WO2024102968A1 - Uses of ep2/ep4 antagonist compounds for treating familial adenomatous polyposis (fap) - Google Patents

Abstract

The present disclosure relates generally to the use of EP2/EP4 antagonist compounds for treating familial adenomatous polyposis (FAP).

Description

USES OF EP2/EP4 ANTAGONIST COMPOUNDS FOR TREATING FAMILIAL

ADENOMATOUS POLYPOSIS (FAP)

CROSS-REFERENCE

[0001] This application claims the benefit of U. S. Provisional Application Serial No. 63/383,132 filed November 10, 2022, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Familial Adenomatous Polyposis (FAP) is a rare genetic condition in which a single allele of the APC gene is mutated. FAP presents as a disease of the GI tract and is characterized by hundreds to thousands of intestinal and rectal polyps forming on average by age 16. Patients with FAP will succumb to tumor burden in their fourth decade of life if untreated, and have a 100% risk of colorectal cancer (CRC). Regular surgical resection of these polyps, along with colostomy and pharmacological therapy are effectively utilized to ameliorate disease burden and extend life, but constitute significant burden to patients quality of life. Pharmacological therapies that are safe and effective present the possibility of extending time before colostomy and between surgical resections, as well as to extend the overall life expectancy.

[0003] In FAP and CRC, malignant progression is associated with induced cyclooxygenase-2 (COX- 2) expression and increased synthesis of prostaglandin E2 (PGE2) from arachidonic acid intermediates. PGE2 signals through four consecutively named homologous cell surface receptors, EPl to EP4, that are differentially expressed depending on cell lineage and environmental cues. The intracellular signal delivered in response to binding of PGE2 to each of its four targeted EP receptors depends on the alpha subunit of the E-prostanoid G-protein-coupled receptor and informs the development of new therapeutics which selectively target the prostaglandin pathway. In general, EPl and EP3 signaling potentiates immunity by increasing calcium flux and decreasing cAMP levels. In contrast, EP2 and EP4 signaling suppresses immunity by stimulating cAMP production and downregulating T cell and myeloid cell activation by signaling through protein kinase A (PKA).

[0004] PGE2 promotes tumorigenesis, tumor cell survival and metastasis, angiogenesis, and fibroblast development in the tumor microenvironment (TME) through both autocrine and paracrine signaling. Tumor- or stroma-produced PGE2 is profoundly immune suppressive and facilitates evasion of immune recognition by inhibiting the effector functions of myeloid cell and lymphocyte populations and enhancing the function of suppressive immune cell populations including myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and M2 macrophages. Elevated COX-2 levels are associated with tumor progression in both human cancers and animal tumor models and have diverse etiologies including inflammation, tumor-driver mutations, radiation, and chemotherapy-induced immunogenic cell death. Recent investigations have suggested that elevated COX-2 production and expression of EP2 and EP4 in tumor cells may be associated with adaptive immune resistance to immune checkpoint inhibitor therapies. These observations indicate that PGE2 production can be a critical factor in mediating evasion of immune recognition and tumor proliferation, pointing to its downstream signaling receptors as possible critical targets for development of new therapies.

[0005] Despite the well-established linkage between PGE2 production and tumor progression, there are no FDA approved cancer therapeutics targeting the prostaglandin pathway. Broad inhibition of PGE2 signaling pathways with non-steroidal anti-inflammatory drugs (NSAIDs) that block both COX-1 and COX-2 (e.g., naproxen), or those that block only COX-2 (e.g., celecoxib), results in anti-tumor effects in mouse syngeneic tumor models. Epidemiological and prospective studies indicate that inhibition of the PGE2 pathway can translate to clinical benefit. Indeed, regular use of NSAIDs after diagnosis with colorectal cancer (CRC) and head and neck cancer has been associated with improved outcomes in PIK3CA- mutated cancers. However, gastro-intestinal, renal, and cardiac toxicities are associated with chronic usage of COX inhibitors due to imbalances in prostacyclin and thromboxane levels limiting both tolerable dose levels and their long-term use for treatment of malignancies. The clinical results with available COX inhibitors indicate a need for the development of therapies which selectively target EP signaling pathways which promote tumor progression.

[0006] The linkage between PGE2 and tumor progression in tumor formation in APCmin/+ mice, as well as the development of CRC serves as the principal rationale for treating FAP patients with a dual EP2/EP4 antagonist.

SUMMARY OF THE INVENTION

[0007] Disclosed herein is a method for treating familial adenomatous polyposis (FAP) in a subject in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

[0008] Also disclosed herein is a method for preventing precancerous polyps in a subject in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

[0009] Also disclosed herein is a method for maintaining a low polyp burden in a subject in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

[0010] In some embodiments of a method disclosed herein, the polyps are located in the colon, the stomach, the duodenum, or the small intestine.

[0011] In some embodiments of a method disclosed herein, the polyps are rectal polyps.

[0012] In some embodiments of a method disclosed herein, the polyps are located in the colon.

[0013] Also disclosed herein is a method of treating or preventing a pre- malignant condition in a subject in need thereof, the method comprising administering to the subject an EP2/EP4 dual antagonist.

[0014] In some embodiments of a method disclosed herein, the pre-malignant condition is present in any one of a plurality of regions of the gastrointestinal tract, and in particular of the colon and/or rectum. [0015] In some embodiments of a method disclosed herein, the pre-malignant condition is present in the colon and/or the rectum.

[0016] In some embodiments of a method disclosed herein, the pre-malignant condition is manifested in the form of polyps. [0017] In some embodiments of a method disclosed herein, the polyps are located in the colon, the stomach, the duodenum, or the small intestine.

[0018] In some embodiments of a method disclosed herein, the polyps are rectal polyps.

[0019] In some embodiments of a method disclosed herein, the polyps are located in the colon.

[0020] Also disclosed herein is a method for preventing colorectal cancer in a subj ect in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

[0021] Also disclosed herein is a method for minimizing the risk of developing colorectal cancer in a subject in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

[0022] Also disclosed herein is a method for preventing colostomy in a subject in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

[0023] Also disclosed herein is a method for delaying the need for colostomy in a subj ect in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

[0024] Also disclosed herein is a method for delaying the need for polypectomy in a subject in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

[0025] In some embodiments of a method disclosed herein, the subject in need thereof has been diagnosed with familial adenomatous polyposis (FAP).

[0026] In some embodiments of a method disclosed herein, the EP2/EP4 dual antagonist is

pharmaceutically acceptable salt thereof.

[0027] In some embodiments of a method disclosed herein, the method further comprises administering to the subject in need thereof an additional therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the TNFa ELISA results from a murine whole blood assay. Whole blood was treated with increasing amounts of compound 1 (dual EP2/4 inhibitor) followed by treatment with lOnM or 500nM PGE2 and 0.5pg/ml LPS (“whole blood assay”).

FIG. IB shows the TNFa ELISA results from a murine whole blood assay. Whole blood was treated with increasing amounts of E7046 (single EP4 inhibitor) followed by treatment with lOnM or 500nM PGE2 and 0.5pg/ml LPS (“whole blood assay”). FIG. 1C shows the TNFa ELISA results from a murine whole blood assay. Whole blood was treated with increasing amounts of PF 04418948 (single EP2 inhibitor) followed by treatment with lOnM or 500nM PGE2 and 0.5pg/ml LPS (“whole blood assay”).

FIG. ID shows the IC50 results from whole blood assay described in FIG. 1A, IB, and 1C.

FIG. ID

FIG. IE shows the percent recovery results from whole blood assay described in FIG. 1A, IB, and 1C. FIG. IF shows the TNFa ELISA results from human whole blood assays. Whole blood was treated with increasing amounts of compound 1 (dual EP2/4 inhibitor) followed by treatment with lOnM or 500nM PGE2 and 0.5pg/ml LPS (“whole blood assay”).

FIG. 1G shows the TNFa ELISA results from human whole blood assays. Whole blood was treated with increasing amounts E7046 (Single EP4 inhibitor) followed by treatment with lOnM or 500nM PGE2 and 0.5pg/ml LPS (“whole blood assay”).

FIG. 2A shows the IFNy ELISA from supernatants of CD8+ enriched PBMC stimulated with CEF peptides and compound 1.

FIG. 2B shows the IFNy ELISA from supernatants of CD8+ enriched PBMC stimulated with CEF peptides and PF04418948.

FIG. 2C shows the IFNy ELISA from supernatants of CD8+ enriched PBMC stimulated with CEF peptides and E7046.

FIG. 2D shows the IFNy Elisa from the same assay as in FIG. 2A, 2B, and 2C treated with increasing amounts of EPl and EP3 inhibition. Results are representative of multiple donors.

FIG. 3A shows Lewis Lung Carcinoma (LLC) Tumors treated with listed EP antagonists. Tumors were implanted in flanks of animals on day -11, then assorted into groups where tumor volumes averaged 200mm³ and treated with listed EP antagonists.

FIG. 3B shows the tumor count in small intestines of APCmin/+ mice treated for 3 weeks, starting at 13 weeks of age with orally administered compound 1 at lOOmpk BID, E7046 at 150mpk QD, PF04418948 at lOOmpk QD, or celecoxib at 60mpk QD.

FIG. 3C shows the Survival of APCmin/+ animals treated beginning at 12 weeks of age with listed EP antagonists. All antagonists were administered PO for 6 weeks. Statistics were calculated by the Gehan- Breslow- Wilcoxon test.

FIG. 3D shows the survival of APCmin/+ animals treated beginning at 6 weeks of age with orally administered QD regimens of compound 1 (lOOmpk, 50mpk, lOmpk).

FIG. 4A shows the quantification by a blinded pathologist of immunohistochemistry (IHC) of resected hyperplasias from small intestines of APCmin/+ mice treated for 3 weeks, starting at 13 weeks of age with orally administered compound 1 at lOOmpk BID, E7046 at 150mpk QD, PF04418948 at lOOmpk QD, or celecoxib at 60mpk QD (CD8a).

FIG. 4B shows the quantification by a blinded pathologist of immunohistochemistry (IHC) of resected hyperplasias from small intestines of APCmin/+ mice treated for 3 weeks, starting at 13 weeks of age with orally administered compound 1 at lOOmpk BID, E7046 at 150mpk QD, PF04418948 at lOOmpk QD, or celecoxib at 60mpk QD (CD4).

