained upregulated expression of many major melanocyte differentiation markers—including tyrosinase, tyrosinase-related proteins, MC1R, Melan-A, and dopachrome tautomerase. Figure 3 ).

[0061] This finding helps explain the clinical observation that women with melanoma generally have a better prognosis than men with the same tumor in other respects. Furthermore, women who had been pregnant and thus exposed to high levels of the GPER agonist estrogen had a better prognosis at diagnosis of melanoma than age-matched women who had never given birth, and this protection increased with the number of previous pregnancies. Without being bound by any specific theory, this suggests that the hormonal effects of pregnancy reduce the risk of melanoma.

[0062] In some respects, treatment with estrogen, G-1, or any other GPCR agonist (e.g., selective GPER agonists), including short-term treatment, reduces the long-term melanoma risk in both women and men. According to the method of the invention, the beneficial anti-melanoma effects of pregnancy can be captured in both sexes without having to endure actual pregnancy.

[0063] In some respects, GPCR agonist therapy in melanoma patients can help slow tumor growth and prolong overall survival.

[0064] In some respects, subjects were further treated with histone deacetylase inhibitors—such as, but not limited to, valproic acid, vorinostat (SAHA), romidasin, trichostatin A (TSA), JQ1, or other bromine domain-targeting inhibitors. These compounds unexpectedly enhanced the effects of GPCR agonists—including G-1 as a selective GPER agonist.

[0065] In some respects, subjects are further treated with anticancer therapies—such as, but not limited to, chemotherapy, engineered chimeric antigen receptor (CAR) T-cells, any immunotherapeutic agents (e.g., immune checkpoint inhibitors), and / or radiation therapy. CARs targeting melanoma cells can be based on the extracellular domains of proteins—including but not limited to MC1R, HGFR (Met), MART-1, VEGFR, ganglioside GD3, GP100, tyrosinase, and NY-ESO-1. The invention also contemplates vaccines targeting differentiation proteins—such as, but not limited to, MC1R, MART, TYR, and / or DCT.

[0066] Immune checkpoint therapies (e.g., but not limited to inhibitors targeting PD-1 and / or CTLA4) work by activating cytotoxic T cells that recognize antigens on tumor cells. These antigens on melanoma typically include melanocyte differentiation markers (tyrosinase, tyrosinase-associated protein, MC1R, Melan-A, and dopachrome tautomerase). Treatment of subjects with estrogen, G-1, or any other GPCR agonist (e.g., a GPER agonist) increases the expression of these antigenic proteins. In some embodiments, treatment of subjects with estrogen and / or a GPER agonist increases the efficacy of immunotherapies—including current standard of care regimens with immune checkpoint inhibitors and / or vaccines targeting differentiation proteins.

[0067] In some embodiments, subjects at increased risk of melanoma (e.g., subjects with a history of melanoma, a family history of melanoma, and / or a transplant recipient), or any other type of GPCR-expressing (e.g., GPER-expressing) and / or Myc-expressing cancer, may benefit from treatment with estrogen and / or a GPCR agonist (e.g., a selective GPER agonist). In other embodiments, general subjects may benefit from treatment with estrogen and / or a GPCR agonist (e.g., a selective GPER agonist) as prophylactic or maintenance therapy to prevent future development of GPCR-expressing (e.g., GPER-expressing) and / or Myc-expressing cancer. In yet another embodiment, current cancer patients may benefit from treatment with estrogen and / or a GPCR agonist (e.g., a selective GPER agonist) alone, in combination with HDAC, and / or in combination with any other therapy—including, but not limited to, immunotherapy (using, for example, immune checkpoint inhibitors), targeted chemotherapy, conventional non-selective chemotherapy, or radiotherapy.

[0068] In some embodiments, estrogen and / or a GPER agonist (e.g., G-1) and / or any other GPCR agonist are formulated for oral administration (e.g., as a pill), intravenous or intramuscular administration, and / or topical administration (e.g., topical cream, gel, and / or ointment). In other embodiments, estrogen, a GPER agonist (e.g., G-1), and / or other GPCR agonists are used to reduce the likelihood that a benign nevus (i.e., a melanocytic nevus) will develop into melanoma.

[0069] This invention should not be construed as limiting the use of GPER agonists in the treatment of cancers (e.g., but not limited to melanoma) that express GPCRs (e.g., GPER-expressing) and / or Myc-expressing. In addition to GPER, melanocytes and tumors in other tissue types also express other G protein-coupled receptors (melanocyte GPCRs highlighted in Table 1), which, upon activation, signal via the same downstream pathways as GPER to similarly drive differentiation. Figure 9 In some embodiments, agonists of these receptors, alone and / or in combination with other therapeutic agents—including, but not limited to, immunotherapeutic agents—have antitumor activity. Non-limiting examples of such receptor ligands include MSH peptide derivatives, such as afanotide, also known as melanotide II or NDP-α-MSH. Figure 10A-10DThis diagram illustrates how certain agonists of inflammatory melanocyte GPCRs regulate melanin synthesis in human melanocytes. Therefore, GPCR agonists that increase melanocyte differentiation are useful in this invention. In some embodiments, non-limiting examples of such GPCR agonists include agonists targeting MC1R, CYSLTR2, F2R, HRH2, LPAR2 / 3 / 6, PTGER1, S1PR2, S1PR3, and / or TBXA2R. In other embodiments, non-limiting examples of such GPCR agonists include agonists targeting F2R, PTGER1, and / or TBXA2R. In other embodiments, non-limiting examples of agonists that can be used in this invention include afanotide (N-acetyl-L-seryl-L-tyrosyl-L-seryl-L-leucyl-L-α-glutamyl-L-histyl-D-phenylalanyl-L-arginyl-L-tryptophanylglycyl-L-lysyl-L-prolyl-L-valamid), N-methylLTC4 (N-methyl-5S-hydroxy-6R-(S-glutathione)-7E,9E,11Z,14Z-eicosatetraenoic acid), TFLLR-NH2 (Thr-Phe-Leu-Leu-Arg-NH2), and impromide (2-[3-(1H-imidazol-5-yl)propyl]-1-[2-[(5-methyl-1H-imidazol-4-yl)- [(aminocarbonyl)oxy]-N,N,N-trimethylethylammonium chloride, thioprostone ((Z)-7-[(1R),3R)-3-hydroxy-2-[(E,3R)-3-hydroxy-4-phenoxybut-1-enyl]-5-oxocyclopentyl]-N-methylsulfonylhept-5-enamide), FTY720 (2-amino-2-[2-(4-octyl-phenyl)-ethyl]-propane-1,3-diol hydrochloride or fingomod hydrochloride) and U46619 ((E)-7-((1R,4R,5S,6R)-6-((S,Z)-3-hydroxyoct-1-en-1-yl)-2-oxabicyclo[2.2.1]hept-5-yl)hept-5-enic acid).

[0070] Table 1. GPCRs in melanocytes that promote cell differentiation and melanin production when activated by natural or synthetic ligands.

[0071]

[0072] definition

[0073] As used herein, each of the following terms has the meaning relevant to this section.

[0074] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Typically, the nomenclature and laboratory procedures used herein in cell culture, molecular genetics, and chemistry are those well-known and commonly used in the art.

[0075] The articles “a” and “an” used in this article refer to one or more (i.e., at least one) grammatical object. For example, “an element” means one or more elements.

[0076] As used herein, the term “about” will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. As used herein, when referring to measurable values ​​such as quantity, duration, etc., the term “about” means including variations from a specific value of ±20% or ±10%, ±5%, ±1% or ±0.1%, as such variations are suitable for performing the disclosed methods.

[0077] As used herein, unless otherwise stated, the term "alkoxy" used alone or in combination with other terms refers to an alkyl group having a specified number of carbon atoms, as defined elsewhere herein, connected to the remainder of the molecule by an oxygen atom, such as methoxy, ethoxy, 1-propoxy, 2-propoxy (or isopropoxy), and higher homologues and isomers. Specific examples are (C1-C3)alkoxy groups, such as, but not limited to, ethoxy and methoxy.

[0078] Unless otherwise stated, the term "alkyl" as used herein, either on its own or as part of another substituent, refers to a straight-chain or branched hydrocarbon having a specified number of carbon atoms (i.e., C1-C1). 10 (Representing 1 to 10 carbon atoms) and including straight-chain, branched, or cyclic substituents. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. Specific embodiments are (C1-C4) alkyl groups, such as, but not limited to, ethyl, methyl, isopropyl, n-butyl, isobutyl, tert-butyl, and cyclopropylmethyl.

[0079] "Disease" is a state of health in animals where they are unable to maintain homeostasis, and where their health continues to deteriorate if the disease is not treated. Conversely, "disorder" in animals is a state of health in which they are able to maintain homeostasis, but their health is not as good as it would be without the disorder. Disorders, if left untreated, do not necessarily lead to a further decline in the animal's health.

[0080] The terms “effective amount” or “therapeutic effective amount” are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition as described herein that effectively achieves specific biological results or provides therapeutic or preventative benefits. These results may include, but are not limited to, antitumor activity as determined by any means suitable in the art.

[0081] As used in this article, “endogenous” means any material that originates from or is produced within an organism, cell, tissue, or system.

[0082] As used herein, “estrogen” or “oestrogen” refers to any substance, natural or synthetic (including estrogen analogs and derivatives), that mimics the effects of the natural hormone—estrogen. Types of estrogens include, but are not limited to, estrone (E1), estradiol (E2), estriol (E3), estradiol (E4), 17β-estradiol, 27-hydroxycholesterol, dehydroepiandrosterone (DHEA), 7-oxo-DHEA, 7α-hydroxy-DHEA, 16α-hydroxy-DHEA, 7β-hydroxyepiandrosterone, and Δ… 4 -androstenedione, Δ 5 -Androstenediol, 3α-androstanediol, 3β-androstanediol, 2-hydroxyestradiol, 16-hydroxyestradiol, estradiol cyclopentylpropionate, estradiol valerate, estradiol acetate, estradiol benzoate, ethinylestradiol (EE), mesestrol, mokestrol, ethinylestradiol, diethylstilbestrol, phenylestradiol, diethylstilbestrol acetate, diethylstilbestrol dipropionate, phosphostrol, hexanestilbestrol, mesestrol dipropionate, exogenous estrogens, phytoestrogens and / or mycoestrogens.

[0083] Unless otherwise stated, the terms “halogenated” or “halogen” as used herein, alone or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0084] As used in this article, the term "GPCR" refers to G protein-coupled receptor.

[0085] As used in this article, the term “GPER” refers to the G protein-coupled estrogen receptor, which is a type of GPCR.

[0086] As used herein, “guidance material” includes publications, records, charts, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. For example, guidance material for a kit of the present invention may be attached to a container containing the nucleic acids, peptides, and / or compositions of the present invention, or may be shipped together with a container containing the nucleic acids, peptides, and compositions. Alternatively, guidance material may be shipped separately from the container, with the intention that the guidance material and the compound be used collaboratively by the recipient.

