Cysteamides, therapeutic compositions thereof, and related methods

Abstract

The invention relates to bile acid cysteamides, acting as FXR receptor modulators, therapeutic compositions containing such cysteamides and methods of using the same.

Description

[0001] CYSTEAMIDES, THERAPEUTIC COMPOSITIONS THEREOF, AND RELATED METHODS

[0002] Frank Schroeder

[0003] David Artis

[0004] Tae Hyung Won

[0005] Marissa Anna Fontaine

[0006] Chun- Jun Guo

[0007] Mohammad Arifuzzaman

[0008] Christopher Parkhurst

[0009] This application claims priority under 35 U. S. C. §119(e) to U. S. Provisional Patent Application No. 63 / 741,653, filed January 3, 2025. The foregoing application is incorporated by reference herein.

[0010] GOVERNMENT SUPPORT

[0011] This invention was made with Government support under Grant Nos. R35GM131877 DK126871, AI151599, AI095466, AI095608, AI142213, AR070116, AI172027, DK132244 and U2CES030167 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.

[0012] FIELD OF THE INVENTION

[0013] This invention pertains to the field of small molecule therapeutics and provides therapeutic compositions and pharmacologically active analogs of compounds as well as methods of using the same therapeutically.

[0014] BACKGROUND OF THE INVENTION

[0015] Secondary metabolites derived from plants, fungi and microbes are among the richest sources of therapeutically useful chemical compounds. For example, in the decade between 2000 and 2010, approximately 50% of all NCEs (new chemical entities) approved by the US FDA for use as human drugs were natural products or derivatives of natural products (J Nat Prod. 2012 Mar 23; 75(3): 311-335).

[0016] Vertebrates harbor diverse communities of bacteria, protozoa, fungi and viruses, collectively referred to as the microbiota. Co-evolution with these microbes has resulted in theestablishment of host-microbiota cross-talk via complex metabolic networks. One such example is the metabolism of bile acids, which play a central role in vertebrate physiology as ligands of the farnesoid X receptor (FXR), a conserved nuclear hormone receptor that controls cholesterol and BA biosynthesis as well as fat metabolism and glucose homeostasis. BAs are produced in the liver where they are conjugated to taurine and to amino acids by BA-CoA: amino acid N-acyltransferase (BAAT). Conjugated BAs are then secreted via the biliary system into the intestine where they undergo extensive modification by microbial metabolism, including diverse dihydroxylation and oxidation reactions as well as deconjugation, which recovers free, unconjugated BAs that are efficiently reabsorbed and transported back to the liver. While BA-taurine conjugates are inactive or have weak agonistic or antagonistic activities free BAs act as potent FXR agonists that negatively regulate BA production. BA-taurine conjugation by the host and microbial deconjugation thus form a negative feedback loop that tightly regulates BA levels and BA-dependent physiology. However, the requirement for adaptation to changing metabolic demands and feeding states and the ubiquity of feed-forward signaling in biological systems raises the question whether there exist FXR antagonists that might act as positive regulators of BA production.

[0017] Recent investigations by have demonstrated that mammals are an unexpected and rich source of small molecules with diverse biological activities. Meanwhile, as the underlying mechanisms of aging, and a wide range of human health disorders becomes better understood, the need for more selective and efficacious therapeutic and pharmaceutical treatments has never been greater,

[0018] The present invention addresses these and other related needs.

[0019] SUMMARY OF THE INVENTION

[0020] Among other things, the present invention encompasses the discovery of a family of novel small molecule metabolites produced in mice. Important additional discoveries have been made regarding the production and function of these metabolites including: that specific microbiota in the mouse gut that are associated with production of the novel metabolites (and the relevance of gut microbiota to human health and their connection to certain health conditions and diseases); the distribution of the new metabolites within the producing organisms’ bodies; and the changing levels of absolute and / or relative production, accumulation or consumption of thesemetabolites in response to diverse metabolic, dietary and / or environmental stimuli. Based on these discoveries, it has been recognized that administering compositions containing the identified metabolites (or analogs thereof) provides a useful strategy to improve the health of animals including humans and / or to treat certain diseases and disorders.

[0021] Untargeted metabolomics was utilized to compare germ-free (GF) and microbiota-replete specific pathogen-free (SPF) mice to uncover a host-mediated BA modification that generates molecules that are potent FXR antagonists which act as intestinal regulators of BA metabolism. In addition to realizing therapeutic applications of these newly discovered endogenous molecules, it has been recognized that the methylcysteamides of a broader class of synthetic bile acid congeners and other molecules resembling them also have therapeutic applications.

[0022] The emerging field of metabolomics is providing profound insights into the structures and functions of the small molecule metabolites that regulate life processes in all creatures including humans. A comparative metabolomic study in mice (Mus musculis) has been undertaken to evaluate the influence of their diet on the small molecule metabolites produced by their gut microbiome. These efforts have led to the identification of a family of previously undescribed molecules comprising amides of cysteamine.

[0023] A particular class of these molecules are bile acid methyl cysteamide conjugates (BA-MCYs). The biochemical mechanisms by which mammals may regulate BA activities are of significant interest in the context of human health and disease. In the case of BAs, their taurine conjugation by the host and subsequent deconjugation by intestinal microbiota provide a classical example for the regulation of metabolite abundance via opposing host- and microbiotadependent pathways.

[0024] Our unexpected identification of BA-MCY conjugates as FXR antagonists reveals a previously unrecognized host-dependent layer of BA metabolism that counteracts the physiological functions of free BAs. Whereas free BAs generally act as FXR agonists, it has been discovered that administration of CDCA-MCY to mammals increased total BA production and expression of the enzymes that catalyze the rate limiting steps in BA biosynthesis. This is in surprising contrast to its parent compound CDCA, which acts as an FXR agonist and reduces overall BA levels. Importantly, CDCA-MCY administration alleviates hepatic lipid accumulation in mice fed a high cholesterol diet (Fig. 5 J, 5K). Thus BA-MCYs represent an important and previously unrecognized component of the feedback mechanisms regulating BAbiosynthesis and other FXR-dependent phenotypes, including diverse aspects of fatty acid metabolism. Given the dysregulation of BA levels in type II diabetes, metabolic syndrome, and the cholestatic diseases, the BA-MCYs have significant therapeutic potential.

[0025] It has also demonstrated that dietary fiber can upregulate production of BA-MCY conjugates in mammals, indicating that therapeutic applications of BA-MCYs may also be enhanced by diet and that bile acid cysteamides may have utility as prebiotic or probiotic supplements. Such combinations of BA-MCYs with a fiber rich diet have translational potential in conditions of dysregulated immune or metabolic homeostasis.

[0026] Additional therapeutic applications of the provided compounds and formulations include modulation of other BA receptors such as TGR5 and LXR and treatment of diseases or disorders dependent on or responsive to activators and / or inhibitors of these receptors.

[0027] Therefore, in one aspect, the present invention encompasses therapeutic compositions comprising a therapeutically effective amount of one or more bile acid cysteamides or derivatives or analogs of such cysteamides. In certain embodiments, provided compositions are characterized in that the sulfur atom of the cysteamide is substituted with an alkyl group. In certain embodiments, such compositions are characterized in that the sulfur atom of the cysteamide is substituted with a C1.40 alkyl group (referred to herein as S-alkyl cysteamides, or SACAs). In certain embodiments, provided compositions comprise bile acid S-methylcysteamides (BA-MCYs).

[0028] In certain embodiment, provided compositions comprise a sulfoxide derivative of a bile acid cysteamide (hereafter referred to as “S-oxide cysteamides” or SOCAs). In certain embodiment, provided compositions comprise a sulfoxide derivative of a S-alkyl bile acid cysteamide (hereafter referred to as “as S-oxide alkyl cysteamides, or SOACAs). In certain embodiments, provided compositions comprise bile acid SOACAs that are enantioenriched at the chiral sulfoxide sulfur atom. In certain embodiments, provided therapeutic compositions comprise sulfoxides of S-methylcysteamides (BA-MCYOs)

[0029] In certain embodiments, provided compositions comprise a sulfone derivative of a bile acid cysteamide (hereafter referred to as cysteamide sulfones or CASOs). In certain embodiments, provided therapeutic compositions comprise 5-alkyl cysteamide sulfones (SACASOs). In certain embodiments, provided therapeutic compositions comprise of bile acid S -methyl cysteamide sulfones (BA-MCY02s).In another aspect, the present invention encompasses methods of improving the health of an animal or of treating or ameliorating a health disorder in an animal by administering to the animal an effective amount of any one or more of the compounds or therapeutic or compositions described herein. In certain embodiments, the methods comprise administering such a composition to a mammal. In certain embodiments, the methods comprise administering such a composition to a human.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Fig. 1 A. Shows the roles of gut microbiota in bile acids (BAs) metabolism and a schematic overview of the analytical strategy for comparison of GF and SPF mice.

[0032] Fig. 1B. Shows Volcano plots of differential metabolites detected in serum of GF (n = 12) and SPF (n = 9) mice. Dots represent metabolites five- or more-fold downregulated or upregulated in GF relative to SPF mice at P < 0.05, as calculated by unpaired two-sided t- test.

[0033] Fig. 1C. Shows a partial representation of the MS2 network (cosine>0.7) for mouse serum in ESI+ and ESI- showing clusters representing free BAs, BA-taurine conjugates, and previously unannotated BA-MCY conjugates. Nodes are downregulated and upregulated in serum of GF compared to SPF mice.

[0034] Fig. 1D. Shows the MS2 spectrum of CA-MCY. MS2 fragments represent the MCY group; some MS2 fragments are derived from water loss.

[0035] Fig. 1E. Shows the structures of BAMCY conjugates identified in mouse serum.

[0036] Fig. 1F. Shows the extracted ion chromatograms (EICs) of BA-MCY conjugates in serum of GF and SPF mice and comparison with synthetic standards analyzed in ESI+.

[0037] Fig. 1G. Shows the scheme for the synthesis of CA-MCY conjugates from CA.

[0038] Fig. 2A. Shows the relative abundances of CA-MCY conjugates as well as corresponding free CA in serum of SPF mice (n = 11), GF mice (n =12), and mice that received FMT (n = 10, the three donors are represented by triangles, circles, and crosses, n = 3-4 for each donor). Data are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction.

[0039] Fig. 2B. Shows the relative abundances of βMCA-MCY conjugates as well as corresponding free βMCA-MCY in serum of SPF mice (n = 11), GF mice (n =12), and mice that receivedFMT (n = 10, the three donors are represented by triangles, circles, and crosses, n = 3-4 for each donor). Data are mean ± s.e.m. P values were calculated by unpaired two-sided t- test with Welch’s correction.

[0040] Fig. 2C. Shows the relative abundances of CA-MCY conjugates as well as corresponding free CA in serum of mice fed control (n = 8) or inulin fiber diet (n = 7). Data are mean ± s.e.m. P values were calculated by unpaired two sided t-test with Welch’s correction, ****p < 0.0001.

[0041] Fig. 2D. Shows the relative abundances of 0MCA-MCY conjugates as well as corresponding free PMCA-MCY in serum of mice fed control (n = 8) or inulin fiber diet (n = 7). Data are mean ± s.e.m. P values were calculated by unpaired two sided t-test with Welch’s correction, ****p < 0.0001.

[0042] Fig. 2E. Shows the relationship between abundances of free BAs and BA-MCY conjugates in liver of SPF mice (n = 11), SPF control mice for the inulin fiber diet study (n = 4), and inulin fiber diet fed SPF mice (n = 5).

[0043] Fig. 2F. Shows the relative abundance of CA-MCY conjugates or CDCA-MCY conjugates in human serum (n = 19).

[0044] Fig. 3A. Shows ratio of total BA-taurine or BA-MCY conjugates to corresponding free BAs in feces of SPF (n = 14) and GF (n = 16) mice. Data are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction, ****p < 0.0001.

[0045] Fig. 3B. Shows total amounts of free BAs and BA-MCY conjugates in feces of GF (n =16) mice.

[0046] Data are mean ± s.e.m. P values were calculated by paired two-sided t-test.

[0047] Fig. 3C. Shows HRMS analysis of feces of mice fed CDCA-d5-MCY revealed deconjugation of supplemented CDCA-d5-MCY, represented by peaks in the CDCA mass spectrum highlighted in red. Endogenously produced CDCA can be distinguished, highlighted in green. CA remained unlabeled.

[0048] Fig. 3D. Shows deconjugation of supplemented CDCA-d5-MCY in feces of SPF (n = 8), GF (n = 3), and ABX (n =15) mice. Data are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction.

[0049] Fig. 3E. Shows deconjugation of MCY or taurine conjugates of BA in feces of GF monocolonized with WT (n = 3) or BSH-deleted B. ovatus (n =3) (WT Bo or Absh Bo,respectively). Data are mean ± s.e.m. P values were calculated by unpaired two-sided t- test with Welch’s correction, ****p < 0.0001.

[0050] Fig. 3F. Shows roles of host and microbiota in the biosynthesis and metabolism of BA taurine and MCY conjugates.

[0051] Figs. 4A-4D. Show in vitro data demonstrating that BA-MCY conjugates are FXR antagonists.

[0052] Compounds were tested against a cell-based protein-protein interaction assays in both agonist and antagonist modes. CDCA-MCY (Fig. 4A), CA-MCY (Fig. 4B), and 0MCA- MCY (Fig. 4C) showed strong FXR antagonistic effects to GW4604-mediated activation of FXR, whereas CA-MCYO (Fig. 4D) showed no FXR antagonistic effects. None of the BA-MCY conjugates showed FXR agonistic effects in the assay. Assays were performed in duplicate for each concentration.

[0053] Fig. 5A. Shows a schematic overview of BA biosynthesis, highlighting the separate CA and CDCA pathways. Cyp8bl, sterol 12a-hydroxylase, and Cyp7al, cholesterol 7a- hydroxylase, are under the control of hepatic FXR and intestinal FXR-FGF15 pathways, which is regulated by levels of free BAs. Compounds highlighted in blue and green belong to the CA and CDCA pathways, respectively.

[0054] Fig. 5B. Shows gene expression ratio of Shp, Cyp8bl and Cyp7al to Hprtl reference control in liver of mice administered CDCA-MCY (n = 4) or control (com oil, n = 3). Data are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction. Fig. 5C. Shows FGF15 levels in serum of mice administered CDCA-d5-MCY or control (n = 12). Data are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction, ****p < 0.0001.

[0055] Fig. 5D. Shows gene expression ratio of Shp to Hprtl reference control in small intestine of mice administered CDCA-d5-MCY (n = 7) or control (n = 7). Data are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction.

[0056] Fig. 5E. Shows gene expression ratio of Slcl0a2 to Hprtl reference control in small intestine of mice administered CDCA-d5-MCY (n = 3) or control (n = 3). Data are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction.

[0057] Figs. 5F and 5G. Show abundances of endogenously produced BAs are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction.Fig. 5H. Shows total endogenously produced BAs in feces of ABX mice administered CDCA- d5-MCY. Shown are total amounts of BAs (n = 13 for control and n = 14 for CDCA-MCY fed mice). Data are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction.

[0058] Fig. 51. Shows abundances of endogenously produced BAs in feces of WT or Nrlh4’ ' mice administered CDCA-d5-MCY daily for 14 days. Shown are total amounts of BAs (n = 8 for control and n = 8 for CDCA-MCY fed mice). Data are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction.

[0059] Figs. 5 J. Shows representative photomicrographs of oil red O staining of liver sections of mice treated with the indicated conditions. Mice were fed control (n = 4 for vehicle and n = 4 for CDCA-MCY) or high cholesterol diet (n = 4 for vehicle and n = 4 for CDCA-MCY). CDCA-MCY was delivered by oral gavage at a rate of 50 mg / kg body weight per day for two weeks. Scale bars, 50 pm.

[0060] Fig. 5K. Shows the average measured Oil red O area. Data are mean ± s.e.m. P values were calculated by one-way ANOVA with Tukey’s correction.

[0061] Fig. 5L. Shows a proposed rheostat model for FXR-dependent regulation of BA metabolism by BA-MCYs. BA-taurine conjugates are secreted into the intestine and deconjugated by microbial BSH into free BAs that function as FXR agonists and decrease BA production. Fig. 6. Shows MS2 networks of microbiota-dependent differential features in mouse serum in ESI-. Nodes represent downregulated and upregulated features for GF (n = 9) relative to SPF (n = 11) mice in ESI-. Subnetworks for differential metabolites were annotated corresponding to their molecular families.

[0062] Figs. 7A-7E: Show MS2 spectra of MCY conjugates of BAs. MS2 spectra of CA-MCY (Fig.

[0063] 7A), CDCA-MCYO (Fig. 7B), CA-MCYO (Fig. 7C), 7KDCA-MCY0 (Fig. 7D), and CA- MCY02 (Fig. 7E) are provided. The listed BA-MCY conjugates in each panel produced MS2 spectra very similar to the shown examples. Arrows indicate inferred fragmentation. MS2 fragments and structure parts highlighted represent MCY groups. Certain fragments are derived from water loss.

[0064] Fig. 8. Shows MS2 networks of microbiota-dependent differential features in mouse serum in ESI+. Nodes represent downregulated and upregulated features for GF (n = 9) relative toSPF (n = 11) mice in ESI+. Subnetworks for differential metabolites were annotated corresponding to their molecular families.

[0065] Fig. 9 Shows an enlarged view of part of the MS2 networks of microbiota-dependent differential features in mouse serum. Enlarged partial MS2 networks shown in Fig. 1C for mouse serum in ESI- (top) and ESI+ (bottom), showing clusters representing bile acids derivatives.

[0066] Figs. 10A-10E. Show metabolite identification using authentic standards. Figs. 10A-10D:

[0067] Extracted ion chromatograms (EICs) of BA-MCY and BA-MCYO conjugates (Figs. 10A- 10C), and BA-MCY02 (Fig. 10D) in serum of GF and SPF mice and comparison with synthetic standards analyzed in ESI+. Fig. 10E: Scheme for the synthesis of CA-MCY conjugates from CA. For syntheses of other BA-MCY conjugates.

[0068] Figs. 11A-11D. Show microbial deconjugation of BA-MCYs in vivo. Figs. 11A and 11B: EICs for CDCA-MCY conjugates (Fig. 11A) and free BAs (Fig. 11B) with molecular ion peaks (black) and four deuterium isotope peaks from HPLC-HRMS analysis of feces of SPF mice administered CDCA-d5-MCY. Figs. 11C and 11D: EICs for CDCA-MCY conjugates (Fig. 11C) and free BAs (Fig. 11D) with molecular ion peaks (black) and four deuterium isotope peaks from HPLC-HRMS analysis of feces of GF mice administered CDCA-d5-MCY.

[0069] Fig. 12. Shows ¹H (600 MHz) NMR spectrum of CA-MCY in methanol-d₄.

[0070] Fig. 13. Shows the dqfCOSY spectrum of CA-MCY in methanol-d₄.

[0071] Fig. 14. Shows the HSQC spectrum of CA-MCY in methanol-d₄.

[0072] Fig. 15. Shows the HMBC spectrum of CA-MCY in methanol-d₄.

[0073] Fig. 16. Shows ¹H (600 MHz) NMR spectrum of CA-MCYO in methanol-d₄.

[0074] Fig. 17. Shows the dqfCOSY spectrum of CA-MCYO in methanol-d₄.

[0075] Fig. 18. Shows the HSQC spectrum of CA-MCYO in methanol-d₄.

[0076] Fig. 19. Shows the HMBC spectrum of CA-MCYO in methanol-d₄.

[0077] Fig. 20. Shows ¹H (600 MHz) NMR spectrum of CA-MCYO2 in methanol-d₄.

[0078] Fig. 21. Shows the dqfCOSY spectrum of CA-MCYO2 in methanol-d₄.

[0079] Fig. 22. Shows the HSQC spectrum of CA-MCYO2 in methanol-d₄.

[0080] Fig. 23. Shows the HMBC spectrum of CA-MCYO2 in methanol-d₄.

[0081] Fig. 24. Shows ’H (600 MHz) NMR spectrum of [3MCA-MCY, in methanol-^.Fig. 25. Shows the dqfCOSY spectrum of βMCA-MCY (6), in methanol-d₄. Fig. 26. Shows the HSQC spectrum of βMCA-MCY in methanol-d₄.

[0082] Fig. 27. Shows the HMBC spectrum of βMCA-MCY in methanol-d₄.

[0083] Fig. 28. Shows ¹H (600 MHz) NMR spectrum of βMCA-MCYO in methanol-d₄. Fig. 29. Shows the dqfCOSY spectrum of βMCA-MCYO in methanol-d₄.

[0084] Fig. 30. Shows the HSQC spectrum of βMCA-MCYO in methanol-d₄.

[0085] Fig. 31. Shows the HMBC spectrum of βMCA-MCYO in methanol-d₄.

[0086] Fig. 32. Shows ¹H (600 MHz) NMR spectrum of βMCA-MCYO2 in methanol-d₄. Fig. 33. Shows the dqfCOSY spectrum of βMCA-MCYO2 in methanol-d₄. Fig. 34. Shows the HSQC spectrum of βMCA-MCYO2 in methanol-d₄.

[0087] Fig. 35. Shows the HMBC spectrum of βMCA-MCYO2 in methanol-d₄.

[0088] Fig. 36. Shows ’H (600 MHz) NMR spectrum of CDCA-MCY in methanol-^. Fig. 37. Shows the dqfCOSY spectrum of CDCA-MCY in methanol-d₄.

[0089] Fig. 38. Shows the HSQC spectrum of CDCA-MCY in methanol-d₄.

[0090] Fig. 39. Shows the HMBC spectrum of CDCA-MCY in methanol-d₄.

[0091] Fig. 40. Shows ¹H (600 MHz) NMR spectrum of CDCA-MCYO in methanol-d₄. Fig. 41. Shows the dqfCOSY spectrum of CDCA-MCYO in methanol-d₄. Fig. 42. Shows the HSQC spectrum of CDCA-MCYO in methanol-d₄.

[0092] Fig. 43. Shows the HMBC spectrum of CDCA-MCYO in methanol-d₄.

[0093] Fig. 44. Shows ¹H (600 MHz) NMR spectrum of UDCA-MCY in methanol-d₄. Fig. 45. Shows the dqfCOSY spectrum of UDCA-MCY in methanol-d₄.

[0094] Fig. 46. Shows the HSQC spectrum of UDCA-MCY in methanol-d₄.

[0095] Fig. 47. Shows the HMBC spectrum of UDCA-MCY in methanol-d₄.

[0096] Fig. 48. Shows ¹H (600 MHz) NMR spectrum of UDCA-MCYO in methanol-d₄. Fig. 49. Shows the dqfCOSY spectrum of UDCA-MCYO in methanol-d₄.