FIG. 4C shows the quantification by a blinded pathologist of immunohistochemistry (IHC) of resected hyperplasias from small intestines of APCmin/+ mice treated for 3 weeks, starting at 13 weeks of age with orally administered compound 1 at lOOmpk BID, E7046 at 150mpk QD, PF04418948 at lOOmpk QD, or celecoxib at 60mpk QD (FoxP3).

FIG. 5A shows significant log2 fold changes in differentially expressed genes from RNA sequencing of tumors resected from APCmin/+ mice in FIG. 3B.

FIG. 5B shows significant log2 fold changes in differentially expressed genes from RNA sequencing of tumors resected from APCmin/+ mice in FIG. 3B.

FIG. 5C shows significant log2 fold changes in differentially expressed genes from RNA sequencing of tumors resected from APCmin/+ mice in FIG. 3B.

FIG. 5D shows a volcano plot of selected genes from samples in (FIG. 5A-5C) for compound 1.

FIG. 5E shows a volcano plot of selected genes from samples in (FIG. 5A-5C) for E7046.

FIG. 5F shows a volcano plot of selected genes from samples in (FIG. 5A-5C) for PF 04418948.

FIG. 5G shows a volcano plot of selected genes from samples in (FIG. 5A-5C) for Celecoxib.

FIG. 6A shows Compound 1 EPl receptor PGE2 antagonist values in CA++ flux assays.

FIG. 6B shows Compound 1 EP2 receptor PGE2 antagonist values in CA++ flux assays.

FIG. 6C shows Compound 1 EP3 receptor PGE2 antagonist values in CA++ flux assays.

FIG. 6D shows Compound 1 EP4 receptor PGE2 antagonist values in CA++ flux assays.

FIG. 7 shows increased efficacy with Compound 1 and a-PD-1 combination therapy in CT26 model.

FIG. 8A shows TNFa concentrations and quantification of percent recovery.

FIG. 8B shows TNFa concentrations and quantification of percent recovery at select concentrations.

FIG. 9 shows Compound 1 displays T cell-independent anti-tumor efficacy in CT26.

FIG. 10A shows the lesion area percentage in histopathological analysis of resected APCmin/+ colon hyperplasia.

FIG. 10B shows the number of adenoma lesions histopathological analysis of resected APCmin/+ colon hyperplasia.

FIG. 10C shows the number of carcinoma lesions histopathological analysis of resected APCmin/+ colon hyperplasia.

DETAILED DESCRIPTION OF THE INVENTION

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise. [0029] The term “halo,” “halogen” or “halide” as used herein and unless otherwise indicated, refers to any radical of fluorine, chlorine, bromine, or iodine.

[0030] The term “alkyl” as used herein and unless otherwise indicated, refers to a saturated hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms or otherwise having from one to ten, one to eight, one to six or one to four carbon atoms, and which is attached to the rest of the molecule by a single bond. In certain embodiments, the hydrocarbon chain is optionally deuterated. For example, Ci-Ce alkyl indicates that the group may have from 1 to 6 (inclusive) carbon atoms in it. In some embodiments, an alkyl is a Ci-Ce alkyl which represents a straight- chain or branched saturated hydrocarbon radical having 1 to 6 carbon atoms. Examples of alkyl include without limitation methyl, ethyl, «-propyl, isopropyl, «-butyl, isobutyl, sec-butyl, and tert-butyl.

[0031] The term “cycloalkyl” as used herein and unless otherwise indicated, refers to a monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group having the indicated number of ring carbon atoms or otherwise having three to ten carbon atoms and which are fully saturated or partially unsaturated Multicyclic cycloalkyl may be fused, bridged or spiro-ring systems. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, and partially unsaturated hydrocarbon rings such as cyclobutylene, cyclopentene and cyclohexene. In some embodiments, cycloalkyl is a monocyclic Cs-Cs cycloalkyl.

[0032] The term “haloalkyl” as used herein and unless otherwise indicated, refers to an alkyl group in which at least one hydrogen atom is replaced by a halogen. In some embodiments, more than one hydrogen atom (e.g., 2, 3, 4, 5 or 6) are replaced by halogens. In these embodiments, the hydrogen atoms can each be replaced by the same halogen (e.g., fluoro) or the hydrogen atoms can be replaced by a combination of different halogens (e.g., fluoro and chloro). “Haloalkyl” also includes alkyl moieties in which all hydrogens have been replaced by halogens (sometimes referred to herein as perhaloalkyl, e.g., perfluoroalkyl, such as trifluoromethyl).

[0033] The term “alkoxy” as used herein and unless otherwise indicated, refers to a group of formula -O-(alkyl). Alkoxy can be, for example, methoxy, ethoxy, w-propoxy. isopropoxy, w-butoxy. isobutoxy, sc -butoxy. /7-pentoxy. 2-pentoxy, 3-pentoxy. or hexyloxy. Likewise, the term “thioalkoxy” refers to a group of formula -S-(alkyl). The terms “haloalkoxy” and “thiohaloalkoxy” refer to -O-(haloalkyl) and -S-(haloalkyl), respectively.

[0034] The term “aralkyl” as used herein and unless otherwise indicated, refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. One of the carbons of the alkyl moiety serves as the point of attachment of the aralkyl group to another moiety. Non-limiting examples of “aralkyl” include benzyl, 2-phenylethyl, and 3 -phenylpropyl groups.

[0035] The term “alkenyl” as used herein and unless otherwise indicated, refers to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms or otherwise having from two to ten, two to eight or two to six carbon atoms, having one or more carbon-carbon double bonds and which is attached to the rest of the molecule by a single bond or a double bond. Alkenyl groups can include, e.g., vinyl, allyl, 1-butenyl, and 2-hexenyl. In some embodiments, an alkenyl is a C2-C6 alkenyl.

[0036] The term “alkynyl” as used herein and unless otherwise indicated, refers to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms or otherwise having from two to ten, two to eight or two to six carbon atoms and having one or more carbon-carbon triple bonds. Alkynyl groups can include, e.g., ethynyl, propargyl, 1-butynyl, and 2-hexynyl. In some embodiments, an alkynyl is a C2-C6 alkynyl.

[0037] The term “cycloalkylalkyl” as used herein and unless otherwise indicated, refers to a monovalent alkyl group substituted with cycloalkyl.

[0038] The term “deuterium” as used herein and unless otherwise indicated, refers to the heavy isotope of hydrogen represented by the symbol D or ²H. As used herein, when a particular position in a compound is designated as “deuterated” or as having deuterium, it is understood that the compound is an isotopically enriched compound and the presence of deuterium at that position in the compound is substantially greater than its natural abundance of 0.0156%.

[0039] The term “enantiomerically pure” or “pure enantiomer” as used herein denotes that the compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 98.5% by weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by weight, more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by weight or more than 99.9% by weight, of a single enantiomer to the exclusion of its corresponding non-superimposable mirror image.

[0040] The term “heterocycle”, “heterocyclyl” or “heterocyclic” as used herein and unless otherwise indicated, represents a stable 4-, 5-, 6- or 7-membered monocyclic- or a stable 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic heterocyclic ring system which comprises at least one non-aromatic (i.e. saturated or partially unsaturated) ring which consists of carbon atoms and from one to four, preferably up to three, heteroatoms selected from the group consisting of N, O and S, wherein the nitrogen and sulfur atoms may optionally be oxidized as A-oxide, sulfoxide or sulfone, and wherein the nitrogen atom may optionally be quaternized. A heterocycle can be bonded via a ring carbon atom or, if available, via a ring nitrogen atom. Bicyclic heterocyclic ring systems may be fused, bridged, or spiro-bicyclic heterocyclic ring system(s). In some embodiments, heterocyclyl is monocyclic having 4 to 7, preferably 4 to 6, ring atoms, of which 1 or 2 are heteroatoms independently selected from the group consisting of N, O and S. In some embodiments, a heterocyclyl group is bicyclic, and in which case, the second ring may be an aromatic or a non-aromatic ring which consists of carbon atoms and from one to four, preferably up to three, heteroatoms independently selected from the group consisting of N, O and S, or the second ring may be a benzene ring, or a “cycloalkyl,” or a “cycloalkenyl”, as defined herein. Examples of such heterocyclic groups include, but are not limited to azetidine, chroman, dihydrofuran, dihydropyran, dioxane, dioxolane, hexahydroazepine, imidazolidine, imidazoline, indoline, isochroman, isoindoline, isothiazoline, isothiazolidine, isoxazoline, isoxazolidine, morpholine, oxazoline, oxazolidine, oxetane, piperazine, piperidine, dihydropyridine, tetrahydropyridine, dihydropyridazine, pyran, pyrazolidine, pyrazoline, pyrrolidine, pyrroline, tetrahydrofuran, tetrahydropyran, thiamorpholine, tetrahydrothiophene, thiazoline, thiazolidine, thiomorpholine, thietane, thiolane, sulfolane, 1,3 -di oxolane, 1,3 -oxazolidine, 1,3- thiazolidine, tetrahydrothiopyran, tetrahydrotriazine, 1,3-di oxane, 1,4-di oxane, hexahydrotriazine, tetrahydro- oxazine, tetrahydropyrimidine, perhydroazepine, perhydro- 1,4-diazepine, perhydro- 1,4- oxazepine, 7-azabicyclo[2.2.1]heptane, 3-azabicyclo[3.2.0]heptane, 7-azabicyclo[4.1.0]heptane, 2,5- diazabicyclo[2.2. l]heptane, 2-oxa-5-azabicyclo[2.2.1]heptane, tropane, 2-oxa-6-azaspiro[3.3]heptane, dihydrobenzofuran, diydrobenzimidazolyl, dihydrobenzoxazole, and dihydrobenzothiazolyl, and V-oxides or sulfones or sulfoxides thereof.

[0041] The term “heterocyclylalkyl” as used herein and unless otherwise indicated, refers to a monovalent alkyl group substituted with heterocyclyl.

[0042] The term “aryl” as used herein and unless otherwise indicated, is intended to mean any stable monocyclic or bicyclic carbon ring of up to 6 members in each ring, wherein at least one ring is aromatic. Examples of aryl include phenyl, naphthyl, tetrahydronaphthyl, indanyl, or biphenyl.