[0087] As used herein, “immune checkpoint inhibitor” refers to drugs (e.g., small molecules, peptides, and / or antibodies) that trigger the immune system to attack cancer cells. Examples of immune checkpoint inhibitors include, but are not limited to, antibodies, PD-1 inhibitors (i.e., pembrolizumab, nivolumab, anti-PD-1), PD-L1 inhibitors (i.e., atezolizumab, anti-PD-L1), CTLA-4 inhibitors (i.e., ipilimumab, anti-B7-1 / B7-2, anti-CTLA-4), indoleamine (2,3)-dioxygenase (IDO1 / 2) inhibitors, B7 homology 3 (B7-H3) inhibitors, lymphocyte activation gene 3 (LAG3) inhibitors, and TIGIT (a T-cell immune receptor with Ig and ITIM domains) targeting antibodies and reagents.

[0088] As used herein, the term "modified" refers to an altered state or structure of the molecules or cells of the present invention. Molecules can be modified in many ways—including chemically, structurally, and functionally. Cells can be modified by introducing nucleic acids.

[0089] As used herein, the term "modulation" refers to a detectable increase or decrease in response level in a subject compared to response levels in subjects without treatment or the compound, and / or compared to response levels in other identical but untreated subjects. This term includes perturbing and / or influencing natural signals or responses, thereby mediating a beneficial therapeutic response in the subject, preferably in a human.

[0090] As used herein, the term "Myc-expressing cancer" refers to a type of cancer whose origin and / or proliferation depend on Myc activation, dysregulation, mutation, and / or abnormal function and / or be accelerated by Myc activation, dysregulation, mutation, and / or abnormal function. Non-limiting examples of Myc-expressing cancers include melanoma, Burkitt lymphoma, leukemia, sarcoma, lymphoma, multiple myeloma, brain cancer, neuroblastoma, medulloblastoma, astrocytoma, glioblastoma, ovarian cancer, cervical cancer, uterine cancer, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, thyroid cancer, liver cancer, prostate cancer, esophageal cancer, kidney cancer, bladder cancer, and gallbladder cancer.

[0091] "Parenteral" administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.

[0092] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful in this invention with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. Pharmaceutical compositions facilitate the application of compounds to a living organism. Various techniques for administering compounds exist in the art, including but not limited to: intravenous, oral, aerosol, parenteral, ocular, pulmonary, intracranial, transdermal, and topical administration. In some embodiments, administration includes topical application.

[0093] As used herein, the term “pharmaceutically acceptable” means a material, such as a carrier or diluent, that does not eliminate the biological activity or properties of the composition and is relatively non-toxic, i.e., that the application of the material to an individual will not cause undesirable biological effects or interact with any component of the composition comprising it in a harmful manner.

[0094] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, which participates in the delivery or transport of compounds useful to the present invention within or to a subject, enabling them to perform their intended function. Typically, such an construct is delivered or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation, which includes compounds useful in the present invention and harmless to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragali powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; and other non-toxic and compatible substances used in pharmaceutical formulations. As used herein, "pharmaceuticalally acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delay agents that are compatible with the activity of compounds useful in this invention and are physiologically acceptable to the subject. Additional active compounds may also be incorporated into the composition. "Pharmaceuticalally acceptable carrier" may further include pharmaceutically acceptable salts of compounds usable in this invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of this invention are known in the art and described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0095] As used herein, the term “pharmaceutically acceptable salt” means a salt of an applied compound prepared from a pharmaceutically acceptable nontoxic acid and / or base, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates (including hydrates) and their inclusion complexes.

[0096] As used herein, a “pharmaceutical effective amount,” “therapeutic effective amount,” or “effective amount” of a compound is an amount of the compound sufficient to provide a beneficial effect to a subject who has been given the compound.

[0097] As used herein, the terms “prevent,” “preventing,” or “prevention” refer to the avoidance or delay of the onset of symptoms associated with a subject’s disease or condition, provided that the subject has not experienced these symptoms at the start of administration of the drug or compound. Disease, condition, and disorder are used interchangeably here.

[0098] The term “specifically bind” or “specifically binds” as used in this article means that the first molecule preferentially binds to the second molecule (e.g., a specific receptor or enzyme), but not necessarily only to the second molecule.

[0099] The term "subject" is intended to include any living organism (e.g., a mammal) that can elicit an immune response. As used herein, "subject" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as sheep, bovines, pigs, canines, felines, and rodents. Preferably, the subject is a human.

[0100] As used herein, “substantially purified” cells are cells that are substantially free of other cell types. Substantially purified cells also refer to cells that have been isolated from other cell types normally associated with their natural state. In some embodiments, a substantially purified cell population refers to a homologous cell population. In other embodiments, the term refers only to cells that have been isolated from cells naturally associated with their natural state. In yet another embodiment, the cells are cultured in vitro. In yet another embodiment, the cells are not cultured in vitro.

[0101] As used in this article, the term "therapeutic agent" refers to treatment and / or prevention. Therapeutic effects are achieved by suppressing, alleviating, or eradicating a disease state.

[0102] As used in this article, “topical application” or “external application” refers to the application of a drug to the surface of the body, such as the skin or mucous membranes.

[0103] As used herein, “treatment” refers to reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. As used herein, the terms “treatment” or “treating” are defined as the application or administration of a therapeutic agent, i.e., a composition available in this invention (alone or in combination with another agent), to a subject, or the application or administration of a therapeutic agent to an isolated tissue or cell line (e.g., for diagnostic or in vitro application) from a subject suffering from a disease or disorder, having symptoms of a disease or disorder, or having the potential to develop a disease or disorder, with the aim of curing, restoring, alleviating, reducing, altering, remedying, improving, ameliorating, or influencing the disease or disorder, its symptoms, or the potential to develop a disease or disorder. These treatments can be specifically tailored or modified based on knowledge gained from the field of pharmacogenomics.

[0104] As used in this article, the term "UV" refers to ultraviolet light.

[0105] "Heterogeneous" refers to any substance derived from animals of different species.

[0106] Throughout this disclosure, various aspects of the invention can be presented in a scope format. It should be understood that the scope format description is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, it should be considered that the scope description specifically discloses all possible sub-ranges and individual numerical values ​​within those ranges. For example, it should be considered that a description of a range such as 1 to 6 specifically discloses sub-ranges, such as from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0107] Compounds and Compositions

[0108] In one aspect, the present invention contemplates the binding and activation of estrogen and / or other small molecules to GPCRs—such as the G protein-coupled estrogen receptor GPER. This induces cellular signaling events that increase the differentiation state of tumor cells. This slows tumor cell proliferation, slows overall tumor growth, and makes tumor cells more visible to immune cells and / or more susceptible to immunotherapy. In some embodiments, the present invention contemplates other molecules that act as agonists of other GPCRs. These molecules induce cell differentiation by participating in many of the same downstream pathways activated by GPER.

[0109] In some embodiments, the compound of the present invention, or a salt, solvate, tautomer, enantiomer, or diastereomer thereof, is at least one of a compound of formula (I):

[0110] In (I):

[0111] R1 is selected from =O, =N-OH, =N-NHC(=O) (p-methoxyphenyl), =N-NHC(=O)CH(OMe)phenyl and =N-NH(5-iodopyridin-2-yl);

[0112] R2 is selected from C1-C4 alkyl and C1-C4 haloalkyl (e.g., but not limited to -CF3);

[0113] Bond a is either a single bond or a double bond, such that:

[0114] If bond a is a double bond, then R3 and R4 are H bonds, and

[0115] If bond a is a single bond, then R3 is selected from H, -OH, -OAc and halogens; R4 is selected from H, -OH, -OAc and -S (o-nitrophenyl); or R3 and R4 combine to form a dimer selected from -CH2-, -OCH2O-, -OCH(CH3)O- and -OC(CH3)2O-.

[0116] R5 is selected from H, benzyl, C1-C4 alkyl, and acetyl;

[0117] R6 is selected from H, halogen, -NO2, C1-C4 alkyl, -C≡CH, -C≡C-Si(CH3)3 (or -C≡C-TMS), -O-benzyl, -OH, -OAc, C1-C4 alkoxy, -COOH and -COO (C1-C4 alkyl);

[0118] R7 is selected from H, halogens, -NO2, C1-C4 alkyl, -OH, -OAc, and C1-C4 alkoxy.

[0119] R8 is selected from H, halogen, -NO2, C1-C4 alkyl, -O-benzyl, -N(R)(R), -SR, -COOH, -COO(C1-C4 alkyl), -OH, -OAc, C1-C4 alkoxy, 3-thionecyclobutadienyl-methoxy, -SO2(morpholino), and -OCH2CH=CH2, wherein each R is independently selected from H and C1-C4 alkyl;

[0120] R9 is selected from H, halogens, -NO2, C1-C4 alkyl, -OH, -OAc, and C1-C4 alkoxy groups.

[0121] Or R8 and R9 combine to form a bimole selected from -OCH2O-, -OCH(CH3)O-, -OC(CH3)2O-, -O(CH2)2O-, -O-CH=CH- and -CH=CH-O-;

[0122] R 10 Selected from H, C1-C4 alkyl groups and halogens;

[0123] Each occurrence of the benzyl group is independently and optionally substituted with at least one group selected from C1-C4 alkyl, -OH, C1-C4 alkoxy, halogen and -NO2.

[0124] In some embodiments, the compound of the present invention or its salt, solvate, tautomer, enantiomer or diastereomer is at least one of the compounds of formula (I-1):

[0125] In (I-1):

[0126] R1 is selected from =O and =N-OH;

[0127] R2 is a C1-C4 alkyl group;

[0128] Bond a is either a single bond or a double bond, such that:

[0129] If bond a is a double bond, then R3 and R4 are H bonds, and

[0130] If bond a is a single bond, then R3 and R4 are independently selected from H and -OH, or R3 and R4 combine to form a dimer selected from -OCH2O-, -OCH(CH3)O- and -OC(CH3)2O-;

[0131] R5 is selected from H, benzyl, and C1-C4 alkyl;

[0132] R6 is selected from H and halogens;

[0133] R8 and R9 are independently selected from H and C1-C4 alkoxy groups, or R8 and R9 are combined to form a dimer selected from -OCH2O-, -OCH(CH3)O- and -OC(CH3)2O-.

[0134] In some embodiments, the compound is not G-1. In other embodiments, the compound is G-1.

[0135] In some implementations, R1 is =0. In other implementations, R1 is =N-OH.

[0136] In some embodiments, R2 is selected from methyl, trifluoromethyl, ethyl, 1-propyl, and 2-propyl. In other embodiments, R2 is methyl or trifluoromethyl.

[0137] In some implementations, bond a is a double bond, and R3 and R4 are H.

[0138] In some embodiments, bond a is a single bond, and R3 and R4 are independently selected from H and -OH. In other embodiments, bond a is a single bond, and R3 and R4 are H. In still other embodiments, bond a is a single bond, and R3 and R4 are -OH. In still other embodiments, bond a is a single bond, and R3 and R4 are -OH and are cis-to- ...

[0139] In some implementations, bond a is a single bond, and R3 and R4 combine to form -OC(CH3)2O-.

