Aminoethyl thioesters, therapeutic compositions thereof, and related methods

Abstract

The invention relates to aminoethyl thioesters, therapeutic compositions containing such aminoethyl thioesters and methods of using the same.

Description

[0001] AMINOETHYL THIOESTERS, THERAPEUTIC COMPOSITIONS THEREOF, AND RELATED METHODS

[0002] Frank Schroeder

[0003] David Artis

[0004] Tae Hyung Won

[0005] Bingsen Zhang

[0006] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 741,660, filed January 3, 2025. The foregoing application is incorporated by reference herein.

[0007] GOVERNMENT SUPPORT

[0008] This invention was made with Government support under Grant Nos. R35GM131877 DK126871, AH51599, AI095466, AI095608, AH42213, AR070116, AH72027, DK132244 and U2CES030167 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.

[0009] FIELD OF THE INVENTION

[0010] 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.

[0011] BACKGROUND OF THE INVENTION

[0012] 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).

[0013] Recent investigations 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

[0014] The present invention addresses these and other related needs.SUMMARY OF THE INVENTION

[0015] Among other things, the present invention encompasses the surprising discovery that a recently discovered family of carboxylic acids that are modified with a methyl cysteamide moiety are biosynthetically accessible from aminothioester derivatives via intramolecular rearrangement. This is particularly surprising given the fact that taurine derivatives of carboxylic acids (e.g. derivative of the formula R-CONCH2CH2SO3H) are well known. As such, it would be logical to assume that the newly discovered cysteamide derivatives of carboxylic acids would most likely arise from reduction of taurine derivatives, however this is not the case. Instead, it appears cysteamide biosynthesis proceeds via the pantetheinase VNNl / Vanin 1. VNNl / Vanin l’s primary function is to hydrolyze pantetheine into cysteamine and pantothenic acid (Fig. 3D), as part of coenzyme A recycling, and recent studies have shown that VNNl / Vanin 1 plays important roles in the regulation of metabolism, inflammation, and associated diseases. It has been found that VNN1 is also capable of hydrolyzing bile acid-pantetheine or bile acid CoA conjugates. The resulting S-linked BA-cysteamine derivatives then rearrange to the N-linked isomer, which following S-methylation produce bile acid methyl cysteamide (BA-MCY) conjugates (Fig. 3D).

[0016] Based on these findings, the present invention provides therapeutic compositions comprising aminoethylthioesters, as well as novel, non-naturally occurring aminoethyl thioester compounds, and methods of treating health disorders by administering the provided compounds or therapeutic compositions. In another aspect, the invention provides new treatments for health conditions related to VNNl / Vanin function (or dysfunction).

[0017] Discoveries have been made regarding the production and function of cysteamides including the fact that administering compositions containing cysteamides (or analogs thereof) provides a useful strategy to improve the health of animals including humans and / or to treat certain diseases and disorders (WO 2023 / 168249A2). The present invention builds on these results by providing certain ethylamino thioesters of carboxylic acids as therapeutic agents and / or precursors to therapeutic agents such as the disclosed cysteamides. In certain embodiments, the provided compounds described herein that are capable of rearranging in vivo to provide pharmacologically active cysteamides (or precursors thereof).

[0018] In certain embodiments, the provided compounds and compositions comprise aminoethyl thioesters of short chain fatty acids (SFCA’s). Without being bound by theory or limiting claims of the present invention, it is believed these SFCA derivatives, and / or their rearrangement products and related derivatives thereof constitute an important class of signaling molecules that operate between the gut microbiome and other organ systems withinthe mouse (and by extension humans). Perhaps most notably, certain SFCA cysteamide derivatives have been found to efficiently cross the blood brain barrier and these molecules may provide a useful method to modulate the signaling pathways mediating interactions between the gut microbiome and brain chemistry. By extension, such molecules may provide a useful method to modulate broader physiological and / or cognitive states associated with changes in diet. These discoveries have led to the invention of new therapeutic compositions comprising one or more SFCA aminoethyl thioesters (or related synthetic derivatives and / or analogs) as well as methods of administering such compositions to improve the health or wellbeing of an animal and / or of treating, ameliorating, or curing diseases in an animal or a human.

[0019] SFCA’s are known to mediate signaling between the gut microbiome and other organ systems within the mouse (and by extension humans). Perhaps most notably, certain SFCA cysteamide derivatives have been found to efficiently cross the blood brain barrier and these molecules provide a useful method to modulate the signaling pathways mediating interactions between the gut microbiome and brain chemistry. By extension, the aminoethyl thioesters provided herein provide a useful method to modulate broader physiological and / or cognitive states associated with changes in diet. These discoveries have led to the invention of new therapeutic compositions comprising one or more SFCA aminoethyl thioesters (or related synthetic derivatives and / or analogs) as well as methods of administering such compositions to improve the health or wellbeing of an animal and / or of treating, ameliorating, or curing diseases in an animal or a human.

[0020] In certain embodiments, the provided compounds and therapeutic compositions comprise aminoethyl thioesters of bile acids (BA’s). Bile acids (BAs) represent gut microbiota-dependent metabolites whose pervasive effects on human physiology are among the most well studied. Correspondingly, the biochemical mechanisms by which the host may regulate their activities are of substantial 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-dependent and microbiota-dependent pathways.

[0021] Our identification of VNN1 -dependent BA-MCY conjugates as FXR antagonists reveals a previously unrecognized host-dependent layer of BA metabolism that counteracts the physiological functions of free BAs (Fig. 5K). In vitro protein-protein interaction assays demonstrated strong FXR antagonistic activity for CA-MCY, CDCA-MCY and PMCA-MCY (Figs. 4A, 4B), whereas free BAs generally act as FXR agonists (Wang, et al., Mol. Cell3:543-553 (1999)). Supplementation of mice with CDCA-MCY increased total BA production and expression of the enzymes that catalyze the rate-limiting steps in BA biosynthesis, consistent with CDCA-MCY functioning as an FXR antagonist in vivo. This is in contrast to its parent compound CDCA, which acts as an FXR agonist and reduces overall BA levels (Einarsson, et al., Hepatology 33:1189-1193 (2001)). BA-MCYs were most abundant in intestinal tissues (Fig. 3 A), where VNN1 is highly expressed, and appeared to get oxidized quickly into the inactive BA-MCYO derivatives when entering the general circulation. Similar to other intestinal FXR antagonists (Jiang, et al., Nat. Commun. 6:10166 (2015); Ito, et al., J. Clin. Invest. 115:2202-2208 (2005)), CDCA-MCY supplementation alleviated hepatic lipid accumulation in mice fed a HCD (Fig. 51, 5J). Thus, it seems likely that, following their reuptake in the intestine, conversion of free BAs (FXR agonists) into BA-MCYs (FXR antagonists) via the pantetheinase VNN1 represents an important component of the feedback mechanisms regulating BA biosynthesis and other FXR-dependent phenotypes, including diverse aspects of fatty acid metabolism (Li, et al., Front. Pharmacol. 11:1247 (2020); Ali, et al., Ann. Transl. Med. 3:5 (2015); Claudel, et al.

[0022] Arterioscler. Thromb. Vase. Biol. 25:2020-2031 (2005)). Given the dysregulation of BA levels in type II diabetes, metabolic syndrome and the cholestatic diseases (Molinaro, et al., Trends Endocrinol. Metab. 29:31-41 (2018); Ferrell, et al., Diabetes Metab. J. 43:257-272 (2019); Li, et al., Adv. Pharmacol. 74:263-302 (2015)), BA-MCYs have substantial therapeutic potential. We further demonstrate that dietary fiber can upregulate production of BA-MCY conjugates in mice, indicating that the levels of these conjugates can be regulated by diet and prebiotic or probiotic supplements, which have translational ability in conditions of dysregulated immune or metabolic homeostasis. The profound effects of MCY conjugation on the biological activity of BAs led us to investigate the roles of both host and microbiota in the production of the MCY conjugates. Levels of unconjugated, free BAs are largely dependent on microbiota, as the vast majority of free BAs in SPF mice is derived from microbial deconjugation of the corresponding taurine conjugates. Using a series of stable isotope supplementation studies, we have shown that BA-MCYs are derived from conjugation with cysteamine or another CoA-derived metabolite, instead of reduction of corresponding taurine derivatives. Consistent with the idea that BA-taurine and BA-MCY conjugates have distinct biosynthetic origins, BA-MCY conjugates accumulate in mice deficient in the enzyme conjugating BAs with taurine (BAAT) (Neugebauer, et al., J. Lipid Res. 63, 100297 (2022)). The intriguing connection to CoA breakdown metabolism led us to uncover the role of the pantetheinase VNN1 for BA-MCY production, demonstrating that ahost enzyme that is highly expressed in the intestine, the site of BA reuptake, has a central role for BA-MCY production (Fig. 3D and 4T). BA-MCY biosynthesis is strongly reduced but not fully abolished in Vnnl- / - mice, indicating that other pantetheinases (VNN3 in mouse or VNN2 in humans) may also contribute. Furthermore, although bacteria have no close homologues of vertebrate pantetheinases, it is possible that other bacterial hydrolases or peptidases have similar activity and also contribute to the residual amounts of BA-MCYs observed in Vnnl- / - mice.