[0043] The term “heteroaryl”, as used herein and unless otherwise indicated, represents a stable 5-, 6- or 7-membered monocyclic- or stable 9- or 10-membered fused bicyclic ring system which comprises at least one aromatic ring, which consists of carbon atoms and from one to four, preferably up to three, heteroatoms selected from the group consisting of N, O and S wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. In the case of a “heteroaryl” which is a bicyclic group, the second ring need not be aromatic and need not comprise a heteroatom. Accordingly, bicyclic “heteroaryl” includes, for example, a stable 5- or 6-membered monocyclic aromatic ring consisting of carbon atoms and from one to four, preferably up to three, heteroatoms, as defined immediately above, fused to a benzene ring, or a second monocyclic “heteroaryl”, or a “heterocyclyl”, a “cycloalkyl”, or a “cycloalkenyl”, as defined above. Examples of heteroaryl groups include, but are not limited to, benzimidazole, benzopyrazole, benzisothiazole, benzisoxazole, benzofuran, isobenzofuran, benzothiazole, benzothiophene, benzotriazole, benzoxazole, furan, furazan, imidazole, indazole, indole, indolizine, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, phthalazine, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, quinazoline, quinoline, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazine, triazole, benzimidazole, benzothiadiazole, isoindole, pyrrol opyri dines, imidazopyri dines such as imidazo[l,2-a] pyridine, pyrazolopyridine, pyrrolopyrimidine and A- ox ides thereof.

The term “hydrate” as used herein and unless otherwise indicated, refers to a compound provided herein or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. [0044] The term "treating", "treat", or "treatment" refers generally to controlling, alleviating, ameliorating, slowing the progress of, or eliminating a named condition once the condition has been established. In addition to its customary meaning, the term "preventing", “prevent,” or “prevention” also refers to delaying the onset of, or reducing the risk of developing a named condition or of a process that can lead to the condition, or the recurrence of symptoms of a condition.

[0045] The term "therapeutically effective amount" or "effective amount" is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. An effective amount is typically sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.

[0046] Unless stated otherwise or specifically described, it is understood that substitutions where present can occur on any atom of the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl groups. [0047] Unless specifically stated otherwise, where a compound may assume alternative tautomeric or stereoisomeric forms, all alternative isomers are intended to be encompassed within the scope of the claimed subject matter. For example, unless specifically stated otherwise, the compounds provided herein may be enantiomerically pure, or be enantiomeric mixtures.

[0048] In the description herein, if there is any discrepancy between a chemical name and chemical structure, the chemical structure controls.

[0049] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an agent” includes a plurality of such agents, and reference to “the cell” includes reference to one or more cells (or to a plurality of cells) and equivalents thereof known to those skilled in the art, and so forth. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range, in some instances, will vary between 1% and 15% of the stated number or numerical range. The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, “consist of’ or “consist essentially of’ the described features. [0050] As used herein, the term “therapeutic” means an agent utilized to treat, combat, ameliorate, prevent, or improve an unwanted condition or disease of a patient. In some embodiments, a therapeutic agent such as a compound 1 is directed to the treatment and/or the amelioration of cancers.

[0051] “Administering” when used in conjunction with a therapeutic means to administer a therapeutic systemically or locally, as directly into or onto a target tissue, or to administer a therapeutic to a patient whereby the therapeutic positively impacts the tissue to which it is targeted. Thus, as used herein, the term “administering,” when used in conjunction with a composition described herein, can include, but is not limited to, providing a composition into or onto the target tissue; providing a composition systemically to a patient by, e.g., oral administration whereby the therapeutic reaches the target tissue or cells.

“Administering” a composition may be accomplished by injection, topical administration, and oral administration or by other methods alone or in combination with other known techniques.

[0052] The term “animal” as used herein includes, but is not limited to, humans and non-human vertebrates such as wild, domestic and farm animals. As used herein, the terms “patient,” “subject” and “individual” are intended to include living organisms in which certain conditions as described herein can occur. Examples include humans, monkeys, cows, sheep, goats, dogs, cats, mice, rats, and transgenic species thereof. In a preferred embodiment, the patient is a primate. In certain embodiments, the primate or subject is a human. In certain instances, the human is an adult. In certain instances, the human is child. In further instances, the human is under the age of 12 years. In certain instances, the human is elderly. In other instances, the human is 60 years of age or older. Other examples of subjects include experimental animals such as mice, rats, dogs, cats, goats, sheep, pigs, and cows. The experimental animal can be an animal model for a disorder, e.g., a transgenic mouse with hypertensive pathology.

[0053] By “pharmaceutically acceptable,” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0054] The term “pharmaceutical composition” shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

[0055] A “therapeutically effective amount” or “effective amount” as used herein refers to the amount of active compound or pharmaceutical agent that elicits a biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following: (1) preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease, (2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), and (3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).

[0056] The terms “treat,” “treated,” “treatment,” or “treating” as used herein refers to both therapeutic treatment in some embodiments and prophylactic or preventative measures in other embodiments, wherein the object is to prevent or slow (lessen) an undesired physiological condition, disorder, or disease, or to obtain beneficial or desired clinical results. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. A prophylactic benefit of treatment includes prevention of a condition, retarding the progress of a condition, stabilization of a condition, or decreasing the likelihood of occurrence of a condition. As used herein, “treat,” “treated,” “treatment,” or “treating” includes prophylaxis in some embodiments.

[0057] The term “substantially the same as” as used herein, refers to a powder x-ray diffraction pattern or differential scanning calorimetry pattern that is non-identical to those depicted herein, but that falls within the limits of experimental error, when considered by one of ordinary skill in the art.

Methods of Treatment - Familial Adenomatous Polyposis (FAP)

[0058] Germline mutations leading to loss of the tumor suppressor Adenomatous Polyposis Coli (APC) result in Familial Adenomatous Polyposis (FAP), an autosomal dominant disorder characterized by the early development of hundreds to thousands of adenomatous polyps throughout the colon and rectum. Polyposis develops in affected individuals very early in life, at a mean age of 16 years but as young as 8 years. Unless the colon and rectum are removed by prophylactic colectomy the lifetime risk of colorectal cancer approaches 100%, with the average age of cancer diagnosis 39 years. Importantly, colectomy is preventative only for the intestine that is removed, and patients may still develop cancer anywhere in the small bowel (most notably the duodenum) and in any part of the rectum that is retained (e.g., the rectal pouch in the setting of ileo-rectal anastomosis to avoid colostomy). While the risk of rectal cancer can be eliminated by complete removal of the rectum via total proctocolectomy with ileostomy, or proctocolectomy with ileal-pouch-anal anastomosis (IPAA) these surgeries have increased morbidity and detriment to quality of life - the latter particularly impactful in the young patients being treated for FAP syndrome. Retained at-risk areas of intestine are managed by a lifetime program of regular endoscopic surveillance and polypectomy which reduces but does not eliminate the risk of developing cancer. There also is risk of non-intestinal cancers associated with loss of the APC tumor suppressor, including thyroid, stomach, pancreas, biliary and liver cancers, as well as non-cancerous tumors including osteomas and desmoid tumors. Thus, for individuals with FAP syndrome, standard of care management entails a morbid surgery performed at young age with significant detriment to quality of life followed by lifelong frequent surveillance that unfortunately does not fully eliminate the risk of developing a life-threatening cancer. Chemoprevention to reduce adenoma development and cancer in patients with FAP syndrome is a highly desirable approach with clear goals of improved survival and improved quality of life at all stages of the disease. [0059] Targeting of cyclooxygenases (COX) using either non-selective (COX-1 and COX- 2) or COX-2 selective non-steroidal anti-inflammatory drugs (NSAIDS) significantly decreases polyp burden and improves survival in the APCmin preclinical mouse model of FAP that, like the human disease, is characterized by inactivation of APC. In humans with FAP, treatment with non-selective or COX-2- selective NS AIDs demonstrated reduced adenoma burden compared to placebo in randomized, controlled trials. Expression of COX-2, the inducible COX isoform, is upregulated by loss of APC function. Celecoxib, a selective COX-2 inhibitor, was evaluated in a multi-institutional randomized double-blinded placebo-controlled study in patients with FAP syndrome randomized to receive celecoxib at 400 mg BID, 100 mg BID, or a placebo (2:2:1). The mean reduction in colorectal polyp count was 28% on the celecoxib 400 mg BID arm, 11.9% on the celecoxib 100 mg BID arm and 4.5% on the placebo arm, and the higher celecoxib dose was associated with a statistically superior mean reduction in polyp count compared to placebo. This study led to the accelerated approval of celecoxib to be used as an adjunctive therapy in addition to the standard of care (e.g., endoscopic surveillance and prophylactic surgery) for FAP syndrome. Despite clear evidence of anti-polyp activity, enthusiasm for celecoxib as chemoprophylaxis for patients with FAP has been tempered by cardiovascular toxicity associated with the high dose of celecoxib employed for polyp reduction. At this time there are no FDA approved therapies that meet the critical need to reduce the incidence of adenomas in patients with FAP syndrome.

[0060] Prostaglandin E2 (PGE2) is a key bioactive lipid produced by multistep metabolism of arachidonic acid. PGE2 supports multiple tumor-promoting processes, including cell proliferation and migration, growth factor signaling, and angiogenesis. PGE2 reduces immune function by inhibiting T- effector cell activity while enhancing production of suppressive immune cell populations. In the APCmin mouse model of FAP driven by loss of APC function, PGE2 administration increases adenoma growth and number while selective inhibition of PGE2 blocks this effect. Notably, the growth inhibitory effect of NS AIDs in the APCmin model can be attenuated by administration of PGE2 or by direct agonists of the PGE2 signaling receptors, suggesting that PGE2 is a critical adenoma-supporting signal downstream of COX enzymes in the APCmin model.