[0097] Fig. 50. Shows the HSQC spectrum of UDCA-MCYO in methanol-d₄.

[0098] Fig. 51. Shows the HMBC spectrum of UDCA-MCYO in methanol-d₄.

[0099] Fig. 52. Shows ¹H (600 MHz) NMR spectrum of DCA-MCY in methanol-d₄. Fig. 53. Shows the HSQC spectrum of DCA-MCY in methanol-d₄.

[0100] Fig. 54. Shows the dqfCOSY spectrum of DCA-MCY in methanol-d₄.

[0101] Fig. 55. Shows the HMBC spectrum of DCA-MCY in methanol-d₄.Fig. 56. ShowsXH (600 MHz) NMR spectrum of DCA-MCYO in methanol-^.

[0102] Fig. 57. Shows the dqfCOSY spectrum of DCA-MCYO in mcthanol-< Y.

[0103] Fig. 58. Shows the HSQC spectrum of DCA-MCYO in methanol-c / ,.

[0104] Fig. 59. Shows the HMBC spectrum of DCA-MCYO in methanol-^.

[0105] Fig. 60. Shows 'H (600 MHz) NMR spectrum of KDCA-MCY in methanol -ch

[0106] Fig. 61. Shows the dqfCOSY spectrum of KDCA-MCY in methanol-c / j.

[0107] Fig. 62. Shows the HSQC spectrum of KDCA-MCY in methanol -ch.

[0108] Fig. 63. Shows the HMBC spectrum of KDCA-MCY in methanol -ch.

[0109] Fig. 64. Shows 'H (600 MHz) NMR spectrum of KDCA-MCYO in methanol-t.

[0110] Fig. 65. Shows the dqfCOSY spectrum of KDCA-MCYO in methanol-t / v.

[0111] Fig. 66. Shows the HSQC spectrum of KDCA-MCYO in methanol-cL.

[0112] Fig. 67. Shows the HMBC spectrum of KDCA-MCYO in methanol-^.

[0113] Fig. 68. Shows 'H (600 MHz) NMR spectrum of CA-CY in methanol-cL.

[0114] Fig. 69. Shows the dqfCOSY spectrum of CA-CY in methanol-tL.

[0115] Fig. 70. Shows the HSQC spectrum of CA-CY in methanol-< Y.

[0116] Fig. 71. Shows the HMBC spectrum of CA-CY in methanol- / .

[0117] Figs. 72A and 72B. Show FXR antagonism by DCA-MCY (Fig. 72A) and CDCA-CY (Fig.

[0118] 72B).

[0119] Fig. 73. Shows TCF-1 expression in CD8+T cells exposed to the indicated compounds (CA- MCY, CDCA-MCY, DCA-MCY, DCA-MCYO, or DCA-CY; 10 pM each). DMSO is control.

[0120] Figs. 74A, 74B, and 74C. Show GPBAR1 agonist activity by CA-MCY (Fig. 74A), DCA-MCY (Fig. 74B), and DCA-MCYO (Fig. 74C).

[0121] DETAILED DESCRIPTION OF THE INVENTION

[0122] Metabolites derived from the intestinal microbiota, including bile acids (BAs), extensively modulate vertebrate physiology, including development (Robertson, et al., Trends Microbiol. 27:131-147 (2019)), metabolism, immune responses, and cognitive function.

[0123] However, to what extent host responses balance the physiological effects of microbiota-derived metabolites remains unclear. Employing untargeted metabolomics of mouse tissues, a family of bile acid-methylcysteamine (BA-MCY) conjugates have been identified that are particularlyabundant in the intestine. This host-dependent MCY conjugation inverts BA function in the hepatobiliary system. Whereas microbiota derived free BAs function as agonists of the farnesoid X receptor (FXR) and negatively regulate BA production, it has unexpectedly been found that BA-MCYs act as potent antagonists of FXR in vitro and promote expression of BA biosynthesis genes in vivo. Surprisingly, supplementation of mice with stable-isotope labeled BA-MCY increased BA production in an FXR-dependent manner and BA-MCY supplementation in a mouse model of hypercholesteremia decreased lipid accumulation in the liver, consistent with BA-MCYs acting as intestinal FXR antagonists. Levels of BA-MCYs and free BAs were reduced in microbiota deficient mice and restored by transplantation of human fecal microbiota. Dietary intervention with inulin fiber further increased levels of both free BAs and BA-MCY levels, indicating that BA-MCY production by the host is regulated by levels of microbiota-derived free BAs. We have further shown that diverse BA-MCYs are also present in human serum. Taken together, our results indicate that BA-MCY conjugation by the host balances host-and microbiota-dependent metabolic pathways that regulate FXR-dependent physiology and that BA-MCYs and related molecules can be used as new therapeutic agents to treat a range of health disorders.

[0124] Therapeutic Compositions

[0125] In one aspect, the present invention encompasses therapeutic compositions comprising a therapeutically effective amount of one or more bile acid cysteamides or derivatives or analogs of such cysteamides. The term “bile acid” in this context encompasses any carboxylic acid having a steroid carbon skeleton in its structure, e.g. any carboxylic acid containing the

[0126] XX>

[0127] substructure, or compounds derivable from such a substructure (for example by breaking one or more of the ring bonds) or addition of additional moieties or rings. Examples of the former include compounds such as 9,11 seco cholesterol:

[0128] 8S,11-seco-Cholesterol

[0129]

[0130] formally derived from oxidative cleavage of a ring bond in the steroid skeleton.

[0131] In certain embodiments, provided therapeutic compositions are characterized in that the sulfur atom of the cysteamide is substituted with an alkyl group. Such molecules are referred to herein as S-alkyl cysteamides, or BA-SACAs for short. In certain embodiments, the sulfur atom of the BA-SACA is substituted with a C1-40 aliphatic group. In certain embodiments, provided compositions are characterized in that the sulfur atom of the cysteamide is substituted with a Ci-24 alkyl group, a C1-12 alkyl group, a C1-8 alkyl group, a C1-6 alkyl group, a C1.5 alkyl group, a C1-4 alkyl group, a C1-3 alkyl group, a C1-2 alkyl group, or with a methyl group. In certain embodiments, such cysteamides are characterized in that they comprise a straight chain alkyl group attached to the sulfur atom. In certain embodiments, such cysteamides are characterized in that they comprise a saturated alkyl group attached to the sulfur atom. In certain embodiments, such cysteamides are characterized in that they comprise a straight-chain saturated alkyl group attached to the sulfur atom. In certain embodiments, such cysteamides are characterized in that the sulfur atom is substituted with a branched alkyl group, a mono- or poly-unsaturated alkyl group, an optionally substituted alkyl group, or an alkyl group having any two or more of these features in combination.

[0132] In certain embodiments, provided therapeutic compositions comprise a sulfoxide derivative of a bile acid cysteamide (hereafter referred to as “S-oxide cysteamides” or BA-SOCAs). In certain embodiments, provided therapeutic compositions are characterized in that the sulfur atom of a provided cysteamide sulfoxide is substituted with an alkyl group. Such molecules are referred to herein as bile acid 5-oxide alkyl cysteamides, (BA- SOACAs). In certain embodiments, the sulfur atom of the BA-SOACA is substituted with a C1-40 aliphatic group. In certain embodiments, provided compositions are characterized in that the sulfur atom of the BA-SOACA is substituted with a C1-24 alkyl group, a C1-12 alkyl group, a C1-8 alkyl group, a Ci-6 alkyl group, a C1-5 alkyl group, a C1.4 alkyl group, a C1-3 alkyl group, a C1-2 alkyl group, or with a methyl group. In certain embodiments, such BA-SOACAs are characterized in that they comprise a straight chain alkyl group attached to the sulfur atom. In certain embodiments, such BA-SOACAs are characterized in that they comprise a saturated alkyl group attached to the sulfur atom. In certain embodiments, such BA-SOACAs are characterized in that they comprise a straight-chain saturated alkyl group attached to the sulfur atom. In certain embodiments, such BA-SOACAs are characterized in that the sulfur atom is substituted with a branched alkyl group,a mono- or poly-unsaturated alkyl group, an optionally substituted alkyl group, or an alkyl group having any two or more of these features in combination.

[0133] In certain embodiments, such compositions comprise a SOACA of an endogenous carboxylic acid. In certain embodiments, such compositions comprise a SOACA of a microbiome-derived carboxylic acid. In certain embodiments, such compositions comprise a SOACA of a bile acid.

[0134] The BA-SOACs and BA-SOACAs described above contain a chiral center on the sulfur atom. In certain embodiments, the provided therapeutic compositions comprise a BA-SOAC or a BA-SOACA that is enantioenriched with respect to the chiral sulfur atom. In certain embodiments, such compositions are characterized in that the contained BA-SOACs and / or BA-SOACAs comprise greater than 70% of one stereoisomer of the sulfur-based chiral center. In certain embodiments, such compositions are characterized in that the contained BA-SOACs and / or SOACAs comprise greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 92%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or greater than 99.5%, of one stereoisomer of the sulfurbased chiral center, such compositions are characterized in that the contained BA-SOACs and / or BA-SOACAs comprise essentially a single enantiomer with respect to the sulfur-based chiral center. In certain embodiments where the BA-SOAC and / or BA-SOACAs are entantioenriched at the sulfur-based chiral center, the composition is enriched in the compound having the S configuration at the Sulfur-based center. In certain embodiments where the BA-SOAC and / or BA-SOACAs are entantioenriched at the sulfur-based chiral center, the configuration of the dominant enantiomer is R.

[0135] In certain embodiments, provided therapeutic compositions comprise a sulfone derivative of a bile acid cysteamide (hereafter referred to bile acid cysteamide sulfones or BA-CASOs). In certain embodiments, provided therapeutic compositions are characterized in that the sulfur atom of the cysteamide sulfone is substituted with an alkyl group. Such molecules are referred to herein as S-alkyl cysteamide sulfones, (BA-SACASOs). In certain embodiments, the sulfur atom of the BA-SACASO is substituted with a C1-40 aliphatic group. In certain embodiments, provided compositions are characterized in that the sulfur atom of the BA-SACASO is substituted with a Ci-24 alkyl group, a C1-12 alkyl group, a Ci-s alkyl group, a C1-6 alkyl group, a C1-5 alkyl group, a C1.4 alkyl group, a C1-3 alkyl group, a C1.2 alkyl group, or with a methyl group. In certainembodiments, such SACASOs are characterized in that they comprise a straight chain alkyl group attached to the sulfur atom. In certain embodiments, such BA-SACASOs are characterized in that they comprise a saturated alkyl group attached to the sulfur atom. In certain embodiments, such BA-SACASOs are characterized in that they comprise a straight-chain saturated alkyl group attached to the sulfur atom. In certain embodiments, such BA-SACASOs are characterized in that the sulfur atom is substituted with a branched alkyl group, a mono- or poly-unsaturated alkyl group, an optionally substituted alkyl group, or an alkyl group having any two or more of these features in combination.

[0136] In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula I:

[0137] R1

[0138] A

[0139] Y

[0140]

[0141] 0iz

[0142] 5

[0143] wherein:

[0144] A- is a bile acid;

[0145] R1is selected from -H, and an optionally substituted C1-40 aliphatic group;

[0146] Z is selected from -H, and an optionally substituted C1-40 aliphatic group; and

[0147] y is 0, 1, or 2.

[0148] In certain embodiments, A- comprises a C24 bile acid. In certain embodiments, A-comprises a C27 bile acid.

[0149] In certain embodiments, A- is selected from the group consisting of: cholic acid (CA), -muricholic acid (PMCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), deoxycholic acid (DCA), and 7-ketodeoxycholic acid (7-KDCA).

[0150] In certain embodiments, A- is selected from the group consisting of: lithocholic acid (LCA), isolithocholic acid, 3-oxolithocholic acid, allolithocholic acid, 3-oxoallolithocholic acid, isoallolithocholic acid, 7a,12a-dihydroxy-3-oxochol-4-en-24-oic acid, 7a-hydroxy-3-oxochol-4-en-24-oic acid and 12a-hydroxy-3-oxochol-4,6-dien-24-oic acid, ursodeoxycholic acid, and 3a, 601, 7a, 12a-tetrahydroxy-5l3-cholan-24-oic acid.In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula II:

[0151] R1

[0152] Y

[0153]

[0154] ° I,

[0155] wherein each of A, R1, and Z is as defined above and in the genera and subgenera herein. In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula III:

[0156]

[0157] wherein each of A, R1, and Z is as defined above and in the genera and subgenera herein. In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of a compound selected from formulae Illa, Illb, and IIIc:

[0158]

[0159] wherein each of A, R1, and Z is as defined above and in the genera and subgenera herein. In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula IV:R1

[0160] I

[0161]

[0162] wherein each of A, R1, and Z is as defined above and in the genera and subgenera herein. In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula IV:

[0163]

[0164] wherein each of A and R1is, independently, as defined above and in the genera and subgenera herein.

[0165] In certain embodiments, provided therapeutic compositions comprise one or more compounds of formulae I, II, III, Illa, Illb, IIIc, IV, or V, wherein the moiety -R1is -H.

[0166] In certain embodiments, provided therapeutic compositions comprise one or more compounds of formulae I, II, III, Illa, Illb, IIIc, IV, or V, wherein the moiety -R1comprises an optionally substituted C1-40 aliphatic group. In certain embodiments, the moiety -R1comprises a straight chain C3-24 aliphatic group. In certain embodiments, the moiety -R1comprises a an unsaturated C3-24 aliphatic group. In certain embodiments, the moiety -R1comprises a C1.40 alkyl group. In certain embodiments, the moiety -R1comprises a C1-32 alkyl group. In certain embodiments, the moiety -R1comprises a straight chain C3-18 alkyl group. In certain embodiments, the moiety -R1comprises a straight chain C3-12 alkyl group. In certain embodiments, the moiety -R1comprises a C1-8 alkyl group. In certain embodiments, the moiety -R1comprises a C1-6 alkyl group. In certain embodiments, the moiety -R1comprises a C1-4 alkyl group. In certain embodiments, the moiety -R1is -CH2CH2CH2CH2CH3. In certain embodiments, the moiety -R1is -CH2CH2CH2CH3. In certain embodiments, the moiety -R1is -CH2CH(CH3)2. In certain embodiments, the moiety -R1is -CH(CH3)CH2CH3. In certain embodiments, the moiety -R1is -CH2CH2CH3. In certain embodiments, the moiety -R1is -CH(CH3)2. In certain embodiments, the moiety -R1is -CH2CH3.In certain embodiments, the moiety -R1is -CH3. In certain embodiments, -R1is selected from the group consisting of: -H, / / -propyl, ethyl, methyl, and combinations of two or more of these. In certain embodiments, provided therapeutic compositions comprise one or more compounds of formulae I, II, III, Illa, Illb, IIIc, or IV, wherein the moiety -Z comprises -H. In certain embodiments, provided therapeutic compositions comprise one or more compounds of formulae I, II, III, Illa, Illb, IIIc, or IV, wherein the moiety -Z is other than -H.

[0167] In certain embodiments, provided therapeutic compositions comprise one or more compounds of formulae I, II, III, Illa, Illb, IIIc, or IV, wherein the moiety -Z comprises -CH3.

[0168] In certain embodiments, provided therapeutic compositions comprise one or more compounds of formulae I, II, III, Illa, Illb, IIIc, or IV, wherein the moiety -Z comprises an optionally substituted C1-40 aliphatic group. In certain embodiments, the moiety -Z comprises a straight chain C3-24 aliphatic group. In certain embodiments, the moiety -Z comprises a an unsaturated C3-24 aliphatic group. In certain embodiments, the moiety -Z comprises a C1.40 alkyl group. In certain embodiments, the moiety -Z comprises a C1-32 alkyl group. In certain embodiments, the moiety -Z comprises a straight chain C3-18 alkyl group. In certain embodiments, the moiety -Z comprises a straight chain C3-12 alkyl group. In certain embodiments, the moiety -Z comprises a Ci-s alkyl group. In certain embodiments, the moiety -Z comprises a Ci-6 alkyl group. In certain embodiments, the moiety -A comprises a C1.4 alkyl group. In certain embodiments, the moiety -Z is -CH2CH2CH2CH2CH3. In certain embodiments, the moiety -Z is -CH2CH2CH2CH3. In certain embodiments, the moiety -Z is -CH2CH(CH3)2. In certain embodiments, the moiety -Z is -CH(CH3)CH2CH3. In certain embodiments, the moiety -Z is -CH2CH2CH3. In certain embodiments, the moiety -Z is -CH(CH3)2. In certain embodiments, the moiety -Z is -CH2CH3. In certain embodiments, -Z is selected from the group consisting of: / / -propyl, ethyl, methyl, and combinations of two or more of these.

[0169] In certain embodiments, provided therapeutic compositions comprise one or more compounds of formulae I, II, III, Illa, Illb, IIIc, IV, or V, wherein the moiety -R1is -CH3 and the moiety -A comprises an endogenous bile acid.

[0170] In certain embodiments, provided therapeutic compositions comprise one or more compounds of formulae I, II, III, Illa, Illb, IIIc, IV, or V, wherein the moiety -R1is -CH3 and the moiety A-C(O)- comprises an acyl group derived from an endogenous bile acid. In certain embodiments, provided therapeutic compositions comprise one or more compounds of formulaeI, II, III, Illa, ITIb, IIIc, IV, or V, wherein the moiety -R1is -CH3 and the moiety A-C(O)-comprises an acyl group derived from a bile acid.

[0171] In embodiments where provided therapeutic compositions comprise bile acid cysteamide derivatives or oxides or sulfones thereof having an 5-alkyl substituent, the substituent may be any of those described above and in the genera and subgenera herein.

[0172] In certain embodiments, provided therapeutic compositions comprise compounds conforming to Formula VI:

[0173] R1

[0174] I

[0175] stY

[0176]

[0177] wherein:

[0178] -L- comprises an optionally substituted, optionally unsaturated C1-24 linker where any one or more carbon atoms may be optionally replaced with -NRy-, -C(O)-, -O-, -S-, -S(O)-, -S(O)2-, -C(=NRy)-, and -C(=S)-;

[0179] -St comprises a moiety having a steroid carbon skeleton

[0180]

[0181] ); and each of R1, Z, andy is as defined above and in the genera and subgenera herein.

[0182] In certain embodiments, where provided therapeutic compositions comprise a compound of Formula VI, the moiety -L- comprises an optionally substituted linker moiety comprising a chain of 2 to 20 carbon atoms separating the moiety -St from the acyl linkage to the cysteamide. In certain embodiments, -L- comprises an optionally substituted linker moiety comprising a chain of 2 to 12 carbon atoms separating the moiety -St from the acyl linkage to the cysteamide. In certain embodiments, -L- comprises a chain of 3 to 7 carbon atoms separating the moiety -St from the acyl linkage.

[0183] In certain embodiments, the moiety -L- comprises

[0184]

[0185] where * represents the site of attachment to the moiety -St.

[0186] In certain embodiments, the moiety -L- comprises

[0187]

[0188] , where * represents the site of attachment to the moiety -St.In certain embodiments, the moiety -St comprises a steroid moiety linked to -L- through ring-D. In certain embodiments, the moiety -St comprises a steroid moiety linked to -L- through

[0189]

[0190] In certain embodiments, the steroid ring of the moiety -St is substituted with 1 or more hydroxyl groups. In certain embodiments, the steroid ring of the moiety -St is substituted with 3 hydroxyl groups. In certain embodiments, the steroid ring of the moiety -St is substituted with 2 hydroxyl groups. In certain embodiments, the steroid ring of the moiety -St is substituted 1 hydroxyl group. In certain embodiments, at least one hydroxyl substituent is at carbon 3, 7, or 12

[0191] 12

[0192] C c Tpy

[0193] o r L A J iL B J i -7

[0194] of the steroid ring.

[0195]

[0196] In certain embodiments, the moiety -St-L- in Formula VI is selected from the group consisting of:

[0197]

[0198] In certain embodiments, the moiety Z in Formula VI is methyl. In certain embodiments, R1in Formula V is -H. In certain embodiments, Z1in Formula V is methyl, the moiety R1is -H, and the moiety -St-L- is selected from the group consisting of:

[0199]

[0200] In certain embodiments, provided therapeutic compositions comprise a compound selected from Table 1:

[0201] TABLE 1

[0202]

[0203] In certain embodiments, provided therapeutic compositions comprise a compound of formula:

[0204] o

[0205]

[0206] In certain embodiments, provided therapeutic compositions comprise a compound of formula:

[0207]

[0208] In certain embodiments, provided therapeutic compositions comprise a compound of formula:

[0209]

[0210] H

[0211] In certain embodiments, the compound is selected from the groups consisting of CA-MCY, CA-MCYO, CA-MCY02, aMCA-MCY, aMCA-MCYO, 0MCA-MCY, £MCA-MCYO, PMC A-MCYO2, coMCA-MCY, wMCA-MCYO, CDCA-MCY, CDCA-MCYO, UDCA-MCY, UDCA-MCYO, DCA-MCY, DCA-MCYO, KDCA-MCY, KDCA-MCYO, CDCA-d5-MCY, and CA-CY. In certain embodiments, the compound is selected from the groups consisting of CA-MCY, CDCA-CY, DCA-MCY, DCA-MCYO, CDCA-MCY, DCA-CY, and 7-KDCA-MCY.

[0212] In certain embodiments, the present invention provides therapeutic compositions comprising a mixture of any of the cysteamides described above in combination with an S-oxidized congener of the same compound. In certain embodiments, such compositions comprise a mixture of an un-oxidized cysteamide and a sulfoxide of that cysteamide. In certain embodiments, such compositions comprise a mixture of an un-oxidized cysteamide and a sulfone of that cysteamide. In certain embodiments, such compositions comprise a mixture of an un-oxidized cysteamide, in combination with the sulfoxide and the sulfone of that cysteamide.

[0213] In another aspect, the present invention provides pharmaceutical compositions containing cysteamides (e.g. any one or more compounds conforming to Formulae I, II, III, Illa, Illb, IIIc, IV, V, or VI described above). In certain embodiments, the invention encompasses a pharmaceutical composition or a single unit dosage form of any of the cysteamide compoundsdescribed above. Tn certain embodiments, pharmaceutical compositions and single unit dosage forms of the invention comprise a prophylactically or therapeutically effective amount of one or more of the cysteamides describe above, or their pro-drugs, and typically one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment and in this context, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in " Remington's Pharmaceutical Sciences" by E. W. Martin.