[0023] VNN1 serves diverse functions in lipid metabolism and forms an important link between lipid accumulation and hepatic diseases (Bartucci, et al., Int. J. Mol. Sci. 20:3891 (2019); Yu, et al., Eur. J. Pharmacol. 962:176220 (2024)), which is of particular interest in light of our finding that BA-MCY supplementation alleviated lipid accumulation in the liver of HCD mice (Fig. 51, 5 J). Clarifying the role of VNN1 for BA-MCY production and other aspects of BA metabolism in the intestine and other tissues may provide new insights in associated phenotypes. More generally, the identification of the role of VNN1 in BA-MCY production reveals an intriguing connection between BA signaling and CoA breakdown pathways, which are extensively regulated by nutritional state, feeding back on many other aspects of metabolism (Naquet, et al., Prog. Lipid Res. 78:101028 (2020)). BA-MCY conjugates can be hydrolyzed by microbial BSH, albeit perhaps less efficiently than BA-taurine conjugates. To what extent microbial deconjugation of BA-MCYs is physiologically relevant is unclear; however, it may represent an additional mechanism by which the microbiota contribute to regulating the balance of FXR agonists and antagonists.

[0024] Together, our results indicate that MCY conjugation of BAs by the host balances microbiota-dependent taurine deconjugation, as part of a metabolic network integrating host-derived and microbiota-derived pathways that regulates FXR-dependent BA production, fat metabolism, CoA metabolism and possibly other processes downstream of BAs.

[0025] Without being bound by theory or limiting claims of the present invention, it is believed these BA derivatives, (and / or their rearrangement products and related derivatives thereof) constitute an important class of molecules that operate to maintain balance in BA-related processes. BAs are important physiological agents for intestinal nutrient absorption and biliary secretion of lipids, toxic metabolites, and xenobiotics. BAs and their derivatives are signaling molecules and metabolic regulators that activate nuclear receptors and G protein-coupled receptor (GPCR) signaling to regulate hepatic lipid, glucose, and energy homeostasis and maintain metabolic homeostasis. Conversion of cholesterol to bile acids is critical for maintaining cholesterol homeostasis and preventing accumulation of cholesterol,triglycerides, and toxic metabolites, and injury in the liver and other organs. Enterohepatic circulation of bile acids and their derivatives from the liver to intestine and back to the liver plays a central role in nutrient absorption and distribution, and metabolic regulation and homeostasis. This physiological process is regulated by a complex membrane transport system in the liver and intestine regulated by nuclear receptors. Bile acid-sensitive nuclear and GPCR signaling protects against inflammation in liver, intestine, and macrophages. Disorders in bile acid metabolism cause cholestatic liver diseases, dyslipidemia, fatty liver diseases, cardiovascular diseases, and diabetes. As such, the provided compounds and compositions are promising therapeutic agents for treating chronic liver diseases, obesity, and diabetes in humans.

[0026] In another aspect, the present invention encompasses novel compositions of matter including compositions of novel molecules. While some of the aminoethyl thioesters described herein may be naturally occurring molecules, pure samples of these molecules and, in particular, bulk samples of pure aminoethyl thioesters free from other biological materials are not found in nature. Additionally, many of the aminoethyl thioesters and related compounds described herein 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 provided compounds constitute novel non-natural compositions of matter.

[0027] In another aspect, the present invention comprises methods of making therapeutic compositions, the methods comprising formulating an effective amount of one or more purified or synthetically produced aminoethyl thioester derivatives described herein (or a pharmaceutically-acceptable salt, prodrug or derivative thereof) into a therapeutic composition. In certain embodiments, such therapeutic compositions are selected from the group consisting of: an injectable liquid, a tablet, a capsule, a pill, a solution or suspension for oral administration, a solid dosage form for suspension or dissolution into a drinkable- or injectable liquid, a dermal patch, an eye drop, a cream, an ointment, a gel, a powder, a spray, an inhalable composition, and a nasal spray.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] Fig. IB: Shows Volcano plots of differential metabolites detected in serum of GF (n = 12) and SPF (n = 9) mice. Blue and red dots represent metabolites five- or more-folddownregulated or upregulated in GF relative to SPF mice at P < 0.05, as calculated by unpaired two-sided t-test.

[0031] 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. Shown nodes are downregulated or upregulated in serum of GF compared to SPF mice.

[0032] Fig. ID: Shows the MS2 spectrum of CA-MCY. Dark grey MS2 fragments represent the MCY group; light grey MS2 fragments are derived from water loss.

[0033] Fig. IE: Shows the structures of BAMCY conjugates identified in mouse serum.

[0034] Fig. IF: 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+.

[0035] Fig. 1G: Shows the scheme for the synthesis of CA-MCY conjugates from CA.

[0036] Fig. 1H: 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.

[0037] Fig. II: 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.

[0038] Fig. 1J: 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.

[0039] Figs. 1K-1O: Shows MS2 spectra of MCY conjugates of BAs. MS2 spectra of CA-MCY (Fig. IK), CDCA-MCYO (Fig. IL), CA-MCYO (Fig. IM), 7KDCA-MCYO (Fig. IN), and CA-MCYO2 (Fig. 10) are provided. The listed BA-MCY conjugates in each panel produced MS2 spectra very similar to the shown examples. Blue arrows indicate inferred fragmentation. MS2 fragments and structure parts highlighted in red represent MCY groups. Green fragments are derived from water loss.

[0040] 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 foreach donor). Data are mean ± s.e.m. P values were calculated by unpaired two-sided t- test with Welch’s correction.

[0041] Fig. 2B: Shows the relative abundances of bMCA-MCY conjugates as well as corresponding free bMCA-MCY 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, (a) and

[0042] 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.

[0043] Fig. 2D: Shows the relative abundances of bMCA-MCY conjugates as well as corresponding free bMCA-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.

[0044] 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).

[0045] Fig. 2F: Shows the relative abundance of CA-MCY conjugates or CDCA-MCY conjugates in human serum (n = 19).

[0046] Fig. 2G: Shows pathways considered for the origin of BAMCY conjugates. MCY conjugates could originate either from reduction of BA taurine conjugates produced by the liver enzyme BAAT or conjugation of BAs with cysteamine or another cysteamine derivative from coenzyme A and pantetheine degradation. Oxidation of MCY conjugates produces the corresponding MCYO and MCY02 conjugates; BSH: bile salt hydrolase.

[0047] Fig. 2H: Shows administration of taurine-d4 in SPF mice resulted in deuterium incorporation in all detected taurine conjugates, but not in any MCY conjugates. Shown are EICs for the m / z of molecular ions of unlabeled (black) and deuterium-labeled versions of the different conjugates in serum of mouse fed taurine-d4.

[0048] Fig. 21: Shows administration of deuterium-labeled L-cysteine (L-cys-d2) in SPF mice resulted in deuterium incorporation in the MCY conjugates of BAs. Shown are EICs for the m / z of the molecular ions of the unlabeled (black) and the deuterium labeled versions of BA-MCY conjugates detected in serum of mice fed L-cys-d2.Figs. 2J-2M: Show metabolite identification using authentic standards. Extracted ion chromatograms (EICs) of BA-MCY and BA-MCYO conjugates (Figs. 2J and 2K), and BA-MCY02 (Fig. 2L) in serum of GF and SPF mice and comparison with synthetic standards analyzed in ESI+. Fig. 2M: Scheme for the synthesis of CA-MCY conjugates from CA.

[0049] Figs. 2N-2T: Shows microbiota dependent production of BA-taurine and BA-MCY conjugates. Fig. 2N: Relative abundances of BA-MCYO conjugates of less abundant BAs in serum of SPF (n = 11) and GF (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). Figs. 20 and 2P: Relative abundances of CA-MCY conjugates (Fig. 20) and PMCA-MCY conjugates (Fig. 2P) as well as the corresponding free BAs in feces of SPF mice (n = 3), GF mice (n = 3), and mice that received FMT (n = 10, the three donors are represented by triangles, circles, and crosses, n = 3-4 for each donor). N.D., not detected. Fig. 2Q: Relative abundances of BA-MCYO conjugates of less abundant BAs in feces of SPF (n = 3) and GF (n = 3), and mice that received FMT (n = 10, the three donors are represented by triangles, circles, and crosses, n = 3-4 for each donor). Fig. 2R: Relative abundances of BA-taurine conjugates in serum or feces of SPF (n = 11 for serum and n = 3 for feces) and GF (n = 12 for serum and n = 3 for feces), and mice that received FMT (n = 10, the three donors are represented by triangles, circles, and crosses, n = 3-4 for each donor). Figs. 2S and 2T: Relative abundances of free BAs in serum (Fig. 2S) or feces (Fig. 2T) of SPF (n = 11 for serum and n = 3 for feces) and GF (n = 12 for serum and n = 3 for feces), 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 using unpaired two-sided t-test with Wei ch ’ s correcti on .

[0050] Fig. 3 A: Shows ICs of BA-MCY conjugates in liver of WT and Baat- / - mice and comparison with synthetic standards analyzed in ESI+.

[0051] Fig. 3B: Shows relative abundances of BA-MCY conjugates in liver of WT (n = 4) or Baat- / - (n = 5) mice. Data are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction, ****p < 0.0001. N.D., not detected.