[0061] While the COX enzymes are necessary for production of PGE2, they act upstream of PGE2 synthesis and are not specific to PGE2 production. Thus, inhibition of COX enzymes has pleotropic metabolic effects independent of PGE2. The cardiovascular toxicity of COX-2 inhibition is thought to result from perturbations of multiple hemodynamically active arachidonic acid metabolites, including thromboxanes, prostacyclins and leukotrienes. One potential means to achieve the benefit of PGE2 inhibition without the toxicity of COX inhibition is to specifically inhibit PGE2 signaling rather than the COX enzymes. PGE2 exerts its effects via four homologous E-prostanoid G-protein coupled receptors - EPl, EP2, EP3, and EP4 - which activate distinct signal transduction pathways. Accumulating evidence suggests that optimal anti-tumor activity is achieved by specifically inhibiting the growth-stimulating and immune-suppressing EP2 and EP4 receptors, while sparing the immune-stimulating EPl and EP3 receptors. [0062] Compound 1 is dual antagonist of the PGE2 receptors EP2 and EP4. In some embodiments, the effect of dual EP2/EP4 inhibition with Compound 1 on formation and/or growth of intestinal lesions in APCmin/+ mice has been evaluated in several experiments, including direct comparisons to the COX-2 inhibitor celecoxib, and to single antagonists of the EP2 and EP4 receptors. In some embodiments, treatment of APCmin mice with Compound 1 resulted in a statistically significant reduction in adenomas, and improvement in survival duration, compared to celecoxib or to single antagonists of EP2 and EP4. In some embodiments, the clear superiority of Compound 1 over celecoxib in the APCmin model is particularly promising as the effect of celecoxib in the APCmin model correlates with benefit in patients with FAP syndrome. In some embodiments, superiority of Compound 1 over celecoxib in the APCmin model supports the potential for Compound 1 to be more effective than celecoxib in patients with FAP syndrome. In some embodiments, Compound 1 does not have cardiac toxicities consistent with the drug mechanism specifically targeting PGE2 signaling rather than COX-2 activity.

[0063] Disclosed herein is a method of treating familial adenomatous polyposis (FAP) in a subject in need thereof, the method comprising administering to the subject an EP2/EP4 dual antagonist.

[0064] Also disclosed herein is a method of treating or preventing a pre- malignant condition in a subject in need thereof, the method comprising administering to the subject an EP2/EP4 dual antagonist. In some embodiments, the pre-malignant condition is present in any one of a plurality of regions of the gastrointestinal tract, and in particular of the colon and/or rectum.

[0065] In some embodiments, the pre-malignant condition is manifested in the form of polyps that can be present in small numbers, as in the sporadic onset of the same, in large numbers, as in familial adenomatous polyposis (“FAP”), or not be present at all, as in certain forms of hereditary non-polyposis colorectal cancer, also known as Lynch Syndrome.

[0066] Also disclosed herein are methods of treating a subject suffering from stomacho-intestinal system cancer or colorectal cancer, the method comprising administering to the subject an EP2/EP4 dual antagonist. The stomach, small intestine, and large intestine (the latter including the colon, rectum, and anus) are here collectively referred to as the stomacho-intestinal system (“SIS”) and cancer in any of such organs is referred to as stomacho-intestinal system cancer (“SISC”). Cancer in any of the colon and rectum is referred to as colorectal cancer (“CRC”).

[0067] Also disclosed herein is a method of treating, delaying, or preventing any manifestation of disease in a subject suffering from any SISC-related cancer predisposition syndrome, such as familial adenomatous polyposis (FAP), the method comprising administering to the subject an EP2/EP4 dual antagonist. In some embodiments, the method includes delaying the need for surgical procedures such as colectomy, which prophylactically removes the colon in order to lower the risk of future colorectal cancer development. The term FAP is used herein to encompass what is sometimes referred to as subtypes of the same, such as attenuated familial adenomatous polyposis (AFAP). Unless indicated otherwise or referred to expressly as any specific subtype, the term FAP includes subtypes such as AFAP. [0068] In some aspects, a method of reducing the number of polyps or polyp burden (the latter defined by considering both the numbers and individual sizes of polyps) in a subject comprises administering to the subject an EP2/EP4 dual antagonist. In some embodiments, reducing the number of polyps comprises reducing the number of polyps in the colon, rectum, post-surgical J-pouch and/or duodenum after administration of the EP2/EP4 dual antagonist. In some embodiments, the number of polyps that are > 2 mm in diameter in the colon, rectal, post-surgical J-pouch and duodenum are reduced. In some embodiments, number of polyps that are > 5 mm in diameter in the colon, rectal, post-surgical J-pouch and/or duodenum are reduced. In some embodiments, the decrease in the number of polyps after administration of the EP2/EP4 dual antagonist is by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more, or any range(s) in between.

[0069] Also disclosed herein is a method of treating or preventing the onset of SISC at any stage of disease evolution, and especially the onset of CRC, comprising administering to the subject an EP2/EP4 dual antagonist.

[0070] Also disclosed herein is a method of treating or preventing any chronic or acute inflammation disorder in the stomacho-intestinal system in a subject, comprising administering to the subject anEP2/EP4 dual antagonist.

[0071] Additional aspects relate to the use of an EP2/EP4 dual antagonist for administration to a subject who has or has had at least one of sporadic stomacho-intestinal polyp formation, FAP (or other inherited polyposis syndromes) and Lynch Syndrome (or other inherited nonpolyposis syndromes).

[0072] Additional aspects are methods of treating a subject suffering from or diagnosed with at least one form of localized or metastatic SISC, including at least one of gastric, small intestine, colorectal and anus cancer by administering a an EP2/EP4 dual antagonist.

[0073] Additional aspects of the invention include a method of SISC interception in a subject comprising administering an EP2/EP4 dual antagonist. Further aspects are given by any one of the foregoing in which SISC interception is FAP SISC interception. Further aspects are given by any one of the foregoing aspects in which SISC interception is Lynch syndrome SISC interception. Further aspects are given by any one of the foregoing aspects in which SISC interception is sporadic polyp interception. Further aspects are given by any one of the foregoing aspects in which SISC interception is gastric cancer interception. Further aspects are given by any one of the foregoing aspects in which SISC interception is small intestine cancer interception. Further aspects are given by any one of the foregoing aspects in which SISC interception is anus cancer interception.

[0074] In some SISC interception scenarios, a drug delivery along the entire or a large portion of the intestinal tract may be desirable. In other scenarios, it may be desirable to increase local concentration at any given portion of the gastrointestinal tract. Still in other scenarios, a combination of these two forms of delivery at different sites in the intestinal tract could be desirable. [0075] In some embodiments, the subject has APC mutation or genetic diagnosis with confirmed APC mutation.

[0076] In some embodiments, the subject has genetic or clinical FAP.

[0077] In some embodiments, the subject has genetic or clinical FAP and has undergone prior colectomy with IRA.

[0078] In some embodiments, the subject with attenuated FAP was previously treated with colectomy and IRA.

[0079] In some embodiments, the subject has a clinical diagnosis of classic FAP with > 100 colorectal adenomas status post colectomy and a family history of FAP.

[0080] In some embodiments, the method reduces rectal polyps.

[0081] In some embodiments, the method reduces rectal polyp burden.

[0082] In some embodiments, the method reduces rectal polyp burden in patients with FAP.

[0083] In some embodiments, the method reduces duodenal polyp.

Familial Adenomatous Polyposis

[0084] Familial adenomatous polyposis (FAP) is a rare inherited cancer predisposition syndrome characterized by hundreds to thousands of precancerous colorectal polyps (adenomatous polyps). If left untreated, affected individuals inevitably develop cancer of the colon and/or rectum at a relatively young age. FAP is inherited in an autosomal dominant manner and caused by abnormalities (mutations) in the APC gene. Mutations in the APC gene cause a group of polyposis conditions that have overlapping features: familial adenomatous polyposis, Gardner syndrome, Turcot syndrome and attenuated FAP.

[0085] Classic FAP is characterized by hundreds to thousands of colorectal adenomatous polyps, with polyps appearing on average at age 16 years. Without colectomy, affected individuals usually develop colorectal cancer by the third or fourth decade of life. FAP is also associated with an increased risk for cancer of the small intestine including the duodenum, and cancer of the thyroid, pancreas, liver (hepatoblastoma), central nervous system (CNS), and bile ducts, although these typically occur in less than 10% of affected individuals.

[0086] Individuals with CNS tumors and colorectal polyposis have historically been defined as Turcot syndrome. Two-thirds of cases of Turcot syndrome develop from mutations in the APC gene. The remaining cases develop from mutations in the genes that cause hereditary non-polyposis colorectal cancer (HNPCC) also known as Lynch syndrome. Mutations in the APC gene are more commonly associated with medulloblastoma; mutations in the genes that cause HNPCC are more commonly associated with glioblastoma.

[0087] Extracolonic manifestations are variably present in FAP, including polyps of the stomach, duodenum, and small bowel; and osteomas (bony growths), dental abnormalities, congenital hypertrophy of the retinal pigment epithelium (CHRPE), and soft tissue tumors including epidermoid cysts, fibromas and desmoid tumors. About 5% of individuals with FAP experience morbidity and/or mortality from desmoid tumors. The term Gardner syndrome is often used when colonic polyposis is accompanied by clinically obvious osteomas and soft tissue tumors.

[0088] Attenuated FAP is a variant of familial adenomatous polyposis. The disorder is characterized by an increased risk for colorectal cancer (although lower risk than classical FAP) but with fewer polyps (average of 30) and later age of onset of polyps and cancer than is typically seen in classic FAP. Extracolonic manifestations are also associated with attenuated FAP.

[0089] Familial adenomatous polyposis is caused by germline (present in the first cell of the embryo) mutations in the APC gene and is inherited in an autosomal dominant manner, meaning that on average 50% of children of an affected parent will have the disease passed on to them.

[0090] Dominant genetic disorders occur when only a single copy or allele of a specific gene is mutated, thereby causing a particular disease. The abnormal gene can be inherited from either parent or can be the result of anew mutation (gene change) in the affected individual. The risk of passing the abnormal gene from affected parent to offspring is 50% for each pregnancy. The risk is the same for males and females. [0091] Familial adenomatous polyposis affects males and females in equal numbers. It occurs in approximately one in 5,000 to 10,000 individuals in the United States and accounts for about 0.5% of all cases of colorectal cancer. One estimate suggests that familial adenomatous polyposis affects 50,000 American families. According to national registries, familial adenomatous polyposis occurs in 2.29-3.2 per 100,000 individuals.

[0092] Features of the following disorders can be similar to those of the APC gene-associated polyposis conditions. Comparisons may be useful for a differential diagnosis:

[0093] MYH gene-associated polyposis (MAP) is an autosomal recessive cancer predisposition syndrome with a colonic phenotype similar to attenuated FAP. Mutations in the MYH gene are associated with this condition.

[0094] Hereditary non-polyposis colon cancer (HNPCC) or Lynch syndrome is an autosomal dominant cancer predisposition syndrome that causes a very high risk for colorectal and endometrial cancer in addition to an increased risk for cancers of the ovary, stomach, small intestine, hepatobiliary tract, upper urinary tract, brain, and skin. Individuals with Lynch syndrome most often exhibit only one or several precancerous polys of the colon. Thus, Lynch syndrome is sometimes difficult to distinguish from attenuated FAP, as some individuals with attenuated FAP may have a low number of polyps.