[0214] Typical pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are well-known to those skilled in the art of pharmacy, and non -limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient and the specific active ingredients in the dosage form. The composition or single unit dosage form, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0215] Lactose-free compositions of the invention can comprise excipients that are well known in the art and are listed, for example, in the U. S. Pharmacopeia (USP) SP (XXI) / NF (XVI). In general, lactose-free compositions comprise an active ingredient, a binder / filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts. Preferred lactose-free dosage forms comprise an active ingredient, microcrystalline cellulose, pre-gelatinized starch, and magnesium stearate.This invention further encompasses anhydrous pharmaceutical compositions and dosage forms comprising active ingredients (e.g. any of the cysteamides described above), since water can facilitate the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical arts as a means of simulating long-term storage to determine characteristics such as shelf-life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, NY, N. Y., 1995, pp. 379-80. In effect, water and heat accelerate the decomposition of some compounds. Thus, the effect of water on a formulation can be of great significance since moisture and / or humidity are commonly encountered during manufacture, handling, packaging, storage, shipment, and use of formulations.

[0216] Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine are preferably anhydrous if substantial contact with moisture and / or humidity during manufacturing, packaging, and / or storage is expected.

[0217] An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0218] The invention further encompasses pharmaceutical compositions and dosage forms that comprise any one or more cysteamides and one or more compounds that reduce the rate by which an active ingredient will decompose. Such compounds, herein referred to as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[0219] The pharmaceutical compositions and single unit dosage forms can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions and dosage forms will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent preferably in purifiedform, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In certain embodiments, the pharmaceutical compositions or single unit dosage forms are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.

[0220] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), intranasal, transdermal (topical), transmucosal, intra-tumoral, intra-synovial and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to human beings. In certain embodiments, a pharmaceutical composition is formulated in accordance with routine procedures for subcutaneous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocane to ease pain at the site of the injection. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

[0221] The composition, shape, and type of dosage forms of the invention will typically vary depending on their use. For example, a dosage form used in the acute treatment of inflammation or a related disorder may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the chronic treatment of the same disease. Also, the therapeutically effective dosage form may vary among different types of cancer. Similarly, a parenteral dosage form may contain smaller amounts of one or more of the active ingredients itcomprises than an oral dosage form used to treat the same disease or disorder. These and other ways in which specific dosage forms encompassed by this invention will vary from one another will be readily apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).

[0222] Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. Typical dosage forms of the invention comprise a compound of the invention, or a pharmaceutically acceptable salt, solvate or hydrate thereof lie within the range of from about 1 mg to about 10,000 mg per day, given as a single once-a-day dose in the morning but preferably as divided doses throughout the day taken with food.

[0223] Pharmaceutical compositions of the invention that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).

[0224] Typical oral dosage forms of the invention are prepared by combining the active ingredient(s) in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.

[0225] For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0226] Examples of excipients that can be used in oral dosage forms of the invention include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, com starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

[0227] Examples of fdlers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions of the invention is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.

[0228] Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. An specific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or low moisture excipients or additives include AVICEL -PH-103. TM. and Starch 1500 LM.

[0229] Disintegrants are used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form solid oral dosage forms of the invention. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, specifically from about 1 to about 5 weight percent of disintegrant.

[0230] Disintegrants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.

[0231] Lubricants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured by W. R. Grace Co. of Baltimore, Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, Mass.), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated.

[0232] Delayed Release Dosage Forms

[0233] Active ingredients of the invention can be administered by controlled release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, butare not limited to, those described in U. S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which is incorporated herein by reference. Such dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients of the invention. The invention thus encompasses single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled-release.

[0234] All controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects.

[0235] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.

[0236] Parenteral Dosage Forms

[0237] Parenteral dosage forms can be administered to patients by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, andintraarterial. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

[0238] Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0239] Compounds that increase the solubility of one or more of the active ingredients disclosed herein can also be incorporated into the parenteral dosage forms of the invention.

[0240] TransdermaL Topical & Mucosal Dosage Forms

[0241] Transdermal, topical, and mucosal dosage forms of the invention include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art. See, e g., Remington's Pharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa. (1980 & 1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treating mucosal tissues within the oral cavity can be formulated as mouthwashes or as oral gels. Further, transdermal dosage forms include "reservoir type" or "matrix type" patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredients.

[0242] Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal, topical, and mucosal dosage forms encompassed by this invention are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied. With that fact in mind, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propyleneglycol, butane-l,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form lotions, tinctures, creams, emulsions, gels or ointments, which are non-toxic and pharmaceutically acceptable. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa. (1980 & 1990).

[0243] Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with active ingredients of the invention. For example, penetration enhancers can be used to assist in delivering the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water-soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

[0244] The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, may also be adjusted to improve delivery of one or more active ingredients. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients so as to improve delivery. In this regard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery-enhancing or penetration-enhancing agent. Different salts, hydrates or solvates of the active ingredients can be used to further adjust the properties of the resulting composition.

[0245] Dietary Supplement and Functional Food Forms

[0246] In certain embodiments, compounds of the present invention can be added to food or compounded with food additives and provided as a part of a subject’s diet. In certain embodiments, a cysteamide derivative of the present invention (e.g. any one or more compounds conforming to Formulae I, II, III, Illa, Illb, IIIc, IV, V, or VI described above) is added to a prepared or packaged food item or included in a vitamin or dietary supplement. Representative examples include adding the cysteamides to formula, milk or other dairy products, powderedmilk, protein powder, beverages, health drinks, teas, juices and the like. In certain embodiments, the cysteamides are added to multivitamins or to other dietary supplements and similar products.

[0247] Chemical Compositions of Matter

[0248] In another aspect, the present invention encompasses novel compositions of matter including compositions of novel molecules. While some of the cysteamides described above are naturally occurring molecules that have been detected in the bodies of mice or other animals or have been shown to be produced by organisms in the microbiomes of mice or other creatures, pure samples of these molecules and, in particular, bulk samples of pure bile acid cysteamides free from other biological materials are not found in nature. Additionally, many of the Cysteamides and related compounds described above have not been detected in nature, even with the aid of highly sensitive and selective analytical techniques such as HPLC-coupled high resolution mass spectroscopy. As such, many of the compounds described above constitute novel non-natural compositions of matter.

[0249] In certain embodiments, the present invention provides pure samples of any of the cysteamides, S-alkyl cysteamides, cysteamide S-oxides, 5-alkyl cysteamide -oxides, cysteamide sulfones, and S-alkyl cysteamide sulfones described above and in the genera and subgenera herein. In certain embodiments, the present invention provides samples comprising bulk quantities of such molecules in substantially pure form. In certain embodiments, the present invention provides samples comprising at least 100mg, at least 1g, at least 10g, at least 50g, at least 200g, at least 500g, or at least 1kg of such molecules in substantially pure form.

[0250] In certain embodiments, the present invention provides novel compositions comprising one or more compounds depicted in Table 1. In certain embodiments, the present invention provides novel compositions comprising mixtures of between two and ten different cysteamides.

[0251] In certain embodiments, the present invention provides novel compositions comprising a mixture of an un-oxidized cysteamide as described herein and an S-oxidized congener of the same cysteamide. In certain embodiments, such compositions comprise a mixture of an unoxidized cysteamide and a sulfoxide of that cysteamide. In certain embodiments, such compositions comprise a mixture of an un-oxidized cysteamide and a sulfone of that cysteamide. In certain embodiments, such compositions comprise a mixture of an un-oxidized cysteamide, in combination with the sulfoxide and the sulfone of that cysteamide. In certain embodiments, thepresent invention provides compositions comprising a mixture of sulfoxide of a cysteamide as described herein and a sulfone of that cysteamide.

[0252] Therapeutic Methods

[0253] In another aspect, the present invention encompasses methods of improving the health of an animal or of treating or ameliorating a health disorder in an animal by administering to the animal an effective amount of any one or more of the compounds and / or therapeutic compositions described above (e.g. a composition comprising any one or more compounds conforming to Formulae I, II, III, Illa, Illb, IIIc, IV and V described above). In certain embodiments, the method comprises administering such a composition to a mammal. In certain embodiments, the method comprises administering such a composition to a human.

[0254] The ligand-regulated nuclear receptor FXR plays a central role in lipid and glucose metabolism as well as cholesterol homeostasis. High cholesterol levels underlie a wide range of human diseases, including blood clots, cardiovascular disease, peripheral arterial disease, type 2 diabetes, and stroke.

[0255] In certain embodiments, the present invention comprises a method of lowering cholesterol levels, including low-density lipoprotein and triglyceride levels. In certain embodiments, the present invention comprises a method for treating or preventing hyperlipidemia, blood clots, cardiovascular disease, fatty liver disease, peripheral arterial disease, and stroke. In addition, the present invention in certain embodiments comprises a method for treating or preventing hyperglycemia and / or type 2 diabetes.

[0256] In certain embodiments, methods are provided for treating metabolic and liver diseases (i.e. via the activity of the provided compounds as FXR antagonists). In certain embodiments, the method comprises treating a patient with non-alcoholic fatty liver disease (NAFLD) by treatment with an effective amount of any or more of the compounds or pharmaceutical compositions above. In certain embodiments, the effect of such treatment is a reduced accumulation of fat in the liver. In certain embodiments, the method comprises treating a patient with non-alcoholic steatohepatitis (NASH). In certain embodiments, such treatments result in decreased inflammation and / or hepatocyte injury. In certain embodiments, such treatments result in decreased fibrosis and cirrhosis.In certain embodiments, methods are provided for modulating the lipid and / or glucose metabolism of a patient by treatment with an effective amount of any or more of the compounds or pharmaceutical compositions above. In certain embodiments provided methods result in one or more of: reduced hepatic fat accumulation, reduced inflammation, and reduced fibrosis. In certain embodiments, methods are provided for modulation of lipid and / or glucose metabolism in a person in need of such therapy, the method comprising the step of administering one or more of the bile acid cysteamides described herein (or a pharmaceutically acceptable precursor, pro-drug or salt thereof).

[0257] In certain embodiments, methods are provided for treating cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). These diseases are characterized by impaired bile flow, leading to bile acid accumulation and liver damage. Without being bound by theory, it is believed that treatment with the compounds provided herein effectively inhibit FXR activation and thereby reduce bile acid levels, alleviating cholestasis and its associated liver damage. In certain embodiments, methods are provided for treatment of cholestatic liver diseases, the method comprising the step of administering one or more of the bile acid cysteamides described herein (or a pharmaceutically acceptable precursor, pro-drug or salt thereof).

[0258] In certain embodiments, methods are provided for treating obesity or providing weight management for a patient by treatment (e.g., administration) with an effective amount of any or more of the compounds or pharmaceutical compositions of the instant invention. Intestinal FXR antagonism prevents the inhibition of bile acid synthesis, altering the bile acid pool. This metabolic shift has been shown to reduce intestinal ceramide production and promote beige fat thermogenesis (browning of white adipose tissue), leading to increased energy expenditure and weight loss.

[0259] In certain embodiments, methods are provided for treating, inhibiting, and / or preventing Type 2 Diabetes (T2D) in a patient by treatment (e.g., administration) with an effective amount of any or more of the compounds or pharmaceutical compositions of the instant invention. By blocking intestinal FXR, these agents can enhance the secretion of Glucagon-Like Peptide-1 (GLP-1), an incretin hormone that improves insulin sensitivity and glucose homeostasis.

[0260] Research indicates that FXR normally suppresses GLP-1. Thus, antagonism releases this "brake," thereby offering a mechanism similar to established diabetes drugs.In certain embodiments, methods are provided for treating, inhibiting, and / or preventing Non-Alcoholic Fatty Liver Disease (NAFLD) and / or Metabolic Dysfunction-Associated Steatohepatitis (MASH) in a patient by treatment (e.g., administration) with an effective amount of any or more of the compounds or pharmaceutical compositions of the instant invention. In NAFLD and MASH, intestinal FXR antagonists have demonstrated the ability to reduce hepatic steatosis (fat accumulation) and inflammation, thereby offering an alternative or complement to systemic FXR agonists.

[0261] In certain embodiments, methods are provided for treating certain types of cancers. FXR has been implicated in the regulation of cell proliferation and apoptosis, making it a target for cancer therapy. In particular, FXR antagonists have shown promise in preclinical studies for the treatment of hepatocellular carcinoma, a common type of liver cancer. Therefore, in certain embodiments, methods are provided for treatment of FXR-dependent cancers, the method comprising the step of administering one or more of the bile acid cysteamides described herein (or a pharmaceutically acceptable precursor, pro-drug or salt thereof).

[0262] BA cysteamides have applications in cardiovascular diseases. FXR activation is involved in the regulation of lipid metabolism, and its dysregulation is associated with atherosclerosis and other cardiovascular conditions. Therefore, in certain embodiments, methods are provided for treatment of atherosclerosis and other cardiovascular conditions associated with dysregulation of lipid metabolism, the method comprising the step of administering one or more of the bile acid cysteamides described herein (or a pharmaceutically acceptable precursor, pro-drug or salt thereof). In certain embodiments, the method comprises treating a patient at risk of cardiovascular disease with one or more of the bile acid cysteamides described herein (or a pharmaceutically acceptable precursor, pro-drug or salt thereof) to improve lipid profiles and reduce cardiovascular risk.

[0263] It has unexpectedly found that administration of BA cysteamides selectively inhibits FXR receptors in the intestine with little or no effect on FXR receptors in other tissues. This surprising result distinguishes the activity of the provided compounds from other known FXR inhibitors which are not tissue selective and are known to have side effects some of which are likely due to FXR interactions in non-disease related tissues. This discovery enables novel methods wherein administration of the compounds or pharmaceutical compositions describe above leads to selective antagonism of intestinal FXR receptors. Without being bound by theory, it is believedthis can provide therapeutics with lower risks of side effects. By selectively interacting with FXR receptors in the intestine, such compounds and compositions provide superior treatments for diseases associated with glucose, lipid, and / or cholesterol metabolism or the regulation or dysfunction thereof. Intestinal Farnesoid X Receptor (FXR) antagonists are emerging as a promising therapeutic class, primarily targeting metabolic and liver diseases. Unlike systemic FXR modulators, these intestine-specific agents aim to harness the gut-liver axis without causing hepatic side effects like cholestasis.

[0264] In certain embodiments, such methods are characterized in that antagonism of intestinal FXR receptors resulting from treatment with the provided compounds or compositions is at least 1.5 times greater, at least 2 times greater, at least 5 times greater, or at least 10 times greater than antagonism of FXR receptors in at least one non-intestinal tissue. In certain embodiments, such methods are characterized in that antagonism of intestinal FXR receptors is at least 1.5 times greater, at least 2 times greater, at least 5 times greater, or at least 10 times greater than antagonism of FXR receptors in the liver. In certain embodiments, such methods comprise oral administration of BA cysteamides (or a pharmaceutically acceptable precursor, pro-drug or salt thereof). In certain embodiments, such methods comprise oral administration of a provided BA cysteamide formulation characterized in that it is stable / protected from degradation or hydrolysis in the stomach.

[0265] In a related vein, it has been demonstrated that BA cysteamides are rapidly oxidized to S-oxides and S-dioxides outside of the intestine. This allows for developing pro-drugs for oxidized BA cysteamides that target receptors outside of the intestine, oral administration of the unoxidized BA cysteamide would then lead to circulation of the active BA cysteamide oxides and / or dioxides outside of the intestine where they will interact with the target non-intestinal receptors.

[0266] BA-MCYs also have effects on the microbiota. This is based on the finding that microbiota of VNN1 KO mice (which lack BA-MCYs) have an altered gut microbiome.

[0267] BA-MCYs also have effects on IBD / colitis. This is based on the finding that VNN1 overexpression is protective of these diseases.

[0268] In another aspect, the present invention provides methods of modulating the gut microbiome of a mammal (e.g. a human). An unexpected modulation of the composition of the gut microbiome by BA cysteamides has been discovered. Therefore, BA cysteamides may beused as a therapeutic, or probiotic to modulate the gut microbiome composition to improve the health of a mammal. In certain embodiments, the method comprises treating a mammal with one or more of the BA cysteamides described herein (or a pharmaceutically acceptable precursor, pro-drug or salt thereof) to modulate the composition of the mammal’s gut microbiome. In certain such embodiments, the modulation of the gut microbiome comprises a change in the relative abundances of microbes composing the mammal’s gut microbiome. In certain such embodiments, the modulation of the gut microbiome comprises a change in the metabolism or a change in the production of one or more metabolites by one or more microbes composing the mammal’s gut microbiome. In certain such embodiments the modulation of the gut microbiome results in changes of the mammal’s metabolism.

[0269] In related embodiments, the invention provides methods of treating GI disorders as well as metabolic disorders, including but not limited to non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), obesity, type-2 diabetes, metabolic syndrome, cardiovascular diseases, hypertriglyceridemia, cholestasis, hypocholesteremia, that have some degree of microbiota dependence. It has been demonstrated that BA cysteamides can alter the gut microbiome; as such, the compositions described herein also have utility in treating diseases such as IBD, colitis, NAFLD, and other metabolic disorders. In certain embodiments, the method comprises treating a patient with a GI or metabolic disorder with one or more of the BA cysteamides described herein (or a pharmaceutically acceptable precursor, pro-drug or salt thereof). In certain embodiments, the GI disorder comprises colitis. In certain embodiments, the GI disorder comprises IBD. In certain embodiments, the metabolic disorder comprises NAFLD or NASH. In certain embodiments, the metabolic disorder comprises cholestasis or hypocholesteremia. In certain embodiments the treatment comprises adding BA cysteamides to the patient’s diet (e.g., as a probiotic, or nutritional supplement). In certain embodiments, such methods are characterized in that they ameliorate the GI or metabolic disorder at least partially via modulation of the patient’s gut microbiome.

[0270] In another aspect, the present invention includes methods of modulating known bile acid receptors other than FXR. For example, in certain embodiments, provided methods comprise treating a mammal with BA cysteamides, (or their oxides or dioxides) to modulate TGR5 receptors. In certain embodiments, such treatments reduce body weight. In certain embodiments, such treatments increase GLP-1 secretion and improve insulin sensitivity and / or postprandialglycemic control. In certain embodiments, such treatments reduce hypertriglyceridemia. In certain embodiments, such treatments are provided as anti-inflammatory agents since TGR5 agonism switches macrophages away from the pro-inflammatory Ml phenotype towards the anti-inflammatory M2 phenotype. In certain embodiments, such treatments reduce or inhibit LPS-induced cytokine secretion in macrophages, and inhibit LPS-induced production of proinflammatory cytokines such as tumor necrosis factor-a (TNF-a), interleukin (IL)-la, IL-ip, IL-6 and IL-8 in these cells. These effects can help suppress constitutive low-grade inflammation which can lead to arthritis and contributes to the development of the metabolic syndrome.

[0271] In another aspect, the present invention includes methods of activating LXR by treating a mammal with BA cysteamides, (or their oxides or dioxides). The resulting LXR modulation can have beneficial effects, such as reduced fat accumulation and promotion of fat tissue browning. LXR activation increases cholesterol excretion and decreases its absorption therefore such treatments can lower cholesterol levels.

[0272] In accordance with another aspect of the instant invention, methods of modulating (e.g., increasing or decreasing) sternness and / or making a cell into a stem cell and / or increasing stem cell properties (e.g., ability to self-renew and / or differentiate into other cell types (e.g., compared to prior to treatment or to a control) in a cell are provided. The methods comprise contacting a cell with a compound or composition of the instant invention. In certain embodiments, the cell is a T cell. In certain embodiments, the cell is a CD8+ T cell. The method may be performed in vitro or in vivo (e.g., administered to a subject comprising the cell). In certain embodiments, the compound is CA-MCY, CDCA-MCY, or DCA-CY.

[0273] In certain embodiments, the method comprises contacting a cell or administering to a subject the compound or composition of the instant invention as a cancer immunotherapy. In certain embodiments, the compound or composition of the instant invention is administered with an checkpoint inhibitor or blockade (e.g., PD-1 and / or PD-L1 inhibitors). The efficacy ofPD-1 / PD-L1 inhibitors relies heavily on a subset of “stem-like” exhausted T cells. Drugs that preserve this sternness (e.g., TCF1 stabilizers, metabolic modulators like metformin, or IL-21) can prevent terminal exhaustion, replenish the effector pool, and significantly extend the durability of tumor remission. Accordingly, the compounds of the instant invention can be used to reduce or prevent terminal exhaustion, replenish the effector pool, and / or significantly extend the durability of tumor remission.In certain embodiments, the method comprises contacting a cell or administering to a subject the compound or composition of the instant invention for adoptive cell transfer and / or CAR-T therapy. Culturing T cells with agents that enforce sternness (like IL-15, IL-21, or specific epigenetic modifiers) before infusion creates CAR-T cells with superior persistence, proliferative capacity, and resistance to exhaustion, leading to better long-term tumor control. Thus, in certain embodiments, CAR-T cells are contacted with a compound or composition of the instant invention prior to administration of the CAR-T cells to a subject.

[0274] In certain embodiments, the method comprises contacting a cell or administering to a subject the compound or composition of the instant invention for treating, inhibiting, and / or preventing a viral infection, particularly a chronic viral infection. In certain embodiments, the viral infection is an HIV infection. In certain embodiments, the viral infection is a hepatitis B infection. In persistent infections like HIV and Hepatitis B, T cell exhaustion leads to viral escape. Promoting sternness allows the CD8+ T cell pool to self-renew and sustain antiviral pressure over time, preventing the depletion of virus-specific T cells and aiding in functional cure strategies. In certain embodiments, the method comprises administering the compound or composition of the instant invention to a subject who has a viral infection. In certain embodiments, the method comprises administering the compound or composition of the instant invention to a subject prior to a viral infection.

[0275] In certain embodiments, the method comprises contacting a cell or administering to a subject the compound or composition of the instant invention for improving vaccine efficacy. Agents that promote stem-like memory properties during vaccination can generate long-lived central memory T cells, providing more robust and durable protection against future pathogen encounters compared to short-lived effector responses. In certain embodiments, the methods comprise administering a compound or composition of the instant invention with a vaccine (in the same composition or separate composition; at the same time and / or sequentially).

[0276] In accordance with another aspect of the instant invention, methods for stimulating and / or increasing the activity of TGR5 are provided. In certain embodiments, the method comprises contacting a cell or administering to a subject a compound or composition of the instant invention. TGR5 (GPBAR1) is a G-protein coupled receptor that is known as a target of other bile acids. TGR5 (GPBAR1) agonists are potent therapeutic targets for metabolic, inflammatory,and liver diseases, primarily by leveraging the gut-liver-immune axis. In certain embodiments, the compound is CA-MCY, DCA-MCY, or DCA-MCYO.