[0052] Fig. 3C: Shows total amounts of BA-MCY conjugates and corresponding free BAs in liver, small intestine, and cecum of SPF (n = 11) and GF (n = 12 for liver and n = 13 for small intestine and cecum) mice. Data are mean ± s.e.m. P values were calculated by unpaired two sided t-test with Welch’s correction.Fig. 3D: Shows established function of VNN1 in pantetheine hydrolysis (box) and proposed role of VNN1 in CA-pantetheine hydrolysis, followed by re-arrangement and methylation to form CA-MCY.

[0053] Fig. 3E: Shows production of pantothenic acid from a range of concentrations of CA-pant and pantetheine incubated with recombinant VNN1 in vitro. Reactions with both substrates follow saturation kinetics. Enzyme concentration was 0.01 pM. The reactions were incubated at 37°C for 15 minutes. Number of independent assays using the same batch of enzyme (n = 3). Data are mean ± s.d.

[0054] Fig. 3F: Shows relative abundances of BA-MCY conjugates in small intestine, liver, serum and feces of WT (n = 5) or Vnnl- / - (n = 5) mice. Data are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction, ****p < 0.0001. Fig. 3G: 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. Fig. 3H: Shows total amounts of free BAs and BA-MCY conjugates in feces of GF (n =16) mice. Data are mean ± s.e.m. P values were calculated by paired two-sided t-test. Fig. 31: 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.

[0055] Fig. 3 J: 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.

[0056] Fig. 3K: 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

[0057] Fig. 3L: Shows roles of host and microbiota in the biosynthesis and metabolism of BA taurine and MCY conjugates.

[0058] Figs. 3M-3S: Shows the relationship between abundances of free BAs and BA-MCY conjugates. Figs. 3M and 3N: Relative abundances of CA-MCY (Fig. 3M) and PMCA-MCY conjugates (Fig. 3N) as well as corresponding free BAs in serum of mice fed control (n = 8) or inulin fiber diet (n = 7). Fig. 30: Relative abundances of BA- MCYO conjugates as well as corresponding free BAs in serum of mice fed control (n =8) or inulin fiber diet (n = 7). Fig. 3P Relationship between abundances of free BAs and BA-MCY conjugates in feces of SPF mice used as control for the FMT study (n = 25), FMT mice (n = 10), SPF mice fed a control diet for the inulin fiber diet study (n = 6), and inulin fiber diet fed SPF mice (n = 8). Fig. 3Q Relationship between abundances of free BAs and BA-MCY conjugates in serum of SPF as control for the FMT study (n = 11), FMT mice (n = 10), SPF mice fed a control diet for the inulin fiber diet study (n = 8), and inulin fiber diet fed SPF mice (n = 7). Figs. 3R and 3S: Relative abundances of free BAs (Fig. 3R) and corresponding BA-MCY conjugates (Fig. 3 S) in serum of human (n = 19). Data are mean ± s.e.m. P values were calculated using unpaired two-sided t-test with Welch’s correction.

[0059] Figs. 3T-3W: Shows the analysis of stable-isotope feeding experiments. Fig. 3T:

[0060] Administration of taurine-d4 in SPF mice resulted in deuterium incorporation in all detected taurine conjugates, but not in any MCY conjugates. Shown are EICs for the m / z of molecular ions of unlabeled (black) and deuterium-labeled versions of the different conjugates in serum of mouse fed taurine-d4. Fig. 3U: Administration of deuterium-labeled L-cysteine (L-cys-d2) in SPF mice resulted in deuterium incorporation in the MCY conjugates of BAs. Shown are EICs for the m / z of the molecular ions of the unlabeled (black) and the deuterium -lab eled versions of BA- MCY conjugates detected in serum of mice fed L-cys-d2. Fig. 3 V and 3W:

[0061] Administration of deuterium-labeled L-cysteine (L-cys-d2) in SPF mice resulted in deuterium incorporation in taurine conjugates of BAs (Fig. 3 V) and pantetheine (Fig.

[0062] 3W). EICs for molecular ion peaks (black) and deuterium isotope peaks of taurine conjugates of BAs (Fig. 3 V) and pantetheine (Fig. 3W) in serum of mouse fed L-cys- d2.

[0063] Figs. 4A-4D: Show in vitro data demonstrating that BA-MCY conjugates are FXR antagonists. Compounds were tested against a cell-based protein-protein interaction assays in both agonist and antagonist modes. CDCA-MCY (Fig. 4A), CA-MCY (Fig.

[0064] 4B), and PMCA-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.

[0065] Figs. 4E-4J: Shows the analysis of Baat- / - mice. Figs. 4E-4H: Extracted ion chromatograms (EICs) of BA-MCY and BA-MCYO conjugates (Figs. 4E-4G) and BAMCY02 conjugates (Fig. 4H) in liver of Baat- / - mice and comparison with synthetic standardsanalyzed in ESI + . Figs. 41 and 4J: Relative abundances of BA-MCY conjugates (Fig.

[0066] 41) and corresponding free BAs (Fig. 4J) in liver of WT (n = 4) or Baat- / - (n = 5) mice. Data are mean ± s.e.m. P values were calculated using unpaired two-sided t-test with Welch’s correction. N.D., not detected.

[0067] Figs. 4K-4P: Show abundances of BA-MCY conjugates in different tissues. Figs. 4K and 4L:

[0068] Abundances of CA-MCY conjugates, TCA and CA (Fig. 4K), and PMCA-MCY conjugates, TpMCA and PMCA (Fig. 4L) in liver, small intestine, and cecum of SPF (n = 11) and GF (n = 12 for liver and n = 13 for small intestine and cecum) mice. Data are mean ± s.e.m. Figs. 4M-4P: Abundances of UDCA-MCY conjugates and UDCA (Fig. 4M), CDCA-MCY conjugates and CDCA (Fig. 4N), DCA-MCY conjugates and DCA (Fig. 40), and 7-KDCA-MCY conjugates and 7-KDCA (Fig. 4P) in liver, small intestine, and cecum of SPF (n = 11) and GF (n = 12 for liver and n = 13 for small intestine and cecum) mice. Data are mean ± s.e.m. P values were calculated using unpaired two-sided t-test with Welch’s correction. N.D., not detected.

[0069] Figs. 4Q-4T: Show the role of VNN1 in production of BA-MCY conjugates. Fig. 4Q: Steadystate kinetic analysis of CA-pant and pantetheine hydrolysis catalyzed by recombinant human VNN1 (AN490aa truncated) revealed both reactions follow saturation kinetics. The steady-state kinetic parameters Km and Vmax are determined by HPLC-HRMS for pantothenic acid formation to be 39.78 ± 20.31 pM and 1.53 ± 0.20 min-1 for CA- pant, and 74.07 ± 41.52 pM and 2.13 ± 0.37 min-1 for pantetheine. The reaction mixtures contain 0.01 pM VNN1. Number of independent assays using the same batch of enzyme (n = 3). Data are mean ± s.d. Fig. 4R: EICs of CA-CY in ileum of Baat- / - mice, extracts of in vitro reaction of VNN1 hydrolyses CA-pantetheine, and comparison with a synthetic standard analyzed in ESI +. Fig. 4S: EICs of CA-pant in small intestine of Vnnl- / - mice and comparison with a synthetic standard analyzed in ESI + . Fig. 4T: Relative abundances of CA-pant in small intestine, feces, liver, and serum of WT (n = 5) and Vnnl- / - (n = 5) mice. Data are mean ± s.e.m. P values were calculated using unpaired two-sided t-test with Welch’s correction. N.D., not detected. Fig. 5 A: 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 belong to the CA and CDCA pathways.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 (corn oil, n = 3). Data are mean ± s.e.m. P values were calculated by unpaired two-sided t-test with Welch’s correction.

[0070] 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.

[0071] 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.

[0072] 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. 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.

[0073] 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.

[0074] 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.

[0075] 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 mm.

[0076] 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

[0077] 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.Figs. 6A-6J: Show microbial deconjugation of BA-MCYs in SPF, GF, and ABX mice. Figs.

[0078] 6A and 6B: Total amounts of free BAs (Fig. 6A) or BA-MCY conjugates (Fig. 6B) in feces of SPF (n = 14) and GF (n = 16) mice. Data are mean ± s.e.m. P values were calculated using unpaired two-sided t-test with Welch’s correction. Figs. 6C-6E: Total amounts of labeled free BAs (Fig. 6C), BA-MCY conjugates (Fig. 6D), and BA- taurine conjugates (Fig. 6E) in feces of SPF (n = 14), ABX (n = 15), and GF (n = 3) mice administered CDCA-d5-MCY. Fig. 6F: Volcano plot of differential metabolites detected in liver of SPF mice administered control (com oil) (n = 4) or CDCA-d5- MCY (n = 5). Bubble sizes reflect peak areas. P values were calculated using unpaired two-sided t-test. Figs. 6G-6I: Total amounts of labeled free BAs (Fig. 6G), BA-MCY conjugates (Fig. 6H), and BA-taurine conjugates (Fig. 61) in liver of SPF (n = 5) and ABX (n = 5) mice administered CDCA-d5-MCY. Fig. 6J: Volcano plot of differential metabolites detected in liver of ABX mice administered control (com oil) (n = 4) or CDCA-d5-MCY (n = 5). Bubble sizes reflect peak areas. P values were calculated using unpaired two-sided t-test.