[0095] Peutz-Jeghers syndrome is an autosomal dominant genetic condition characterized by multiple benign hamartomatous polyps (Peutz-Jeghers polyps) in the gastrointestinal system. Hamartomatous polyps have a much lower risk of becoming cancerous compared to adenomatous polyps. These polyps occur most often in the small intestine but also occur in the stomach and large intestine. Affected individuals also have dark skin discoloration, like freckles or spots around the lips and on the face but much darker. These pigmented spots often presents in childhood and can also be seen around the eyes, and nostrils, and on the mucous membranes of the mouth and in the perianal area. Affected individuals have an increased risk for intestinal and other cancers. This condition can be distinguished from FAP by clinical features and histology (microscopic examination) of the polyps.

[0096] Juvenile polyposis syndrome (JPS) is an autosomal dominant genetic condition characterized by a predisposition to gastrointestinal polyps. The term “juvenile” refers to the type of polyp as opposed to the age of onset. Polyps are usually diagnosed by 20 years of age and are usually benign, although malignant transformation can occur. JPS is associated with mutations in the SMAD4 and BMPR1A genes.

[0097] Cronkhite- Canada disease is a very rare acquired (not inherited) disease and is characterized by intestinal polyps, loss of taste and hair, and nail growth problems. It is difficult to treat because of malabsorption that accompanies the polyps. Cronkhite-Canada disease occurs primarily in older people (the average age is 59). There have been fewer than 400 cases reported in the past 50 years, primarily in Japan but also in the U.S. and other countries.

[0098] Classical FAP is diagnosed clinically when an individual has 100 or more adenomatous colorectal polyps (typically occurring by the third decade of life) or fewer than 100 polyps and a relative with FAP. Genetic testing for mutations in the APC gene is available to confirm the diagnosis of FAP and the associated conditions. Younger individuals may have fewer polyps. A diagnosis is made in younger people by the presence of the typical polyps and in immediate relative with FAP or by genetic testing.

[0099] Partial or complete removal of the colon (colectomy) is usually recommended for individuals with classical FAP at an appropriate age, usually between the late teens and late 30s. Sulindac is a nonsteroidal antiinflammatory drug (NSAID) usually used for arthritis, but is sometimes prescribed for individuals with FAP who have had a colectomy to treat polyps in the remaining rectum. Polyps may regress, but it is uncertain if the cancer risk is changed, so surveillance must be continued.

[00100] Removal of duodenal polyps is sometimes recommended if they cause symptoms, are large or contain large numbers of abnormal cells (dysplasia). This is to prevent them from becoming cancerous. [00101] Desmoid tumors are benign, but may cause problems, including life threatening, by compressing organs and/or blood vessels in the abdomen. These are treated variously with surgery, NSAIDs, antiestrogen medications, chemotherapy and/or radiation depending on the details in each case. They are sometimes just followed when they do not grow.

[00102] In various embodiments of the method, the method further comprises administering to the subject in need thereof an additional therapy.

[00103] In some embodiments, the additional therapy is a PD-1 inhibitor or a PD-L1 inhibitor. [00104]In some embodiments, the additional therapy is a NSAID.

Compounds

[00105] Described herein are methods of using compounds of Formula (I) that are EP2/EP4 dual antagonists. These compounds and compositions comprising these compounds are useful for the treatment of cancer, especially FAP. [00106] In various embodiments of the methods described herein, the EP2/EP4 dual antagonist compound is a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

Formula (I) wherein:

X¹ is N or CH;

X³ is N or CR³;

X⁴ is N or CR⁴;

X⁵ is N or CR⁵; wherein no more than two of X³, X⁴ and X⁵ are N;

L¹ is -(CR^b ₂)_t-;

Ring A is optionally deuterated aryl; each R¹ is independently C2-C9 alkyl, C2-C9 alkenyl, C2-C9 alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or -OR⁸', wherein C2-C9 alkyl, C2-C9 alkenyl, C2-C9 alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally deuterated and optionally substituted with one, two, or three R^y; or two R¹ substituents on adjacent atoms of Ring A are taken together with the atom to which they are attached to form an aromatic or non-aromatic ring containing 0-2 heteroatoms selected from the group consisting of -O-, =N-, -NR¹⁰-, -S-, and -S(O)2-, wherein the aromatic or non-aromatic ring is optionally deuterated and optionally substituted with one, two, or three R^y; each R⁸' is independently hydrogen, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with one, two, or three groups selected from halogen, alkyl, and haloalkyl; each R^y is independently alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, halogen, -OR⁸, -NR⁸R⁹, -CN, -C(O)Rⁿ, -C(O)NR⁸R⁹, -NR⁸C(O)Rⁿ, -NR⁸C(O)OR⁹, - NR¹⁰C(O)NR⁸R⁹, -OC(O)NR⁸R⁹, -S(O)2Rⁿ, -S(O)Rⁿ, -SR⁸, -S(O)₂NR⁸R⁹, -S(O)NR⁸R⁹, -NR⁸S(O)Rⁿ, -NR⁸S(O)2Rⁿ, or -NR¹⁰S(O)2NR⁸R⁹; wherein alkyl is optionally substituted with -OR⁸ or -NR⁸R⁹ and wherein cycloalkyl and heterocyclyl are optionally substituted with one, two, or three groups selected from halogen, alkyl, and haloalkyl; each R^x is independently halogen, methyl, Cihaloalkyl, or -CN;

R², R³, R⁴, and R⁵ are each independently hydrogen, alkyl, halogen, -OR⁸, -NR⁸R⁹, -SR⁸, - S(O)Rⁿ, -S(O)2Rⁿ, -CN, cycloalkyl, orhaloalkyl;

R⁶ is hydrogen, deuterium, alkyl, or haloalkyl;

R⁷ is hydrogen, deuterium, halogen, alkyl, alkoxy, haloalkoxy, hydroxyl, or haloalkyl; each R⁸ and each R⁹ are independently hydrogen, deuterium, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with one, two, or three groups selected from halogen, alkyl, and haloalkyl; or

R⁸ and R⁹, together with the atom or atoms to which they are attached, form a heterocyclyl optionally substituted with one, two, or three groups selected from halogen, alkyl, and haloalkyl; each R¹⁰ is independently hydrogen, deuterium, alkyl, or deuterated alkyl; each R¹¹ is independently alkyl, deuterated alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with one, two, or three groups selected from halogen, alkyl, and haloalkyl;

Y¹ and Y² are each independently a bond or -(CR^a2)n-, provided that Y¹ and Y² are not both a bond;

Z¹ and Z² are each -CR^a2-;

L² is -(CR^c ₂)m-;

G is -C(O)OR¹², -C(O)NHOH, -SO₃H, -SO2NH2, -SO₂NHR^d, -SO₂NHC(O)R^d, - NHC(0)NHS02R^d, -177-tetrazolyl, -P(O)(OH)2, -l,2,4-oxadiazol-5(477)-one, -tetrazol-5(477)-one, or - C(O)NHSO₂R^d; each R^a is independently hydrogen, deuterium, optionally deuterated alkyl, halogen, or haloalkyl; each R^b is independently hydrogen, deuterium, optionally alkyl or haloalkyl, or two R^bs, together with the carbon atom to which they are attached, form optionally cycloalkyl or heterocyclyl; each R^c is independently hydrogen or halogen;

R^dis alkyl, haloalkyl, cycloalkyl, aryl, or heteroaryl;

R¹² is selected from hydrogen, Ci-Ce alkyl, aryl, aralkyl, CH(R¹³)OC(=O)R¹⁴, CH(R¹³)OC(=O)OR¹⁴ and a (5-alkyl-2-oxo-l,3-dioxolen-4-yl)methyl group having the following formula:

wherein R^e is Ci-Ce alkyl;

R¹³ is hydrogen or Ci-Ce alkyl;

R¹⁴ is Ci-Ce alkyl or Cs-Ce -cycloalkyl; m is 0, 1, or 2; each n is independently 1, 2, or 3; p is 1 or 2; q is 0, 1, or 2; and t is 1, 2 or 3.

[00107] In some embodiments of the methods, the compound of Formula (I) is a compound of Formula (II), or a pharmaceutically acceptable salt thereof:

[00108] In some embodiments of the methods, the compound is selected from:

[00109] In some embodiments, the EP2/EP4 dual antagonist compound is:

(compound 1), or a pharmaceutically acceptable salt thereof. Dosing

[00110] In one aspect, the compounds and compositions described herein are used for the treatment of diseases and conditions described herein. In addition, a method for treating any of the diseases or conditions described herein in a subject in need of such treatment, involves administration of compositions in therapeutically effective amounts to said subject.

[00111] Dosages of compositions described herein can be determined by any suitable method. Maximum tolerated doses (MTD) and maximum response doses (MRD) for the compound described herein, or a pharmaceutically acceptable salt thereof can be determined via established animal and human experimental protocols as well as in the examples described herein. For example, toxicity and therapeutic efficacy of a compound described herein, or a pharmaceutically acceptable salt thereof, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. Additional relative dosages, represented as a percent of maximal response or of maximum tolerated dose, are readily obtained via the protocols.

[00112] In some embodiments, the amount of a given formulation comprising a compound described herein, or a pharmaceutically acceptable salt thereof that corresponds to such an amount varies depending upon factors such as the particular salt or form, disease condition and its severity, the identity (e.g., age, weight, sex) of the subject or host in need of treatment, but can nevertheless be determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the liquid formulation type, the condition being treated, and the subject or host being treated.

[00113] In some embodiments, compound 1 is administered in an amount that is between about 5 mg and about 1200 mg. In some embodiments, compound 1 is administered in an amount that is about 50 mg and about 800 mg. In some embodiments, compound 1 is administered in an amount that is about 100 mg and about 600 mg.

[00114] In some embodiments, compound 1 is administered in an amount that is about 5 mg. In some embodiments, compound 1 is administered in an amount that is about 10 mg. In some embodiments, compound 1 is administered in an amount that is about 15 mg. In some embodiments, compound 1 is administered in an amount that is about 25 mg. In some embodiments, compound 1 is administered in an amount that is about 50 mg. In some embodiments, compound 1 is administered in an amount that is about 100 mg. In some embodiments, compound 1 is administered in an amount that is about 200 mg. In some embodiments, compound 1 is administered in an amount that is about 400 mg. In some embodiments, compound 1 is administered in an amount that is about 800 mg. In some embodiments, compound 1 is administered in an amount that is about 1200 mg.