[0140] In some embodiments, R5 is selected from H, benzyl, methyl, ethyl, 1-propyl, and 2-propyl.

[0141] In some embodiments, R6 is selected from H, F, Cl, Br, and I. In some embodiments, R5 is selected from H, Cl, and Br.

[0142] In some embodiments, R8 and R9 are independently selected from H and C1-C4 alkoxy groups. In other embodiments, R8 and R9 are independently selected from H, methoxy, ethoxy, 1-propoxy, and 2-propoxy groups. In still other embodiments, R8 and R9 are independently selected from H and methoxy groups.

[0143] In some embodiments, R8 and R9 combine to form a bimole selected from -OCH2O-, -O(CH2)2O-, -OCH(CH3)O- and -OC(CH3)2O-.

[0144] In some embodiments, the compound or its salt, solvate, tautomer, enantiomer, or diastereomer is at least one selected from the group consisting of:

[0145]

[0146] In some embodiments, the compound or its salt, solvate, tautomer, enantiomer, or diastereomer is at least one selected from the group consisting of:

[0147]

[0148]

[0149] In some embodiments, compound A1. In other embodiments, compound A2. In still other embodiments, compound A3. In still other embodiments, compound A4. In still other embodiments, compound A5. In still other embodiments, compound A6. In still other embodiments, compound A7. In still other embodiments, compound A8. In still other embodiments, compound A9. In still other embodiments, compound A10. In still other embodiments, compound A11. In still other embodiments, compound A12. In still other embodiments, compound A13. In still other embodiments, compound A14. In still other embodiments, compound A15.

[0150] In some embodiments, the compound is not A1. In other embodiments, the compound is not A2. In still other embodiments, the compound is not A3. In still other embodiments, the compound is not A4. In still other embodiments, the compound is not A5. In still other embodiments, the compound is not A6. In still other embodiments, the compound is not A7. In still other embodiments, the compound is not A8. In still other embodiments, the compound is not A9. In still other embodiments, the compound is not A10. In still other embodiments, the compound is not A11. In still other embodiments, the compound is not A12. In still other embodiments, the compound is not A13. In still other embodiments, the compound is not A14. In still other embodiments, the compound is not A15.

[0151] In some embodiments, the compound or its salt, solvate, tautomer, enantiomer, or diastereomer is at least one selected from the group consisting of:

[0152]

[0153]

[0154]

[0155]

[0156] In some embodiments, compound A16 is used. In other embodiments, compound A17 is used. In still other embodiments, compound A18 is used. In still other embodiments, compound A19 is used. In still other embodiments, compound A20 is used. In still other embodiments, compound A21 is used. In still other embodiments, compound A22 is used. In still other embodiments, compound A23 is used. In still other embodiments, compound A24 is used. In still other embodiments, compound A25 is used. In still other embodiments, compound A26 is used. In still other embodiments, compound A27 is used. In still other embodiments, compound A28 is used. In still other embodiments, compound A29 is used. In still other embodiments, compound A30 is used. In still other embodiments, compound A31 is used. In still other embodiments, compound A32 is used. In still other embodiments, compound A33 is used. In still other embodiments, compound A34 is used. In still other embodiments, compound A35 is used. In still other embodiments, compound A36 is used. In still other embodiments, compound A37 is used. In yet another embodiment, the compound is A38. In yet another embodiment, the compound is A39. In yet another embodiment, the compound is A40. In yet another embodiment, the compound is A41. In yet another embodiment, the compound is A42. In yet another embodiment, the compound is A43. In yet another embodiment, the compound is A44. In yet another embodiment, the compound is A45. In yet another embodiment, the compound is A46. In yet another embodiment, the compound is A47. In yet another embodiment, the compound is A48. In yet another embodiment, the compound is A49. In yet another embodiment, the compound is A50. In yet another embodiment, the compound is A51. In yet another embodiment, the compound is A52. In yet another embodiment, the compound is A53. In yet another embodiment, the compound is A54. In yet another embodiment, the compound is A55. In yet another embodiment, the compound is A56. In yet another embodiment, the compound is A57. In yet another embodiment, the compound is A58. In yet another embodiment, the compound is A59. In yet another embodiment, the compound is A60. In yet another embodiment, the compound is A61. In yet another embodiment, the compound is A62. In yet another embodiment, the compound is A63. In yet another embodiment, the compound is A64. In yet another embodiment, the compound is A65. In yet another embodiment, the compound is A66. In yet another embodiment, the compound is A67. In yet another embodiment, the compound is A68. In yet another embodiment, the compound is A69. In yet another embodiment, the compound is A70.In yet another embodiment, the compound is A71. In yet another embodiment, the compound is A72. In yet another embodiment, the compound is A73. In yet another embodiment, the compound is A74. In yet another embodiment, the compound is A75. In yet another embodiment, the compound is A76. In yet another embodiment, the compound is A77. In yet another embodiment, the compound is A78. In yet another embodiment, the compound is A79. In yet another embodiment, the compound is A80. In yet another embodiment, the compound is A81. In yet another embodiment, the compound is A82. In yet another embodiment, the compound is A83. In yet another embodiment, the compound is A84. In yet another embodiment, the compound is A85. In yet another embodiment, the compound is A86. In yet another embodiment, the compound is A87. In yet another embodiment, the compound is A88. In yet another embodiment, the compound is A89. In yet another embodiment, the compound is A90. In yet another embodiment, the compound is A91. In yet another embodiment, the compound is A92. In yet another embodiment, the compound is A93. In yet another embodiment, the compound is A94. In yet another embodiment, the compound is A95. In yet another embodiment, the compound is A96. In yet another embodiment, the compound is A97. In yet another embodiment, the compound is A98. In yet another embodiment, the compound is A99. In yet another embodiment, the compound is A100. In yet another embodiment, the compound is A101. In yet another embodiment, the compound is A102. In yet another embodiment, the compound is A103. In yet another embodiment, the compound is A104.

[0157] In some embodiments, the compound is not A16. In other embodiments, the compound is not A17. In still other embodiments, the compound is not A18. In still other embodiments, the compound is not A19. In still other embodiments, the compound is not A20. In still other embodiments, the compound is not A21. In still other embodiments, the compound is not A22. In still other embodiments, the compound is not A23. In still other embodiments, the compound is not A24. In still other embodiments, the compound is not A25. In still other embodiments, the compound is not A26. In still other embodiments, the compound is not A27. In still other embodiments, the compound is not A28. In still other embodiments, the compound is not A29. In still other embodiments, the compound is not A30. In still other embodiments, the compound is not A31. In still other embodiments, the compound is not A32. In still other embodiments, the compound is not A33. In still other embodiments, the compound is not A34. In still other embodiments, the compound is not A35. In still other embodiments, the compound is not A36. In still other embodiments, the compound is not A37. In yet another embodiment, the compound is not A38. In yet another embodiment, the compound is not A39. In yet another embodiment, the compound is not A40. In yet another embodiment, the compound is not A41. In yet another embodiment, the compound is not A42. In yet another embodiment, the compound is not A43. In yet another embodiment, the compound is not A44. In yet another embodiment, the compound is not A45. In yet another embodiment, the compound is not A46. In yet another embodiment, the compound is not A47. In yet another embodiment, the compound is not A48. In yet another embodiment, the compound is not A49. In yet another embodiment, the compound is not A50. In yet another embodiment, the compound is not A51. In yet another embodiment, the compound is not A52. In yet another embodiment, the compound is not A53. In yet another embodiment, the compound is not A54. In yet another embodiment, the compound is not A55. In yet another embodiment, the compound is not A56. In yet another embodiment, the compound is not A57. In yet another embodiment, the compound is not A58. In yet another embodiment, the compound is not A59. In yet another embodiment, the compound is not A60. In yet another embodiment, the compound is not A61. In yet another embodiment, the compound is not A62. In yet another embodiment, the compound is not A63. In yet another embodiment, the compound is not A64. In yet another embodiment, the compound is not A65. In yet another embodiment, the compound is not A66. In yet another embodiment, the compound is not A67.In yet another embodiment, the compound is not A68. In yet another embodiment, the compound is not A69. In yet another embodiment, the compound is not A70. In yet another embodiment, the compound is not A71. In yet another embodiment, the compound is not A72. In yet another embodiment, the compound is not A73. In yet another embodiment, the compound is not A74. In yet another embodiment, the compound is not A75. In yet another embodiment, the compound is not A76. In yet another embodiment, the compound is not A77. In yet another embodiment, the compound is not A78. In yet another embodiment, the compound is not A79. In yet another embodiment, the compound is not A80. In yet another embodiment, the compound is not A81. In yet another embodiment, the compound is not A82. In yet another embodiment, the compound is not A83. In yet another embodiment, the compound is not A84. In yet another embodiment, the compound is not A85. In yet another embodiment, the compound is not A86. In yet another embodiment, the compound is not A87. In yet another embodiment, the compound is not A88. In yet another embodiment, the compound is not A89. In yet another embodiment, the compound is not A90. In yet another embodiment, the compound is not A91. In yet another embodiment, the compound is not A92. In yet another embodiment, the compound is not A93. In yet another embodiment, the compound is not A94. In yet another embodiment, the compound is not A95. In yet another embodiment, the compound is not A96. In yet another embodiment, the compound is not A97. In yet another embodiment, the compound is not A98. In yet another embodiment, the compound is not A99. In yet another embodiment, the compound is not A100. In yet another embodiment, the compound is not A101. In yet another embodiment, the compound is not A102. In yet another embodiment, the compound is not A103. In yet another embodiment, the compound is not A104.

[0158] In some embodiments, the compound of the present invention, or its salt, solvate, tautomer, enantiomer, or diastereomer, is selected from at least one of the following compounds:

[0159] In other embodiments, the compound is not A105. In other embodiments, the compound is not A106.

[0160] In some embodiments, the compound of the present invention or its salt, solvate, tautomer, enantiomer, or diastereomer is

[0161] In other embodiments, the compound is not A107.