[0079] Figs. 6K-6N: Shows Microbial deconjugation of BA-MCYs in vivo. Figs. 6K and 6L: EICs for CDCA-MCY conjugates (Fig. 6K) and free BAs (Fig. 6L) with molecular ion peaks (black) and four deuterium isotope peaks from HPLC-HRMS analysis of feces of SPF mice administered CDCA-d5-MCY. Figs. 6M and 6N: EICs for CDCA-MCY conjugates (Fig. 6M) and free BAs (Fig. 6N) with molecular ion peaks (black) and four deuterium isotope peaks from HPLC-HRMS analysis of feces of GF mice administered CDCA-d5-MCY.

[0080] Figs. 7A-7C: Show the microbial deconjugation of BA-MCYs in vitro and gnotobiotic mice.

[0081] Figs 7A and 7B: Deconjugation of CA-MCY conjugates in fecal suspensions obtained from SPF mice (Fig. 7A) (n = 3) and cultured gut bacteria (Fig. 7B) (n = 3). Fig. 7C: Relative abundances of CDCA-d5-MCY conjugates and corresponding free BA in feces of GF monocolonized with WT (n = 3) or BSH-deficient B. ovatus (n = 3) (WT Bo and Absh Bo, respectively). Data are mean ± s.e.m. P values were calculated using unpaired two-sided t-test with Welch’s correction.

[0082] Figs. 7D-7J: Show FXR-related activity of known ligands and BA-MCYs. Fig. 7D: FXR agonistic effect of CDCA as measured in the protein-protein interaction assays. Data were normalized to the maximal and minimal response observed in the presence of control compound (GW4064) and vehicle (DMSO), respectively. Assays were performed in duplicate for each concentration. Figs. 7E and 7F: CDCA-MCY showedFXR antagonistic effects to obeticholic acid (25 pM) (Fig. 7E) or CDCA (25 pM) (Fig.

[0083] 7F) mediated activation of FXR. Data were normalized to the maximal and minimal response observed in the presence of control compound (DY268) and vehicle (DMSO), respectively. Assays were performed in duplicate for each concentration. Figs 7G-7I: Cytotoxicity assays for CDCA-MCY (Fig. 7G), CDCA-MCYO (Fig. 7H), and CDCA (Fig. 71) in a cell-based assay on human primary hepatocytes. Assays were performed in duplicate for each concentration. Fig. 7J: TpMCA did not show FXR antagonistic effects in protein-protein interaction assays at the tested concentrations. DY268 a synthetic FXR antagonist was used as a positive control. Data were normalized to the maximal and minimal response observed in the presence of control compound (DY268) and vehicle (DMSO), respectively. Assays were performed in duplicate for each concentration.

[0084] Figs. 8A-8F: Show Regulation of BA biosynthesis by BA-MCYs in vivo. Figs. 8A and 8B:

[0085] Abundances of endogenously produced BAs in feces of mice administered CDCA- MCY or CDCA-d5-MCY daily for 14 days. Shown are individual amounts of CDCA- derived BAs (Fig. 8A) and CA-derived BAs (Fig. 8B) in feces. Data are mean ± s.e.m. with control (corn oil) (n = 7) and CDCA-MCY fed mice (n = 7 for CDCA-derived pathway and n = 3 for CA-derived pathway). P values were calculated using unpaired two-sided t-test with Welch’s correction. Figs. 8C and 8D: Abundances of CDCA- derived BAs (Fig. 8C) and CA-derived BAs (Fig. 8D) in liver of mice administered CDCA-MCY or CDCA-d5-MCY daily for 14 days. Data are mean ± s.e.m. with control (com oil) (n = 6) and CDCA-MCY fed mice (n = 3 for CDCA-derived pathway and n = 7 for CA-derived pathway). P values were calculated using unpaired two-sided t-test with Welch’s correction. Figs. 8E and 8F: Abundances of CDCA-derived BAs (Fig. 8E) and CA-derived BAs (Fig. 8F) in serum of mice administered CDCA-MCY or CDCA-d5-MCY daily for 14 days. Data are mean ± s.e.m. with control (com oil) (n = 6) and CDCA-MCY fed mice (n = 3 for CDCA-derived pathway and n = 7 for CA- derived pathway). P values were calculated using unpaired two-sided t-test with Wei ch ’ s correcti on .

[0086] Figs. 8G-8J: Show FXR-related activity of BA-MCYs in vivo. Figs. 8G and 8H: Abundances of total BAs in liver (Fig. 8G) and serum (Fig. 8H) of WT and Nrlh4- / - mice administered CDCA-d5-MCY daily for 14 days. Data are mean ± s.e.m. with control (com oil) (n = 4) and CDCA-d5-MCY fed mice (n = 4). P values were calculated using unpaired two-sided t-test with Welch’s correction. Fig. 81: Representativephotomicrographs 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 (HCD) (n = 4 for vehicle and n = 4 for CDCA-MCY). CDCA-MCY was delivered by oral gavage at a rate of 5 mg / kg body weight per day for two weeks. Scale bar, 100 pm. Fig. 8 J Average measured oil red O area of liver sections of mice in Fig. 81. Data are mean ± s.e.m. P values were calculated using oneway ANOVA with Tukey’s correction.

[0087] Fig. 9: Shows 'H (600 MHz) NMR spectrum of CA-pant (21) in methanol-t / 4.

[0088] Fig. 10: Shows the dqfCOSY spectrum of CA-pant (21) in methanol-t / 4.

[0089] Fig. 11 : Shows the HSQC spectrum of CA-pant (21) in methanol-t / 4.

[0090] Fig. 12: Shows the HMBC spectrum of CA-pant (21) in methanol-t / 4.

[0091] DETAILED DESCRIPTION OF THE INVENTION

[0092] Compounds and Therapeutic Compositions

[0093] In one aspect, the present invention encompasses novel compounds and / or therapeutic compositions comprising aminoethyl thioesters or derivatives or analogs of such aminoethyl thioesters.

[0094] In certain embodiments, provided compositions comprise one or more aminoethyl thioesters of a C1-40 carboxylic acid. In certain embodiments, such compositions comprise an aminoethyl thioester of a C1-24 carboxylic acid, a C1-20 carboxylic acid, a C1-16 carboxylic acid, a C1-12 carboxylic acid, a C1-8 carboxylic acid, a C2-12 carboxylic acid or a C2-8 carboxylic acid. In certain embodiments, such compositions comprise aminoethyl thioesters of straight-chain carboxylic acids. In certain embodiments, such compositions comprise aminoethyl thioesters of saturated carboxylic acids. In certain embodiments, such compositions are characterized in that they comprise aminoethyl thioesters of straight-chain saturated carboxylic acids. In certain embodiments, such compositions comprise aminoethyl thioesters of carboxylic acids that are branched, mono- or poly-unsaturated, optionally substituted, or that have two or more of these features in combination. In certain embodiments, such compositions comprise an aminoethyl thioester of an endogenous carboxylic acid. In certain embodiments, such compositions comprise an aminoethyl thioester of a microbiome-derived carboxylic acid.

[0095] In certain embodiments, provided therapeutic compositions comprise aminoethyl thioesters of a short chain fatty acid. In certain embodiments, such compositions comprise aminoethyl thioesters of a C1-6 carboxylic acid. In certain embodiments, such compositions comprise an aminoethyl thioester of a C2-6 straight chain carboxylic acid. In certainembodiments, such compositions comprise an aminoethyl thioester of a C3-6 saturated carboxylic acid, certain embodiments, such compositions comprise an aminoethyl thioester derived from a carboxylic acid selected from the group consisting of / / -butyric acid, propionic acid, acetic acid and formic acid.

[0096] In certain embodiments, provided therapeutic compositions comprise aminoethyl thioesters of a bile acid. 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

[0097] substructure

[0098]

[0099] > , or compounds derivable from such a substructure (for example by breaking one or more of the ring bonds). Examples of the latter include seco-congeners of bile acids, and compounds such as 9,11 seco cholesterol:

[0100]

[0101] In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula I:

[0102]

[0103] wherein:

[0104] A is an optionally substituted C1-40 aliphatic group; and

[0105] Each of R1and R2is independently selected from: -H, an optionally substituted C1-40 aliphatic group, an optionally substituted C1-40 acyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, and a linker moiety attached to the nitrogen atom of one or more additional molecules of formula I, where R1and R2may also be taken together to form a an optionally unsaturated ring.

[0106] In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula II:

[0107]

[0108] wherein each of A, and R2, is as defined above and in the genera and subgenera herein.

[0109] In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula III:

[0110]

[0111] wherein A, is as defined above and in the genera and subgenera herein; and

[0112] R3is selected from the group consisting of: -H, an optionally substituted C1-39 aliphatic group, an optionally substituted aryl group, an optionally substituted heterocyclic group, an amino acid, a peptide, and a linker attached to a molecule of formula I.

[0113] In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula Illb:

[0114]

[0115] (illb), wherein each of A, R1, and R2is as defined above and in the genera and subgenera herein.

[0116] In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula IIIc:

[0117]

[0118] wherein each of A, and R3is 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 Hid:

[0119]

[0120] wherein A, is as defined above and in the genera and subgenera herein.