[0277] In certain embodiments, the methods comprise treating, inhibiting, and / or preventing type 2 diabetes. TGR5 agonists stimulate enteroendocrine L-cells in the gut to secrete Glucagon-Like Peptide-1 (GLP-1). This enhances glucose-dependent insulin secretion and improves glucose homeostasis, similar to existing incretin-based therapies. In certain embodiments, the method comprises administering to a subject in need thereof a compound or composition of the instant invention.

[0278] In certain embodiments, the methods comprise treating, inhibiting, and / or preventing obesity. Activation of TGR5 in brown adipose tissue and muscle promotes energy expenditure by converting inactive thyroid hormone (T4) to active T3. This induces thermogenesis (heat production), helping to reduce body weight and fat accumulation. In certain embodiments, the method comprises administering to a subject in need thereof a compound or composition of the instant invention.

[0279] In certain embodiments, the methods comprise treating, inhibiting, and / or preventing inflammation and / or inflammatory bowel disease. TGR5 is highly expressed on immune cells like monocytes and macrophages. Agonists inhibit the production of pro-inflammatory cytokines (e.g., TNF-a, IL-1 P), offering treatment for inflammatory conditions such

[0280] as inflammatory bowel disease (IBD), colitis, and atherosclerosis. In certain embodiments, the method comprises administering to a subject in need thereof a compound or composition of the instant invention.

[0281] In certain embodiments, the methods comprise treating, inhibiting, and / or preventing liver disease and / or metabolic dysfunction-associated steatohepatitis (MASH). TGR5 agonists reduce hepatic inflammation and improve microcirculation, particularly in MASH. However, systemic activation can cause gallbladder filling. As explained herein, the compounds of the instant invention can act in as tissue-specific or non-systemic agonists to minimize this side effect. In certain embodiments, the method comprises administering to a subject in need thereof a compound or composition of the instant invention.

[0282] DEFINITIONSDefinitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein.

[0283] Additionally, general principles of organic chemistry, as well as specific functional moi eties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March ’s Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0284] Certain compounds of the present invention can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and / or diastereomers. Thus, inventive compounds and compositions thereof may be in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers. In certain embodiments, the compounds of the invention are enantiopure compounds. In certain other embodiments, mixtures of enantiomers or diastereomers are provided.

[0285] Furthermore, certain compounds, as described herein may have one or more double bonds that can exist as either a Z or E isomer, unless otherwise indicated. The invention additionally encompasses the compounds as individual isomers substantially free of other isomers and alternatively, as mixtures of various isomers, e.g., racemic mixtures of enantiomers.

[0286] In addition to the above-mentioned compounds per se, this invention also encompasses compositions comprising one or more compounds.

[0287] As used herein, the term “isomers” includes any and all geometric isomers and stereoisomers. For example, “isomers” include cis- and / ra / is-i somers, E- and Z- isomers, R- and 5-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. For instance, a compound may, in some embodiments, be provided substantially free of one or more corresponding stereoisomers, and may also be referred to as “stereochemically enriched.”Where a particular enantiomer is preferred, it may, in some embodiments be provided substantially free of the opposite enantiomer, and may also be referred to as “optically enriched.” “Optically enriched,” as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain embodiments the compound is made up of at least about 90% by weight of an enantiomer. In some embodiments the compound is made up of at least about 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.8%, or 99.9% by weight of an enantiomer. In some embodiments the enantiomeric excess of provided compounds is at least about 90%, 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.8%, or 99.9%. In some embodiments, enantiomers may be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses. See, for example, Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S. H., et al., Tetrahedron 332125 (1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p.

[0288] 268 (E. L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972).

[0289] The terms “halo” and “halogen” as used herein refer to an atom selected from fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), and iodine (iodo, -I).

[0290] The term “aliphatic” or “aliphatic group”, as used herein, denotes a hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridging, and spirofused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-30 carbon atoms. In certain embodiments, aliphatic groups contain 1-12 carbon atoms. In certain embodiments, aliphatic groups contain 1-8 carbon atoms. In certain embodiments, aliphatic groups contain 1-6 carbon atoms. In some embodiments, aliphatic groups contain 1-5 carbon atoms, in some embodiments, aliphatic groups contain 1-4 carbon atoms, in yet other embodiments aliphatic groups contain 1-3 carbon atoms, and in yet other embodiments aliphatic groups contain 1-2 carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0291] The term "unsaturated", as used herein, means that a moiety has one or more double or triple bonds.The terms “cycloaliphatic”, “carbocycle”, or “carbocyclic”, used alone or as part of a larger moiety, refer to a saturated or partially unsaturated cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having from 3 to 12 members, wherein the aliphatic ring system is optionally substituted as defined above and described herein.

[0292] Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl. In some embodiments, the cycloalkyl has 3-6 carbons. The terms “cycloaliphatic”, “carbocycle” or “carbocyclic” also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl or tetrahydronaphthyl, where the radical or point of attachment is on the aliphatic ring. In some embodiments, a carbocyclic group is bicyclic. In some embodiments, a carbocyclic group is tricyclic. In some embodiments, a carbocyclic group is polycyclic.

[0293] The term “alkyl,” as used herein, refers to saturated, straight- or branched-chain hydrocarbon radicals derived from an aliphatic moiety containing between one and six carbon atoms by removal of a single hydrogen atom. Unless otherwise specified, alkyl groups contain 1-12 carbon atoms. In certain embodiments, alkyl groups contain 1-8 carbon atoms. In certain embodiments, alkyl groups contain 1-6 carbon atoms. In some embodiments, alkyl groups contain 1-5 carbon atoms, in some embodiments, alkyl groups contain 1-4 carbon atoms, in yet other embodiments alkyl groups contain 1-3 carbon atoms, and in yet other embodiments alkyl groups contain 1-2 carbon atoms. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like.

[0294] The term “alkenyl,” as used herein, denotes a monovalent group derived from a straighter branched-chain aliphatic moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Unless otherwise specified, alkenyl groups contain 2-12 carbon atoms. In certain embodiments, alkenyl groups contain 2-8 carbon atoms. In certain embodiments, alkenyl groups contain 2-6 carbon atoms. In some embodiments, alkenyl groups contain 2-5 carbon atoms, in some embodiments, alkenyl groups contain 2-4 carbon atoms, in yet other embodiments alkenyl groups contain 2-3 carbon atoms, and in yet other embodimentsalkenyl groups contain 2 carbon atoms. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, and the like.

[0295] The term “alkynyl,” as used herein, refers to a monovalent group derived from a straighter branched-chain aliphatic moiety having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. Unless otherwise specified, alkynyl groups contain 2-12 carbon atoms. In certain embodiments, alkynyl groups contain 2-8 carbon atoms. In certain embodiments, alkynyl groups contain 2-6 carbon atoms. In some embodiments, alkynyl groups contain 2-5 carbon atoms, in some embodiments, alkynyl groups contain 2-4 carbon atoms, in yet other embodiments alkynyl groups contain 2-3 carbon atoms, and in yet other embodiments alkynyl groups contain 2 carbon atoms. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0296] The term “carbocycle” and “carbocyclic ring” as used herein, refers to monocyclic and polycyclic moieties wherein the rings contain only carbon atoms. Unless otherwise specified, carbocycles may be saturated, partially unsaturated or aromatic, and contain 3 to 20 carbon atoms. Representative carbocyles include cyclopropane, cyclobutane, cyclopentane, cyclohexane, bicyclo[2,2,l]heptane, norbornene, phenyl, cyclohexene, naphthalene, spiro[4.5]decane,

[0297] The term “aryl” used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic and polycyclic ring systems having a total of five to 20 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to twelve ring members. The term “aryl” may be used interchangeably with the term “aryl ring”. In certain embodiments of the present invention, “aryl” refers to an aromatic ring system which includes, but is not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term "aryl”, as it is used herein, is a group in which an aromatic ring is fused to one or more additional rings, such as benzofuranyl, indanyl, phthalimidyl, naphthimidyl, phenantriidinyl, or tetrahydronaphthyl, and the like.

[0298] The terms “heteroaryl” and “heteroar-”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to groups having 5 to 14 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. The term “heteroatom” refers to nitrogen,oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quatemized form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl and pteridinyl. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, di benzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4 / 7-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin-3(4H)-one. A heteroaryl group may be mono- or bicyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted. The term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.

[0299] As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7-14-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2 / 7-pyrrolyl), NH (as in pyrrolidinyl), or+NR (as in A-substituted pyrrolidinyl).

[0300] A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle”, “heterocyclyl”, “heterocyclyl ring”, “heterocyclicgroup”, “heterocyclic moiety”, and “heterocyclic radical”, are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3 / -indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[0301] As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

[0302] As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0303] In some chemical structures herein, substituents are shown attached to a bond which crosses a bond in a ring of the depicted molecule. This means that one or more of the substituents may be attached to the ring at any available position (usually in place of a hydrogen atom of the parent ring structure). In cases where an atom of a ring so substituted has two substitutable positions, two groups may be present on the same ring atom. When more than one substituent is present, each is defined independently of the others, and each may have a different structure. In certain cases where the substituent shown crossing a bond of the ring is -R, this has the samemeaning as if the ring were said to be “optionally substituted” as described in the preceding paragraph.

[0304] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH2)o-4R°; -(CH2)o-40R°; -0-(CH2)o-4C(0)OR°; -(CH2)O-4CH(OR°)2; -(CH2)O-4SR°; -(CH2)o-4Ph, which may be substituted with R°; -(CH2)o-40(CH2)o-iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -NO2; -CN; -N3; -(CH2)O-4N(R0)2; -(CH2)O-4N(R0)C(0)R0; -N(R°)C(S)R°; -(CH2)O-4N(R0)C(0)NR°2; -N(R°)C(S)NRO2; -(CH2)O-4N(R°)C(0)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(0)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2)O-4C(0)R°; -C(S)R°; -(CH2)O-4C(0)OR°; -(CH2)O-4C(0)N(R°)2; -(CH2)O-4C(0)SR°; -(CH2)o-4C(0)OSiR°3; -(CH2)o-40C(0)R0; -OC(0)(CH2)O-4SR-, SC(S)SR°; -(CH2)O-4SC(0)R°; -(CH2)O-4C(0)NR02; -C(S)NRO2; -C(S)SR°; -SC(S)SR°, -(CH2)O-40C(0)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)RO; -C(NOR°)R°; -(CH2)O-4SSR°; -(CH2)O-4S(0)2R°; -(CH2)O-4S(0)2OR°; -(CH2)O-40S(0)2R°; -S(O)2NR°2; -(CH2)O-4S(0)R°; -N(RO)S(O)2NR°2; -N(RO)S(O)2R°; -N(OR°)R°; -C(NH)NRO2; -P(O)2R°; -P(0)RO2; -OP(O)RO2; -OP(O)(OR°)2; SiR°3; -(C1-4 straight or branched alkylene)O-N(R°)2; or -(C1-4 straight or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, Ci-s aliphatic, -CH2PI1, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or polycyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0305] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)o-2R*, -(haloR*), -(CH2)O-20H, -(CH2)O.2OR*, -(CH2)O-2CH(OR*)2; -O(haloR’), -CN, -N3, -(CH2)o-2C(O)R’, -(CH2)O-2C(0)OH, -(CH2)O.2C(0)OR*, -(CH2)O-4C(0)N(R°)2; -(CH2)O.2SR’, -(CH2)O- 2SH, -(CH2)O-2NH2, -(CH2)O-2NHR’, -(CH2)O-2NR*2, -NO2, -SiR, -OSiR%, -C(O)SR* -(Ci-4straight or branched alkylene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selectedfrom Ci-4 aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0306] Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0307] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0, =S, =NNR*2, =NNHC(0)R*, =NNHC(0)0R’, =NNHS(0)2R*, =

[0308]

[0309] NR*, =N0R*, -O(C(R*2))2-3O-, or -S(C(R*2))2.3S-, wherein each independent occurrence of R is selected from hydrogen, Ci-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR*2)2-3O-, wherein each independent occurrence of R* is selected from hydrogen, Ci-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0310] Suitable substituents on the aliphatic group of R* include halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR*), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0311] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -R\ -NR^, -C(O)R+, -C(O)ORt, -C(O)C(O)Rt, -C(O)CH2C(O)R+, -S(O)2R+

[0312]

[0313] , -S(O)2NRf2, -C(S)NRf2, -CCNT^NR^, or -N^SCO)^; wherein each:is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R;, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0314] Suitable substituents on the aliphatic group of R:are independently halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR*), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2,wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CH2PI1, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0315] EXAMPLES

[0316] The following Examples are useful to confirm aspects of the disclosure described above and to exemplify certain embodiments of the disclosure.

[0317] Methods

[0318] Metabolite extraction from mouse brain. Intact mouse brain was frozen and stored at -80°C before processing. Frozen brain was crushed and grinded with a pre-chilled mortar and pestle. Dry ice was added to mortar and pestle throughout the homogenization process to prevent thawing. Resulting powdered brain samples were sonicated for 1 minute with 5 mL methanol in 20 mL glass scintillation vials, using 10 pL solvent per mg, followed by another 10 minutes of vigorous stirring. Extracts were pelleted at 5,000 g for 5 min, and supernatants were transferred to another 20 mL glass vials. Remaining pellets were further extracted with another 10 minutes of vigorous stirring in 5 mL ethanol. The supernatants were combined and then dried in a SpeedVac™ (ThermoFisher Scientific) vacuum concentrator. Dried materials were resuspended in 300 pL of methanol. Samples were pelleted at 5,000 g for 5 minutes and clarified extracts were transferred to fresh HPLC vials and stored at -20 °C until analysis.

[0319] Metabolite extraction from mouse serum samples. 800 pL of methanol were added to 200 pL of serum in 1.7 mL Eppendorf tubes. The tubes were sonicated for 1 minutes followed by another 10 minutes of vigorous stirring. Extracts were pelleted at 5,000 g for 5 minutes, and supernatants were transferred to 2 mL HPLC vials. Remaining pellets were further extracted with another 10 minutes of vigorous stirring in 0.5 mL ethanol. Extracts were pelleted at 5,000 g for 5 minutes, and the combined supernatants were then dried in a SpeedVac™ (ThermoFisher Scientific) vacuum concentrator. Samples were then resuspended in 150 pL of methanol.

[0320] Samples were pelleted at 5,000 g for 5 minutes and clarified extracts were transferred to fresh HPLC vials and stored at -20 °C until analysis.Analytical methods and equipment overview, (a) Mass spectrometry: High resolution LC-MS was performed on a Thermo Fischer Scientific Vanquish™ UHPLC system coupled with a Thermo Q-Exactive™ HF hybrid quadrupole-orbitrap high-resolution mass spectrometer equipped with a HESI ion source. Metabolites were separated using a water-acetonitrile gradient on a Thermo Scientific Hypersil GOLD™ C18 column (150 mm x 2.1 mm, particle size 1.8 pm) maintained at 40°C; solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile. The AZB gradient started at 1% B for 3 minutes after injection and increased linearly to 100% B at 20 minutes, then 100% B for 5 minutes, and down to 1% B for 3 minutes using a flow rate of 0.5 mL / minute was applied for a Cl 8 column. Mass spectrometer parameters: spray voltage 3.5 kV, capillary temperature 380°C, prober heater temperature 400°C; 60 sheath flow rate, 20 auxiliary flow rate, and one spare gas; S-lens RF level 50, resolution 240,000, AGC target 3 x 106. The instrument was calibrated weekly with positive and negative ion calibration solutions (ThermoFisher). Each sample was analyzed in negative and positive ionization modes using a m / z range of 100 to 800. (b) NMR spectroscopy: NMR spectroscopy was performed on a Varian INOVA 600 MHz NMR spectrometer (600 MHz 'H reference frequency, 151 MHz for13C) equipped with an HCN indirect-detection probe. Non-gradient phase-cycled dqfCOSY spectra were acquired using the following parameters: 0.6 s acquisition time; 400-600 complex increments; 8, 16 or 32 scans per increment. HSQC and HMBC spectra were acquired with these parameters: 0.25 s acquisition time, 200-500 increments, 8-64 scans per increment. 'H,13C-HMBC spectra were optimized for, / H, C = 6 Hz. HSQC spectra were acquired with or without decoupling. NMR spectra were processed and baseline corrected using MestreLabs MNOVA software packages.

[0321] Feature detection and characterization. LC-MS RAW fdes for all brain and serum samples were converted to mzXML format (centroid mode) using MSconvert (ProteoWizard), followed by analysis using the XCMS analysis feature in Metaboseek (metaboseek.com) based on the centWave XCMS algorithm to extract features1,2. Peak detection values were set as: 4 ppm, 3 to 20 peakwidth, 3 snthresh, 3 and 100 prefdter, FALSE fitgauss, 1 integrate, TRUE firstBaselineCheck, 0 noise, wMean mzCenterFun, -0.005 mzdiff XCMS feature grouping values were set as: 0.2 minfrac, 2 bw, 0.002 mzwid, 500 max, 1 minsamp, FALSE usegroup. Metaboseek peak filling values set as: 5 ppm m, 5 rtw, TRUE rtrange. Resulting tables of all detected features were then processed with the Metaboseek data explorer. To select differentialfeatures, we applied a filter retaining entries with peak area ratios smaller than 1 / 3 (down in GF mice) or larger than 3 (up in GF mice), with a retention time window of 1 to 20 min, and >0.97 Peak Quality as calculated by METABOseek3. We manually curated the resulting list to remove false positive entries, i.e., features that upon manual inspection of raw data were not differential. For verified differential features, we examined elution profiles, isotope patterns, and MSI spectra to find molecular ions and remove adducts, fragments, and isotope peaks. Remaining masses were put on the inclusion list for MS / MS (ddMS2) characterization. Positive and negative ionization mode data were processed separately. To acquire MS2 spectra, we ran a top-10 data dependent MS2 method on a Thermo Q-Exactive™-HF mass spectrometer with MSI resolution 60,000, AGC target 1 x 106, maximum IT (injection time) 50 ms, MS2 resolution 45,000, AGC target 5 x 105, maximum IT 80 ms, isolation window 1.0 mz, stepped NCE (normalized collision energy) 10 and 30 for positive and negative ionization mode, dynamic exclusion 3 s.

[0322] References. The following references are relevant to certain aspects of the Examples presented herein. The entirety of each of these references, and their supporting information is incorporated herein by reference.

[0323] 1. Tautenhahn, R., Bottcher, C. & Neumann, S. Highly sensitive feature detection for high resolution LC / MS. BMC Bioinformatics 9, 1-16 (2008).

[0324] 2. Wang, M. et al. Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nat. Biotechnol. 34, 828-837 (2016).

[0325] 3. Helf, M. J., Fox, B. W., Artyukhin, A. B., Zhang, Y. K. & Schroeder, F. C. Comparative metabolomics with Metaboseek reveals functions of a conserved fat metabolism pathway in C. elegans. Nat. Commun. 13, 782 (2022).

[0326] 4. Shannon, P. et al. Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks. Genome Res. 13, 2498-2504 (2003).

[0327] Example 1. Identification of certain compounds of the invention using MS2-based molecular networking

[0328] Here we utilize untargeted metabolomics to compare germ-free (GF) and microbiota-replete specific pathogen-free (SPF) mice to uncover a host-mediated BA modification generating potent FXR antagonists that act as intestinal regulators of BA metabolism.Comparative metabolomics reveals unannotated BA derivatives Lack of microbial deconjugation results in increased levels of BA-taurine conjugates in GF mice, while abundances of free BAs are dramatically reduced. We hypothesized that abundances of yet unannotated BA derivatives may be similarly affected by the absence of the microbiota. To discover such unannotated compounds, we first obtained a comprehensive overview of microbiota-dependent changes in the mouse serum metabolome via high-resolution mass spectrometry-(HRMS-) based comparative metabolomics of samples from microbiota-replete specific pathogen-free (SPF) and GF mice (Fig.lA). The resulting datasets were processed using the xcms-based (Tautenhahn, et al., Anal. Chem. 84:5035-5039 (2012)) Metaboseek platform, which facilitates identification of MS features whose abundances differ significantly between different conditions (Fig. IB). This untargeted comparison revealed stark differences between GF and SPF mice. In total we detected more than 40,000 MS features from combined analyses of serum samples in positive and negative ionization modes, of which -10% were significantly differential (at p < 0.05) between GF and SPF mice. To prioritize among the large number of differential features, we focused on compounds that were robustly detected in all replicates (see Methods) and at least five-fold different in GF relative to SPF samples. Using these stringent criteria, we detected several hundred microbiota-dependent metabolites in the serum metabolome. For further characterization, we acquired tandem mass spectrometry (MS2) data for all differential metabolites. To focus on BAs and BA derivatives, we applied a permissive molecular formula filter that required the presence of MS2 fragments containing a complete 24-carbon backbone, which would indicate the presence of a steroid backbone (see Methods). MS2 networking revealed three major clusters of microbiota-dependent metabolites with fragmentation patterns indicating they represent BAs or BA-derivatives (Fig. 1C, and Fig. 6). Two of these clusters represented BA-taurine conjugates and free BAs, whose abundances were greatly increased and decreased in GF mice, respectively, as expected, given the lack of microbial taurine deconjugation. In contrast, the third cluster appeared to represent a family of previously unannotated BA derivatives, whose abundances, similar to free BAs, were reduced in GF mice.

[0329] Detailed analysis of the MS isotope patterns and MS2 spectra of the putative BA derivatives indicated the presence of an S-methylcysteamine (MCY) moiety (Fig. ID and Fig. 7). The MS2 spectra further indicated that these MCY derivatives belong to three different series, representing putative bile acid methylcysteamides (BA-MCY), corresponding sulfoxides (BA-MCYO) and sulfodioxides (BA-MCY02) (Fig. 7). Structures of these BA-MCYs were proposed based on the relative abundances and retention times of free bile acids in the analyzed samples and confirmed via synthesis of authentic standards, which led to the identification of a total of 18 BA derivatives, including the MCY, MCYO, and MCY02 derivatives of cholic acid (CA), 0-muricholic acid (0MCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), deoxycholic acid (DCA), and 7-ketodeoxy cholic acid (7-KDCA) (Figs. IE, IF, 1G, and 8, and Table 2, see Examples 5 and 6 for synthetic procedures and NMR data). Taken together, our untargeted metabolomic comparison of SPF and GF mice revealed BA-MCYs as a previously unannotated family of microbiota-dependent BA derivatives.

[0330] Compound Observed Calculated ion Calculated Ret. time Schym.