Administration

[00115] Administration of the compound, or a pharmaceutically acceptable salt thereof, described is at a dosage described herein or at other dose levels and compositions determined and contemplated by a medical practitioner. In certain embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, the compound, or a pharmaceutically acceptable salt thereof, is administered to a patient already suffering from a disease in an amount sufficient to cure the disease or at least partially arrest or ameliorate the symptoms. Amounts effective for this use depend on the age of the patient, severity of the disease, previous therapy, the patient's health status, weight, and response to the compositions, and the judgment of the treating physician. Therapeutically effective amounts are optionally determined by methods including, but not limited to, a dose escalation clinical trial.

[00116] In prophylactic applications, the compositions described herein are administered to a patient susceptible to or otherwise at risk of a particular disease, e.g., cancer. Such an amount is defined to be a “prophylactically effective amount or dose.” In this use, the precise amounts also depend on the patient's age, state of health, weight, and the like. When used in a patient, effective amounts for this use will depend on the risk or susceptibility of developing the particular disease, previous therapy, the patient's health status and response to the compositions, and the judgment of the treating physician.

[00117] In certain embodiments wherein the patient’s condition does not improve, upon the doctor’s discretion the administration of a composition described herein are administered chronically, that is, for an extended period of time, including throughout the duration of the patient’s life in order to ameliorate or otherwise control or limit the symptoms of the patient’ s disease. In other embodiments, administration of a composition continues until complete or partial response of a disease.

[00118] In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered once a day (QD). In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered twice a day (BID).

[00119] In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 5 mg QD. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 10 mg QD. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 15 mg QD. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 25 mg QD. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 50 mg QD. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 75 mg QD. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 100 mg QD.

[00120] In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 25 mg BID. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 50 mg BID. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 100 mg BID. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 200 mg BID. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 400 mg BID. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered in an amount that is about 600 mg BID.

[00121] The length of a treatment cycle depends on the treatment being given. In some embodiments, the length of a treatment cycle ranges from two to six weeks. In some embodiments, the length of a treatment cycle ranges from three to six weeks. In some embodiments, the length of a treatment cycle ranges from three to four weeks.

[00122] In some embodiments, the length of a treatment cycle is three weeks (or 21 days). In some embodiments, the length of a treatment cycle is four weeks (28 days).

[00123] In some embodiments, a treatment cycle lasts one, two, three, or four weeks. In some embodiments, a treatment cycle lasts three weeks. In some embodiments, a treatment cycle lasts four weeks. The number of treatment doses scheduled within each cycle also varies depending on the drugs being given.

[00124] In some embodiments of the methods, the compound, or a pharmaceutically acceptable salt thereof, is administered in 28-day cycles. In some embodiments of the methods, the compound, or a pharmaceutically acceptable salt thereof, is administered for multiple 28-day cycles. In some embodiments of the methods, the compound, or a pharmaceutically acceptable salt thereof, is administered for at least one 28-day cycle. In some embodiments of the methods, the compound, or a pharmaceutically acceptable salt thereof, is administered for at least two 28-day cycles. In some embodiments of the methods, the compound, or a pharmaceutically acceptable salt thereof, is administered for at least three 28-day cycles. In some embodiments of the methods, the compound, or a pharmaceutically acceptable salt thereof, is administered for at least four 28-day cycles. In some embodiments of the methods, the compound, or a pharmaceutically acceptable salt thereof, is administered for at least five 28-day cycles. In some embodiments of the methods, the compound, or a pharmaceutically acceptable salt thereof, is administered for at least six 28-day cycles. In some embodiments of the methods, the compound, or a pharmaceutically acceptable salt thereof, is administered for at least twelve 28-day cycles.

[00125] In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered every day of each 28-day cycle. [00126] In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered on an intermittent dosing schedule.

[00127] In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is administered on day 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 22, 23, 24, 25, and 26 of a 28-day cycle.

[00128] In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is not administered on days 6, 7, 13, 14, 20, 21, 27, and 28 of the 28-day cycle.

EXAMPLES

Example 1

[00129] FIG. 1A - FIG. ID, FIG. 8A - FIG. 8B, and FIG. 2A - FIG. 2D describe the immunological activity of compound 1 by in vitro stimulation of human monocytes and T cells with LPS (monocytes) or peptides (T Cells) after suppression with PGE2. Compound 1 is more effective than a single EP4, EP2, or Cox antagonists in mice. Compound 1 shows potency in overcoming PGE-2-mediated immune suppression in human enriched T cells, though not whole blood monocytes (FIG. 8A - FIG. 8B). These results attest to the general immune activity of compound 1.

FIG. 1A shows the TNFa ELISA results from a murine whole blood assay. Whole blood was treated with increasing amounts of compound 1 (dual EP2/4 inhibitor) followed by treatment with lOnM or 500nM PGE2 and 0.5pg/ml LPS (“whole blood assay”).

FIG. IB shows the TNFa ELISA results from a murine whole blood assay. Whole blood was treated with increasing amounts of E7046 (single EP4 inhibitor) followed by treatment with lOnM or 500nM PGE2 and 0.5pg/ml LPS (“whole blood assay”).

FIG. 1C shows the TNFa ELISA results from a murine whole blood assay. Whole blood was treated with increasing amounts of PF 04418948 (single EP2 inhibitor) followed by treatment with lOnM or 500nM PGE2 and 0.5pg/ml LPS (“whole blood assay”).

FIG. ID shows the IC50 results from whole blood assay described in FIG. 1A, IB, and 1C.

FIG. IE shows the percent recovery results from whole blood assay described in FIG. 1A, IB, and 1C. FIG. IF shows the TNFa ELISA results from human whole blood assays. Whole blood was treated with increasing amounts of compound 1 (dual EP2/4 inhibitor) followed by treatment with lOnM or 500nM PGE2 and 0.5pg/ml LPS (“whole blood assay”).

FIG. 1G shows the TNFa ELISA results from human whole blood assays. Whole blood was treated with increasing amounts E7046 (Single EP4 inhibitor) followed by treatment with lOnM or 500nM PGE2 and 0.5pg/ml LPS (“whole blood assay”).

FIG. 8A shows TNFa concentrations and quantification of percent recovery. Whole blood was treated with increasing amounts of compound 1 (dual EP2/4 inhibitor) followed by treatment with lOnM or 500nM PGE2 and 0.5pg/ml LPS (“whole blood assay”). Graphs show data that is representative of N=2 technical replicates from 1 experiment.

FIG. 8B shows TNFa concentrations and quantification of percent recovery at select concentrations. Whole blood was treated with increasing amounts of compound 1 (dual EP2/4 inhibitor) followed by treatment with lOnM or 500nM PGE2 and 0.5pg/ml LPS (“whole blood assay”). Graphs show data that is representative of N=2 technical replicates from 1 experiment.

FIG. 2A shows the IFNy ELISA from supernatants of CD8+ enriched PBMC stimulated with CEF peptides and compound 1.

FIG. 2B shows the IFNy ELISA from supernatants of CD8+ enriched PBMC stimulated with CEF peptides and PF04418948.

FIG. 2C shows the IFNy ELISA from supernatants of CD8+ enriched PBMC stimulated with CEF peptides and E7046.

FIG. 2D shows the IFNy Elisa from the same assay as in FIG. 2A, 2B, and 2C treated with increasing amounts of EPl and EP3 inhibition. Results are representative of multiple donors. Treatment of tumors with compound 1 leads to greater therapeutic effect than with single antagonists.

FIG. 3A shows Lewis Lung Carcinoma (LLC) Tumors treated with listed EP antagonists. Tumors were implanted in flanks of animals on day -11, then assorted into groups where tumor volumes averaged 200mm^A3 and treated with listed EP antagonists.

FIG. 3B shows the tumor count in small intestines of APCmin/+ mice treated for 3 weeks, starting at 13 weeks of age with orally administered compound 1 at lOOmpk BID, E7046 at 150mpk QD, PF04418948 at lOOmpk QD, or celecoxib at 60mpk QD. Treatment of APCmin/+mice with compound 1 results in significant reduction of tumor burden in the small intestines of these mice, compared to both vehicle and to other COX2 pathway blocking agents.

FIG. 3C shows the Survival of APCmin/+ animals treated beginning at 12 weeks of age with listed EP antagonists. All antagonists were administered PO for 6 weeks. Statistics were calculated by the Gehan- Breslow- Wilcoxon test. At this age, animals have established tumors and will begin to succumb to tumor burden around 19-20 weeks of age. Treatment with compound 1 administered orally BID or QD extends the life of mice by 7 weeks, while treatment with other COX2 pathway inhibitors does not significantly extend the life of these animals.

FIG. 3D shows the survival of APCmin/+ animals treated beginning at 6 weeks of age with orally administered QD regimens of compound 1 (lOOmpk, 50mpk, lOmpk). In this experiment, mice were treated daily until death or humane endpoints were reached, for the full length of the experiment. This experiment is still ongoing, but the regimen appears to be safe and effective after treatment up to 150 days.

FIG. 4A shows the quantification by a blinded pathologist of immunohistochemistry (IHC) of resected hyperplasias from small intestines of APCmin/+ mice treated for 3 weeks, starting at 13 weeks of age with orally administered compound 1 at lOOmpk BID, E7046 at 150mpk QD, PF04418948 at lOOmpk QD, or celecoxib at 60mpk QD (CD8a).

FIG. 4B shows the quantification by a blinded pathologist of immunohistochemistry (IHC) of resected hyperplasias from small intestines of APCmin/+ mice treated for 3 weeks, starting at 13 weeks of age with orally administered compound 1 at lOOmpk BID, E7046 at 150mpk QD, PF04418948 at lOOmpk QD, or celecoxib at 60mpk QD (CD4).

FIG. 4C shows the quantification by a blinded pathologist of immunohistochemistry (IHC) of resected hyperplasias from small intestines of APCmin/+ mice treated for 3 weeks, starting at 13 weeks of age with orally administered compound 1 at lOOmpk BID, E7046 at 150mpk QD, PF04418948 at lOOmpk QD, or celecoxib at 60mpk QD (FoxP3).