[0121] In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula Hie:

[0122]

[0123] (Hie),

[0124] wherein A, is as defined above and in the genera and subgenera herein, and

[0125] -Z is selected from R1,

[0126]

[0127] In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula IV:

[0128]

[0129] wherein A- is, independently at each occurrence, as defined above and in the genera and subgenera herein;

[0130] R4is independently at each occurrence, selected from the group consisting of -H, an optionally substituted C1-40 aliphatic group, an optionally substituted C1-40 acylgroup, an optionally substituted aryl group, and an optionally substituted heterocyclic group;

[0131] -L- is a multidentate linker moiety containing at least 2 carbon atoms and optionally containing one or more sites of unsaturation, rings, and / or heteroatoms; and is an integer from 1 to 100.

[0132] In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula V:

[0133]

[0134] wherein each of A, and R2is as defined above and in the genera and subgenera herein, and

[0135] R5may be absent, or is selected from -H, a metal cation, an organic cation, an optionally substituted C1-40 aliphatic group, an optionally substituted C1-40 acyl group, an optionally substituted aryl group, and an optionally substituted heterocyclic group.

[0136] In certain embodiments, in any of the compounds of Formulae I through V, the moiety A- is derived from, or is identical to, an endogenous carboxylic acid.

[0137] In certain embodiments, in any of the compounds of Formulae I through V, the moiety A- comprises a short-chain fatty acid.

[0138] In certain embodiments, in any of the compounds of Formulae I through V, the moiety A- is propionic acid, butyric acid, or valeric acid. In certain embodiments, in any of the compounds of Formulae I through V, the moiety A- is butyric acid.

[0139] In certain embodiments, in any of the compounds of Formulae I through V, the moiety A- is St-L’- wherein:

[0140] -L’- comprises an optionally substituted, optionally unsaturated Ci-24 linker where any one or more carbon atoms may be optionally replaced with -N(Ry)2-, -C(O)-, -C(O)O-, -C(O)NRy-, -NRyC(O)-, -NRyCO2-, - CO2NRy-, -O-, -S- , -S(O)-, -S(O)2-, -C(=NRy)-, and -C(=S)-, where Ryis, -H, or an optionally substituted aliphatic, aryl, heteroaliphatic or heteroaryl group; and

[0141] -St comprises a moiety having a steroidal structure.In certain embodiments, where provided therapeutic compositions comprise a moiety St-L’-, 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 thioester. 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 thioester. In certain embodiments, -L’- comprises a chain of 3 to 7 carbon atoms separating the moiety -St from the acyl linkage.

[0142] In certain embodiments, the moiety -L’- comprises

[0143]

[0144] where * represents the site of attachment to the moiety -St.

[0145] In certain embodiments, the moiety -L’- comprises

[0146]

[0147] where * represents the site of attachment to the moiety -St.

[0148] In certain embodiments, the moiety -St comprises a steroid moiety linked to -L’-through ring-D.

[0149] In certain embodiments, the moiety -St comprises a steroid moiety linked to -L’-

[0150]

[0151] 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

[0152] is at carbon 3, 7, or 12 of the steroid ring.

[0153]

[0154] In certain embodiments, the moiety -St-L’- in Formula VI is selected from the group consisting of:

[0155]

[0156] In certain embodiments, in any of the compounds of Formulae I through V, the moiety A- comprises a bile acid. In certain embodiments, A- comprises a C24 bile acid. In certain embodiments, A- comprises a C27 bile acid. In certain embodiments, A- is selected from the group consisting of: cholic acid (CA), a-muricholic acid (aMCA), P-muricholic acid (PMCA), w-muricholic acid (wMCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), deoxycholic acid (DCA), and 7-ketodeoxycholic acid (7-KDCA).

[0157] 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, 6a, 7a, 12a-tetrahydroxy-5B-cholan-24-oic acid.

[0158] In certain embodiments, for compounds of Formula I, at least one of R1and R2is -H. In certain embodiments, both R1and R2are -H.

[0159] In certain embodiments, for compounds of Formula I, neither of R1and R2is -H. In certain such embodiments, R1and R2are the same. In certain such embodiments, R1and R2are different. In certain such embodiments, R1and R2are taken together to form a ring. In certain such embodiments, R1and R2are taken together to form a 3- to 12- membered ring. In certain such embodiments, R1and R2are taken together to form a 5- to 7- membered ring. In certain such embodiments, R1and R2are taken together to form a 5-membered ring. In certain such embodiments, R1and R2are taken together to form a 6-membered ring. In certain embodiments, where neither R1nor R2are -H, at least one of these substituents is labile under physiological conditions. In certain embodiments, where neither R1nor R2are -H, at least one of these substituents is an acyl group. In certain embodiments, where neither R1or R2are -H, at least one of these substituents is an amino acid or a peptide.

[0160] In certain embodiments, for compounds of Formulae I, II, Illb, or V, R2is an optionally substituted moiety selected from the group consisting of: a C1-24 alkyl group, a Ci-12 alkyl group, a Ci-s alkyl group, a C1-6 alkyl group, a C1-5 alkyl group, a C1-4 alkyl group, aCi-3 alkyl group, a C1-2 alkyl group, and a methyl group. In certain embodiments, for compounds of Formulae I, II, Illb, or V, R2is a C1-12 alkyl group. In certain embodiments, for compounds of Formulae I, II, Illb, or V, R2is a C1-6 alkyl group. In certain embodiments, for compounds of Formulae I, II, Illb, or V, R2is a C1-4 alkyl group.

[0161] In certain embodiments, for compounds of Formulae I, II, Illb, or V, R2is a C1-30 acyl moiety. In certain embodiments, for compounds of Formulae I, II, Illb, or V, R2is a Ci-12 acyl group. In certain embodiments, for compounds of Formula e I, II, Illb, or V, R2is a C1-6 acyl group. In certain embodiments, for compounds of Fo rmulae I, II, Illb, or V, R2is a C1-4 acyl group. In certain embodiments, for compounds of Formulae I, II, Illb, or V, R2is acetate. In certain embodiments, for compounds of Formulae I, II, Illb, or V, R2is propionate. In certain embodiments, for compounds of Formulae I, II, Illb, or V, R2is butyrate.

[0162] In certain embodiments, for compounds of Formulae I, II, Illb, or V, R2is an amino acid or a peptide. In certain embodiments, for compounds of Formulae I, II, Illb, or V, R2is an acyl moiety that is labile under physiological conditions.

[0163] In certain embodiments, for compounds of Formula Illb, R2is selected from the group consisting of:

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

[0165] p is an integer of 1-3.

[0166] . 1

[0167] In certain embodiments, for compounds of Formula Illb, R2is

[0168] In certain embodiments, a compound of Formula I is selected from the molecules shown in Table 1.

[0169] TABLE 1

[0170]

[0171] > &

[0172] >

[0173] &

[0174] "

[0175] <

[0176] >< < "

[0177]

[0178] In certain embodiments, a compound of Formula I is selected from the molecules shown in Table 2.

[0179] TABLE 2

[0180] "

[0181]

[0182] &

[0183] &

[0184]

[0185] In certain embodiments, a compound of Formula I is selected from the molecules shown in Table 3.

[0186] TABLE 3

[0187] >

[0188] <&

[0189] &

[0190] > <

[0191]

[0192] In certain embodiments, a compound of Formula I is selected from the molecules shown in Table 4.

[0193] TABLE 4

[0194] >

[0195] >

[0196] "

[0197] "

[0198]

[0199] &

[0200] <

[0201] "

[0202] "

[0203] < &

[0204] &

[0205] <

[0206]

[0207] In Tables 3 and 4, the bile acid abbreviations CA, aMCA, PMC A, coMCA, CDCA, UDCA, DC A, and KDCA correspond to the following structures:

[0208] <

[0209] <

[0210]

[0211] In another aspect, the present invention provides pharmaceutical compositions containing aminoethyl thioesters (e.g. any one or more compounds conforming to Formulae I, II, III, Illa, Illb, IIIc, Hid, Hie, IV, or V, described above). In certain embodiments, the invention encompasses a pharmaceutical composition or a single unit dosage form of any of the aminoethyl thioesters compounds described above. In 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 aminoethyl thioesters 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 orsynthetic 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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 longterm storage to determine characteristics such as shelf-life or the stability of formulations overtime. 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 lactoseand 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 purified form, 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 forintravenous 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 it comprises 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.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).

[0223] 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.

[0224] 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 fillers 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. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl 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, com oil, and soybean oil), zinc stearate, ethyl oleate, ethyllaureate, 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, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;

[0234] 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.

[0235] 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.

[0236] 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 orprophylactic 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.

[0237] Parenteral Dosage Forms

[0238] Parenteral dosage forms can be administered to patients by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. 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.

[0239] 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, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0240] 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.

[0241] Transdermal, Topical & Mucosal Dosage Forms

[0242] 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.

[0243] 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, propylene glycol, butane- 1,3 -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).

[0244] 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).

[0245] 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.Chemical Compositions of Matter

[0246] In another aspect, the present invention encompasses novel compositions of matter including compositions of novel molecules. While some of the aminoethyl thioesters described above may be 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 aminoethyl thioesters free from other biological materials are not found in nature. Additionally, many of the aminoethyl thioesters 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.

[0247] In certain embodiments, the present invention provides pure samples of any of the aminoethyl thioesters described above (e.g. compounds conforming to Formulae I-IV) and in the genera and subgenera described 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 lOOmg, 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.

[0248] 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 one or more compounds depicted in Table 2. In certain embodiments, the present invention provides novel compositions comprising one or more compounds depicted in Table 3. In certain embodiments, the present invention provides novel compositions comprising one or more compounds depicted in Table 4.