[0331] (m / z) Ion formula m / z (min) ID level CA-MCY (1) 482.3294 [M+H]+C2? H48NO4S+482.3299 12.4 Level 1 CA-MCYO (2) 498.3250 [M+H]+C27H4BNO5S+498.3248 10.8 Level 1 CA-MCYO2 (3) 514.3207 [M+H]+C27H48NO6S+514.3197 11.3 Level 1 aMCA-MCY (4) 482.3294 [M+H]+C27H48NO4S+482.3299 11.6 Level 2 aMCA-MCYO (5) 498.3250 [M+H]+C27H48NOSS+498.3248 10.0 Level 2 pMCA-MCY (6) 482.3294 [M+H]+C27H48NO4S+482.3299 11.7 Level 1 pMCA-MCYO (7) 498.3250 [M+H]+C27H48NOSS+498.3248 10.2 Level 1 PMCA-MCYO2 (8) 514.3207 [M+H]+C27H48NOgS+514.3197 10.8 Level 1 wMCA-MCY (9) 482.3294 [M+H]+C27H48NO4S+482.3299 11.5 Level 2 wM CA-MCYO (10) 498.3250 [M+H]+C27H48NOSS+498.3248 9.8 Level 2 CDCA-MCY (11) 466.3349 [M+H]+C27H48NO3S+466.3349 13.9 Level 1 CDCA-MCYO (12) 482.3294 [M+H]+C27H48NO4S+482.3299 11.9 Level 1 UDCA-MCY (13) 466.3349 [M+H]+C27H48NO3S+466.3349 12.5 Level 1 UDCA-MCYO (14) 482.3294 [M+H]+C27H48NO4S+482.3299 10.7 Level 1 DCA-MCY (15) 466.3349 [M+H]+C27H4SNO3S+466.3349 14.1 Level 1 DCA-MCYO (16) 482.3294 [M+H]+C27H48NO4S+482.3299 12.1 Level 1 KDCA-MCY (17) 480.3141 [M+H]+C27H4eNO4S+480.3142 12.8 Level 1 KDCA-MCYO (18) 496.3093 [M+H]+C27H46NOSS+496.3091 10.1 Level 1 CDCA-cfe-MCY (19) 471.3654 [M+H]+C27H43D5NO3S+471.3663 13.9 Level 1 CA-CY (20) 468.3142 [M+H]+C26H46NO4S+468.3141 12.5 Level 1 CA-pant (21) 669.4140 [M+H]+ C35H6IN20SS+ 669.4143 12.6 Level 1

[0332]

[0333] Table 2. HPLC-HRMS data for compounds (1-21).

[0334] Example 2. Discovery of the Microbiota-dependence of BA-MCY conjugatesAbundances of BA-MCYs were strongly reduced in GF compared to SPF serum samples, but not abolished (Figs. 2A, 2B, and 10A). Similarly, BA-MCY levels were decreased in feces of GF mice compared to SPF mice (Fig. 10B-10E).

[0335] To better understand the relationship between BA-MCY levels and the presence of the microbiota, we next tested whether introduction of human microbiota into GF mice would affect BA-MCY production. For this purpose, we performed human fecal microbiota transfer (FMT) from healthy individuals into GF mice (Arifuzzaman, et al., Nature doi.org / 10.1038 / s41586-022-05380-y (2022)), and then profded BAs and BA conjugates in serum and feces. We found that abundances of both free BAs and BA-MCY conjugates were greatly increased in serum and feces of mice that received human FMT (Figs. 2A, 2B, and 10A-10D).

[0336] The results from our comparison of GF with SPF and human FMT mice indicated that BA-MCY levels are governed in part by levels of the corresponding free BAs. Changes in the microbiota induced by supplementation with inulin fiber can dramatically increase levels of free BAs in serum of SPF mice. We took advantage of this to test whether such a dietary intervention-based increase of free BAs would also affect BA-MCY levels. We observed that BA-MCY-levels were greatly increased in SPF mice fed an insulin-based high fiber diet (Figs.

[0337] 2C, 2D, and 5H), indicating that expansion of the free-BA pool leads to increased BA conjugation with MCY. Moreover, we found that levels of free BAs and BA-MCY conjugates in SPF mice fed different diets are generally correlated (Figs. 2E, 51, and 5 J). Finally, to determine whether BA-MCYs are also present in humans, we analyzed human serum samples, which revealed MCY derivatives of all major bile acids common in humans (Fig. 2F, 5K, and 5L). Collectively, these data indicate that BA-MCY conjugates are present in mouse and human, and that increases of free BA levels, following human FMT into GF mice or as a result of supplementation with dietary fiber, are associated with parallel increases of the corresponding BA-MCY conjugates.

[0338] Microbial and host metabolism of BA-MCY conjugates

[0339] Taurine and glycine BA conjugates are efficiently deconjugated in the gut by microbial bile salt hydrolases (BSHs), generating free BAs. Correspondingly, the ratio of BA-taurine conjugates to free BAs was dramatically increased in feces of GF compared to SPF mice (Fig. 3 A). Considering the possibility that the gut microbiota may play a role also in the deconjugationof BA-MCYs, we noted that, even though BA-MCY levels are reduced in GF mice, the ratio of BA-MCY conjugates to free BAs was greatly increased in GF compared to SPF fecal samples (Fig. 3 A). In fact, BA-MCY levels were similar to or exceeded levels of free BAs in feces of GF mice (Fig. 3B).

[0340] To determine whether BA-MCYs can indeed be deconjugated by the microbiota, we analyzed fecal and liver samples from SPF and GF mice supplemented with stable-isotope labeled CDCA-d5-MCY for the presence of free labeled CDCA and other labeled BAs that can be derived from CDCA. To broadly survey metabolism of BA-MCYs, we additionally compared supplemented and control mice via untargeted metabolomics using the Label Finder approach in the Metaboseek platform. Targeted analysis of fecal samples from CDCA-d5-MCY-supplemented SPF mice revealed CDCA-d5-MCY as well as d4- and d5-labeled free CDCA (Fig. 3C), indicating that BAMCYs can be deconjugated in the gut. In addition, we detected labeled versions of other free BAs that can be derived from CDCA whereas CA, DCA, and other CA-derived BAs remained unlabeled, consistent with their separate biosynthetic pathway.

[0341] Analysis of fecal and liver samples from CDCAd5- MCY-supplemented SPF mice further indicated that labeled free BAs derived from supplemented CDCA-d5-MCY are partly reconjugated with taurine. Label Finder analysis additionally revealed that the remainder of supplemented CDCA-d5-MCY that was not deconjugated was converted into its oxidized derivative, CDCA-d4 / 5-MCYO, and, to a lesser extent, CDCA-d4 / 5-MCYO2. In fact, only trace amounts of CDCA-d5-MCY could be detected in the liver of supplemented mice, indicating that supplemented CDCA-d5-MCY is quickly oxidized to CDCA-d4 / 5-MCYO(2).

[0342] In contrast to SPF mice, deconjugation-derived, labeled CDCA or other labeled free BAs were not detected in GF mice supplemented with CDCA-d5-MCY (Fig. 3D), indicating that deconjugation of CDCA-MCY is microbiota-dependent. Similarly, suppression of microbiota in SPF mice treated with antibiotics (ABX) resulted in significantly reduced deconjugation of supplemented CDCA-d5-MCY compared to SPF mice (Fig. 3D). In ABX and GF mice, supplemented CDCA-d5-MCY was instead primarily converted into the corresponding oxidation products, CDCAd4 / 5-MCYO and CDCA-d4 / 5-MCYO2.

[0343] Next, we demonstrated that BA-MCY conjugates are deconjugated by fecal suspensions obtained from SPF mice and individual gut bacteria known to harbor the BSH gene. To determine whether BSH is required for BA-MCY deconjugation, we tested gnotobiotic micecolonized with Bacteroides ovatus ATCC 8483 (WT Bo) or a B. ovatus mutant strain in which we deleted the BSH-encoding gene B0 02350 (Absh Bo). We found that CDCA-d5-MCY was partially deconjugated in the gnotobiotic mice colonized with WT Bo, but was not deconjugated in mice colonized with mutant Absh Bo (Fig. 3E), where the supplemented CDCA-d5-MCY was exclusively converted to the oxidized CDCA-d4 / 5-MCYO(2), as in GF mice. These results indicate that BSH of gut microbiota can deconjugate BA-MCY conjugates, albeit less efficiently than the corresponding taurine conjugates (Fig. 3E), and that, in the absence of microbiota, BA-MCY conjugates are metabolized by the host into the corresponding BA-MCYO and BA-MCY02 derivatives (Fig. 3F).

[0344] Example 3. Discovery that BA-MCYs act as potent FXR antagonists in vitro

[0345] For functional evaluation of the BA-MCY conjugates, we focused on the FXR, one of the major endogenous targets of BAs in vertebrates. Free BAs, e.g., the broadly conserved CDCA, CA, and DCA14, as well amino acid conjugates of CA43, function as potent FXR agonists that negatively regulate BA production. In contrast, it is unclear whether there are any conserved endogenous FXR antagonists that would promote BA production. To test for potential FXR agonist or antagonist activity of the identified BA-MCY conjugates, we selected four derivatives, CA-MCY, CAMCYO, 0MCA-MCY, and CDCA-MCY based on their relative abundance in SPF mouse serum samples and considering FXR agonist activity of the corresponding free BAs. We assayed these four compounds in agonist and antagonist modes using a protein-protein interaction assay between the full-length human FXR protein and a Steroid Receptor Coactivator Peptide- (SRCP-) derived nuclear fusion protein. Whereas none of the tested conjugates showed agonist activity at any of the tested concentrations (Fig. 4), very surprisingly, CDCA-MCY, CA-MCY, and PMCA-MCY showed potent antagonistic activity with IC50 values of 1.68, 19.9, and 104.5 pM, respectively, against GW4604-mediated activation of FXR (Fig. 4A-4C). In contrast, CA-MCYO was inactive, indicating that sulfur oxidation abolished antagonistic activity (Fig. 4D). CDCA-MCY also inhibited FXR activation mediated by CDCA and the more potent synthetic BA, obeticholic acid. In parallel, we also tested T0MCA, which is a weak, murine-specific FXR antagonist. However, T0MCA was inactive at the tested range of concentrations in this assay. These results demonstrate that BA-MCY conjugates function as endogenous FXR antagonists.Example 4. Discovery that BA-MCY supplementation shows FXR antagonistic effects in vivo BA biosynthesis in the liver is controlled by a complex signaling network regulated by hepatic and intestinal FXR via distinct pathways (Pandak, et al., Liver Res. 3:88-98 (2019); Chiang, J. Y., J. Lipid Res. 50:1955-1966 (2009); Al-Dury, et al., J. Hepatol. 64: S436 (2016)) (Fig. 5A). In the liver, FXR agonists promote expression of short heterodimer partner (SHP), which in turn antagonizes expression of Cyp7al, a cytochrome P450 enzyme required for the first and rate-limiting step in BA synthesis (Pandak, et al., Liver Res. 3:88-98 (2019); Chiang, J. Y., J. Lipid Res. 50:1955-1966 (2009); Al-Dury, et al., J. Hepatol. 64: S436 (2016)) (Fig. 5A). In addition, SHP expression suppresses Cyp8bl, which catalyzes the conversion of the BA precursor 7a-hydroxy-4-cholesten-3-one into 7a,12a-dihydroxy-4-cholesten-3-one and thereby controls the balance between the relative amounts of bile acids that are 12a-hydroxylated (such as CA) and bile acids that are not 12a-hydroxylated (such as CDCA). In contrast, intestinal FXR activation promotes production of ileal hormone fibroblast growth factor 15 (FGF15, FGF19 in humans), a signaling peptide that via the enterohepatic circulation travels to the liver to suppress expression of Cyp7al. Conversely, FXR antagonists promote BA synthesis by relieving repression of Cyp7al and Cyp8bl by suppressing SHP and FGF15 / 19 expression (Sayin, et al., Cell Metab. 17:225-235 (2013); Yu, et al., Eur. J. Pharmacol., 962:176220 (2024); Yao, et al., eLife doi.org / 10.7554 / eLife.37182 (2018); Al-Dury, et al., J. Hepatol. 64: S436 (2016)) (Fig. 5A).

[0346] To determine whether BA-MCYs affect FXR-dependent regulation of BA biosynthesis in vivo, we supplemented SPF mice via oral gavage with CDCA-MCY, which had shown the highest potency in our in vitro FXR antagonist assay (Fig. 4A). Gene expression analysis indicated that Cyp7al and Cyp8bl expression in the liver was significantly increased (Fig. 5B). In addition, we found that ileal Shp mRNA and serum FGF15 levels were significantly decreased in mice supplemented with CDCAMCY (Fig. 5C, 5D), indicating that increased expression of Cyp7al is in part due to antagonism of the intestinal FXR-FGF15 pathway. Increased Cyp8bl expression, which is largely independent of the intestinal FXRFGF15 pathway, indicates that liver FXR may also be affected by CDCA-MCY supplementation, or that other pathways contribute to FXR-dependent ileum-to-liver signaling. Because we found that BAMCY production may be dependent on reabsorption of free BAs from the ileum, we additionally testedwhether CDCA-MCY supplementation affects expression of Slcl 0a2, the transporter mediating BA re-uptake from the gut; however, Slcl0a2 expression was unchanged (Fig. 5E).

[0347] To measure effects of CDCA-MCY on BA production in vivo, we conducted additional supplementation studies using stable isotope labeled CDCA-d5-MCY. The use of labeled CDCA-d5-MCY avoided potentially confounding effects arising from deconjugation and further metabolism of the supplemented CDCA-MCY, as it allowed us to distinguish unambiguously between BAs derived from the supplemented, labeled CDCA-d5-MCY and de novo-produced, unlabeled BAs (Fig. 31). Quantification of BA levels from CDCA-d5-MCY-supplemented animals showed a strong increase of unlabeled CDCA-derived BAs in fecal samples (Fig. 5F). Similarly, levels of CA-derived BAs were increased in fecal samples of animals supplemented with CDCA-d5-MCY or CDCA-MCY (Fig. 5G). Fecal BA levels were also increased by CDCA-d5-MCY-supplementation in ABX mice (Fig. 5H). Given that levels of unlabeled BAs in liver and serum of supplemented mice were not significantly changed, the large increase in fecal excretion of both CA- and CDCA-family BAs indicates strong upregulation of BA production in CDCA-MCY-supplemented animals, in line with the increased expression of BA biosynthesis genes (Fig. 5B) (Inagaki, et al., Cell Metab. 2:217-225 (2005); Kim, et al., J. Lipid Res. 48:2664-2672 (2007); Cheng, et al., Oncotarget 9:25572-25585 (2018); Ito, et al., J. Clin. Invest.

[0348] 115:2202-2208 (2005)).

[0349] Next we tested whether upregulation of BA synthesis by CDCA-MCY supplementation is in fact FXR dependent. We found that CDCA-MCY supplementation increased fecal BA abundance in WT mice but not in FXR-deficient (Nrlh4- / -) mice (Fig. 51). BA levels in liver and serum of WT and Nrlh4- / - mice were not significantly affected by CDCA supplementation, consistent with results from our initial supplementation study.

[0350] These data demonstrate that CDCA-MCY supplementation increases BA biosynthesis in an FXR-dependent manner. Given that intestinal FXR antagonists have been shown to alleviate hepatic steatosis in mouse models of obesity, we asked whether CDCA-MCY supplementation could improve lipid accumulation in the liver of mice fed a high cholesterol diet (HCD). Liver histology and oil red-0 staining revealed greatly decreased hepatic lipid accumulation in CDCA-MCY-supplemented HCD-fed mice compared to untreated HCD-fed mice (Fig. 5 J, 5K), consistent with studies of synthetic compounds acting as intestinal FXR antagonists (Jiang, et al.,Nat. Commun, 6:10166 (2015); Kuang, et al., Cell Metab. 35:1752-1766.e8 (2023)). We observed similar effects when CDCA-MCY was supplied at a 10-fold lower dose.

[0351] Taken together, our results support a model in which host-derived BA-MCY conjugates act as intestinal FXR antagonists that balance the FXR agonistic activity of microbiota-derived free BAs, as part of a regulatory circuitry that fine tunes BA signaling within the hepatobiliary system (Fig. 5L).

[0352] Example 5. Synthesis of certain compounds of the invention

[0353] General synthetic procedures. Unless noted otherwise, all chemicals and reagents were purchased from Sigma-Aldrich. Solutions and solvents sensitive to moisture and oxygen were transferred via standard syringe and cannula techniques. Acetic acid (AcOH), acetonitrile (ACN), dichloromethane (DCM), and methanol (MeOH) used for chromatography and as a reagent or solvent were purchased from Fisher Scientific. Flash chromatography was performed using Teledyne Isco CombiFlash systems and Teledyne Isco RediSep Rf silica and C18 columns.

[0354] Example 5.1 Synthesis of CA-MCYs (1, 6, 11, 13, 15, and 17) and BA-MCYOs (2, 7, 12, 14, 16, and 18)

[0355] 1a R1= H, R2= OH, a R3= OH, I R1= H, R2= OH, a R3= OH, 2 R1= H, R2= OH, a R3= OH, 6a R1- OH, R2- OH, p R3= H, 6 R1= OH, R2= OH, p R3= H, 7 R1= OH, R2= OH, p R3= H, 11a R1= H, R2= OH, a R3= H, II R1= H, R2= OH, a R3= H, 12 R1= H, R2= OH, a R3= H, 13a R1= H, R2= OH, p R3= H, 13 R1= H, R2= OH, p R3= H, 14 R1= H, R2= OH, p R3= H, 15a R1= H, R2= H, R3= OH 15 R1= H, R2= H, R3= OH 16 R1= H, R2= H, R3= OH

[0356]

[0357] 17a R1= H, R2= O(-H2), R3= OH 17 R1= H, R2= O(-H2), R3= OH 18 R1= H, R2= O(-H2), R3= OH (a) To a stirred solution of cholic acid (la, Sigma, 204 mg, 0.5 mmol), 4-dimethylaminopyridine (Sigma, 244 mg, 2 mmol, 4.0 equiv.), and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (TCI, 192 mg, 1 mmol, 2.0 equiv.) in 1 mL of di chloromethane was added 5-methyl cysteamine (lb, Sigma, 55 mg, 0.6 mmol, 1.1 equiv ). The reaction mixture was stirred at room temperature for 24 hr and concentrated in vacuo. Purification by flash chromatography on a reverse-phase column (Cl 8) using a gradient of 30-80 % acetonitrile in 0.1% acetic acid afforded CA-MCY (1, 200 mg, 82 %). The same procedure was performed with 0-muricholic acid (6a, Sigma, 15 mg, 0.037 mmol), chenodeoxycholic acid (Ila, Sigma, 186 mg, 0.5 mmol), ursodeoxycholic acid (13a, Sigma, 186 mg, 0.5 mmol), deoxycholic acid (15a, Sigma, 93 mg, 0.25 mmol), and 7-ketodeoxycholic acid (17a, Sigma, 20 mg, 0.05 mmol) as an alternative to cholic acid for the synthesis of 0MCA-MCY (6, 17 mg, 94 %), CDCA-MCY (11, 200 mg, 82 %), UDCA-MCY (13, 200 mg, 82 %), DCA-MCY (15, 95 mg, 79 %), and KDCA-MCY (17, 21 mg, 86 %), respectively.

[0358] CA-MCY (1): HRMS (ESI) m / z: [M+H]+calcd for C27H48NO4S+482.3299; found 482.3294. 0MCA-MCY (6): HRMS (ESI) m / z: [M+H]+calcd for C27H48NO4S+482.3299; found 482.3294. CDCA-MCY (11): HRMS (ESI) m'z: [M+H]+calcd for C27H48NO3S+466.3349; found 466.3345. UDCA-MCY (13): HRMS (ESI) m / z: [M+H]+calcd for C27H48NO3S+466.3349; found 466.3344. DCA-MCY (15): HRMS (ESI) m / z: [M+H]+calcd for C27H48NO3S+466.3349; found 466.3342. KDCA-MCY (17): HRMS (ESI) z: [M+H]+calcd for C27H46NO4S+480.3142; found 480.3137. See FIGS. 12-71 for NMR spectroscopic data.

[0359] (b) To a stirred solution of CA-MCY (1, 90 mg, 0.19 mmol) in 1 mL of dichloromethane was added te / 7-butyl hydroperoxide solution 5.0-6.0 M in decane (Sigma, 0.3 ml, 1.5 mmol, 7 equiv.). The reaction mixture was stirred at room temperature for 24 hr and concentrated in vacuo. Purification by flash chromatography on a reverse-phase column (Cl 8) using a gradient of 20-80 % acetonitrile in 0.1 % acetic acid afforded CA-MCYO (2, 80 mg, 85 %). The same procedure was performed with 0MCA-MCY (6, 49 mg, 0.1 mmol), CDCA-MCY (11, 48 mg, 0.1 mmol), UDCA-MCY (13, 48 mg, 0.1 mmol), DCA-MCY (15, 24 mg, 0.05 mmol), and KDCA-MCY (17, 12 mg, 0.025 mmol) as an alternative to CA-MCY for synthesis of 0MCA-MCYO (7, 45 mg, 89 %), CDCA-MCYO (12, 44 mg, 89 %), UDCA-MCYO (14, 42 mg, 85 %), DCA-MCYO (16, 19 mg, 80 %), andKDCA-MCYO (18, 11 mg, 88 %), respectively.

[0360] CA-MCYO (2): HRMS (ESI) m / z: [M+H]+calcd for C27H48NO5S+498.3248; found 498.3250.

[0361] 0MCA-MCYO (7): HRMS (ESI) m z: [M+H]+calcd for C27H48NO5S+498.3248; found 498.3251. CDCA-MCYO (12): HRMS (ESI) m / z: [M+H]+calcd for C27H48NO4S+482.3299; found 482.3294.

[0362] UDCA-MCYO (14): HRMS (ESI) m^z: [M+H]+calcd for C27H48NO4S+482.3299; found 482.3294.

[0363] DCA-MCYO (16): HRMS (ESI) m / z: [M+H]+calcd for C27H48NO4S+482.3299; found 482.3294.KDCA-MCYO (18): HRMS (EST) m / r. [M+H]+calcd for C27H46NO5S+496.3091; found 496.3093.

[0364] See Example 6 and Figs. 12-71 for NMR spectroscopic data.

[0365] Example 5.2 Synthesis of CA-MCYO2 (3) and pMCA-MCYO2 (8)

[0366] 1a R1= H, R2= OH, a R3= OH, 3 R1= H, R2= OH, a R3= OH, 6

[0367]

[0368] a R1= OH, R2= OH, p R3= H, 8 R1= OH, R2= OH, p R3= H, To a stirred solution of cholic acid (la, Sigma, 204 mg, 0.5 mmol), 4-dimethylaminopyridine (Sigma, 366 mg, 3 mmol, 6.0 equiv.), and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (TCI, 192 mg, 1 mmol, 2.0 equiv.) in 1 mL of dimethylformamide was added 2-(methylsulfonyl)ethanamine hydrochloride (1c, Synthonix, 96 mg, 0.6 mmol, 1.1 equiv.). The reaction mixture was stirred at room temperature for 24 hr and concentrated in vacuo. Purification by flash chromatography on a reverse-phase column (Cl 8) using a gradient of 30-80 % ACN in 0.1 % acetic acid afforded CA-MCYO2 (3, 180 mg, 75 %). The same procedure was performed with PMC A (6a, Sigma, 41 mg, 0.1 mmol) as an alternative to CA for synthesis of PMCA-MCYO2 (8, 40 mg, 83 %).