[0162] In some embodiments, the compound is selected from at least one of the following:

[0163] G1 or G-1 (rel-1-[4-(6-bromo-1,3-benzodioxacyclopenten-5-yl)-3aR,4S,5,9bS-tetrahydro-3H-cyclopentadieno[c]quinolin-8-yl]-acetone):

[0164]

[0165] CMPD4(rel-1-((3aS,4R,9bR)-5-benzyl-4-(6-bromobenzo[d][1,3]m-dioxacyclopenten-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopentadien[c]quinoline-8-yl) ethyl-1-one):

[0166]

[0167] CMPD5(rel-1-((3aS,4R,9bR)-4-(2-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopentadieno[c]quinolin-8-yl)ethyl-1-one):

[0168]

[0169] CMPD6(rel-1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]m-dioxacyclopenten-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopentadien[c]quinolin-8-yl) ethyl-1-one oxime):

[0170]

[0171] CMPD7(rel-1-((3aS,4R,9bR)-4-(2-bromo-4,5-dimethoxyphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopentadieno[c]quinolin-8-yl)ethyl-1-one):

[0172]

[0173] CMPD8(rel-1-((3aS,4R,9bR)-4-(6-chlorobenzo[d][1,3]m-dioxacyclopenten-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopentadien[c]quinolin-8-yl) ethyl-1-one):

[0174]

[0175] CMPD9(rel-1-((6R,6aS,7aS,10aR,10bR)-6-(6-bromobenzo[d][1,3]m-dioxacyclopenten-5-yl)-9,9-dimethyl-6,6a,7,7a,10a,10b-hexahydro-5H-[1,3]m-dioxacyclopenten-[4',5':3,4]cyclopentadien-[1,2-c]quinolin-2-yl) ethyl-1-one):

[0176]

[0177] CMPD10(rel-1-((1R,2S,3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]m-dioxacyclopenten-5-yl)-1,2-dihydroxy-2,3,3a,4,5,9b-hexahydro-1H-cyclopentadien[c]quinoline-8-yl)acet-1-one):

[0178]

[0179] CMPD11(rel-1-((3aS,4R,9bR)-4-(2-bromo-4,5-dimethoxyphenyl)-2,3,3a,4,5,9b-hexahydro-1H-cyclopentadieno[c]quinolin-8-yl)ethyl-1-one):

[0180]

[0181] In some embodiments, the compound of the present invention or its salt, solvate, tautomer, enantiomer, or diastereomer is at least one compound of formula (II):

[0182] in:

[0183] R1 is selected from =O and =N-OH;

[0184] R2 is a C1-C4 alkyl group;

[0185] R5 is selected from H, benzyl, and C1-C4 alkyl;

[0186] R8 and R9 are independently selected from H and C1-C4 alkoxy groups, or R8 and R9 are combined to form a dimer selected from -OCH2O-, -OCH(CH3)O- and -OC(CH3)2O-.

[0187] In some embodiments, the compound of formula (II) is

[0188] CMPD12(rel-1-((4S,5aS,6R,11aR)-4,5,5a,6,11,11a-hexahydro-4,6-bridged methylene[1,3]m-dioxacyclopenteno[4',5':5,6]benzo[1,2-c]acrid-8-yl)ethyl-1-one):

[0189]

[0190] In some embodiments, the compound or its salt, solvate, enantiomer, or diastereomer that can be used in the methods of the present invention may be at least one GPER agonist as described in U.S. Patent Application Publications US 2008 / 0167334 and US 2011 / 0092533—the entire contents of which are incorporated herein by reference:

[0191]

[0192] in:

[0193] X is = N-, O, S, or NR, provided that when X is NR and R is a bond, N and R... 1 Together they form 5- to 7-membered, optionally substituted heterocyclic groups;

[0194] R is a bond, H, -OH, -NO2, optionally substituted C1-C6 hydrocarbon group (e.g., optionally substituted alkyl), optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted -C(O)-(C1-C6)alkyl (amide), optionally substituted -C(O)-O-(C1-C6)alkyl (ethyl carbamate), optionally substituted -C(O)-NH(C1-C6)alkyl (urea), optionally substituted -C(O)-N(C1-C6)dialkyl, optionally substituted -C(O)-NH (aryl), optionally substituted -C(O)-N (diaryl), optionally substituted -C(O)-NH (heteroaryl), optionally substituted -C(O)-N (diheteroaryl), optionally substituted -C(O)-NH (heterocyclic) or optionally substituted -C(O)-N (diheterocyclic);

[0195] R 1 R 2 and R 5Each group is independently selected from H, -OH, -NO2, halogens, C1-C6 optionally substituted carboxylic acid groups, optionally substituted O-(C1-C6)alkyl groups, optionally substituted C1-C6 hydrocarbon groups, optionally substituted aryl groups, optionally substituted heteroaryl groups, optionally substituted heterocyclic groups, optionally substituted -C(O)-(C1-C6)alkyl (ketones), optionally substituted -C(O)-O-(C1-C6)alkyl (esters), optionally substituted OC(O)-(C1-C6)alkyl ( Ester), optionally substituted -C(O)-NH(C1-C6)alkyl (urea), optionally substituted -C(O)-N(C1-C6)dialkyl, optionally substituted -C(O)-NH (aryl), optionally substituted -C(O)-N (diaryl), optionally substituted -C(O)-NH (heteroaryl), optionally substituted -C(O)-N (diheteroaryl), optionally substituted -C(O)-NH (heterocyclic) or optionally substituted -C(O)-N (diheterocyclic);

[0196] R 3 and R 4 Each group is independently selected from H, -OH, -NO2, halogen, optional C1-C6 substituted carboxylic acid groups, optional substituted O-(C1-C6)alkyl groups, optional substituted C1-C6 hydrocarbon groups, optional substituted aryl groups, optional substituted heteroaryl groups, optional substituted heterocyclic groups, optional substituted -C(O)-(C1-C6)alkyl (ketones), optional substituted -C(O)-O-(C1-C6)alkyl (esters), and optional OC(O)-(C1-C6)alkyl (esters). ), optionally substituted -C(O)-NH(C1-C6)alkyl (urea), optionally substituted -C(O)-N(C1-C6)dialkyl, optionally substituted -C(O)-NH (aryl), optionally substituted -C(O)-N (diaryl), optionally substituted -C(O)-NH (heteroaryl), optionally substituted -C(O)-N (diheteroaryl), optionally substituted -C(O)-NH (heterocyclic) or optionally substituted -C(O)-N (diheteroaryl) or R 3 and R 4 Together they form a 5- or 6-membered optionally substituted carbocyclic ring (which may be saturated or unsaturated), an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted heterocyclic group.

[0197] R 6 and R 7Each of the following groups is independently absent or selected from H, -OH, -NO2, halogen, optional C1-C6 substituted carboxylic acid groups, optional substituted O-(C1-C6)alkyl groups, optional substituted C1-C6 hydrocarbon groups, optional substituted aryl groups, optional substituted heteroaryl groups, optional substituted heterocyclic groups, optional substituted -C(O)-(C1-C6)alkyl (ketones), optional substituted -C(O)-O-(C1-C6)alkyl (esters), and optional OC(O)-(C1-C6)alkyl groups. (ester), optionally substituted -C(O)-NH(C1-C6)alkyl (urea), optionally substituted -C(O)-N(C1-C6)dialkyl, optionally substituted -C(O)-NH (aryl), optionally substituted -C(O)-N (diaryl), optionally substituted -C(O)-NH (heteroaryl), optionally substituted -C(O)-N (diheteroaryl), optionally substituted -C(O)-NH (heterocyclic) or optionally substituted -C(O)-N (diheteroaryl), or R 6 and R 7 Together they form a 4-, 5-, 6-, or 7-membered optionally substituted carbocyclic ring (which may be saturated or unsaturated), an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted heterocyclic group, or a 5- to 9-membered optionally substituted carbocyclic or heterocyclic bicyclic group, provided that R 7 'and R 7 When neither R nor R exists 7 It does not exist;

[0198] R 6 'For non-existent, H, C1-C6 optionally substituted hydrocarbon groups (such as H, CH3 or CH2CH3) or with R 6 Together they form = O;

[0199] R 7 'is absent, H, optionally substituted hydrocarbon group (such as H, CH3 or CH2CH3), or with R 7 Together they form = O;

[0200] R 7"For non-existent, H, -OH, halogen, optionally substituted O-(C1-C6)alkyl, optionally substituted C1-C6 hydrocarbon, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted -C(O)-(C1-C6)alkyl (ketone), optionally substituted -C(O)-O-(C1-C6)alkyl (ester), optionally substituted OC(O)-(C1-C6)alkyl (ester), optionally substituted -C( -C(O)-NH(C1-C6)alkyl (urea), optionally substituted -C(O)-N(C1-C6)dialkyl, optionally substituted -C(O)-NH (aryl), optionally substituted -C(O)-N (diaryl), optionally substituted -C(O)-NH (heteroaryl), optionally substituted -C(O)-N (diheteroaryl), optionally substituted -C(O)-NH (heterocyclic) or optionally substituted -C(O)-N (diheteroaryl);

[0201] R 8 'Does not exist (when R) 8 'Connected carbon and R 6 (When the connected carbon atoms form an optional double bond), H, CH3, or CH2CH3;

[0202] R 10 R 11 R 12 and R 13 Each of the following is independently H, -OH, -NO2, halogen, C1-C6 optionally substituted carboxylic acid group, optionally substituted O-(C1-C6)alkyl, optionally substituted C1-C6 hydrocarbon group, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted -C(O)-(C1-C6)alkyl (ketone), optionally substituted -C(O)-O-(C1-C6)alkyl (ester), optionally substituted OC(O)-(C1-C6)alkyl (ester) ), optionally substituted -C(O)-NH(C1-C6)alkyl (urea), optionally substituted -C(O)-N(C1-C6)dialkyl, optionally substituted -C(O)-NH (aryl), optionally substituted -C(O)-N (diaryl), optionally substituted -C(O)-NH (heteroaryl), optionally substituted -C(O)-N (diheteroaryl), optionally substituted -C(O)-NH (heterocyclic) or optionally substituted -C(O)-N (diheteroaryl);

[0203] R 14It is H, -OH, -NO2, halogen, C1-C6 optionally substituted carboxylic acid group, optionally substituted O-(C1-C6)alkyl, optionally substituted C1-C6 hydrocarbon group, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic, optionally substituted -C(O)-(C1-C6)alkyl (ketone), optionally substituted -C(O)-O-(C1-C6)alkyl (ester), optionally substituted OC(O)-(C1-C6)alkyl (ester), etc. Optionally substituted -C(O)-NH(C1-C6)alkyl (urea), optionally substituted -C(O)-N(C1-C6)dialkyl, optionally substituted -C(O)-NH (aryl), optionally substituted -C(O)-N (diaryl), optionally substituted -C(O)-NH (heteroaryl), optionally substituted -C(O)-N (diheteroaryl), optionally substituted -C(O)-NH (heterocyclic) or optionally substituted -C(O)-N (diheterocyclic), or with R 7 The linked carbon atoms together form a 5-, 6-, or 7-membered optionally substituted carbon ring (which may be saturated or unsaturated), optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocycle.

[0204] Y is an optional substitution of (CH2). n Groups—where n is 0, 1 or 2, optionally substituted =CH- group, C=O group, O, S, optionally substituted N-(C1-C6)alkyl, optionally substituted N-aryl, optionally substituted N-heteroaryl, optionally substituted N-heterocyclic, optionally substituted NC(O)-(C1-C6)alkyl, optionally substituted NC(O)-aryl, optionally substituted NC(O)-heteroaryl, optionally substituted NC(O)-heterocyclic;

[0205] Z is an optional substitution of (CH2). n Groups – where n is 1 or 2, optionally substituted =CH- group, C=O group, O, S, optionally substituted N-(C1-C6)alkyl, optionally substituted N-aryl, optionally substituted N-heteroaryl, optionally substituted N-heterocyclic, optionally substituted NC(O)-(C1-C6)alkyl, optionally substituted NC(O)-aryl, optionally substituted NC(O)-heteroaryl, optionally substituted NC(O)-heterocyclic.