[0249] In certain embodiments, the present invention provides novel compositions comprising mixtures of between two and ten different aminoethyl thioesters.

[0250] Therapeutic Methods

[0251] 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 therapeutic compositions described above (e.g. a composition comprising any one or more compounds conforming to Formulae I, II, III, Illa, Illb, IIIc, Hid, Hie, IV, and V described above). In certain embodiments, themethod comprises administering such a composition to a mammal. In certain embodiments, the method comprises administering such a composition to a human.

[0252] In certain embodiments, provided therapeutic methods comprise administration of aminoethyl thioesters of short-chain fatty acids (SCFAs) (e.g. administration of any of the compounds of formulae I, II, III, Illa, Illb, IIIc, Hid, Hie, IV, and V where -A comprises a SCFA moiety). SFCAs are known to be bacterial fermentation products, which are chemically composed of a carboxylic acid moiety and a small hydrocarbon chain. Among them, acetic, propionic and butyric acids are the most studied, presenting, respectively, two, three and four carbons in their chemical structure. These metabolites are found in high concentrations in the intestinal tract and are taken up by intestinal epithelial cells (lECs). The biological roles of SCFAs in mammals are varied and therefore indicate a number of therapeutic areas where the provided therapeutic compositions have utility.

[0253] Aminoethyl thioesters of SCFAs as therapeutic agents

[0254] The SCFAs are known to modify several cellular processes including gene expression, chemotaxis, differentiation, proliferation and apoptosis. For example, SCFAs are partially used as a source of ATP by IEC cells. In addition, these molecules act as a link between the microbiota and the immune system. Provided compositions can therefore modulate different aspects of lECs and modulate leukocyte development, survival and function (for example, through activation of G protein coupled receptors (e.g. FFAR2, FFAR3, GPR109a and Olfr78) and by modulation of the activity of enzymes and transcription factors (including, for example the histone acetyltransferase and deacetylase, and the recently described stabilization of the hypoxia-inducible factor (HIF) are implicated in their effects (see, for example: Donohoe DR, Collins LB, Wali A, Bigler R, Sun W, Bultman SJ. The Warburg effect dictates the mechanism of butyrate-mediated histone acetylation and cell proliferation. Mol Cell 2012; 48: 612-626; inolo MA, Rodrigues HG, Nachbar RT, Curi R. Regulation of inflammation by short chain Fatty acids. Nutrients 2011; 3: 858-876; Vinolo MA, Hirabara SM, Curi R. G-protein-coupled receptors as fat sensors. Curr Opin Clin Nutr Metab Care 2012; 15: 112-116; and Kelly CJ, Zheng L, Campbell EL, Saeedi B, Scholz CC, Bayless AJ et al. Crosstalk between microbiota-derived short-chain fatty acids and intestinal epithelial HIF augments tissue barrier function. Cell Host Microbe 2015; 17: 662-671, each of which is incorporated herein by reference). SCFAs activate at least four different GPCRs: the free fatty acid receptors (FFAR)-2 and -3, which are also known as GPR43 and GPR41, respectively, the niacin / butyrate receptor GPR109a (alsoknown as HCA2) and the olfatory receptor (Olfr)-78 (see, for example, Pluznick J. A novel SCFA receptor, the microbiota, and blood pressure regulation. Gut Microbes 2014; 5: 202-207; and Thangaraju M, Cresci GA, Liu K, Ananth S, Gnanaprakasam JP, Browning DD et al. GPR109A is a G-protein-coupled receptor for the bacterial fermentation product butyrate and functions as a tumor suppressor in colon. Cancer Res 2009; 69: 2826-2832, each of which is incorporated herein by reference). These receptors show distinct patterns of expression and they have been partially associated with the effects of the SCFAs on leukocytes and intestinal epithelial cells (lECs). More details on the molecular mechanisms and on the effects of SCFAs in other tissues can be found in different reviews at the literature, see for example: den Besten G, van Eunen K, Groen AK, Venema K, Reijngoud DJ, Bakker BM. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. J Lipid Res 2013; 54: 2325-2340 which is also incorporated herein by reference)).

[0255] Considering this, administration of the compositions provided herein (and in particular those containing aminoethyl thioesters of SCFAs) provides valuable new methods to maintain intestinal homeostasis and to treat disorders arising from imbalances in this system including, but not limited to: periodontal disease, bacterial vaginosis, inflammatory bowel disease, rheumatoid arthritis, obesity, asthma / allergy, psoriasis, multiple sclerosis and cancer.

[0256] The SCFAs are known to modify several cellular processes including, but not limited to, innate and adaptive lymphocyte development and function. Considering this, administration of the compositions provided herein (and in particular those containing aminoethyl thioesters of SCFAs) provides valuable new methods to influence innate lymphoid cell functions that can regulate innate and adaptive immune cell responses and acute and chronic inflammatory processes associated with inflammatory bowel disease, rheumatoid arthritis, obesity, psoriasis, asthma / allergy, MS and cancer. In particular, group 3 innate lymphoid cell activation, recruitment and functional potential can be influenced by ligands of GPR43, GPR183 and other family members. In addition, multiple functions of regulatory T cells that limit inflammation can be regulated by the microbiota, their metabolites and signaling via GPCRs.

[0257] SCFA aminoethyl thioesters to treat or prevent cancer and proliferative disorders Taking into account the fact that carboxylic acids in general and SCFAs in particular are important energetic substrates for epithelial cells and the fact that they are regulators oftheir proliferation, treatment with the SCFA aminoethyl thioesters described herein can provide therapeutic value by modulating cellular proliferation. For example, cysteamides of SCFAs, (particularly of butyrate) that may result from treatment with SCFA aminoethyl thioesters, may exert different effects on the growth of normal and tumoral colonocytes. For example, butyrate inhibits the growth of cancerous colonic cells, but not of normal colonocytes and, depending on the concentration, it actually increases the proliferation of this latter cell type. Treatment with SCFA aminoethyl thioesters therefore provides a useful method of modulating proliferation of colonic cells and therefore of treating diseases such as colon cancer.

[0258] Similar patterns features are known to be present in other cancerous cells and therefore treatment with the provided aminoethyl thioesters compositions (e.g. compositions containing one or more compounds conforming to Formulae I, II, III, Illa, Illb, IIIc, Hid, Hie, IV, and V described above) provide a useful therapy for treating or preventing cancer.

[0259] Based on the established role of FXR activation in a wide range of cancers, e.g., esophageal cancer, pancreatic cancer, lung cancer, and head and neck cancer, BA cysteamides as FXR antagonists have utility as therapeutics for the prevention and treatment of diverse cancers and related proliferative disorders. Thus, in certain embodiments, the present invention comprises a method of treating or preventing cancer and other proliferative disorders by treatment via oral or intravenous administration of a composition containing BA aminoethyl thioesters, optionally in combination with other cancer treatments, e.g. surgery, radiation treatment, chemotherapy, hormonal treatment, and / or immunotherapy.

[0260] Aminoethyl thioesters as therapies to modulate FXR

[0261] In certain embodiments, provided therapeutic methods comprise administration of aminoethyl thioesters of bile acids (BAs). BAs and their taurine-conjugated derivatives play diverse roles in human metabolism. As ligands of the nuclear receptor FXR, bile acids regulate cholesterol homeostasis, liver regeneration, and inflammation as well as lipid metabolism and glucose metabolism, and furthermore contribute to inter-organ communication, in addition, regulation of FXR activity plays a role in cancer. For example, regulation of FXR activity by bile acids has been linked to differential outcomes in colorectal cancer. Moreover, FXR expression is positively correlated with tumor size and the proliferative rate of e.g. breast cancer, and FXR expression is significantly increased in some types of lung cancer (Int J Mol Sci. 2018 Jul; 19(7): 2069). FXR has been shown to beassociated with a higher tumor grade, greater tumor size and lymph node metastasis in esophageal adenocarcinomas. In addition, FXR is hypothesized to serve functional roles in the nervous system.

[0262] We have shown that aminoethyl thioesters of BAs can transform under physiological conditions to BA-cysteamide derivatives which can then function as potent antagonists of FXR. FXR antagonists have been shown to protect against liver injury in cholestasis, lower cholesterol, suppresses gluconeogenesis in mouse primary hepatocytes, and improve glucose homeostasis in HFD / STZ-induced T2DM mice. Moreover, FXR antagonists have anti-cancer activities that indicate use in both prevention and treatment of cancer. For example, a natural product-derived FXR antagonist inhibited proliferation of cancer cell lines, and induced cell apoptosis in esophageal cancer, pancreatic cancer, and head and neck cancer, and other cancers.

[0263] 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. Therefore, in certain embodiments, the present invention comprises treating a patient with any of the provided aminoethyl thioesters described herein to lower cholesterol levels, low-density lipoprotein levels, and / or triglyceride levels. In certain embodiments, the present invention comprises a method of treating a patient with bile acid aminoethyl thioesters as described herein, to lower 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.

[0264] In certain embodiments, treatment with the provided aminoethyl thioesters is effective for treating metabolic and liver diseases (i.e. via the activity of the provided compounds — or cysteamides formed as a consequence of such treatment -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, suchtreatments result in decreased inflammation and / or hepatocyte injury. In certain embodiments, such treatments result in decreased fibrosis and cirrhosis.