Example 2

[00130] Compound 1 increased the CD8+ and CD4+ lymphocytic infiltrate and not the FoxP3+ Treg infiltrate into these tumors, while other COX2 pathway inhibitors failed to meet all three of these criteria. FIG. 5A shows significant log2 fold changes in differentially expressed genes from RNA sequencing of tumors resected from APCmin/+ mice in FIG. 3B.

FIG. 5B shows significant log2 fold changes in differentially expressed genes from RNA sequencing of tumors resected from APCmin/+ mice in FIG. 3B.

FIG. 5C shows significant log2 fold changes in differentially expressed genes from RNA sequencing of tumors resected from APCmin/+ mice in FIG. 3B.

FIG. 5D shows a volcano plot of selected genes from samples in (FIG. 5A-5C) for compound 1. Blue coloration represents significantly (Padj <0.05) downregulated genes while red correlation represents significantly upregulated genes. Genes in grey did not meet the significance cutoff.

FIG. 5E shows a volcano plot of selected genes from samples in (FIG. 5A-5C) for E7046. Blue coloration represents significantly (Padj <0.05) downregulated genes while red correlation represents significantly upregulated genes. Genes in grey did not meet the significance cutoff.

FIG. 5F shows a volcano plot of selected genes from samples in (FIG. 5A-5C) for PF 04418948. Blue coloration represents significantly (Padj <0.05) downregulated genes while red correlation represents significantly upregulated genes. Genes in grey did not meet the significance cutoff.

FIG. 5G shows a volcano plot of selected genes from samples in (FIG. 5A-5C) for Celecoxib. Blue coloration represents significantly (Padj <0.05) downregulated genes while red correlation represents significantly upregulated genes. Genes in grey did not meet the significance cutoff.

Example 3

[00131] FIG. 6A - FIG. 6D and Table A describe the exquisite selectivity of compound 1 for EP2 and EP4, demonstrating compound 1 as a dual antagonist of human EP2 and EP4.

FIG. 6A shows Compound 1 EP receptor PGE2 antagonist values in CA++ flux assays. Serial elevenpoint half-log dose response curves shown in 293HEK cells engineered to express a single designated EP receptor linked genetically to a promiscuous G alpha coupled to calcium flux (Eurofins Scientific). Prostaglandin PGE2 was used at a final concentration of 10 nM. Error bars represent standard deviation. FIG. 6B shows Compound 1 EP2 receptor PGE2 antagonist values in CA++ flux assays. Serial elevenpoint half-log dose response curves shown in 293HEK cells engineered to express a single designated EP receptor linked genetically to a promiscuous G alpha coupled to calcium flux (Eurofins Scientific). Prostaglandin PGE2 was used at a final concentration of 10 nM. Error bars represent standard deviation. FIG. 6C shows Compound 1 EP3 receptor PGE2 antagonist values in CA++ flux assays. Serial elevenpoint half-log dose response curves shown in 293HEK cells engineered to express a single designated EP receptor linked genetically to a promiscuous G alpha coupled to calcium flux (Eurofins Scientific). Prostaglandin PGE2 was used at a final concentration of 200 nM. Error bars represent standard deviation. FIG. 6D shows Compound 1 EP4 receptor PGE2 antagonist values in CA++ flux assays. Serial elevenpoint half-log dose response curves shown in 293HEK cells engineered to express a single designated EP receptor linked genetically to a promiscuous G alpha coupled to calcium flux (Eurofins Scientific). Prostaglandin PGE2 was used at a final concentration of 10 nM. Error bars represent standard deviation. [00132] Compound 1 EP receptor IC50 antagonist values (Table A). Percent activity of Compound 1 was determined against the average of the positive control (100%) and negative control (0%). The IC50 of Compound 1 for EP receptors was determined as log inhibitor against normalized response (variable slope) curve fitting. The number of experiments performed to determine the antagonist activity against each EP receptor is shown (as n = x) in the Table. IC50 was determined for each individual experiment. Composite IC50 was determined using the normalized percent activity per experiment results.

Table A

Example 4

[00133] FIG. 7 shows compound 1 significantly enhanced PD-1 therapeutic efficacy in mice bearing CT26 flank tumors, suggesting that primed T cell responses were potentiated by the combination of PD-1 and PGE2 blockade, demonstrating that treatment of mice bearing high PGE2-producing CT26 tumors with compound 1 remodeled the TME as shown by a significant increase of innate and adaptive effector immune cell populations, combined with the reduction of immune suppressive innate and adaptive immune cell populations and significant therapeutic benefit when combined with immune checkpoint inhibitors.

FIG. 7 shows increased efficacy with Compound 1 and a-PD-1 combination therapy in CT26 model. Tumor outgrowth in BALB/c mice implanted with CT26 cells and treated with lOOmg/kg BID PO Compound 1 +/- 200mg Q3D IP ant-PD-1 antibody. Results are representative of 2 experiments with at least 5 mice.

Example 5

[00134] FIG. 9 and Table B show that after 14 days of Compound 1 treatment therapy, tumor outgrowth was inhibited by 33% inhibition when T cells were depleted and 52% without T-cell depletion, suggesting that the observed anti-CT26 tumor response resulted from both T cell-dependent and T cell-independent mechanisms.

FIG. 9 shows Compound 1 displays T cell-independent anti-tumor efficacy in CT26. Tumor outgrowth of CT26 tumors in mice treated for 14 days with lOOmg/kg Compound 1 and/or anti-CD8a depleting antibody.

[00135] Table B below depicts tumor averages and calculated TGI where TGI=100*(Control Volume- Treatment Volume)/Control Volume.

Table B

Example 6

[00136] To determine the differing efficacy in APCmin/+ mice among the prostaglandin pathway inhibitors tested aligned with differences in TME immune infiltrates, the lymphocyte populations in APCmin/+ mice were measured by performing IHC on resected small intestinal areas with tumors or hypertrophy after 3 weeks of prostaglandin pathway inhibitor treatment (FIG. 10A - FIG. IOC). At this timepoint, no adenomatous or carcinomatous tissue could be detected in Compound 1-treated animals by blinded independent pathology assessment. In contrast, either no or a minimal decrease in the number of adenomas and carcinomas was observed in the small intestine of APCmin/+ mice treated with COX-2 or single EP2 or EP4 PGE2 receptor inhibitors. Following the 3-week course of therapy, IHC staining revealed a significant increase in tissue-infiltrating CD8+ T cells in hypertrophic areas of the small intestine in mice treated with Compound 1, E7046, and celecoxib, though not PF04418948. While the level of CD8+ T-cell infiltration in Compound 1-treated mice was greater than with other agents, these differences were nonsignificant. However, CD4+ T-cell infiltration was significantly higher in Compound 1-treated mice. FoxP3 staining of CD4+ T cells increased uniquely in celecoxib-treated mice, suggesting that the increase of CD4+ T cells in Compound 1-treated animals did not include an abundance of regulatory T cells. FIG. 10A shows the lesion area percentage in histopathological analysis of resected APCmin/+ colon hyperplasia. Mean and standard deviation are depicted and the ordinary one-way ANOVA with Tukey's multiple comparisons test was used to assess significance between individual groups. *, p<0.05.

FIG. 10B shows the number of adenoma lesions in histopathological analysis of resected APCmin/+ colon hyperplasia. Mean and standard deviation are depicted and the ordinary one-way ANOVA with Tukey's multiple comparisons test was used to assess significance between individual groups. *, p<0.05. FIG. 10C shows the number of carcinoma lesions is histopathological analysis of resected APCmin/+ colon hyperplasia. Mean and standard deviation are depicted and the ordinary one-way ANOVA with Tukey's multiple comparisons test was used to assess significance between individual groups. *, p<0.05.

[00137] Transcriptional analysis demonstrates that treatment Compound 1 leads to similar but stronger effects than EP4 antagonism in the TME. Only Compound 1 significantly upregulates the IFNy gene signature as found by GSEA, and Compound 1 most strongly upregulates the lymphocyte chemoattractants CXCL9 and CXCL10.

[00138] In mouse and human whole blood assays, dual blockade of EP2 and EP4 receptors with Compound 1 reversed PGE2-mediated suppression of LPS induced TNF-a, while single EP4 receptor antagonists were unable to block suppression at higher PGE2 concentrations. Similarly, in murine and human T cells in vitro, Compound 1 inhibited PGE2-mediated suppression, resulting in a significant increase of IFNy production in response to stimulation with cognate peptide antigen. Compound 1 monotherapy demonstrated a decrease of both the intestinal tumor size and number in Adenomatous Polyposis (APCmin/+) mice, as compared to EP2, EP4 and COX2 antagonism. Immunohistochemistry analysis of resected areas of hyperplasia from the small intestines of treated APCmin/+ revealed increased infiltration of adaptive immune cells after treatment with Compound 1. Additionally, transcriptional analysis of these samples demonstrated that Compound 1 led to a similar transcriptional signature but greater effect on key genes downstream of prostaglandin E2 signaling as compared to single EP2 or EP4 antagonism, reinforcing that redundancy of these two receptors necessitates dual blockade.

Example 7- Multicenter, Open-Label Phase la/lb, Dose-Escalation and Dose Expansion Study of Compound 1 as Single Agent in Subjects with Advanced Solid Tumors

Study Design

[00139] This is a first in human Phase la/lb, multicenter, open-label, dose-escalation and expansion study of Compound 1 administered as a single agent to determine its Maximum Tolerated Dose (MTD) and/or recommended Phase 2 dose (RP2D), safety, tolerability, pharmacokinetics (PK), pharmacodynamics and preliminary anti-tumor activity in subjects with advanced solid tumors. Subjects with all histologic types of solid tumors are eligible for the Dose-Escalation and Schedule and Dose Optimization stages, however, the preferred tumor types for enrollment are colorectal cancer (CRC), non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), urothelial cancer, endometrial cancer, and gastroesophageal junction (GEJ) or gastric adenocarcinoma. Dose expansion cohorts are disease specific indications, as described. To be study-eligible, subjects must have no remaining standard therapy known to confer clinical benefit. Prior to treatment initiation, subjects will undergo screening to determine study eligibility.

Study Objectives

[00140] The primary objective of this study is to determine the MTD and/or RP2D, and the optimum schedule of Compound 1 as a single-agent therapy

[00141] The secondary objectives of this study are to evaluate safety and tolerability profile of Compound 1 as a single-agent therapy, to undertake a preliminary evaluation of the activity in subjects with solid tumors, and to assess the PK of Compound 1.

[00142] The exploratory objectives of this study are to evaluate pharmacodynamics of Compound 1 as single-agent therapy and to investigate immunomodulatory effects in blood and in tumors.