[0206] In some embodiments, the compound of the present invention, or a salt, solvate, enantiomer, or diastereomer thereof, is at least one GPER agonist described in PCT patent application number WO 2016 / 014847—which is incorporated herein by reference in its entirety:

[0207] Among them, in (i)-(iv):

[0208] Ring A is an aromatic or heteroaromatic five- or six-membered ring containing one or more heteroatoms such as N, O, or S;

[0209] R1 is independently selected from SO2NH2 and SO2NR. a R b COOH, CONH2 and CONR a R b And H. In R1, each occurrence of R... a and R b Independently selected from H, alkyl (C1-C6), alkenyl (C2-C6), alkynyl (C2-C6), alkoxy (C2-C6), cycloalkyl (C3-C7), alkylthio, alkylaryl, and aromatic and heteroaromatic rings. Aromatic and heteroaromatic rings may be further substituted with electron-withdrawing and electron-donating groups. R a and R b It can form cyclic rings (C3-C7) or aromatic rings, which optionally contain one or more heteroatoms. These aromatic rings can be further substituted with electron-withdrawing groups such as halogens, -COOH, -CN, -NO2, etc., or with electron-donating groups such as alkyl groups;

[0210] R2 is H, a halogen, or a heteroatom such as N, O, or S;

[0211] Ring A is an aromatic ring or a heteroaromatic ring (5- or 6-membered ring);

[0212] Ring B is a six-membered saturated ring or aromatic ring containing nitrogen at a specified position. The nitrogen in ring B may optionally be substituted with alkyl, aryl, or alkylaryl substituents.

[0213] The carbon ring (C) can be independently a substituted or unsubstituted carbocyclic ring, bicyclic ring, aromatic ring, fused aromatic ring, or heteroaromatic ring. Additionally, when it is a carbocyclic ring, it can contain one or more double bonds and one or more heteroatoms such as N, O, or S. It can also have α-β-unsaturated ketone functional groups.

[0214] R3 is independently selected from H, halogen, -OH, CN, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)alkoxy, (C3-C7)cycloalkyl, (C1-C6) alkylthio, NR a R b R a R b Or a haloalkyl group (e.g., CF3). In R3, each occurrence of R... a and R b Independently hydrogen or (C1-C6) alkyl, or R a and R bForming a saturated or unsaturated heterocycle containing 3-7 ring atoms, the ring optionally containing another heteroatom selected from N, O and S, and optionally substituted by 1-3 groups that may be the same or different and selected from (C1-C4) alkyl, phenyl and benzyl groups; and m is 1-4;

[0215] In some implementations, rings B and C are cis-fused;

[0216] Rings D and B are directly connected or connected via a spacer (C1-C2). When directly connected, their fusion with respect to rings B and C can be cis or trans. Ring D is an aromatic or heteroaromatic ring containing one or more heteroatoms such as N, O, or S. It can optionally be substituted with independently selected R5 and R6 groups or substituted with -R6R5 or R5R6;

[0217] R5 is independently selected from H, halogens, electron-donating groups and electron-withdrawing groups, such as alkyl, haloalkyl, alkoxy, -NO2, -SF5, -CN, etc.

[0218] R6 can be NHC(O)OR c OC(O)NHR c C(O)O(CH2) n R c OC(O)(CH2) n R c C(O)NHR c or NHC(O)R c Where n = 0-4; or R6 can be an alkyl group, a branched alkyl group (C1-C6). 10 ), alkynyl (C1-C) 10 ), carbocyclic rings, alkenyl groups (C1-C) 10 ), halogens, CN, COOH, CONH2, -OH or NH2. c It is an alkyl group, a branched alkyl group (C1-C2). 10 R6 can be alkoxy, alkylamino, acyl, alkynyl (C1-C8), or alkenyl (C1-C6). R6 can also be X(CH2). n E, where X is NH, O, S, C≡C or HC=CH, and n=0-2, and E is independently a substituted or unsubstituted carbocyclic, bicyclic, aromatic, fused aromatic or heterocyclic ring;

[0219] In some embodiments, R1 is selected from carboxyl, formamide, carboxylalkyl, carboxylaryl, cyano, nitro, hydroxyl, sulfonyl, sulfonamide, alkylsulfonamide, arylsulfonamide, alkylsulfonyl, aralkylsulfonamide, trifluoromethylsulfonamide, trifluoromethylsulfonylformamide, and sulfonylurea. In some embodiments, R1 is a sulfonamide, alkylsulfonamide, or arylsulfonamide. R2 can be a 3-, 4-, 5-, or 6-membered saturated or aromatic carbocyclic ring or ring system, optionally containing one or two heteroatoms selected from N, O, and S, the ring or ring system optionally substituted by one or more substituents selected from: cyano, halogen, acyl, acyloxy, alkyl, alkoxy, heteroalkyl, alkyl ester, alkylamide, alkylamino, alkylamino, aryl, aryloxy, aralkyl, aryl ester, azide, alkyl halide, alkenyl, alkynyl, alkyl ether, nitro, thiohalide, and thiocyano. R3 can be H, or a C1-C5 alkyl or cycloalkyl group, optionally substituted with one or more cyano, nitro, and one or more aromatic or heteroaromatic groups containing N, O, or S. A and D can be independently selected from CH, CH2, N, and O, and the bonds connecting them are single or double bonds suitable for the selected atoms. X, Y, and Z are independently selected from no atoms (i.e., they are absent), CH, C-halogen, N, O, and S;

[0220] In some implementations, R2 is derived from -R d R e R f or -R d COR e R f The substituents are represented by R; where R d R e and R f Independently selected from 3-, 4-, 5-, or 6-membered saturated or aromatic carbocyclic or cyclic systems, optionally containing one or two heteroatoms selected from N, O, and S, the ring or cyclic system optionally being substituted by one or more substituents selected from: cyano, halogen, acyl, acyloxy, alkyl, alkoxy, heteroalkyl, alkyl ester, alkylamide, alkylamino, aryl, aryloxy, aralkyl, aryl ester, azide, alkyl halide, alkenyl, alkynyl, alkyl ether, nitro, thiohalide, and thiocyano;

[0221] The compounds of the present invention can be prepared according to the methods described herein, methods known in the art, and / or methods described in certain references, such as, but not limited to: PCT application publications WO 2004 / 072046 and WO 2016 / 014847; U.S. application 10 / 511,083; U.S. patent application publications US 2008 / 0167334 and US 2011 / 0092533; and Burai, et al., 2010, Org. & Biomol. Chem. 8:2252-2259; all of which are incorporated herein by reference in their entirety.

[0222] The compounds of the present invention may have one or more stereocenters, and each stereocenter may exist independently in an (R) or (S) configuration. In some embodiments, the compounds described herein are present in optical or racemic forms. The compounds described herein include racemic, optical, regioisomeric, and stereoisomeric forms or combinations thereof having the therapeutically useful properties described herein. Preparation of the optical form is achieved in any suitable manner—including by means of non-limiting examples, by resolving the racemic form using recrystallization, synthesis from an optically active source, chiral synthesis, or chromatographic separation using a chiral stationary phase. The compounds represented herein by racemic formulations further represent any one of two enantiomers or mixtures thereof, or, in the presence of two or more chiral centers, all diastereomers or mixtures thereof.

[0223] In some embodiments, the compounds of the present invention are present as tautomers. All tautomers are included within the scope of the compounds described herein.

[0224] The compounds described herein also include isotopically labeled compounds, wherein one or more atoms are replaced by atoms having the same number of atoms but with an atomic mass or mass number different from those normally found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include, but are not limited to, those... 2 H, 3 H, 11 C 13 C 14 C 36 Cl、 18 F, 123 I, 125 I, 13 N、 15 N、 15 O、 17 O、 18 O、 32 P and 35S. In some embodiments, substitution with a heavier isotope, such as deuterium, provides greater chemical stability. The isotope-labeled compound is prepared by any suitable method or by using an appropriate isotope-labeled reagent instead of an additionally used unlabeled reagent.

[0225] In some embodiments, the compounds described herein are labeled in other ways, including but not limited to the use of chromophores or fluorescent moieties, bioluminescent labeling, or chemiluminescent labeling.

[0226] Examples of suitable optional substituents in all embodiments provided herein are not intended to limit the scope of the claimed invention. Compounds of the present invention may contain any substituent or combination of substituents provided herein.

[0227] In some embodiments, the invention further provides pharmaceutical compositions comprising at least one compound of the invention and at least one pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical compositions are formulated for inhalation, oral administration, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, intrathecal, intracranial, or intravenous administration. Each formulation of the compounds contemplated by the invention can be used to treat or prevent cancers expressing GPCRs (e.g., GPER-expressing) and / or Myc-expressing, such as, but not limited to, melanoma.

[0228] In some embodiments, the pharmaceutical compositions of the present invention comprise at least one additional anticancer agent and at least one compound of the present invention. Examples of additional anticancer agents include, but are not limited to, chemotherapy and immune checkpoint inhibitors. Non-limiting examples of chemotherapy include, but are not limited to, HDAC, temozolomide, dacarbazine (DTIC), vemurafenib, dabrafenib, and trametinib. Non-limiting examples of checkpoint inhibitors include, but are not limited to, PD-1 inhibitors (i.e., pembrolizumab, nivolumab, anti-PD-1), PD-L1 inhibitors (i.e., atezolizumab, anti-PD-L1), CTLA-4 inhibitors (i.e., ipilimumab, anti-B7-1 / B7-2, anti-CTLA-4), indoleamine (2,3)-dioxygenase (IDO1 / 2) inhibitors, B7 homolog 3 (B7-H3) inhibitors, lymphocyte activation gene 3 (LAG3) inhibitors, and TIGIT (a T-cell immune receptor having Ig and ITIM domains) targeting antibodies and reagents.

[0229] This invention also relates to kits that can be used in any of the methods described herein. Such kits contain components that can be used in any of the methods described herein, including, for example, compositions and methods for treating or preventing cancers—such as, but not limited to, melanoma—in subjects, such as humans, containing one or more containers (e.g., test tubes, cell culture dishes, cell culture plates, cell culture flasks, cell culture bags) and instruction materials for containing components of any embodiment of the invention described elsewhere herein.

[0230] Salt

[0231] The compounds described herein can form salts with acids or bases, and these salts are included in this invention. The term "salt" includes addition salts of free acids or bases that can be used in the methods of this invention. The term "pharmaceutically acceptable salt" refers to a salt having a toxicity profile within the range that provides utility in pharmaceutical applications. In some embodiments, the salt is pharmaceutically acceptable. Nevertheless, pharmaceutically unacceptable salts may have properties such as high crystallinity, which are useful in the practice of this invention, for example, in the synthesis, purification, or formulation of compounds useful in the methods of this invention.

[0232] Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Examples of inorganic acids include sulfates, hydrogen sulfates, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid (including hydrogen phosphate and dihydrogen phosphate). Suitable organic acids can be selected from aliphatic, alicyclic, aromatic, arylaliphatic, heterocyclic, carboxylic, and sulfonic acid organic acids, examples of which include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (or papoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, sulfanilic acid, 2-hydroxyethanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactobionic acid, galacturonic acid, glycerophosphonic acid, and saccharin (e.g., saccharinate, saccharate). With respect to any compound of the present invention, the salt may consist of an acid or base in fractions of a molar equivalent, a molar equivalent, or more than a molar equivalent.