[0265] 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 aminoethyl thioester derivatives described herein (or a pharmaceutically acceptable precursor, pro-drug or salt thereof).

[0266] 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 aminoethyl thioesters described herein (or a pharmaceutically acceptable precursor, pro-drug or salt thereof).

[0267] 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.

[0268] 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 aminoethyl thioesters described herein (or a pharmaceutically acceptable precursor, pro-drug or salt thereof).

[0269] BA aminoethyl thioesters also have applications in treatment of cardiovascular diseases. FXR activation is involved in the regulation of lipid metabolism, and its dysregulation is associated with atherosclerosis and other cardiovascular conditions.

[0270] 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 aminoethyl thioesters described herein (or a pharmaceutically acceptable precursor, pro-drug or saltthereof). In certain embodiments, the method comprises treating a patient at risk of cardiovascular disease with one or more of the bile acid aminoethyl thioesters described herein (or a pharmaceutically acceptable precursor, pro-drug or salt thereof) to improve lipid profiles and reduce cardiovascular risk.

[0271] Administration of BA aminoethyl thioesters 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 believed this 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.

[0272] 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 aminoethyl thioesters (or a pharmaceutically acceptable precursor, pro-drug or salt thereof). In certain embodiments, such methods comprise oral administration of a BA cysteamide formulation characterized in that it is stable / protected from degradation or hydrolysis in the stomach.

[0273] 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 provides the possibility of developing pro-drugs for oxidized BA cysteamides that target receptors outside of the intestine, oral administration of the provided BA aminoethyl thioesters 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.Aminoethyl thioesters as therapies to modulate the immune system

[0274] In certain embodiments, provided compositions (e.g. one or more compounds conforming to Formulae I, II, III, Illa, Illb, IIIc, Hid, Hie, IV, and V, described above), or compositions comprising one or more of these compounds, have utility as therapeutics to modulate the immune system of an animal.

[0275] In certain embodiments, the present invention comprises a method administering one or more of the compounds described herein to reduce or ameliorate chronic inflammatory diseases ranging from the skin (psoriasis and atopic dermatitis), oral cavity (periodontal disease), airways (asthma / allergy), gastrointestinal tract (food allergy, IBD, IBS, celiac disease, cancer), obesity and cancer. The impact of specific compounds on the type of inflammation can be context dependent and dictated by the specific tissue and nature of the inflammatory lesions. Immediate examples include manipulation of T helper subsets (Thl, Th2, Thl7, Treg), innate lymphoid cells (ILC1, 2, 3) and myeloid and granulocyte lineages.

[0276] Aminoethyl thioesters as modulators of histone acetylation

[0277] In certain embodiments, provided aminoethyl thioesters (e.g. one or more compounds conforming to Formulae I, II, III, Illa, Illb, IIIc, Hid, Hie, IV, and V described above), or compositions comprising one or more of these compounds, have utility as therapeutics that act by modulating histone acetylation or deacetylation.

[0278] For example, exposure to butyric acid cysteamide derivatives resulting from treatment with the provided aminoethyl thioesters can modulate histone acetylation (more generally, acylation) levels, either by inhibiting acyl transferases (which attach the acyl groups to the lysines of the histones) or by modulating (likely inhibiting, as an antagonist) sirtuins and other histone deactylases (HDACs). This makes therapeutic agents and methods based on the provided aminoethyl thioesters highly relevant to the treatment of cancer.

[0279] Bile acid aminoethyl thioesters as therapies to regulate metabolic disorders

[0280] The ligand-regulated nuclear receptor FXR plays a central role in lipid and glucose metabolism as well as cholesterol homeostasis, and BA cysteamides are potent FXR antagonists. High cholesterol levels underlie a wide range of human diseases, including blood clots, cardiovascular disease, peripheral arterial disease, type 2 diabetes, and stroke.

[0281] In certain embodiments, the present invention comprises a method of treating a mammal with bile acid aminoethyl thioesters as described herein, to lower cholesterol levels, including low-density lipoprotein and triglyceride levels. In certain embodiments, the presentinvention comprises a method for treating or preventing hyperlipidemia, blood clots, cardiovascular 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.

[0282] In certain embodiments, treatment with the 2-aminoethylthioester of a carboxylic acid as described above, results in rearrangement of the amino thioester or a metabolite thereof to a cysteamide of the carboxylic acid. In certain embodiments, the treatment further results in methylation of the cysteamide. In certain embodiments, further results in S-oxidation of the cysteamide.

[0283] In certain embodiments, the invention provides a method for improving the health of an animal comprising the step of contacting the animal with a compound of Formula V:

[0284]

[0285] wherein each of A, R2, and R5is as defined above and in the genera and subgenera herein.

[0286] In certain embodiments, the provided methods include treating an animal with an oligomeric aminoethyl thioester. In certain such embodiments, an oligomer conforms to Formula IV. In certain embodiments, such methods are characterized in that they result in a long-lasting exposure to cysteamides resulting from degradation of the oligomeric aminoethyl thioester composition.

[0287] 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, it is possible to use BA cysteamides as well as BA aminoethyl thioesters 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 and / or BA aminoethyl thioesters 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 gutmicrobiome. 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.

[0288] 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 aminoethyl thioester compounds 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 aminoethyl thioester 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 aminoethyl thioesters 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.

[0289] 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 aminoethyl thioester, 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 postprandial glycemic 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-gradeinflammation which can lead to arthritis and contributes to the development of the metabolic syndrome.

[0290] In another aspect, the present invention includes methods of activating LXR by treating a mammal with BA aminoethyl thioester. 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.

[0291] DEFINITIONS

[0292] Definitions 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. Additionally, general principles of organic chemistry, as well as specific functional moieties 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.

[0293] 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.

[0294] 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. In addition to the above-mentioned compounds per se, this invention also encompasses compositions comprising one or more compounds.As used herein, the term “isomers” includes any and all geometric isomers and stereoisomers. For example, “isomers” include cis- and / ra / r.s-i somers, E- and Z- isomers, R- and S-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.”

[0295] 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 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972).

[0296] 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).

[0297] 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 spiro-fused 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 notlimited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0298] The term "unsaturated", as used herein, means that a moiety has one or more double or triple bonds.

[0299] 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. 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.

[0300] 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.

[0301] The term “alkenyl,” as used herein, denotes a monovalent group derived from a straight- or 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 2-3carbon atoms, and in yet other embodiments alkenyl groups contain 2 carbon atoms. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, and the like.

[0302] The term “alkynyl,” as used herein, refers to a monovalent group derived from a straight- or 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.

[0303] The term “carbocycle” and “carbocyclic ring” as used herein, refers to monocyclic and polycyclic moi eties 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,

[0304] 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.

[0305] 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, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 477-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.

[0306] 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-2J7-pyrrolyl), NH (as in pyrrolidinyl), or+NR (as in N-substituted pyrrolidinyl).

[0307] 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”, “heterocyclic group”, “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, 377-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, 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.

[0308] 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.

[0309] 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.

[0310] 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 same meaning as if the ring were said to be “optionally substituted” as described in the preceding paragraph.

[0311] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH2)o-4R°; -(CH2)o-4OR°; -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(R°)2; -(CH2)o-4N(R°)C(0)R°; -N(R°)C(S)R°; -(CH2)O-4N(R0)C(0)NR°2; -N(RO)C(S)NR°2; -(CH2)O-4N(RO)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(RO)C(O)NRO2; -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(R0)2; -(CH2)O-4C(0)SR°; -(CH2)o-4C(0)OSiR°3; -(CH2)o-40C(0)R°; -OC(0)(CH2)o-4SR-, SC(S)SR°; -(CH2)o-4SC(0)R°; -(CH2)O-4C(0)NR°2; -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)R°; -C(NOR°)R°; -(CH2)o-4SSR°; -(CH2)o-4S(O)2R°; -(CH2)O-4S(0)2OR0; -(CH2)O-40S(0)2R0; -S(O)2NRO2; -(CH2)O-4S(0)R°; -N(RO)S(O)2NR°2; -N(RO)S(O)2R°; -N(OR°)R°; -C(NH)NRO2; -P(O)2RO; -P(O)RO2; -0P(0)R°2; -OP(O)(ORO)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.

[0312] 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-2OH, -(CH2)O-2OR*, -(CH2)O-2CH(OR*)2; -O(haloR’), -CN, -N3, -(CH2)O-2C(0)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*3, -OSiR*3, -C(O)SR* -(C1-4 straight 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 selected from 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. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0313] 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(O)2R*, =NR*, =N0R*, -O(C(R*2))2.3O-, or -S(C(R*2))2.3S-, wherein eachindependent 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.

[0314] 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, -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] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include - C(O)Rt, -S(O)2Rt,

[0316]

[0317] ; wherein each R: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.

[0318] 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.

[0319] EXAMPLESThe following Examples are useful to confirm aspects of the disclosure described above and to exemplify certain embodiments of the disclosure.

[0320] Methods

[0321] 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 min 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 min and clarified extracts were transferred to fresh HPLC vials and stored at -20 °C until analysis.

[0322] 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 min followed by another 10 minutes of vigorous stirring. Extracts were pelleted at 5,000 g for 5 min, 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 min, and the combined supernatants were then dried in a SpeedVac™ (ThermoFisher Scientific) vacuum concentrator. Samples were then resuspended in 150 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.