[0369] CA-MCYO2 (3) HRMS (ESI) m / r. [M+H]+calcd for C27H48NO6S+514.3197; found 514.3207. P

[0370]

[0371] MCA-MCYO2 (8): HRMS (ESI) [M+H]+calcd for C27H48NO5S+514.3197; found 514.3205.

[0372] See Example 6 and Figs. 12-71 for NMR spectroscopic data.

[0373] Example 5.3 Synthesis of CDCA-cfc-MCY (19)o o

[0374]

[0375] To a stirred solution of chenodeoxycholic acid (2,2,3,4,4-t / s) (19a, Cambridge isotope laboratories, 42 mg, 0.1 mmol), 4-dimethylaminopyridine (Sigma, 49 mg, 0.4 mmol, 4.0 equiv.), and 1 -(3 -dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride (Sigma, 38 mg, 0.2 mmol, 2.0 equiv.) in 1 mL of dichloromethane was added 5-methylcysteamine (1c, Sigma, 20 mg, 0.2 mmol, 2.0 equiv.). The reaction mixture was stirred at room temperature for 24 hr and concentrated in vacuo. Purification by flash chromatography on silica using a gradient of 0-30 % methanol in dichloromethane afforded CDCA-t / s-MCY (19, 42 mg, 84 %).

[0376] CDCA-t / 5-MCY (19) HRMS (ESI) m / z [M+H]+calcd for C27H43D5NO3S+471.3663; found 471.3654.

[0377] CDCA s-MCY (19) 'HNMR, 600 MHz, methanol-^: 8 (ppm) 3.78 (m, 1H), 3.35 (m, 3H), 2.58 (t, 7.0 Hz, 2H), 2.24 (m, 1H), 2.10 (m, 1H), 2.10 (s, 3H), 1.97 (m, 2H), 1.85 (m, 4H), 1.73 (m, 1H), 1.50 (m, 5H), 1.30 (m, 4H), 1.20 (m, 2H), 1.09 (m, 1H), 0.98 (m, 4H), 0.93 (s, 3H), 0.70 (s, 3H).

[0378] Example 5.4 Synthesis of CA-CY (20)

[0379] o

[0380]

[0381] (a) To a stirred solution of solution of cholic acid (la, Sigma, 102 mg, 0.25 mmol), 4-dimethylaminopyridine (Sigma, 30.5 mg, 0.25 mmol, 1.0 equiv.), and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (TCI, 78 mg, 0.5 mmol, 2.0 equiv.) in1 mL of dichloromethane was added cysteamine (20a, Sigma, 39 mg, 0.5 mmol, 2.0 equiv ). The reaction mixture was stirred at room temperature for 24 hours and concentrated in vacuo.

[0382] Purification by flash chromatography on silica using a gradient of 0-30 % methanol in dichloromethane afforded 20b (75 mg, 35 %).

[0383] 20b HRMS (ESI) m / r. [M+H]+calcd for C5oH84N08S+858.5912; found 858.5909.

[0384] 20b 'HNMR, 600 MHz, methanol-c / 4: 8 (ppm) 3.95 (m, 2H), 3.80 (m, 2H), 3.35 (m, 4H), 3.01 (m, 2H), 2.62 (m, 1H), 2.52 (m, 1H), 2.07-2.32 (m, 6H), 1.70-2.04 (m, 15H), 1.50-1.69 (m, 13H), 1.25-1.46 (m, 11H), 1.12 (m, 2H), 1.02 (m, 6H), 0.98 (m, 2H), 0.92 (s, 6H), 0.71 (s, 6H).

[0385] (b) To a stirred solution of 20b (17 mg, 0.02 mmol) in ImL of methanol was added sodium hydroxide (Sigma, IM in water, 0.03mL, 0.03mmol, 1.5 equiv.) followed by ImL of water. The reaction mixture was stirred at room temperature for 24 hr and concentrated in vacuo. Purification by flash chromatography on silica using a gradient of 0-30 % methanol in dichloromethane afforded CA-CY (20, 5.5 mg, 58 %).

[0386] CA-CY (20) HRMS (ESI) m / z: [M+H]1calcd for C26H46NO4S1468.3142; found 468.3141.

[0387] Example 6.1 'H (600 MHz) and13C (151 MHz) NMR spectroscopic data for CA-MCY (structure below) in methanol-^

[0388] Chemical shifts were referenced to 8(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical determined from the acquired1H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0389] s

[0390] No Proto

[0391] 5c 5H (JHH[HZ]) HMBC

[0392] n

[0393] 1 36.2 1-Ha 0.98 (Jla,2a = 13.8 Hz, Jla,2b = 3.3 Hz, Jla,1b 19

[0394] = 13.8 Hz)

[0395]

[0396] 1-Hb 1.81 (m)30.9 2-Ha 1.44 (m) 1a, 4b 2-Hb 1.58 (m)

[0397] 72.6 3-H 3.38 (m) 1, 4b

[0398] 40.2 4-Ha 1.65 (m) 6a

[0399] 4-Hb 2.27 (m)

[0400] 42.9 5-H 1.38 (m) 1, 4b, 6, 19 35.6 6-Ha 1.53 (m) 4b

[0401] 6-Hb 1.96 (m)

[0402] 68.8 7-H 3.80 (m) 6a, 8

[0403] 40.8 8-H 1.54 (m) 6a, 7, 9, 11, 14,

[0404] 15a

[0405] 27.6 9-H 2.26 (m) 1a, 7, 8, 11, 12,

[0406] 14, 19

[0407] 35.6 1, 4b, 5, 6a, 9, 19 29.3 11-H 1.60 (m) 9

[0408] 73.8 12-H 3.94 (m) 11, 14, 17, 18 47.2 11, 12, 14, 17, 18 42.7 14-H 1.99 (m) 8, 12, 15a, 18 24.0 15-Ha 1.11 (Jl5a,16a = 11.5 Hz, Jl5a,16b - 5.5 Hz, 14

[0409] Jl5a,15b = 11.5 Hz, Jl5a,14 = 11.5 Hz)

[0410] 15-Hb 1.76 (m)

[0411] 28.5 16-Ha 1.30 (m) 15a, 17

[0412] 16-Hb 1.90 (m)

[0413] 47.8 17-H 1.86 (m) 18, 21

[0414] 12.7 18-H 0.71 12, 14, 17 22.9 19-H 0.92 1a, 9

[0415] 36.6 20-H 1.42 (m) 17, 21, 22a 17.5 21-H 1.02 (J2I,2O = 6.6 HZ) 22

[0416] 33.1 22-Ha 1.33 (m) 21, 23

[0417] 22-Hb 1.80 (m)

[0418] 33.9 23-Ha 2.12 (m) 22a

[0419] 23-Hb 2.24 (m)

[0420] 176. 22a, 23, 25 7

[0421] 39.2 25-H 3.37 (J25,26 = 7.0 Hz) 26

[0422] 34.0 26-H 2.59 (J26,25 = 7.0 Hz) 25, 27

[0423]

[0424] 14.9 27-H 2.10 26Example 6.2 ¹H (600 MHz) and ¹³C (151 MHz) NMR spectroscopic data for CA-MCYO (2) in methanol-d₄

[0425] Chemical shifts were referenced to 6(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical determined from the acquired1H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0426] o

[0427]

[0428] H 6

[0429] No. 6c Proton 6H (JHH[HZ]) HMBC

[0430] 1 36.2 1-Ha 0.98 (Jla,2a = 3.6 Hz, Jla,2b = 14.1 Hz, Jla,1b 19

[0431] = 14.1 Hz)

[0432] 1-Hb 1.79 (m)

[0433] 2 30.9 2-Ha 1.43 (m) 1a, 4b

[0434] 2-Hb 1.60 (m)

[0435] 3 72.5 3-H 3.36 (m) 1, 4b, 6, 19 4 40.1 4-Ha 1.64 (m) 6a

[0436] 4-Hb 2.28 (m)

[0437] 5 42.8 5-H 1.37 (m) 1, 4b, 6, 19 6 35.5 6-Ha 1.52 (m) 4b

[0438] 6-Hb 1.95 (m)

[0439] 7 68.6 7-H 3.79 (m) 6a, 8

[0440] 8 40.7 8-H 1.55 (m) 6a, 7, 9, 11, 14,

[0441] 15a

[0442] 9 27.6 9-H 2.26 (m) 1a, 7, 8, 11, 12,

[0443] 14, 19

[0444] 10 35.7 1, 4b, 5, 6a, 9, 19 11 29.3 11-H 1.58 (m) 9

[0445] 12 73.7 12-H 3.94 (m) 11, 14, 17, 18 13 47.2 11, 12, 14, 17, 18

[0446]

[0447] 14 42.7 14-H 1.99 (m) 8, 12, 15a, 1815 23.9 15-Ha 1.12 (Jl5a,16a =4.8 Hz, Jl5a,16b = 12.0 Hz, 14

[0448] Jl5a,15b = 12.0 HZ, Jl5a,14 = 12.0 Hz)

[0449] 15-Hb 1.74 (m)

[0450] 16 28.4 16-Ha 1.29 (m) 15a, 17

[0451] 16-Hb 1.89 (m)

[0452] 17 47.6 17-H 1.86 (m) 18, 21

[0453] 18 12.4 18-H 0.72 12, 14, 17

[0454] 19 22.9 19-H 0.92 1a, 9

[0455] 20 36.6 20-H 1.41 (m) 17, 21, 22a

[0456] 21 16.3 21 -H 1.03 (J21,2O = 6.5 Hz) 22a

[0457] 22 32.8 22-Ha 1.34 (m) 21, 23

[0458] 22-Hb 1.78 (m)

[0459] 23 33.7 23-Ha 2.13 (m) 22a

[0460] 23-Hb 2.26 (m)

[0461] 24 176.9 22a, 23, 25

[0462] 25 34.2 25-Ha 3.55 (J25a,25b = 14.1 Hz, J25a,26a = 6.5 Hz, 26

[0463] J25a,26b = 6.5 Hz)

[0464] 25-Hb 3.61 (J25b,25a = 14.1 Hz, J25b,26a = 6.5 Hz,

[0465] J25b,26b = 6.5 Hz)

[0466] 26 54.6 26-Ha 2.92 (J26a,25a = 12.5 Hz, J26a,25b = 6.5 Hz, 25, 27

[0467] J26a,25b = 6.5 Hz)

[0468] 26-Hb 3.05 (J26b,25a = 12.5 Hz, J26b,25a = 6.5 Hz,

[0469] J26a,25a = 6.5 Hz)

[0470]

[0471] 27 38.1 27-H 2.68 26

[0472] Example 6.3 ¹H (600 MHz) and ¹³C (151 MHz) NMR spectroscopic data for CA-MCYO2 (3) in methanol-d₄

[0473] Chemical shifts were referenced to 6(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical determined from the acquired1H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0474]

[0475] No. 5c Proton 6H (JHH[HZ]) HMBC

[0476] 1 36.2 1-Ha 0.98 (Jla,2a = 3.5 Hz, Jla,2b = 14.0 Hz, 19

[0477] Jla,1b = 14.0 Hz)

[0478] 1-Hb 1.81 (m)

[0479] 2 30.9 2-Ha 1.43 (m) 1a, 4b

[0480] 2-Hb 1.60 (m)

[0481] 3 72.6 3-H 3.36 (m) 1, 4b, 6, 19 4 40.2 4-Ha 1.64 (m) 6a

[0482] 4-Hb 2.28 (m)

[0483] 5 42.8 5-H 1.37 (m) 1, 4b, 6, 19 6 35.5 6-Ha 1.52 (m) 4b

[0484] 6-Hb 1.96 (m)

[0485] 7 68.6 7-H 3.80 (m) 6a, 8

[0486] 8 40.7 8-H 1.55 (m) 6a, 7, 9, 11, 14,

[0487] 15a

[0488] 9 27.6 9-H 2.26 (m) 1a, 7, 8, 11, 12,

[0489] 14, 19

[0490] 10 35.7 1, 4b, 5, 6a, 9, 19 11 29.3 11-H 1.58 (m) 9

[0491] 12 73.7 12-H 3.95 (m) 11, 14, 17, 18 13 47.2 11, 12, 14, 17, 18 14 42.7 14-H 1.99 (m) 8, 12, 15a, 18 15 23.9 15-Ha 1.12 (Jl5a,16a = 5.2 Hz, Jl5a,16b = 11.8 Hz, 14

[0492] Jl5a,15b = 11.8 Hz, Jl5a,14 = 11.8 Hz)

[0493] 15-Hb 1.74 (m)

[0494] 16 28.4 16-Ha 1.29 (m) 15a, 17

[0495] 16-Hb 1.90 (m)

[0496] 17 47.6 17-H 1.86 (m) 18, 21

[0497] 18 12.7 18-H 0.71 12, 14, 17

[0498]

[0499] 19 22.9 19-H 0.92 1a, 920 36.6 20-H 1.41 (m) 17, 21, 22a

[0500] 21 17.3 21-H 1.03 (J21.20 = 6.5 Hz) 22a

[0501] 22 32.8 22-Ha 1.34 (m) 21, 23

[0502] 22-Hb 1.78 (m)

[0503] 23 33.7 23-Ha 2.13 (J23a,23b = 13.9 Hz, J23a,22a = 6.6 Hz, 22a

[0504] J23a,22b = 10.0 Hz)

[0505] 23-Hb 2.26 (m)

[0506] 24 176.7 22a, 23, 25

[0507] 25 34.2 25-H 3.63 (J25.26 = 7.0 Hz) 26

[0508] 26 54.0 26-H 3.30 (J26.25 = 7.0 Hz) 25, 27

[0509]

[0510] 27 41.0 27-H 3.00 26

[0511] Example 6.41H (600 MHz) and13C (151 MHz) NMR spectroscopic data for PMCA-MCY (6) in methanol-4 / 4

[0512] Chemical shifts were referenced to 5(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical shifts were determined via HMBC and HSQC spectra.rH, 'H-. / -coupling constants were determined from the acquired1H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0513] \27

[0514]

[0515] No. 5c Proton 6H (JHH[HZ]) HMBC

[0516] 1 36.5 1-Ha 1.05 (m) 19

[0517] 1-Hb 1.73 (m)

[0518] 2 30.6 2-Ha 1.22 (m) 1, 4b

[0519] 2-Hb 1.60 (m)

[0520] 3 71.5 3-H 3.49 (m) 1, 2a, 4b

[0521] 4 36.4 4-Ha 1.38 (m) 6

[0522] 4-Hb 1.63 (m)

[0523]

[0524] 5 49.1 5-H 1.72 (m) 1b, 4b, 6, 196 76.9 6-H 3.57 (m) 4b, 7

[0525] 7 74.1 7-H 3.44 (j7,8= 10.3 Hz, J7,6 = 3.6 6, 8

[0526] Hz)

[0527] 8 39.4 8-H 1.75 (m) 6, 7, 9, 11a, 14, 15a 9 41.0 9-H 1.46 (m) 1a, 8, 11a, 12a, 14, 19 10 34.7 1, 4b, 5, 6, 9, 19

[0528] 11 21.8 11 -Ha 1.36 (m) 9

[0529] 11 -Hb 1.46 (m)

[0530] 12 41.2 12-Ha 1.18 (m) 11a, 14, 17, 18

[0531] 12-Hb 2.04 (m)

[0532] 13 44.5 11a, 12, 14, 15b, 17, 18 14 57.0 14-H 1.24 (m) 7, 8, 12b, 15a, 18 15 28.1 15-Ha 1.45 (m) 14, 16b

[0533] 15-Hb 1.96 (m)

[0534] 16 29.5 16-Ha 1.31 (m) 15a, 17

[0535] 16-Hb 1.88 (m)

[0536] 17 56.4 17-H 1.10 (m) 12a, 16b, 18, 21, 22 18 12.4 18-H 0.72 12a, 14, 17

[0537] 19 25.9 19-H 1.09 1a, 5, 9

[0538] 20 36.7 20-H 1.44 (m) 17, 21, 22a

[0539] 21 18.9 21-H 0.98 (J2i,2o = 6.5 Hz) 17, 22a

[0540] 22 33.1 22-Ha 1.31 (m) 21, 23

[0541] 22-Hb 1.81 (m)

[0542] 23 33.9 23-Ha 2.10 (m) 22a

[0543] 23-Hb 2.24 (m)

[0544] 24 176.6 22a, 23, 25

[0545] 25 39.3 25-H 3.35 (J25,26 = 7.0 Hz) 26

[0546] 26 34.0 26-H 2.58 (J26,25 = 7.0 Hz) 25, 27

[0547]

[0548] 27 14.9 27-H 2.10 26

[0549] Example 6.5 ’ll (600 MHz) and13C (151 MHz) NMR spectroscopic data for PMCA-MCYO (7) in methanol-f / 4

[0550] Chemical shifts were referenced to 6(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical determined from the acquired 'H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.O

[0551]

[0552] OH

[0553] No Proto

[0554] 5c 5H (JHH[HZ]) HMBC

[0555] n

[0556] 1 36.5 1-Ha 1.02 (m) 19

[0557] 1-Hb 1.73 (m)

[0558] 2 30.6 2-Ha 1.22 (m) 1, 4b

[0559] 2-Hb 1.59 (m)

[0560] 3 71.6 3-H 3.49 (m) 1, 2a, 4b

[0561] 4 36.4 4-Ha 1.38 (m) 6

[0562] 4-Hb 1.63 (m)

[0563] 5 49.1 5-H 1.72 (m) 1b, 4b, 6, 19

[0564] 6 76.9 6-H 3.57 (m) 4b, 7

[0565] 7 74.1 7-H 3.44 (j78= 10.3 Hz, J7.6 = 3.6 Hz) 6, 8

[0566] 8 39.4 8-H 1.75 (m) 6, 7, 9, 11a, 14, 15a 9 41.0 9-H 1.47 (m) 1a, 8, 11a, 12a, 14,

[0567] 19

[0568] 10 34.7 1, 4b, 5, 6, 9, 19 11 21.8 11 -Ha 1.37 (m) 9

[0569] 11-Hb 1.46 (m)

[0570] 12 41.2 12-Ha 1.18 (m) 11a, 14, 17, 18

[0571] 12-Hb 2.04 (m)

[0572] 13 44.6 11a, 12, 14, 15b, 17,

[0573] 18

[0574] 14 57.1 14-H 1.24 (m) 7, 8, 12b, 15a, 18 15 28.0 15-Ha 1.45 (m) 14, 16b

[0575] 15-Hb 1.95 (m)

[0576] 16 29.5 16-Ha 1.30 (m) 15a, 17

[0577] 16-Hb 1.88 (m)

[0578] 17 56.3 17-H 1.10 (m) 12a, 16b, 18, 21, 22 18 12.4 18-H 0.71 12a, 14, 17

[0579] 19 25.9 19-H 1.08 1a, 5, 9

[0580]

[0581] 20 36.7 20-H 1.42 (m) 17, 21, 22a21 18.9 21-H 0.97 (J21,20= 6.5 Hz) 17, 22a 22 33.1 22-Ha 1.31 (m) 21, 23

[0582] 22-Hb 1.80 (m)

[0583] 23 33.9 23-Ha 2.10 (m) 22a

[0584] 23-Hb 2.24 (m)

[0585] 24 176. 22a, 23, 25

[0586] 9

[0587] 25 34.3 25-Ha 3.54 (m) 26

[0588] 25-Hb 3.60 (m)

[0589] 26 54.7 26-Ha 2.89 (m) 25, 27

[0590] 26-Hb 3.03 (m)

[0591]

[0592] 27 38.1 27-H 2.67 26

[0593] Example 6.61H (600 MHz) and13C (151 MHz) NMR spectroscopic data for pMCA- MCYO2 (8) in m ethanol- dt

[0594] Chemical shifts were referenced to 5(CHD2OD) = 3.31 and 8(13CHD2OD) = 49.0.13C chemical determined from the acquired 'H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0595]

[0596] No. 6c Proton 6H (JHH[HZ]) HMBC

[0597] 1 36.5 1-Ha 1.04 (m) 19

[0598] 1-Hb 1.73 (m)

[0599] 2 30.5 2-Ha 1.22 (m) 1, 4b

[0600] 2-Hb 1.59 (m)

[0601] 3 71.6 3-H 3.49 (m) 1, 2a, 4b

[0602] 4 36.3 4-Ha 1.38 (m) 6

[0603] 4-Hb 1.63 (m)

[0604]

[0605] 5 49.1 5-H 1.72 (m) 1b, 4b, 6, 196 76.9 6-H 3.57 (m) 4b, 7

[0606] 7 74.1 7-H 3.44 (J7,8= 10.3 Hz, J7,6= 3.6 Hz) 6, 8

[0607] 8 39.4 8-H 1.75 (m) 6, 7, 9, 11a, 14, 15a 9 41.0 9-H 1.47 (m) 1a, 8, 11a, 12a, 14, 19 10 34.7 1, 4b, 5, 6, 9, 19 11 21.8 11 -Ha 1.37 (m) 9

[0608] 11 -Hb 1.46 (m)

[0609] 12 41.2 12-Ha 1.18 (m) 11a, 14, 17, 18

[0610] 12-Hb 2.04 (m)

[0611] 13 44.6 11a, 12, 14, 15b, 17,

[0612] 18

[0613] 14 57.1 14-H 1.24 (m) 7, 8, 12b, 15a, 18 15 28.0 15-Ha 1.45 (m) 14, 16b

[0614] 15-Hb 1.95 (m)

[0615] 16 29.4 16-Ha 1.30 (m) 15a, 17

[0616] 16-Hb 1.88 (m)

[0617] 17 56.3 17-H 1.10 (m) 12a, 16b, 18, 21, 22 18 12.4 18-H 0.71 12a, 14, 17

[0618] 19 25.9 19-H 1.08 1a, 5, 9

[0619] 20 36.7 20-H 1.43 (m) 17, 21, 22a

[0620] 21 18.9 21 -H 0.97 (J21,20= 6.5 Hz) 17, 22a

[0621] 22 33.0 22-Ha 1.31 (m) 21, 23

[0622] 22-Hb 1.80 (m)

[0623] 23 33.7 23-Ha 2.10 (m) 22a

[0624] 23-Hb 2.24 (m)