[0233] Suitable pharmaceutically acceptable base addition salts of the compounds of the present invention include, for example, ammonium salts and metal salts, including alkali metal, alkaline earth metal, and transition metal salts, such as calcium, magnesium, potassium, sodium, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines—such as N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (or N-methylglucosamine), and procaine. All these salts can be prepared from the corresponding compounds by reacting them with, for example, a suitable acid or base.

[0234] method

[0235] This invention provides a method for treating or preventing cancers expressing GPCRs (e.g., GPER-expression) and / or Myc-expression in a subject, such as, but not limited to, melanoma, pancreatic cancer, and / or lung cancer (e.g., but not limited to non-small cell lung cancer). In some embodiments, the method includes administering a therapeutically effective amount of estrogen and / or a GPCR agonist to the subject in need—which increases tumor cell differentiation, thereby treating or preventing cancers expressing GPCRs (e.g., GPER-expression) and / or Myc-expression in the subject.

[0236] In some embodiments, the GPCR is GPER. In other embodiments, the GPER agonist comprises G-1. In some embodiments, the GPER agonist is selected from estradiol (E2), tamoxifen, fulvestrant, and raloxifene (also known as 6-hydroxy-2-(4-hydroxyphenyl)-benzothiophene-3-yl]-[4-[2-(1-piperidinyl)ethoxy]phenyl]-methyl ketone).

[0237] In some embodiments, the estrogen is any natural or synthetic substance that mimics the effects of the natural hormone estrogen. Examples of estrogens contemplated in this invention include, but are not limited to, estrone (E1), estradiol (E2), estriol (E3), estradiol (E4), 17β-estradiol, 27-hydroxycholesterol, dehydroepiandrosterone (DHEA), 7-oxo-DHEA, 7α-hydroxy-DHEA, 16α-hydroxy-DHEA, 7β-hydroxyepiandrosterone, Δ 4 -androstenedione, Δ 5 -Androstenediol, 3α-androstanediol, 3β-androstanediol, 2-hydroxyestradiol, 16-hydroxyestradiol, estradiol cyclopentylpropionate, estradiol valerate, estradiol acetate, estradiol benzoate, ethinylestradiol (EE), mesestrol, mokestrol, ethinylestradiol, diethylstilbestrol, phenylestradiol, diethylstilbestrol acetate, diethylstilbestrol dipropionate, phosphostrol, hexanestilbestrol, mesestrol dipropionate, exogenous estrogens, phytoestrogens and / or mycoestrogens.

[0238] In some embodiments, estrogen and / or GPCR agonists are administered to a subject as a pharmaceutical composition, said pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

[0239] In some embodiments, the subject is further administered at least one anticancer treatment. Examples of anticancer treatments include, but are not limited to, chemotherapy, radiation therapy, surgery, and / or immune checkpoint inhibitors. Examples of chemotherapy include, but are not limited to, HDAC, temozolomide, dacarbazine (DTIC), vemurafenib, dabrafenib, and trametinib. Examples of immune checkpoint inhibitors include, but are not limited to, PD-1 inhibitors (i.e., pembrolizumab, nivolumab, anti-PD-1), PD-L1 inhibitors (i.e., atezolizumab, anti-PD-L1), CTLA-4 inhibitors (i.e., ipilimumab, anti-B7-1 / B7-2, anti-CTLA-4), indoleamine (2,3)-dioxygenase (IDO1 / 2) inhibitors, B7 homolog 3 (B7-H3) inhibitors, lymphocyte activation gene 3 (LAG3) inhibitors, and TIGIT (a T-cell immune receptor with Ig and ITIM domains) targeting antibodies and reagents. In other embodiments, chemotherapy or immune checkpoint inhibitors are administered or co-formulated with estrogen or GPCR agonists.

[0240] In some embodiments, the estrogen or GPCR agonist is administered to the subject via the following routes of administration: inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, intrathecal, intracranial, and intravenous. In other embodiments, the estrogen or GPCR agonist is the sole anticancer agent administered to the subject. In still other embodiments, the estrogen or GPCR agonist is the sole anticancer agent administered to the subject in an amount sufficient to treat or prevent cancer in the subject.

[0241] In some embodiments, the cancer is breast cancer. In other embodiments, the cancer is not breast cancer. In still other embodiments, the cancer is ovarian cancer. In still other embodiments, the cancer is not ovarian cancer. In still other embodiments, the cancer is prostate cancer. In still other embodiments, the cancer is not prostate cancer. In still other embodiments, the cancer is castration-resistant prostate cancer (CRPC). In still other embodiments, the cancer is not CRPC. In still other embodiments, the cancer is endometrial cancer. In still other embodiments, the cancer is not endometrial cancer. In still other embodiments, the subject is a mammal. In still other embodiments, the mammal is a human.

[0242] The present invention further provides a method for selecting patients with cancer who will benefit from treatment with estrogen and / or a GPCR agonist (e.g., a selective GPCR agonist). The method includes obtaining a sample from the subject's cancer and determining whether at least one cancer cell from the sample expresses GPER and / or another GPCR. The detection and / or quantification of GPER and / or another GPCR in the sample can be performed using any method described herein or any method known in the art. In some embodiments, if cancer cells express GPER and / or another GPCR, the subject is advised to receive such cancer treatment comprising estrogen and / or a selective GPER agonist and / or another GPCR agonist, optionally in combination with at least one immunotherapeutic agent and / or an HDAC inhibitor. In other embodiments, if cancer cells express GPER and / or another GPCR, the subject is administered such cancer treatment comprising estrogen and / or a selective GPER agonist and / or another GPCR agonist, optionally in combination with at least one immunotherapeutic agent and / or an HDAC inhibitor. In other embodiments, if cancer cells do not express GPER and / or another GPCR, the subject is advised not to receive such cancer treatment comprising estrogen and / or a selective GPER agonist and / or another GPCR agonist, optionally in combination with at least one immunotherapeutic agent and / or an HDAC inhibitor. In yet another embodiment, if cancer cells do not express GPER, the subject is not given such cancer treatment comprising estrogen and / or a selective GPER agonist and / or another GPCR agonist, optionally in combination with at least one immunotherapeutic agent and / or an HDAC inhibitor.

[0243] Formulation / Application

[0244] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any other components in the pharmaceutical compositions of the present invention will vary depending on the identity, size, and condition of the subject being treated. For example, the composition may contain between about 0.005% and about 100% (w / w) of the active agent or any fraction or multiple thereof.

[0245] In some embodiments, a pharmaceutical composition suitable for carrying out the methods of the present invention may be administered to deliver a dose between 1 ng / kg / day and 100 mg / kg / day, such as, for example, 1-50 mg / kg / day. In other embodiments, a pharmaceutical composition suitable for carrying out the present invention may be administered to deliver a dose between 1 ng / kg / day and 1,000 mg / kg / day.

[0246] Compositions containing the compounds contemplated in this invention can be administered frequently to mammals several times a day, or less frequently—for example, once a day, once a week, once every two weeks, once a month, or even less frequently—for example, once every few months, once a year, or less.

[0247] It should be understood that, in non-limiting instances, the amount of compound administered daily may be daily, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, an every-other-day dose of 0.5-5 mg daily could begin on Monday, with the first subsequent dose of 0.5-5 mg daily administered on Wednesday, the second subsequent dose of 0.5-5 mg daily administered on Friday, and so on. The frequency of dosing is obvious to a technician and depends on many factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

[0248] Although the description of the pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to a wide variety of animals. It is well understood that pharmaceutical compositions suitable for human administration can be modified to make them suitable for administration to a wide variety of animals, and veterinary pharmacologists of ordinary skill can design and perform such modifications simply through ordinary—if any—experiments. Subjects considered for administration of the pharmaceutical compositions of the present invention include, but are not limited to, humans and other primates, mammals—including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0249] In some embodiments, compositions comprising the compounds of consideration in this invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In some embodiments, the pharmaceutical compositions of this invention comprise a therapeutically effective amount of at least one compound of consideration in this invention and a pharmaceutically acceptable carrier. Available pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions, such as phosphates and organic acid salts. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0250] The formulation may be used in combination with conventional excipients—that is, pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, intranasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration known in the art. In some embodiments, administration includes topical application. Pharmaceutical articles may be sterilized and, if desired, may be mixed with adjuvants—such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure buffers, colorants, flavoring agents, and / or aromatic substances. They may also be combined, as needed, with other active agents, such as other analgesics.

[0251] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulators and disintegrants; binders; lubricants; sweeteners; flavorings; colorants; preservatives; physiologically degradable compositions, such as gelatin; aqueous carriers and solvents; oily carriers and solvents; suspending agents; dispersants or wetting agents; emulsifiers, medicaments; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungals; stabilizers; and pharmaceutically acceptable polymers or hydrophobic materials. Other “additional ingredients” that may be included in the pharmaceutical compositions of the present invention are known in the art and described, for example, in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0252] Topical application

[0253] The barrier to topical drug application is the stratum corneum of the epidermis. The stratum corneum is a highly resistant layer composed of proteins, cholesterol, sphingolipids, free fatty acids, and various other lipids, and includes keratinized living cells. One factor limiting the rate (flux) of a compound's penetration through the stratum corneum is the amount of active substance that can be loaded or applied to the skin surface. The greater the amount of active substance applied per unit area of ​​skin, the greater the concentration gradient between the skin surface and the subcutaneous layer, and consequently, the greater the diffusion force of the active substance through the skin. Therefore, formulations containing higher concentrations of active substances are more likely to penetrate the skin, and more active substances penetrate the skin at a more consistent rate compared to formulations with lower concentrations, all other things being equal.

[0254] Formulations suitable for topical application include, but are not limited to, liquid or semi-liquid products such as liniments, lotions, oil-in-water or water-in-oil emulsions—e.g., creams, ointments, or pastes—as well as solutions or suspensions. These formulations can be applied to the skin directly or using swabs, applicators, scrapers, etc., as well as in the form of transdermal patches. In some embodiments, patches minimize drug loss due to washing, rubbing, scraping, and / or massaging the skin. In other embodiments, patches increase drug absorption through the skin while minimizing skin exposure to the drug.

[0255] Permeation enhancers can be used. These materials increase the rate at which drugs penetrate the skin. Typical enhancers in the art include ethanol, glyceryl monolaurate, PGML (polyethylene glycol monolaurate), dimethyl sulfoxide, etc. Other enhancers include oleic acid, oleyl alcohol, ethoxydiethylene glycol, lauryl azone, alkyl carboxylic acid, dimethyl sulfoxide, polar lipids, or N-methyl-2-pyrrolidone. An acceptable carrier for topical delivery of some compositions of the present invention may contain liposomes. The composition and uses of liposomes are known in the art (e.g., U.S. Patent No. 6,323,219).