Dose-Escalation Stage

[00143] In this study, dose escalation will be conducted to determine the single agent maximum tolerated dose (MTD) of Compound 1. Dose levels to be potentially evaluated are 25 mg BID, 50 mg BID, 100 mg BID, 200 mg BID, 400 mg BID and 600 mg BID. The starting dose will be 100 mg orally BID. Subjects will be assessed for DLTs from the first dose through Day 22. Dose escalation, if appropriate, will occur after all subjects in a cohort have completed their 21 -day DLT assessment.

[00144] Three (3) subjects will be initially treated at each dose level. If none of the 3 subjects experience a dose-limiting toxicity (DLT) during the DLT evaluation period, the dose will be escalated to the next dose level. If a DLT is observed in a dose level, an additional 3 subjects will be enrolled. If >2 DLT are observed in 3 to 6 subjects, no further subjects will be dosed at that level and the next lower dose level will be opened or reopened for evaluation, with the enrollment of 3 additional subjects, unless 6 subjects have already been dosed at that cohort. Intermediate dosing cohorts may be added upon agreement with the Safety Monitoring Committee (SMC) and Study Sponsor. The MTD will be the highest dose level at which 0 to 1 of 6 subjects experience a DLT. Table 1 below outlines the dose escalation rules.

Dose Optimization Stage

[00145] The Schedule and Dose Optimization Stage for single-agent Compound 1 will evaluate alternate Compound 1 administration schedules that are less dose intensive than BID administration and alternate doses up to 50 mg, as shown in Table 2 below.

* If the 25 mg dose is tolerated, the dose may be escalated to 50 mg

[00146] Schedule A will comprise once daily continuous dosing and Schedule B once daily intermittent dosing, Days 1 to 5 every 7 days. Additional schedules and intermediate dose levels may be investigated based upon emerging clinical data. The Schedule and Dose Optimization stage for single agent Compound 1 will serve to select a dose and administration schedule for further evaluation in expansion cohorts.

[00147] Schedule A will be opened first, evaluating 5, 15, 25, and 50mg administered once daily on a continuous schedule. Then Schedule B will be opened, evaluating the same dose administered Days 1 to 5 every 7 days.

[00148] Subjects will be enrolled into a given cohort in blocks of 3 subjects and the frequency of DLT will be evaluated; subjects who are not DLT-evaluable will be replaced. Further enrollment at that dose level will be paused until 3 subjects have completed the DLT evaluation period (21 days) or experienced a DLT. Due to the decreasing dose intensity from Schedule A to B and then to sequentially lower doses, once 3 subjects have been enrolled at given schedule and dose, the Sponsor may choose to evaluate any of the lower dose levels and less dose-intensive schedules based upon emerging data. The next de-escalated cohort may open immediately and enroll 3 subjects. Thus, multiple cohorts may be evaluated in parallel with enrollment to a given dose level allowed while subjects in a higher dose level or a more intensive (i.e., continuous QD dosing) schedule are being followed during the DLT evaluation period.

[00149] If <1 DLT is observed in the initial 3 subjects in a given cohort a further 3 subjects may be enrolled to that dose level, and enrollment to that dose level again paused during the DLT evaluation period. If >2 DLT are observed in the 3 to 6 subjects in a dose level, it will be considered to exceed the MTD for that schedule. If <1 DLT is observed in 6 subjects evaluated in the 25 mg dose level of Schedules A and/or B, an intermediate dose > 25 mg and up to the 50 mg dose level, may be opened for evaluation, based upon the tolerability of the 25 mg dose. In the event of dose escalation to a 50 mg dose level, subjects will first be evaluated in Schedule B, followed by Schedule A. The rules to be used in the Schedule and Dose Optimization stage are summarized in Table 3 below. Based on safety and PK data, and following consultation with the SMC, the Sponsor may choose to evaluate higher dose levels than 50 mg

[00150] During the Compound 1 Schedule and Dose Optimization stage up to 6 additional subjects (i.e., “backfill” subjects) may be enrolled into any cohort that has been deemed tolerable (i.e., <1 DLT in up to 6 subject) and with approval of the Medical Monitor, if they have biopsy-accessible tumors and consent to undergo paired pre-treatment and on-treatment biopsies.

Example 8: Phase 2 Study to Evaluate the Safety and Efficacy of Compound 1 in Patients with Familial Adenomatous Polyposis (FAP)

Study Design

[00151] This is a Phase 2 trial evaluating the efficacy and safety of compound 1 in patients with genetic or clinical FAP who have undergone prior colectomy with IRA. The co-primary endpoints are mean percentage change from baseline in rectal polyp burden at 6 months, and safety characterization. The study population will be patients with genetically or clinically confirmed FAP or attenuated FAP previously treated with colectomy and ileorectal anastomosis (IRA).

Study Objectives

Primary Objectives:

[00152] The primary objective is to assess the activity of Compound 1 in reducing duodenal polyp burden in patients with FAP. This will be done by comparing a baseline upper GI endoscopy to the 6- month upper GI endoscopy, respectively.

Secondary Objectives:

[00153] a) The activity of Compound 1 in reducing rectum/IPAA polyp burden in patients with FAP. This will be done by comparing the lower GI endoscopy at baseline and 6 months.

[00154] b) The safety of Compound 1 in participants with FAP. This will be completed by a review of the adverse events throughout the study. [00155] c) Reduction in intestinal polyp burden as a function of immunohistochemical staining at baseline and endpoint of rectal and duodenal tissue samples for COX-2 expression level, beta-catenin, and Ki-67.

Exploratory Objectives:

[00156] a) Changes produced by Compound 1 in tumor associated immunity and microbiome. Samples collected will include peripheral blood, tissue, duodenal aspirate at baseline and at 6 months post treatment. The immune analysis will be done by measuring Treg/TH17 balance in PBMC and in polyps using Flow Cytometry, immunohistology, RNAseq, and cell based functional assays. The microbiome analysis will be done by sequencing.

[00157] b) Assessing potential markers of Compound 1 pharmacodynamic and antiproliferative activity which could include PGE2, PGD2, PGF2a, thromboxane, infiltrating immune cell phenotypes, EPl, EP2, EP3, EP4, and urine PGEM.

Eligibility Criteria

Pre-Registration Inclusion Criteria:

[00158] Diagnosis of familial adenomatous polyposis (FAP), Age > 18, Previously underwent prophylactic colectomy with IRA or IPAA at least 3 months before pre-registration evaluation and without ongoing surgical complication, and willing to discontinue taking NSAIDs for 30 days prior to initiation of and during intervention (Except ASA < 81 mg daily is allowed).

Pre-Registration Exclusion Criteria:

[00159] Use of any other investigational agents < 12 weeks prior to pre-registration, history of allergy to or intolerance of NSAIDs (COX-2 inhibitors and non-selective COX-1 and -2 inhibitors including aspirin), known helicobacter pylori infection unless eradicated with full course of approved therapy, Uncontrolled intercurrent illness or recent surgical procedure that in the opinion of the investigative team would limit compliance with study requirements, history of invasive malignancy < 3 years prior to preregistration (exception: adequately treated carcinoma of the cervix, carcinoma in situ, or basal or squamous cell carcinomas of the skin), and history of any upper GI surgery that does not permit access to or evaluation of a 10 cm segment of the duodenum that includes the duodenal bulb.

Registration Inclusion Criteria:

[00160] ECOG performance status < 1, not pregnant or breast-feeding, willing to use adequate contraception to avoid pregnancy or impregnation until 2 weeks after discontinuing study agent, presence of Spiegelman 2 or 3 duodenal polyposis stage that can be assessed endoscopically, presence of 5 or more rectal and/or pouch polyps 2 mm or more in diameter that can be assessed and adequate bone marrow and organ function:

• Leukocytes (WBC) > 3.000/pL (> 2.500/pL for African American participants)

• Platelet count > 100 x 109/L

• Hemoglobin > 11.5 g/dL

• Total bilirubin < 1.5 x institutional ULN (unless patient has Gilbert’s) • Alkaline phosphatase < 1.5 x institutional ULN

• AST/SGOT < 2 x institutional ULN

• SLT/SGPT < 2 x institutional ULN

• Creatinine < institutional ULN

• Urinary testing results within institutional limits of normal or deemed clinically insignificant.

Registration Exclusion Criteria:

[00161] Any histologically confirmed high grade dysplasia (HGD) or cancer, gastrointestinal bleeding, and requirement for anticoagulation therapy after study start except for low dose aspirin.

Claims

CLAIMS What is claimed is:

1. A method for treating familial adenomatous polyposis (FAP) in a subject in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

2. A method for preventing precancerous polyps in a subj ect in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

3. A method for maintaining a low polyp burden in a subj ect in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

4. The method of claim 2 or 3, wherein the polyps are located in the colon, the stomach, the duodenum, or the small intestine.

5. The method of claim 2 or 3, wherein the polyps are rectal polyps.

6. The method of claim 2 or 3, wherein the polyps are located in the colon.

7. A method of treating or preventing a pre- malignant condition in a subject in need thereof, the method comprising administering to the subject an EP2/EP4 dual antagonist.

8. The method of claim 7, wherein the pre-malignant condition is present in any one of a plurality of regions of the gastrointestinal tract, and in particular of the colon and/or rectum.

9. The method of claim 8, wherein the pre-malignant condition is present in the colon and/or the rectum.

10. The method of claim 7, wherein the pre-malignant condition is manifested in the form of polyps.

11. The method of claim 10, wherein the polyps are located in the colon, the stomach, the duodenum, or the small intestine.

12. The method of claim 10, wherein the polyps are rectal polyps.

13. The method of claim 10, wherein the polyps are located in the colon.

14. A method for preventing colorectal cancer in a subject in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

15. A method for minimizing the risk of developing colorectal cancer in a subj ect in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

16. A method for preventing colostomy in a subject in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

17. A method for delaying the need for colostomy in a subject in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

18. A method for delaying the need for polypectomy in a subject in need thereof, the method comprising administering an EP2/EP4 dual antagonist.

19. The method of any one of claims 1-18, wherein the subject in need thereof has been diagnosed with familial adenomatous polyposis (FAP).

20. The method of any one of claim 1-19, wherein the EP2/EP4 dual antagonist is

pharmaceutically acceptable salt thereof. The method of any one of the preceding claims, wherein the method further comprises administering to the subject in need thereof an additional therapy.