[0625] 24 176.9 22a, 23, 25

[0626] 25 34.3 25-H 3.63 (J25,26 = 6.8 Hz) 26

[0627] 26 54.2 26-H 3.30 (J26.25 = 6.7 Hz) 25, 27

[0628]

[0629] 27 41.1 27-H 2.99 26

[0630] Example 6.7 ’ll (600 MHz) and13C (151 MHz) NMR spectroscopic data for CDCA-MCY (11) in methanol-rf4

[0631] Chemical shifts were referenced to 6(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical shifts were determined via HMBC and HSQC spectra.1H,1H-J-coupling constants were determined from the acquired 'H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0632]

[0633] H 6

[0634] No. 5c Proton 5H (JHH[HZ]) HMBC

[0635] 1 36.3 1-Ha 0.98 (m) 19

[0636] 1-Hb 1.85 (m)

[0637] 2 31.0 2-Ha 1.33 (m) 1, 4b

[0638] 2-Hb 1.60 (m)

[0639] 3 72.5 3-H 3.36 (m) 1, 4b

[0640] 4 40.1 4-Ha 1.64 (m) 5, 6a

[0641] 4-Hb 2.26 (m)

[0642] 5 42.8 5-H 1.37 (m) 1, 4b, 6, 19 6 35.6 6-Ha 1.52 (m) 4b, 5

[0643] 6-Hb 1.95 (m)

[0644] 7 68.7 7-H 3.78 (m) 6a, 8

[0645] 8 40.5 8-H 1.51 (m) 6a, 7, 9, 11, 14,

[0646] 15a

[0647] 9 33.8 9-H 1.86 (m) 1a, 7, 8, 11, 12,

[0648] 14, 19

[0649] 10 33.6 1, 4b, 5, 6, 9, 19 11 21.5 11 -Ha 1.31 (m) 9

[0650] 11-Hb 1.49 (m)

[0651] 12 40.7 12-Ha 1.20 (m) 9, 11, 14, 17, 18

[0652] 12-Hb 2.00 (m)

[0653] 13 43.4 11, 12, 14, 17, 18 14 51.2 14-H 1.49 (m) 8, 12, 15a, 18 15 24.3 15-Ha 1.09 (m) 14, 16b

[0654] 15-Hb 1.73 (m)

[0655] 16 29.0 16-Ha 1.32 (m) 15a, 17

[0656] 16-Hb 1.91 (m)

[0657] 17 57.0 17-H 1.17 (m) 16, 18, 21 18 11.9 18-H 0.70 12, 14, 17

[0658]

[0659] 19 23.1 19-H 0.93 1a, 920 36.6 20-H 1.45 (m) 17, 21, 22a

[0660] 21 18.6 21-H 0.98 (J21.20 = 6.6 Hz) 22

[0661] 22 32.9 22-Ha 1.29 (m) 21, 23

[0662] 22-Hb 1.80 (m)

[0663] 23 33.8 23-Ha 2.10 (m) 22a

[0664] 23-Hb 2.24 (m)

[0665] 24 176.4 22a, 23, 25

[0666] 25 39.1 25-H 3.35 (J25,26 = 7.0 Hz) 26

[0667] 26 33.9 26-H 2.58 (J26,25 = 7.0 Hz) 25, 27

[0668]

[0669] 27 14.8 27-H 2.10 26

[0670] Example 6.8 'II (600 MHz) and13C (151 MHz) NMR spectroscopic data for CDCA-MCYO (12) in methanol-f / 4

[0671] Chemical shifts were referenced to 6(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical shifts were determined via HMBC and HSQC spectra.1H,1H-J-coupling constants were determined from the acquired ’H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0672]

[0673] No. 5c Proton 6H (JHH[HZ]) HMBC

[0674] 1 36.3 1-Ha 1.00 (m) 19

[0675] 1-Hb 1.85 (m)

[0676] 2 31.0 2-Ha 1.33 (m) 1, 4b

[0677] 2-Hb 1.60 (m)

[0678] 3 72.5 3-H 3.36 (m) 1, 4b

[0679] 4 40.1 4-Ha 1.64 (m) 5, 6a

[0680] 4-Hb 2.26 (m)

[0681] 5 42.8 5-H 1.37 (m) 1, 4b, 6, 19

[0682] 6 35.6 6-Ha 1.52 (m) 4b, 5

[0683]

[0684] 6-Hb 1.95 (m)7 68.7 7-H 3.78 (m) 6a, 8

[0685] 8 40.5 8-H 1.51 (m) 6a, 7, 9, 11, 14, 15a 9 33.8 9-H 1.86 (m) 1a, 7, 8, 11, 12, 14,

[0686] 19

[0687] 10 33.7 1, 4b, 5, 6, 9, 19 11 21.5 11 -Ha 1.31 (m) 9

[0688] 11 -Hb 1.49 (m)

[0689] 12 40.7 12-Ha 1.20 (m) 9, 11, 14, 17, 18

[0690] 12-Hb 2.00 (m)

[0691] 13 43.4 11, 12, 14, 17, 18 14 51.2 14-H 1.49 (m) 8, 12, 15a, 18

[0692] 15 24.3 15-Ha 1.11 (m) 14, 16b

[0693] 15-Hb 1.74 (m)

[0694] 16 29.0 16-Ha 1.32 (m) 15a, 17

[0695] 16-Hb 1.93 (m)

[0696] 17 57.0 17-H 1.17 (m) 16, 18, 21

[0697] 18 11.9 18-H 0.69 12, 14, 17

[0698] 19 23.1 19-H 0.93 1a, 9

[0699] 20 36.6 20-H 1.44 (m) 17, 21, 22a

[0700] 21 18.6 21-H 0.98 (J21,20= 6.6 Hz) 22

[0701] 22 32.9 22-Ha 1.31 (m) 21, 23

[0702] 22-Hb 1.80 (m)

[0703] 23 33.6 23-Ha 2.10 (m) 22a

[0704] 23-Hb 2.24 (m)

[0705] 24 176.7 22a, 23, 25

[0706] 25 34.2 25-Ha 3.55 (m) 26

[0707] 25-Hb 3.61 (J25b,25a = 12.5 Hz, J25b,26a = 25, 27

[0708] 6.0 HZ, J25a,26b = 6.5 Hz)

[0709] 26 54.7 26-Ha 2.92 (J26a,26b = 12.0 Hz, J26a,26a = 26

[0710] 6.0 Hz, J26a,26b = 6.5 Hz)

[0711] 26-Hb 3.05 (m) 25, 27

[0712]

[0713] 27 38.2 27-H 2.68 26

[0714] Example 6.9 'II (600 MHz) and13C (151 MHz) NMR spectroscopic data for UDCA-MCY (13) in methanol-t / 4

[0715] Chemical shifts were referenced to 6(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical shifts were determined via HMBC and HSQC spectra.1H,1H-J-coupling constants weredetermined from the acquired 'H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0716] o

[0717]

[0718] No. 5c Proton 6H (JHH[HZ]) HMBC

[0719] 1 35.8 1-Ha 1.03 (m) 2, 19

[0720] 1-Hb 1.82 (m)

[0721] 2 30.7 2-Ha 1.28 (m) 1, 4

[0722] 2-Hb 1.64 (m)

[0723] 3 71.6 3-H 3.47 (m) 1, 2b, 4 4 37.8 4-H 1.62 (m) 5, 6a

[0724] 5 43.8 5-H 1.47 (m) 1, 4, 6, 19 6 38.4 6-Ha 1.57 (m) 4, 5

[0725] 6-Hb 1.83 (m)

[0726] 7 71.8 7-H 3.50 (m) 6, 8

[0727] 8 44.2 8-H 1.45 (m) 6a, 14, 15a 9 40.5 9-H 1.51 (m) 1a, 8, 11, 12a,

[0728] 19

[0729] 10 34.9 1, 4, 5, 6, 9, 19 11 22.1 11 -Ha 1.31 (m) 12

[0730] 11-Hb 1.47 (m)

[0731] 12 41.3 12-Ha 1.20 (m) 11b, 17, 18

[0732] 12-Hb 2.04 (m)

[0733] 13 44.5 12, 14, 17, 18 14 57.3 14-H 1.25 (m) 8, 12a, 15a, 18 15 27.6 15-Ha 1.46 (m) 14, 16b

[0734] 15-Hb 1.91 (m)

[0735] 16 29.4 16-Ha 1.31 (m) 15a, 17

[0736] 16-Hb 1.90 (m)

[0737] 17 56.3 17-H 1.10 (m) 16, 18, 21 18 12.4 18-H 0.72 14, 17

[0738]

[0739] 19 23.7 19-H 0.97 1a20 36.5 20-H 1.44 (m) 17, 21, 22a, 23 21 18.7 21-H 0.98 (J21.20 = 6.6 Hz) 22

[0740] 22 33.1 22-Ha 1.31 (m) 21, 23

[0741] 22-Hb 1.81 (m)

[0742] 23 33.9 23-Ha 2.11 (m) 22a

[0743] 23-Hb 2.24 (m)

[0744] 24 176.4 22a, 23, 25 25 39.2 25-H 3.36 (J25,26 = 7.0 Hz) 26

[0745] 26 34.0 26-H 2.59 (J26,25 = 7.0 Hz) 25, 27

[0746]

[0747] 27 14.9 27-H 2.10 26

[0748] Example 6.101H (600 MHz) and13C (151 MHz) NMR spectroscopic data for UDCA-MCYO (14) in methanol-^

[0749] Chemical shifts were referenced to 6(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical shifts were determined via HMBC and HSQC spectra.1H,1H-J-coupling constants were determined from the acquired ’H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0750] o

[0751]

[0752] No. 5c Proton 6H (JHH[HZ]) HMBC

[0753] 1 35.8 1-Ha 1.03 (m) 2, 19

[0754] 1-Hb 1.82 (m)

[0755] 2 30.7 2-Ha 1.27 (m) 1, 4

[0756] 2-Hb 1.62 (m)

[0757] 3 71.6 3-H 3.50 (m) 1, 2b, 4

[0758] 4 37.8 4-H 1.62 (m) 5, 6a

[0759] 5 43.8 5-H 1.47 (m) 1, 4, 6, 19 6 38.4 6-Ha 1.57 (m) 4, 5

[0760] 6-Hb 1.83 (m)

[0761]

[0762] 7 71.8 7-H 3.50 (m) 6, 88 44.2 8-H 1.45 (m) 6a, 14, 15a 9 40.5 9-H 1.49 (m) 1a, 8, 11, 12a, 19 10 34.9 1, 4, 5, 6, 9, 19 11 22.1 11 -Ha 1.31 (m) 12

[0763] 11 -Hb 1.47 (m)

[0764] 12 41.3 12-Ha 1.20 (m) 11b, 17, 18

[0765] 12-Hb 2.04 (m)

[0766] 13 44.5 12, 14, 17, 18 14 57.3 14-H 1.24 (m) 8, 12a, 15a, 18 15 27.6 15-Ha 1.46 (m) 14, 16b

[0767] 15-Hb 1.91 (m)

[0768] 16 29.4 16-Ha 1.31 (m) 15a, 17

[0769] 16-Hb 1.90 (m)

[0770] 17 56.3 17-H 1.10 (m) 16, 18, 21 18 12.4 18-H 0.72 14, 17

[0771] 19 23.7 19-H 0.97 1a

[0772] 20 36.5 20-H 1.43 (m) 17, 21, 22a, 23 21 18.7 21-H 0.98 (J21,20= 6.5 Hz) 22

[0773] 22 33.1 22-Ha 1.31 (m) 21, 23

[0774] 22-Hb 1.80 (m)

[0775] 23 33.9 23-Ha 2.11 (m) 22a

[0776] 23-Hb 2.24 (m)

[0777] 24 176.6 22a, 23, 25 25 34.2 25-H 3.36 (J25,26 = 7.0 Hz) 26

[0778] 26 54.7 26-Ha 2.92 (m) 25, 27

[0779] 26-Hb 3.05 (m) 26

[0780]

[0781] 27 38.1 27-H 2.68 26

[0782] Example 6.111H (600 MHz) and13C (151 MHz) NMR spectroscopic data for DCA-MCY (15) in methanol-^

[0783] Chemical shifts were referenced to 6(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical shifts were determined via HMBC and HSQC spectra.rH,1H-J-coupling constants were determined from the acquired1H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0784]

[0785] No. 5c Proton 6H (JHH[HZ]) HMBC

[0786] 1 36.1 1-Ha 0.98 (Jla,2a = 3.5 Hz, Jla,2b = 14.0 Hz, 19

[0787] Jla,1b = 14.0 Hz)

[0788] 1-Hb 1.78 (m)

[0789] 2 30.8 2-Ha 1.43 (m) 1a, 4a

[0790] 2-Hb 1.60 (m)

[0791] 3 72.2 3-H 3.53 (m) 1, 4a

[0792] 4 36.9 4-Ha 1.48 (m) 6b

[0793] 4-Hb 1.80 (m)

[0794] 5 43.4 5-H 1.40 (m) 1b, 4a, 6b, 19 6 28.3 6-Ha 1.29 (m) 4a, 7a

[0795] 6-Hb 1.88 (m)

[0796] 7 27.1 7-Ha 1.17 (m) 6, 8

[0797] 7-Hb 1.44 (m)

[0798] 8 37.4 8-H 1.47 (m) 6b, 9, 11, 14,

[0799] 15a

[0800] 9 34.5 9-H 1.91 (m) 1a, 8, 11, 12, 19 10 34.8 1, 4b, 5, 6b, 9,

[0801] 19

[0802] 11 29.6 11-H 1.52 (m) 9

[0803] 12 73.7 12-H 3.96 (m) 11, 17, 18 13 47.3 11, 12, 14, 17,

[0804] 18

[0805] 14 49.0 14-H 1.62 (m) 8, 12, 15a, 18 15 24.6 15-Ha 1.10 (m) 14

[0806] 15-Hb 1.62 (m)

[0807] 16 28.2 16-Ha 1.28 (m) 15a, 17

[0808] 16-Hb 1.89 (m)

[0809] 17 47.8 17-H 1.84 (m) 18, 21

[0810] 18 12.9 18-H 0.72 12, 14, 17

[0811]

[0812] 19 23.4 19-H 0.93 1a, 920 36.6 20-H 1.41 (m) 17, 21, 22a 21 17.4 21-H 1.02 (J21.20 = 6.6 Hz) 22a

[0813] 22 32.9 22-Ha 1.33 (m) 21, 23

[0814] 22-Hb 1.80 (m)

[0815] 23 33.8 23-Ha 2.12 (m) 22a

[0816] 23-Hb 2.25 (m)

[0817] 24 176.5 22a, 23, 25 25 39.1 25-H 3.37 (J25,26 = 7.0 Hz) 26

[0818] 26 34.0 26-H 2.59 (J26,25 = 7.0 Hz) 25, 27

[0819]

[0820] 27 14.9 27-H 2.10 26

[0821] Example 6.12 'll (600 MHz) and13C (151 MHz) NMR spectroscopic data for DCA-MCYO (16) in methanol-f / 4

[0822] Chemical shifts were referenced to 6(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical shifts were determined via HMBC and HSQC spectra.1H,1H-J-coupling constants were determined from the acquired ’H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0823] o

[0824]

[0825] No. 5c Proton 5H (JHH[HZ]) HMBC

[0826] 1 36.1 1-Ha 0.98 (m) 19

[0827] 1-Hb 1.78 (m)

[0828] 2 30.8 2-Ha 1.43 (m) 1a, 4a

[0829] 2-Hb 1.60 (m)

[0830] 3 72.2 3-H 3.53 (m) 1, 4a

[0831] 4 36.9 4-Ha 1.48 (m) 6b

[0832] 4-Hb 1.80 (m)

[0833] 5 43.4 5-H 1.40 (m) 1b, 4a, 6b, 19 6 28.3 6-Ha 1.29 (m) 4a, 7a

[0834]

[0835] 6-Hb 1.88 (m)7 27.1 7-Ha 1.17 (m) 6, 8

[0836] 7-Hb 1.44 (m)

[0837] 8 37.3 8-H 1.46 (m) 6b, 9, 11, 14, 15a 9 34.5 9-H 1.90 (m) 1a, 8, 11, 12, 19 10 34.8 1, 4b, 5, 6b, 9, 19 11 29.6 11-H 1.52 (m) 9

[0838] 12 73.7 12-H 3.95 (m) 11, 17, 18

[0839] 13 47.3 11, 12, 14, 17, 18 14 49.0 14-H 1.62 (m) 8, 12, 15a, 18 15 24.6 15-Ha 1.10 (m) 14

[0840] 15-Hb 1.62 (m)

[0841] 16 28.2 16-Ha 1.28 (m) 15a, 17

[0842] 16-Hb 1.89 (m)

[0843] 17 47.8 17-H 1.84 (m) 18, 21

[0844] 18 12.9 18-H 0.71 12, 14, 17

[0845] 19 23.4 19-H 0.93 1a, 9

[0846] 20 36.6 20-H 1.41 (m) 17, 21, 22a 21 17.4 21-H 1.02 (J21.20 = 6.5 Hz) 22a

[0847] 22 32.9 22-Ha 1.33 (m) 21, 23

[0848] 22-Hb 1.78 (m)

[0849] 23 33.7 23-Ha 2.12 (m) 22a

[0850] 23-Hb 2.25 (m)

[0851] 24 176.7 22a, 23, 25 25 34.2 25-Ha 3.53 (m) 26

[0852] 25-Hb 3.61 (m) 25, 27

[0853] 26 54.7 26-Ha 2.92 (m) 26

[0854] 26-Hb 3.05 (m) 25, 27

[0855]

[0856] 27 38.1 27-H 2.68 26

[0857] Example 6.131H (600 MHz) and13C (151 MHz) NMR spectroscopic data for KDCA-MCY (17) in methanol-i / 4

[0858] Chemical shifts were referenced to 3(CHD2OD) = 3.31 and 8(13CHD2OD) = 49.0.13C chemical shifts were determined via HMBC and HSQC spectra.1H,1H-. / -coupling constants were determined from the acquired ’H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0859]

[0860] No. 5c Proton 6H (JHH[HZ]) HMBC

[0861] 1 34.8 1-Ha 1.21 (m) 19

[0862] 1-Hb 1.81 (m)

[0863] 2 30.9 2-Ha 1.33 (m)

[0864] 2-Hb 1.64 (m)

[0865] 3 71.3 3-H 3.52 (m) 1, 4

[0866] 4 37.9 4-Ha 1.24 (m) 6

[0867] 4-Hb 1.62 (m)

[0868] 5 47.2 5-H 1.92 (m) 1b, 6, 19

[0869] 6 46.1 6-Ha 1.86 (J6a,6b = 12.7 Hz, J6a,5 = 2.0 Hz) 4a

[0870] 6-Hb 2.97 (J6b,6a= 12.7 Hz, Jst>,5 = 5.9 Hz)

[0871] 7 214.5 6, 8

[0872] 8 50.4 8-H 2.55 (m) 6a, 9, 11a, 14,

[0873] 15a

[0874] 9 37.2 9-H 2.29 (m) 1a, 8, 11, 12, 19 10 35.6 1, 4b, 6a, 9, 19 11 30.3 11 -Ha 1.55 (m) 9

[0875] 11-Hb 1.78 (m)

[0876] 12 72.6 12-H 3.99 (m) 11a, 17, 18 13 47.2 11, 12, 14, 17, 18 14 41.6 14-H 1.99 (m) 8, 12, 15a, 18 15 25.1 15-Ha 1.00 (m) 14

[0877] 15-Hb 2.15 (m)

[0878] 16 28.5 16-Ha 1.29 (m) 15a, 17

[0879] 16-Hb 1.90 (m)

[0880] 17 47.1 17-H 1.81 (m) 18, 21

[0881] 18 13.0 18-H 0.72 14, 17

[0882] 19 22.9 19-H 1.22 1a, 6a, 9

[0883] 20 36.3 20-H 1.42 (m) 17, 21, 22a 21 17.5 21-H 1.03 (J2i,2o = 6.5 Hz) 22

[0884]

[0885] 22 33.1 22-Ha 1.34 (m) 21, 2322-Hb 1.80 (m)

[0886] 23 33.7 23-Ha 2.13 (m) 22a

[0887] 23-Hb 2.26 (m)

[0888] 24 176.6 22a, 23, 25 25 39.3 25-H 3.35 (J25,26 = 7.0 Hz) 26

[0889] 26 34.0 26-H 2.59 (J26.25 = 7.0 Hz) 25, 27

[0890]

[0891] 27 14.9 27-H 2.10 26

[0892] Example 6.141H (600 MHz) and13C (151 MHz) NMR spectroscopic data for KDCA-MCYO (18) in methanol-r / 4

[0893] Chemical shifts were referenced to 3(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical shifts were determined via HMBC and HSQC spectra.1H,1H-J-coupling constants were determined from the acquired 'H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0894] o

[0895]

[0896] No. 5c Proton 6H (JHH[HZ]) HMBC

[0897] 1 34.8 1-Ha 1.21 (m) 19

[0898] 1-Hb 1.81 (m)

[0899] 2 30.3 2-Ha 1.33 (m)

[0900] 2-Hb 1.80 (m)

[0901] 3 71.3 3-H 3.52 (m) 1, 4

[0902] 4 37.9 4-Ha 1.24 (m) 6

[0903] 4-Hb 1.62 (m)

[0904] 5 47.2 5-H 1.92 (m) 1b, 6, 19

[0905] 6 46.1 6-Ha 1.86 (J6a,6b = 12.7 Hz, J6a,5 = 2.3 Hz) 4a

[0906] 6-Hb 2.97 (J6b,6a= 12.7 Hz, Jeb, 5 = 5.9 Hz)

[0907] 7 214.4 6, 8

[0908] 8 50.4 8-H 2.55 (J8,14 = 11.6 Hz, JB,9 = 11.6 Hz) 6a, 9, 11a, 14,

[0909]

[0910] 15a9 37.2 9-H 2.29 (m) 1a, 8, 11, 12, 19 10 35.5 1, 4b, 6a, 9, 19 11 30.3 11 -Ha 1.55 (Jii,i2 = Hz, Ju, 9 = Hz) 9

[0911] 11 -Hb 1.78 (Jii,i2 = Hz, JH,9 = HZ)

[0912] 12 72.6 12-H 3.99 (Ji2,n = Hz) 11a, 17, 18

[0913] 13 47.2 11, 12, 14, 17, 18 14 41.6 14-H 1.99 (Jl4,15a = HZ, Jl4,15b = Hz, Ji 4,8 = 8, 12, 15a, 18

[0914] Hz)

[0915] 15 25.1 15-Ha 1.00 (m) 14

[0916] 15-Hb 2.15 (m)