[0256] External application

[0257] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical destruction of the subject’s tissue and administration of the pharmaceutical composition through an opening in the tissue. Therefore, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection, application of the composition through a surgical incision, application of the composition through a non-surgical wound penetrating tissue. Specifically, parenteral administration is considered to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and renal dialysis infusion techniques.

[0258] Controlled-release formulations and drug delivery systems

[0259] In some other embodiments, the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, and controlled formulations such as sustained-release, delayed-release, and pulsatile-release formulations.

[0260] The term sustained release, used in its conventional sense, refers to a pharmaceutical formulation that provides a gradual release of the drug over an extended period of time, and—though not necessarily—results in a substantially constant blood level of the drug over that extended period. This period can be as long as a month or longer, and should be a longer release than the same amount of drug administered in pellet form.

[0261] For sustained release, the compound can be formulated with a suitable polymer or hydrophobic material—which provides the sustained release property. Therefore, compounds suitable for use in the methods of this invention can be administered in particulate form, for example by injection or by implantation in the form of a wafer or disc.

[0262] In one embodiment of the invention, the compound of the invention is administered to a patient alone or in combination with another agent using a sustained-release formulation.

[0263] The term delayed release is used in its conventional sense herein to refer to the initial release of a drug from a pharmaceutical preparation after a delay following drug administration, and—though not required—may include a delay of up to about 10 minutes to about 12 hours.

[0264] The term pulsed release is used in its conventional sense in this document, referring to the delivery of a drug by a pharmaceutical formulation in a manner that produces a pulsed plasma profile of the drug upon administration.

[0265] The term immediate release, used in its conventional sense, refers to the immediate release of a drug from a pharmaceutical preparation after administration.

[0266] As used herein, short term means any time period up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, about 10 minutes, or about 1 minute after drug administration, and any or all or part of such increments after drug administration.

[0267] As used herein, rapid compensation refers to any time period up to and including approximately 8 hours, approximately 7 hours, approximately 6 hours, approximately 5 hours, approximately 4 hours, approximately 3 hours, approximately 2 hours, approximately 1 hour, approximately 40 minutes, approximately 20 minutes, approximately 10 minutes, or approximately 1 minute after drug administration, and any or all or part of such increments.

[0268] Those skilled in the art will recognize or be able to determine many equivalents of the specific procedures, implementations, claims, and embodiments described herein using only conventional experiments. These equivalents are considered to be within the scope of the invention and are covered by the appended claims. For example, it should be understood that modifications to reaction conditions—including, but not limited to, reaction time, reaction size / volume, and experimental reagents—such as solvents, catalysts, pressure, atmospheric conditions such as a nitrogen atmosphere, and reducing / oxidizing agents—using only conventional experiments are within the scope of this application.

[0269] It should be understood that wherever values ​​and ranges are provided herein, all values ​​and ranges covered by such values ​​and ranges are included within the scope of this invention. Furthermore, this application also considers all values ​​falling within these ranges, as well as the upper or lower limits of the range of values.

[0270] The following examples further illustrate various aspects of the invention. However, they are by no means intended to limit the teachings or disclosure of the invention as described herein.

[0271] Example

[0272] The invention will now be described with reference to the following embodiments. These embodiments are provided for illustrative purposes only, and the invention is not limited to these embodiments, but includes all variations that will become apparent from the teachings provided herein.

[0273] Example 1: Estrogen and G-1 treatment in vitro and in vivo slows melanoma cell proliferation and drives differentiation

[0274] Tumor cell proliferation was slowed in both mouse and human melanoma cell lines after treatment with estrogen or G-1—a specific GPER agonist. Figures 4A-4F Treatment with the GPER agonist G-1 (100 nM G1) or its delivery vector was performed. Cell proliferation and differentiation (melanin production) were measured. Results showed that both estrogen and G-1 treatment slowed melanoma cell proliferation and drove differentiation in vitro. Figures 4A-4F ).

[0275] Human and mouse melanoma cells treated with estrogen or the specific GPER agonist G-1 grew more slowly in mice and formed significantly smaller tumors. Figure 5-6 Mouse (B16) and human (WM46) cell lines were pretreated in vitro for one week with estrogen, G-1, or a vector, and then injected in the same amount into the left ventral region (vector-treated) or right ventral region (E2-treated) of mice to induce tumor formation (N=5 mice per group). Figure 5 After 14-16 days, the mice were imaged, and the tumors were harvested and weighed. Pretreatment with estrogen inhibited tumor growth in vivo by approximately 3 times. Figure 5 Similarly, treatment with G-1 inhibits tumor growth in vivo ( Figure 6 ).

[0276] Brief estrogen exposure is sufficient to induce epigenetic memory, which maintains a more differentiated state. Normal human melanocytes were treated with pulsed estrogen therapy—consisting of 4 days of estrogen treatment followed by 8 days of withdrawal. RNA-seq showed that melanocytes briefly treated with estrogen maintained upregulated expression of all major melanocyte differentiation markers—including tyrosinase (TYR), tyrosinase-associated protein (TRP1), melanocortin 1 receptor (MC1R), Melan-A (MLANA), and dopachrome tautomerase (DCT)—and reduced expression of aggressive melanoma markers such as promelanosome protein (PMEL). Figure 3 At the protein level, changes in the amounts of melanocyte differentiation markers were even more pronounced. Estrogen-treated cells also produced more pigment, indicating that they were more differentiated compared to control (carrier-treated) cells. Figure 3 ).

[0277] Example 2: Multiple pregnancies limit melanoma development and drive differentiation

[0278] Cell lines and subcutaneous tumors are frequently used in cancer research because they are rapid and readily available, but they may not be physiologically faithful models of true human disease. This article describes engineered human xenograft models that provide a better model of human melanoma.

[0279] Lentiviral modification of human melanoma xenografts to express known melanoma-associated mutant oncoproteins was used to introduce them into normal human melanocytes. (diBRAf) V600E CDK4 R24C dnp53 R248W and hTERT transduction of primary melanocytes ( Figure 7A Next, organoid skin was created in vitro and then transplanted onto the backs of female mice. Figure 7B Mice were divided into two groups, non-breeding and breeding (N=3 mice per group), and melanomas were formed over the following 15 weeks. Tissue was harvested and the mutated genes were determined. In the non-breeding group, large proliferative nests of melanocytes with upward diffusion were observed, which is characteristic of radially growing melanomas. Figure 7C The limited transfer of melanin to the epidermis indicates that melanocytes no longer perform their differentiation function. In the proliferative group, no large proliferative nests were observed, and an increased amount of melanin transferred to the epidermis was seen, suggesting that melanocytes performed their differentiation function better than the proliferative control. In conclusion, this suggests that multiple pregnancies (3 cases in this experiment) can be used to limit melanoma development and drive differentiation.

[0280] like Figures 8A-8EAs shown, after graft healing, mice were randomized and divided into non-breeding or breeding groups, and then fed a doxycycline diet to induce BRAF in all animals. V600E Oncogenes. Human tissue was harvested and histologically analyzed after 15 weeks of gestation and 3 consecutive pregnancies in the reproductive group (or no pregnancies in the non-reproductive group). Figure 8A Grafts from the non-reproductive group developed into melanocytic tumors, exhibiting hallmark features of human melanoma, including large, mitotically active nests of melanocytes with cellular atypia. In contrast, tissues from the reproductive group were relatively inconspicuous and primarily contained resting, monolithic, non-proliferating melanocytes confined to the basal epidermis. These results suggest that repeated pregnancies inhibit the growth of BRaf-driven human melanoma formation. Figure 8B-8D ).

[0281] The primary function of fully differentiated epidermal melanocytes is to produce melanin, which protects the skin from ultraviolet radiation. As with most cell types, melanocyte differentiation and proliferation are negatively correlated, and melanocytes in normal skin rarely proliferate outside the circulating hair follicles. Melanoma tissue is typically less differentiated than normal melanocytes or benign nevi. In current xenograft studies, pregnancy has been associated with increased melanocyte differentiation compared to non-reproductive groups, as evidenced by a relative lack of proliferating melanocytes and a corresponding increase in epidermal melanin. Although the non-reproductive group developing melanoma had significantly more melanocytes in the transplanted skin compared to the reproductive group, the melanin abundance within the surrounding epidermal keratinocytes was significantly reduced. Figure 8E Therefore, pregnancy inhibits melanoma development by inducing melanocyte differentiation.

[0282] Example 3: GPER signaling drives stable differentiation in normal human melanocytes and melanomas

[0283] To test whether pregnancy-related hormones induce long-term changes in melanocyte differentiation—which may affect their future susceptibility to transformation—primary human melanocytes were transiently exposed to estrogen or progesterone. Figure 11A-11E Estrogen drives differentiation and is associated with increased melanin production, while progesterone has the opposite effect. Figure 11A ).

[0284] Following hormone withdrawal, progesterone-treated cells rapidly returned to baseline levels of melanin production. Conversely, estrogen-treated cells remained more differentiated after estrogen withdrawal and steadily produced more melanin through continuous cell division over the following 50 days. Subsequent treatment with progesterone of a subset of cells differentiated through brief estrogen exposure reversed the effects of estrogen, and melanin production decreased to sub-baseline levels seen at the initial progesterone treatment. Upon progesterone withdrawal, these cells fully recovered to the elevated differentiation state induced by the initial estrogen exposure. Consistent with the increased cell differentiation, estrogen exposure was associated with a steady increase in classical melanocyte differentiation antigens—including tyrosinase and MC1R. Figure 11B These results indicate that transient estrogen induces persistent, long-term differentiation processes in melanocytes.

[0285] To test whether transient GPER signaling induces a sustained differentiation state in melanoma cells—which affects subsequent in vivo tumor growth—melanoma cells were treated in vitro with estrogen, G-1, or a carrier, and then the same number of treated cells were injected into host mice. Figure 11C Pretreatment with estrogen or G-1 significantly reduced subsequent tumor size. Figure 11D-11E This indicates that transient GPER activation has a lasting effect on tumor growth.

[0286] Example 4: GPER signaling leads to the loss of C-Myc in melanoma

[0287] Amplification of c-Myc (a transcription factor that antagonizes differentiation and promotes proliferation, survival, and evasion of immune surveillance) is one of the most common genetic alterations in human cancers, including melanoma. GPER signaling consumes c-Myc protein ( Figure 12 (Image AC).

[0288] Furthermore, GPER signaling induces relative growth arrest, which is associated with Rb phosphorylation in mouse and human melanoma cells. Figure 12 (Image D). Melanoma cells engineered to maintain c-Myc protein in response to GPER activation exhibit resistance to G-1, suggesting that c-Myc loss is a key mediator of the anti-proliferative GPER effect. Figure 12 Image E).

[0289] GPER activation leads to rapid and PKA-dependent loss of c-Myc, indicating that classical stimulatory G protein-coupled receptor signaling destabilizes c-Myc. Figure 12 (Image FG). Consistent with this, the c-Myc half-life was significantly shortened in a proteasome-dependent manner after GPER activation ( Figure 12 (Image HI).