[0917] 16 28.4 16-Ha 1.29 (m) 15a, 17

[0918] 16-Hb 1.90 (m)

[0919] 17 46.9 17-H 1.81 (m) 18, 21

[0920] 18 13.0 18-H 0.72 14, 17

[0921] 19 22.9 19-H 1.22 1a, 6a, 9

[0922] 20 36.3 20-H 1.41 (m) 17, 21, 22a

[0923] 21 17.5 21-H 1.03 (J21.20 = 6.5 Hz) 22

[0924] 22 32.8 22-Ha 1.34 (m) 21, 23

[0925] 22-Hb 1.80 (m)

[0926] 23 33.7 23-Ha 2.13 (m) 22a

[0927] 23-Hb 2.26 (m)

[0928] 24 176.8 22a, 23, 25

[0929] 25 34.2 25-Ha 3.55 (m) 26

[0930] 25-Hb 3.61 (m)

[0931] 26 54.6 26-Ha 3.06 (m) 25, 27

[0932] 26-Hb 2.92 (m)

[0933]

[0934] 27 38.1 27-H 2.68 26

[0935] Example 6.15 ¹H (600 MHz) and13C (151 MHz) NMR spectroscopic data for CA-CY (20) in methanol-t / 4

[0936] Chemical shifts were referenced to 6(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical determined from the acquired1H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0937]

[0938] No. 6c Proton 6H (JHH[HZ]) HMBC

[0939] 1 36.2 1-Ha 0.98 (m) 19

[0940] 1-Hb 1.81 (m)

[0941] 2 30.9 2-Ha 1.44 (m) 1a, 4b

[0942] 2-Hb 1.58 (m)

[0943] 3 72.6 3-H 3.38 (m) 1, 4b

[0944] 4 40.2 4-Ha 1.65 (m) 6a

[0945] 4-Hb 2.27 (m)

[0946] 5 42.9 5-H 1.38 (m) 1, 4b, 6, 19

[0947] 6 35.6 6-Ha 1.53 (m) 4b

[0948] 6-Hb 1.96 (m)

[0949] 7 68.8 7-H 3.80 (m) 6a, 8

[0950] 8 40.7 8-H 1.54 (m) 6a, 7, 9, 11, 14, 15a 9 27.6 9-H 2.26 (m) 1a, 7, 8, 11, 12, 14,

[0951] 19

[0952] 10 35.6 1, 4b, 5, 6a, 9, 19 11 29.1 11-H 1.60 (m) 9

[0953] 12 73.8 12-H 3.94 (m) 11, 14, 17, 18 13 47.2 11, 12, 14, 17, 18 14 42.7 14-H 1.99 (m) 8, 12, 15a, 18 15 24.0 15-Ha 1.11 (m) 14

[0954] 15-Hb 1.76 (m)

[0955] 16 28.4 16-Ha 1.30 (m) 15a, 17

[0956] 16-Hb 1.90 (m)

[0957] 17 47.8 17-H 1.86 (m) 18, 21

[0958] 18 12.7 18-H 0.71 12, 14, 17

[0959] 19 22.9 19-H 0.92 1a, 9

[0960] 20 36.6 20-H 1.42 (m) 17, 21, 22a

[0961] 21 17.5 21 -H 1.02 (J21.20 = 6.6 Hz) 22

[0962] 22 33.0 22-Ha 1.33 (m) 21, 23

[0963]

[0964] 22-Hb 1.80 (m)23 33.9 23-Ha 2.12 (m) 22a

[0965] 23-Hb 2.24 (m)

[0966] 24 176.7 22a, 23, 25

[0967] 25 39.1 25-H 3.45 (J25,26 = 6.8 Hz) 26

[0968]

[0969] 26 38.1 26-H 2.81 (J26.25 = 6.8 Hz) 25

[0970] Example 6.16JH (600 MHz) and13C (151 MHz) NMR spectroscopic data for CA-pant (21) in methanol- 4

[0971] Chemical shifts were referenced to 6(CHD2OD) = 3.31 and 6(13CHD2OD) = 49.0.13C chemical determined from the acquired ’H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0972]

[0973] No. 6c Proton 6H (JHH[HZ]) HMBC

[0974] 1 36.2 1-Ha 0.97 (m) 19

[0975] 1-Hb 1.82 (m)

[0976] 2 30.9 2-Ha 1.41 (m) 1a, 4b

[0977] 2-Hb 1.61 (m)

[0978] 3 72.6 3-H 3.34 (m) 1, 4b

[0979] 4 40.2 4-Ha 1.63 (m) 6a

[0980] 4-Hb 2.26 (m)

[0981] 5 42.9 5-H 1.37 (m) 1, 4b, 6, 19

[0982] 6 35.6 6-Ha 1.52 (m) 4b

[0983] 6-Hb 1.94 (m)

[0984] 7 68.8 7-H 3.77 (m) 6a, 8

[0985] 8 40.7 8-H 1.53 (m) 6a, 7, 9, 11, 14, 15a 9 27.6 9-H 2.23 (m) 1a, 7, 8, 11, 12, 14,

[0986] 19

[0987] 10 35.7 1, 4b, 5, 6a, 9, 19

[0988]

[0989] 11 29.2 11-H 1.56 (m) 912 73.8 12-H 3.91 (m) 11, 14, 17, 18 13 47.3 11, 12, 14, 17, 18 14 42.7 14-H 1.96 (m) 8, 12, 15a, 18 15 24.0 15-Ha 1.09 (m) 14

[0990] 15-Hb 1.74 (m)

[0991] 16 28.4 16-Ha 1.28 (m) 15a, 17

[0992] 16-Hb 1.85 (m)

[0993] 17 47.6 17-H 1.84 (m) 18, 21

[0994] 18 12.7 18-H 0.67 12, 14, 17

[0995] 19 22.9 19-H 0.88 1a, 9

[0996] 20 36.3 20-H 1.41 (m) 17, 21, 22a 21 17.5 21-H 0.97 (721,20 = 6.6 Hz) 22

[0997] 22 32.7 22-Ha 1.40 (m) 21, 23

[0998] 22-Hb 1.79 (m)

[0999] 23 41.6 23-Ha 2.46 (m) 22a

[1000] 23-Hb 2.66 (m)

[1001] 24 200.8 22a, 23, 25 25 28.9 25-H 2.97 (725,26 = 6.7 Hz) 26

[1002] 26 39.8 26-H 3.30 (726,25 = 6.7 Hz) 25, 27

[1003] 27 173.5 26, 28, 29

[1004] 28 36.2 28-H 2.38 (m) 29

[1005] 29 36.1 29-H 3.44 (m) 28

[1006] 30 175.7 29, 31

[1007] 31 77.1 31 -H 3.87 33, 34, 35

[1008] 32 40.0 31, 33, 34, 35 33 70.1 33-Ha 3.35 (733a, 33b = 11.5 Hz) 31, 34, 35

[1009] 33-Hb 3.43 (733b, 33a = 11.5 Hz)

[1010] 34 20.9 34-H 0.88 31, 33, 35

[1011]

[1012] 35 20.9 35-H 0.88 31, 33, 34

[1013] EXAMPLE 7 - FXR antagonism

[1014] FXR antagonism was tested for several compounds. The PathHunter® NHR Protein Interaction (NHR Pro) assay was used to monitor the activation of a nuclear receptor (e.g., nuclear hormone receptor) such as FXR in a homogenous, non-imaging assay format using a technology developed by DiscoverX called Enzyme Fragment Complementation (EFC). The NHR Pro assay is based on detection of protein-protein interactions between an activated, full length NHR protein and a nuclear fusion protein containing Steroid Receptor Co-activatorPeptide (SRCP) domains with one or more canonical LXXLL interaction motifs. The NHR is tagged with the Enzyme Donor (ED) component of an EFC assay system, and the SRCP domain is fused to the Enzyme Acceptor (EA) component expressed in the nucleus. When bound by ligand, the NHR will migrate to the nucleus and recruit the SRCP domain, whereby complementation occurs, generating a unit of active b-Galactosidase (b-Gal) and production of chemiluminescent signal upon the addition of PathHunter® detection reagents.

[1015] Briefly, the method comprised seeded cells in a total volume of 20 pL into white walled, 384-well microplates. Assay media contained charcoal-dextran filtered serum to reduce the level of hormones present. For agonist testing, cells were incubated with a compound to induce response. Intermediate dilution of sample stocks was performed to generate 5X sample in assay buffer. 5 pL of 5X sample was added to cells and incubated at 37°C or room temperature for 3-16 hours. Final assay vehicle concentration was 1%. For antagonist testing, cells were preincubated with antagonist followed by agonist challenge at the EC80 concentration.

[1016] Intermediate dilution of sample stocks was performed to generate 5X sample in assay buffer. 5 pL of 5X sample was added to cells and incubated at 37°C or room temperature for 60 minutes. Vehicle concentration was 1%. 5 pL of 6X EC80 agonist in assay buffer was added to the cells and incubated at 37°C or room temperature for 3-16 hours.

[1017] For signal detection, a single addition of 15 pL (50% v / v) of PathHunter® Detection reagent cocktail, followed by a one hour incubation at room temperature. Microplates were read following signal generation with a PerkinElmer Envision™ instrument for chemiluminescent signal detection. Compound activity was analyzed using CBIS data analysis suite (Chemlnnovation, CA). For agonist assays, percentage activity was calculated using the following formula: % Activity = 100% x (mean RLU of test sample - mean RLU of vehicle control) / (mean MAX control ligand - mean RLU of vehicle control). For antagonist mode assays, percentage inhibition was calculated using the following formula: % Inhibition = 100% x (1 - (mean RLU of test sample - mean RLU of vehicle control) / (mean RLU of EC 80 control -mean RLU of vehicle control)).

[1018] Compounds DCA-MCY and CDCA-CY were tested for FXR antagonism. As seen in Figures 72A and 72B, DCA-MCY (IC50 = 10.915 pM) and CDCA-CY (IC50 = 36.349 pM) are potent FXR antagonists.EXAMPLE 8 - CD8+ T cell sternness

[1019] Drugs that maintain or promote CD8+ T cell sternness (characterized by markers like T cell factor 1 (TCF1) and reduced exhaustion) are emerging as critical tools for improving longterm immune responses (Wen et al., J. Leukoc. Biol. (2021) 110(3):585-590. Herein, it is shown that BA-MCYs regulate sternness of CD8+ T cells, i.e., stem cell-like properties (self-renewal, long-term persistence, high proliferation) found in a subset of CD8+ T cells, marked by TCF1 expression (TCF1+), which act as progenitors, generating potent effector cells for sustained immune responses, especially against chronic infections and tumors, making them vital targets in cancer immunotherapy.

[1020] Briefly, naive CD8+T cells were stimulated with plate-bound anti-CD3s (5 pg / mL) and soluble anti-CD28 (1 pg / mL) for 2 days in the presence of the CA-MCY, CDCA-MCY, DCA-MCY, DCA-MCYO, or DCA-CY (10 pM each). After 2 days of co-stimulation, TCF-1 expression was analyzed to evaluate the effects of these compounds on CD8+T cell sternness by flow cytometry. As seen in Figure 73, CA-MCY, CDCA-MCY, DCA-MCY, DCA-MCYO, and DCA-CY all affected TCF-1 expression. Notably, CA-MCY, CDCA-MCY, and DCA-CY all significantly increased TCF-1 expression (DCA-MCY increases TCF-1 expression to a lesser extent). DCA-MCYO significantly decreased TCF-1 expression.

[1021] EXAMPLE 9 - TGR5 agonists

[1022] Takeda G protein-coupled receptor 5 (TGR5) (also known as G-Protein-Coupled Bile Acid Receptor 1 (GPBAR1)) agonists are emerging as potent therapeutic targets for metabolic, inflammatory, and liver diseases, primarily by leveraging the gut-liver-immune axis. TGR5 (GPBAR1) is a G-protein coupled receptor that is known as a target of other bile acids. The ability of certain compounds to act as agonists of TGR5 (GPBAR1) is shown herein.

[1023] TGR5 modulation activity was measured using a GPCR cAMP modulation assay.

[1024] Briefly, cAMP Hunter cell lines were seeded in a total volume of 20 pL into white walled, 384-well microplates and incubated at 37°C. cAMP modulation was determined using the DiscoverX HitHunter® cAMP XS+ assay. For Gs agonist determination, cells were incubated with sample to induce response. Media was aspirated from cells and replaced with 15 pL HBSS / lOmM Hepes. Intermediate dilution of sample stocks was performed to generate 4X sample in assay buffer. 5 pL of 4x sample was added to cells and incubated at 37°C for 30 minutes. Final assayvehicle concentration was 1%. For GI agonist determination, cells were incubated with sample in the presence of EC80 forskolin to induce response. Media was aspirated from cells and replaced with 10 pL HBSS / lOmM Hepes. Intermediate dilution of sample stocks was performed to generate 4X sample in assay buffer and 5 pL of 4X sample was added to cells. 5 pL of 4X EC80 forskolin diluted in HBSS / HEPES was added and incubated at 37°C for 30 minutes. Final assay vehicle concentration was 1%.

[1025] For antagonist determination, cells were pre-incubated with sample followed by agonist challenge at the EC80 concentration. Media was aspirated from cells and replaced with 10 pL HBSS / Hepes. 5 pL of 4X compound was added to the cells and incubated at 37°C for 30 minutes. 5 pL of 4X EC80 agonist was added to cells, diluted in either HBSS / HEPES (Gs) or diluted in 4X EC80 Forskolin (Gi) and incubated at 37°C for 30 minutes. After appropriate compound incubation, assay signal was generated through incubation with 5pL cAMP XS+ Ab reagent followed by 20 pL cAMP XS+ ED / CL lysis cocktail for one hour at room temperature.

[1026] 20 pL cAMP XS+ EA reagent for two hours at room temperature. Microplates were read following signal generation with a PerkinElmer Envision™ instrument for chemiluminescent signal detection. Compound activity was analyzed using CBIS data analysis suite (Chemlnnovation, CA). For Gs agonist mode assays, percentage activity is calculated using the following formula: % Activity =100% x (mean RLU of test sample - mean RLU of vehicle control) / (mean RLU of MAX control - mean RLU of vehicle control). For Gs antagonist mode assays, percentage inhibition is calculated using the following formula: % Inhibition =100% x (1 - (mean RLU of test sample - mean RLU of vehicle control) / (mean RLU of EC 80 control -mean RLU of vehicle control)). For Gi agonist mode assays, percentage activity is calculated using the following formula: % Activity = 100% x (1 - (mean RLU of test sample - mean RLU of MAX control) / (mean RLU of vehicle control - mean RLU of MAX control)). For Gi antagonist mode assays, percentage inhibition is calculated using the following formula: % Inhibition = 100% x (mean RLU of test sample - mean RLU of EC 80 control) / (mean RLU of forskolin positive control - mean RLU of EC80 control).

[1027] As seen in Figs. 74A-74C, CA-MCY (EC50 = 41.61 pM), DCA-MCY (EC50 = 2.2802 pM) and DCA-MCYO (EC50 = 6.6654 pM) are potent GPBAR1 agonists.A number of publications and patent documents are cited throughout the foregoing specification in order to describe the state of the art to which this invention pertains. The entire disclosure of each of these citations is incorporated by reference herein.

[1028] While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method for improving the health of an animal, the method comprising the step of administering to the animal an effective amount of a therapeutic composition comprising a cysteamide of a bile acid.

2. The method of claim 1, wherein the administration results in a benefit selected from the group consisting of: lowering cholesterol levels, lowering low-density lipoprotein levels, lowering triglyceride levels, and combinations thereof.

3. The method of claim 1, wherein the improved health comprises ameliorating or curing a disorder selected from the group consisting of: blood clots, cardiovascular disease, peripheral arterial disease, fatty liver disease, stroke, and combinations thereof.

4. The method of claim 1, wherein the improved health comprises ameliorating the effects of or curing a disorder selected from the group consisting of: hyperglycemia and / or type 2 diabetes.

5. The method of claim 1, wherein the improved health comprises ameliorating treating metabolic or liver diseases.

6. The method of claim 5, wherein the method comprises treating a patient who suffers from non-alcoholic fatty liver disease (NAFLD).

7. The method of claim 6, characterized in that the administration results in a reduced accumulation of fat in the liver of the patient.

8. The method of claim 5, wherein the method comprises treating a patient suffering from non-alcoholic steatohepatitis (NASH).

9. The method of claim 8, characterized in that the administration results in decreased inflammation and / or hepatocyte injury.

10. The method of claim 8, characterized in that the administration results in decreased fibrosis and cirrhosis.

11. The method of claim 5, wherein the method comprises treating a patient who suffers from cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).

12. The method of claim 11, characterized in that the administration of bile acid cysteamides results in improved bile flow, and / or reduced bile acid accumulation and / or reduced liver damage.

13. The method of claim 1, wherein the improved health comprises ameliorating or curing cancer.

14. The method of claim 13, wherein the cancer is FXR dependent.

15. The method of claim 13, wherein the cancer comprises liver cancer.

16. The method of claim 15, wherein the liver cancer comprises hepatocellular carcinoma.

17. A method of selectively modulating FXR receptors in a patient’s intestine comprising the step of treating the patient with one or more bile acid cysteamides.

18. The method of claim 17 characterized in that modulation of intestinal FXR receptors is at least 1.5 times greater, at least 2 times greater, at least 5 times greater, or at least 10 times greater than modulation of FXR receptors in at least one non-intestinal tissue.

19. The method of claim 18, wherein the at least one non-intestinal tissue is liver tissue.

20. A method of modulating the gut microbiome of a mammal comprising the step of administering to the mammal an effective amount of a bile acid cysteamide.

21. The method of claim 20, wherein the modulation of the gut microbiome comprises a change in the relative abundances of microbes composing the mammal’s gut microbiome.

22. The method of claim 21, wherein the modulation of the gut microbiome comprises a change in microbe metabolism or a change in the production of one or more secondary metabolites by one or more microbes composing the mammal’s gut microbiome.

23. The method of claim 20, wherein the modulation of the gut microbiome relieves or cures a disease.

24. The method of claim 23, wherein the disease comprises IBD or colitis.

25. The method of claim 20, wherein the step of administering comprises adding BA cysteamides to the patient’s diet.

26. A method of alleviating or curing a disease or disorder in a patient comprising the step of administering one or more bile acid cysteamides resulting in the modulation of a bile acid receptor other than FXR.

27. The method of claim 26, wherein the bile acid receptor comprises TGR5.

28. The method of claim 27, wherein the disease or disorder is selected from the group consisting of: obesity, insulin sensitivity, postprandial glycemic control, and hypertriglyceridemia.

29. The method of claim 27, wherein the disease or disorder is an inflammatory condition.

30. The method of claim 29, wherein the treatment reduces production of proinflammatory cytokines such as tumor necrosis factor-a (TNF-a), interleukin (IL)-la, IL-1, IL-6 and / or IL-8.

31. The method of claim 27, wherein the disease or disorder is arthritis or metabolic syndrome.

32. The method of claim 26, wherein the bile acid receptor comprises LXR.

33. The method of claim 32, wherein the treatment results in reduced fat accumulation and / or promotion of fat tissue browning.

34. The method of claim 32, wherein the treatment lower cholesterol levels.

35. The method of claim 32, wherein the treatment increases cholesterol excretion.

36. The method of claim 32, wherein the treatment decreases cholesterol absorption.

37. A method for treating, inhibiting, and / or preventing a disease or disorder associated with famesoid X receptor (FXR), said method comprising administering an effective amount of a therapeutic composition comprising a cysteamide of a bile acid, wherein said cysteamide of a bile acid is an FXR antagonist.

38. The method of claim 37, wherein said disease or disorder is obesity, type 2 diabetes, nonalcoholic fatty liver disease, or metabolic dysfunction-associated steatohepatitis.

39. A method of increasing sternness in a cell, said method comprising contacting the cell with a cysteamide of a bile acid.

40. The method of claim 39, wherein said cells are CD8+ T cells.

41. The method of claim 39, wherein said cysteamide of a bile acid is administered to a subject.

42. The methods of claim 39, wherein said cysteamide of a bile acid is administered with a checkpoint blockade, CAR-T therapy, an antiviral, or a vaccine.

43. A method for treating, inhibiting, and / or preventing a disease or disorder associated with TGR5, said method comprising administering an effective amount of a therapeutic composition comprising a cysteamide of a bile acid, wherein said cysteamide of a bile acid is an TGR5 agonist.

44. The method of claim 43, wherein said disease or disorder is type 2 diabetes, obesity, inflammation, inflammatory bowel disease, or metabolic dysfunction associated steatohepatitis.

45. The method of any one of the preceding claims, wherein the cysteamide of a bile acid comprises a compound of Formula I:R1IA, N.Y ■'-XXS(O)J,° IZwherein:A is a bile acid;R1is selected from -H, and an optionally substituted C1-40 aliphatic group;Z is -H, or an optionally substituted C1-40 aliphatic group; andy is 0, 1, or 2.

46. The method of claim 45, wherein the moiety A is derived from an endogenous bile acid.

47. The method of claim 45, wherein the bile acid comprises a C24 bile acid.

48. The method of claim 45, wherein the bile acid comprises a C27 bile acid.

49. The method of claim 45, wherein the bile acid is selected from the group consisting of: cholic acid (CA), -muricholic acid (PMCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), deoxycholic acid (DCA), and 7-ketodeoxycholic acid (7-KDCA).

50. The method of claim 45, wherein the bile acid is selected from the group consisting of: lithocholic acid (LCA), isolithocholic acid, 3-oxolithocholic acid, allolithocholic acid, 3-oxoallolithocholic acid, isoallolithocholic acid, 7a,12a-dihydroxy-3-oxochol-4-en-24-oic acid, 7a-hydroxy-3-oxochol-4-en-24-oic acid and 12a-hydroxy-3-oxochol-4,6-dien-24-oic acid, ursodeoxycholic acid, and 3a, 6a, 7a, 12a-tetrahydroxy-5B-cholan-24-oic acid.

51. The method of claim 45, wherein R1is -H.

52. The method of claim 45, wherein Z is other than -H.

53. The method of claim 45, wherein Z is an optionally substituted moiety selected from the group consisting of: a C1-24 alkyl group, a C1-12 alkyl group, a Ci-s alkyl group, a C1-6 alkyl group, a C1-5 alkyl group, a C1-4 alkyl group, a C1.3 alkyl group, a C1-2 alkyl group, and a methyl group.

54. The method of claim 45, wherein Z is methyl.

55. The method of claim 45, wherein y is 0.

56. The method of claim 45, wherein y is 1 or 2.

57. The method of claim 45, wherein the composition comprises a mixture of two or more compounds that differ only in the value of58. The method of claim 45, wherein R1is -H, Z is methyl, and y is 0.

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