[0290] Example 5: In vivo G-1 treatment alters immunomodulatory proteins

[0291] In addition to its role in proliferation and differentiation, c-Myc also positively regulates the expression of various inhibitory immune checkpoint regulators, including PD-L1. Transient pharmacological GPER activation in melanoma cells leads to a parallel decrease in both c-Myc and PD-L1. Figures 13A-13C In cells engineered to maintain c-Myc in the presence of GPER agonists, PD-L1 is preserved. Figure 13G ).

[0292] Example 6: Transient GPER activation inhibits proliferation and increases response to immunotherapy

[0293] As demonstrated in this paper, GPER signaling induces stable changes in tumor cells, antagonizes tumor proliferation, and reduces the expression of immunosuppressive proteins in tumor cells. Therefore, this study investigated whether GPER activation enhances the antitumor activity of immune checkpoint blockade inhibitors.

[0294] To determine whether tumor cell-intrinsic GPER signaling affects melanoma's vulnerability to immune checkpoint therapy, this study was conducted based on the observation that GPER-driven differentiation is long-term. In murine B16F10 melanoma cells, G-1 was used to activate GPER and drive differentiation in vitro. Figure 13D Then, the same number of vector- or G-1-treated tumor cells were injected into genotypical C57BL / 6 mice, and the animals were treated with αPD-1 antibody or isotype antibody as a control.

[0295] Compared to the control, G-1 pretreatment alone inhibited subsequent tumor growth and prolonged survival. AlphaPD-1 antibody monotherapy also prolonged survival. However, the combination of G-1 pretreatment and alphaPD-1 antibody significantly prolonged survival, exceeding that seen with either agent alone, suggesting that GPER activity in tumor cells induces sustained changes in the tumor sufficient to improve the antitumor activity of systemic alphaPD-1 therapy. Figure 13E-13F ).

[0296] Example 7: Treatment of mice with melanoma with G-1 and aPD-1 immunotherapy significantly prolonged survival.

[0297] To determine whether G-1 has therapeutic efficacy as a systemic delivery agent with or without immune checkpoint inhibitors, syngeneic mice carrying B16F10 melanoma were treated with subcutaneous G-1, αPD-1 antibody, or both, and their survival was compared with matched mice treated with a vector and an isotype antibody control (specifically, a nonspecific isotype control antibody 2A3). Figure 14AThe total tumor volume is assessed after 5-50 days.

[0298] G-1 was well tolerated in mice, and monotherapy with it prolonged survival to the same extent as αPD-1. Each monotherapy and combination therapy with αPD-1 and G-1 slowed tumor growth. Figure 14B Notably, the combination therapy of αPD-1 and G-1 prolonged survival by 7 times compared to either agent alone, demonstrating a significant synergistic effect. Figure 14C ).

[0299] Although B16F10 melanoma is the most commonly used model in melanoma immunology research, and experimental results have been largely transferred to humans, B16F10 lacks the BRaf or NRas oncodriver gene mutations present in most human melanomas. To test whether GPER signaling has similar anti-melanoma activity in potentially more medically relevant models, genetically defined melanoma cells from the Yale University Mouse Melanoma Collection (YUMM) were used. This resource contains melanoma lines generated from established genetically engineered mouse models, backcrossed specifically with a C57BL / 6 background to facilitate immunological research. YUMM 1.7 cells (BRafV600E / wt Pten- / -Cdkn2- / -) were injected into C57BL / 6 mice, and G-1 treatment was initiated with and without αPD-1 after tumor diameter reached 3 to 4 mm (day 14). Figure 14D ).

[0300] Similar to the results observed with B16F10 melanoma, G-1 or αPD-1 monotherapy resulted in a modest but significant increase in survival, while combination therapy further significantly prolonged survival, including long-term survivors. Figure 14E-14F ).

[0301] Example 8: In vivo G-1 treatment alters tumor-infiltrating immune cells

[0302] These results indicate that GPER's antitumor activity is independent of tumor oncogenes. Consistent with the hypothesis that GPER activation alters the nature of immune infiltration, G-1 treatment in melanoma-carrying mice increased several immune cell subsets—including T cells and NK cells—suggesting a more robust inflammatory response. Figures 15A-15C ).

[0303] Example 9: G-1 treatment drives histone acetylation and synergizes with HDAC inhibitors in melanoma.

[0304] To begin identifying possible epigenetic changes following stable melanocyte differentiation after GPER activation, a comprehensive analysis of post-translational histone modifications in estrogen-treated MCs was performed using mass spectrometry. Consistent with mechanisms involved in CREB activity, significant increases were observed in numerous histone acetylation markers regulated by CREB binding to mate-body CBP / P300—including H3K122, H3K23, and H3K18 (in three independent biological replicates). Figure 16A ).

[0305] In contrast, H3K9ac markers not written by CBK / P300 decreased after estrogen treatment. Since post-translational histone modifications—including acetylation—mediate heritable transcriptional memory in other environmental mediators, in some non-limiting embodiments, CBP / P300-regulated histone modifications are responsible for maintaining an enhanced differentiated melanocyte state across cell division. Higher levels of CBP-written histone acetyl markers were observed in benign human nevi and non-tumorigenic melanoma cells compared to melanoma tissue and tumorigenic melanoma cell lines. Taken together, this suggests that histone acetylation can maintain the differentiated, anti-proliferative, non-tumorigenic state of GPER-stimulated melanoma cells.

[0306] Histone acetylation markers are removed by several histone deacetylases (HDACs)—which are aberrantly regulated in many cancers. Although HDAC inhibitors are approved as anticancer agents for cutaneous lymphoma, myeloma, and pancreatic cancer, their applicability in melanoma remains unclear. In human trials, HDAC inhibitors (HDACi) have typically demonstrated modest antimelanoma activity as single agents. HDAC potency may be limited by the fact that trials are conducted in the absence of differentiation drivers to promote the formation of acetylation markers on histones or other key oncoproteins (e.g., c-Myc)—which are then stabilized by HDACi. Without being bound by any theoretical limitations, the anticancer effects of HDAC inhibitors can be enhanced by combination drug regimens—which also promote histone acetylation at functionally critical sites. HDACi and GPER agonists independently promote melanoma cell differentiation and also cooperate to enhance each other's activity. Figure 16B-16C ).

[0307] Example 10: GPER signaling reduces PDAC cell proliferation in vitro and in vivo

[0308] Pancreatic cancer is less common in women than men, and also less common in users of hormone therapy using estrogen alone, suggesting that GPER may also have a tumor-blocking effect in pancreatic cancer. Using a newly obtained syngeneic pancreatic ductal adenocarcinoma (PDAC) cell line, the effect of GPER activation on c-Myc protein levels was examined. Upon exposure to G-1, c-Myc and pRB were reduced in these cell lines, consistent with changes observed in melanoma cell lines. Figure 17A ).

[0309] In addition to changes in the levels of these signaling molecules, the proliferation rate of these cell lines was also inhibited by G-1 GPER activation. Figure 17B ).

[0310] To demonstrate that systemic G-1 administration alters tumor growth in vivo, PDAC tumors were grown in mice for 18 days, followed by administration of a standard G-1 dose for 3 consecutive days. Figure 17C ).

[0311] Even during a short follow-up period of 4 days post-treatment, tumors were significantly smaller in both independent PDAC cell lines, suggesting that GPER activation may have a potent antitumor effect in PDAC. Figure 17D ).

[0312] Example 11: GPER signaling reduces NSCLC cell proliferation in vitro and in vivo and has a combination effect with αPD-1 immunotherapy.

[0313] Reproductive history can also influence lung cancer. A woman's reduced risk of lung cancer is associated with the number of children she has. To demonstrate that G-1 / GPER signaling is active in the NSCLC line LLC1, the line was treated with G-1, and elevated pCREB levels were observed after 30 minutes. Figure 18A ).

[0314] Long-term treatment of LLC1 with G-1 resulted in reduced proliferation. Figure 18B ) and decreased c-Myc protein ( Figure 18C ).

[0315] c-Myc is consumed rapidly. Figure 18D ), and the same signal transduction effect also occurs in another NSCLC series TC-1. Figure 18E ).

[0316] To test whether G-1 has in vivo activity and synergistic effects with PD-1 immunotherapy, LLC1 tumors were grown in mice treated subcutaneously with G-1, αPD-1 antibodies, or both, and their survival was compared with matched mice treated with a vector and an allotype antibody control. Initially, G-1 or αPD-1 monotherapy slowed tumor growth (…). Figure 18FHowever, it did not significantly alter survival. Figure 18G ) Further slowing tumor growth with combination therapy of G-1 and αPD-1 ( Figure 18F And significantly prolongs survival time. Figure 18G ).

[0317] The disclosure of each patent, patent application, and publication cited herein is incorporated herein by reference in its entirety. While the invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the invention can be devised without departing from its true spirit and scope. The appended claims are intended to be construed as encompassing all such embodiments and equivalent variations.

Claims

1. Use of a G-protein-coupled estrogen receptor (GPER) specific agonist and an αPD-1 antibody in the preparation of a pharmaceutical remedy for treating or preventing cancers expressing non-canonical steroid hormone receptor GPER in subjects, wherein the GPER specific agonist and the αPD-1 antibody are each present in a therapeutically effective amount. Among the cancers expressing the GPER expression, melanoma is present; and The GPER-specific agonist mentioned therein is G-1 or a pharmaceutically acceptable salt thereof.

2. The use according to claim 1, wherein the GPER-specific agonist and the αPD-1 antibody are co-administered to the subject.

3. The use according to claim 2, wherein the GPER-specific agonist and the αPD-1 antibody are co-formulated.

4. The use according to claim 1, wherein the pharmaceutical product comprises a pharmaceutical composition, the pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

5. The use according to claim 1, wherein the subject is further administered at least one additional anticancer treatment.

6. The use according to claim 5, wherein the at least one additional anticancer treatment comprises chemotherapy, engineered chimeric antigen receptor (CAR) T-cells, or immune checkpoint inhibitors.

7. The use according to claim 6, wherein the chemotherapy is selected from histone deacetylase inhibitors, temozolomide, dacarbazine, vemurafenib, dabrafenib, and trametinib.

8. The use according to claim 1, wherein the GPER-specific agonist is administered to the subject via at least one of the following routes of administration: inhalation and topical application.

9. The use according to claim 1, wherein the drug is administered to the subject over a period of 3 weeks or less.

10. The use according to claim 9, wherein the GPER-specific agonist is administered to the subject over a period of 2 weeks or less.

11. The use as claimed in claim 10, wherein the GPER-specific agonist is administered to the subject over a period of one week or less.

12. The use as claimed in claim 1, wherein the subject is a mammal.

13. The use as claimed in claim 12, wherein the mammal is a human.

14. The use according to claim 1, wherein the GPER-specific agonist is formulated in a pharmaceutical composition, the pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

15. The use according to claim 1, wherein the GPER-specific agonist is administered to the subject via at least one of the following routes of administration: oral, rectal, vaginal, transdermal, pulmonary, intranasal, buccal, ocular, intrathecal, intracranial, and intravenous.

16. The use according to claim 1, wherein the GPER-specific agonist is administered parenterally to the subject.

17. The use according to claim 1, wherein the GPER-specific agonist is administered orally to the subject.

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