[0323] 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 A / B gradient started at 1% B for 3 min afterinjection and increased linearly to 100% B at 20 min, then 100% B for 5min, and down to 1% B for 3 min using a flow rate of 0.5 mL / min 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 * 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 JH,C = 6 Hz. HSQC spectra were acquired with or without decoupling. NMR spectra were processed and baseline corrected using MestreLabs MNOVA software packages.

[0324] Feature detection and characterization. LC-MS RAW files 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 prefilter, FALSE fitgauss, 1 integrate, TRUE firstBaselineCheck, 0 noise, wMean mzCenterFun, -0.005 mzdifif 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 differential features, 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 negativeionization 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 m / z, stepped NCE (normalized collision energy) 10 and 30 for positive and negative ionization mode, dynamic exclusion 3 s.

[0325] MS2-based molecular networking. A MS2 molecular network was created using Metaboseek version 0.9.7 and visualized in Cytoscape4. Features were matched with their respective MS2 scan within an m / z window of 5 ppm and a retention time window of 15 s, using the MS2scans function. To construct the molecular network, tolerance of the fragment peaks was set to m / z of 0.002 or 5 ppm, minimum number of peaks was set to 3, with a 2% noise level. Once the network was constructed, a cosine value of 0.7 was used, and the number of possible connections was constrained to 6 for both negative ion mode and positive ion mode.

[0326] 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.

[0327] 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.

[0328] 1. Tautenhahn, R., Bottcher, C. & Neumann, S. Highly sensitive feature detection for high resolution LC / MS. BMC Bioinformatics 9, 1-16 (2008).

[0329] 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).

[0330] 3. Helf, M. J., Fox, B. W ., Artyukhin, A. B., Zhang, Y. K. & Schroeder, F. C.

[0331] Comparative metabolomics with Metaboseek reveals functions of a conserved fat metabolism pathway in C. elegans. Nat. Commun. 13, 782 (2022).4. Shannon, P. et al. Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks. Genome Res. 13, 2498-2504 (2003).

[0332] EXAMPLE 1 Comparative metabolomics reveals BA-MCYs

[0333] In this example we utilize untargeted metabolomics to compare germ-free and microbiota-replete specific pathogen-free (SPF) mice to uncover a host-mediated bile acid (BA) modification. Lack of microbial deconjugation results in increased levels of BA-taurine conjugates in germ-free mice, whereas 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 SPF and germ-free mice (Fig. 1 A). The resulting datasets were processed using the xcms-based Metaboseek platform, which facilitates identification of mass spectrometry features whose abundances differ significantly between different conditions (Fig. IB). This untargeted comparison revealed stark differences between germ-free and SPF mice. In total, we detected more than 40,000 mass spectrometry features from combined analyses of serum samples in positive and negative ionization modes, of which approximately 10% were significantly differential (at P < 0.05) between germ-free 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 fivefold different in germ-free 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. MS2 networking revealed three major clusters of microbiota-dependent metabolites with fragmentation patterns, indicating that they represent BAs or BA derivatives (Figs. 1C and 1H-1 J). Two of these clusters represented BA-taurine conjugates and free BAs, whose abundances were greatly increased and decreased in germ-free mice, respectively, as expected, given the lack of microbial taurine deconjugation. By contrast, the third cluster appeared to represent a family of previously unannotated BA derivatives, whose abundances, similar to free BAs, were reduced in germ-free mice.Detailed analysis of the mass spectrometry isotope patterns and MS2 spectra of the putative BA derivatives indicated the presence of a methyl cysteamide (MCY) moiety (Figs. ID and 1K-1O). The MS2 spectra further indicated that these MCY derivatives belong to three different series, representing putative BA-MCYs, corresponding sulfoxides (BA-MCYO) and sulfodioxides (BA-MCY02; Figs. 1K-1O). Structures of these BA-MCYs were proposed based on the relative abundances and retention times of free BAs 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, P-muricholic acid (PMCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), deoxycholic acid (DCA) and 7-ketodeoxycholic acid (7-KDCA; Figs. IE, IF, and 2J-2M; Table 5). Together, the untargeted metabolomic comparison of SPF and germ-free mice revealed BA-MCYs as a previously unannotated family of microbiota-dependent BA derivatives.

[0334]

[0335] Table 5: HPLC-HRMS data for compounds.

[0336] EXAMPLE 2 Microbiota dependence of BA-MCY levels

[0337] Abundances of BA-MCYs were strongly reduced in germ -free compared with SPF serum samples, but not abolished (Figs. 2A, 2B, and 2N-2S). Similarly, BA-MCY levels were decreased in feces of germ-free mice compared with SPF mice (Figs. 2O-2Q). To betterunderstand the relationship between BA-MCY levels and the presence of the microbiota, we next tested whether introduction of human microbiota into germ-free mice would affect BA-MCY production. For this purpose, we performed human fecal microbiota transplantation (FMT) from healthy individuals into germ-free mice, and then profiled BAs and BA conjugates in serum and faces. 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 2N-2S). The results from our comparison of germ-free 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 the serum of SPF mice. We took advantage of this to test whether such a dietary intervention-based increase of free BA levels would also affect BA-MCY levels. We observed that BA-MCY levels were greatly increased in SPF mice fed an inulin-based high-fiber diet (Figs. 3M-3O), 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 (Fig. 2C and Figs. 3P, 3 Q). Finally, to determine whether BA-MCYs are also present in humans, we analyzed human serum samples, which revealed MCY derivatives of all major BAs common in humans (Fig. 2D and Figs. 3R, 3 S). 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 germ-free mice or as a result of supplementation with dietary fiber, are associated with parallel increases of the corresponding BA-MCY conjugates.

[0338] EXAMPLE 3 Biochemical origin of BA-MCY conjugates

[0339] To further clarify the roles of host and microbiota for the production of the BA-MCY conjugates, we next investigated the in vivo origin of the cysteamine moiety. Cysteamine is produced primarily via degradation of CoA, and oxidation of cysteamine in the liver and other tissues produces taurine, which is then conjugated with BAs in the liver, producing BA-taurine conjugates that are secreted into the intestine. Therefore, we considered two different models for the origin of the MCY moi eties in the BA-MCYs (Fig. 2E). First, these compounds could originate from conjugation of BAs with cysteamine or a cysteamine derivative derived from breakdown of CoA. Alternatively, the BA-MCY conjugates could be derived from reduction of corresponding BA-taurine derivatives by the host or the gut microbiota (Fig. 2E). To distinguish between these scenarios, we performed a series of stable-isotope labelling experiments with taurine-d4 and l-cysteine-3,3-d2 in SPF mice (Figs. 3T-3 V). High-performance liquid chromatography (HPLC)-HRMS analysis of serum from SPF mice supplemented with taurine-d4 revealed extensive labelling of taurine conjugates of BAs, as expected (Fig. 3T). However, none of the MCY conjugates was labelled (Fig. 3T), indicating that the biosynthetic pathways of taurine and MCY conjugates are distinct. Next, to test whether the MCY conjugates originate from incorporation of a cysteine-derived cysteamine moiety, we analyzed serum samples from SPF mice supplemented with 1-cysteine-3,3-d2 (Figs. 3U, 3 V). HPLC-HRMS analysis revealed incorporation of deuterium in both the BA-taurine and the BA-MCY conjugates (Fig. 3U, 3 V), consistent with a cysteine origin of both taurine and the MCY moiety. As expected, we also observed deuterium incorporation into the CoA breakdown product pantetheine (Fig. 3W).

[0340] These results support a model in which the BA-MCY conjugates are derived from acylation of a cysteamine derivative other than taurine. Next, we considered whether the conjugation is likely to be mediated by the microbiota or the host. Although the abundances of the BA-MCY conjugates were strongly microbiota dependent, their production was not abolished in germ-free animals (Fig. 2A, 2B, and Figs. 2N-2Q), indicating that the conjugation reaction itself does not require the microbiota. Therefore, we next investigated whether BA-MCY production is related to the biosynthesis of BA-taurine conjugates.

[0341] Conjugation of BAs with taurine and amino acids in the liver is mediated by BAAT, a type 1 acyl-CoA thioesterase (ACOT). Members of this gene family catalyze a wide range of acyl transfer reactions (Hunt, et al., Biochim. Biophys. Acta 1822:1397-1410 (2012)); putative conjugates of BAs with cysteamine and methylcysteamine have recently been reported to accumulate in Baat- / - mice (Neugebauer, et al., J. Lipid Res. 63:100297 (2022)). We confirmed that the BA cysteamine derivatives accumulating in Baat- / - mice are identical with the compounds that we identified (Figs. 4E-4H), demonstrating that BA-MCY biosynthesis is distinct from BA-taurine conjugation. Lack of taurine conjugation in Baat- / -mice results in greatly increased levels of both free BAs and BA-MCY conjugates (Fig. 41 and 4 J), consistent with parallel increase of free BAs and BA-MCY conjugates observed in mice fed a high-fiber diet. Suppression of microbiota in Baat- / - mice does not diminish the elevated levels of BA-MCY in this mutant strain. Together, our results indicate that BA-MCYs are derived from a host-dependent conjugation pathway that exists in parallel with conjugation of BAs with taurine.

[0342] Next, we considered potential sites of BA-MCY biosynthesis. Although BA-taurine conjugates are produced in the liver, the fact that BA-MCY levels are governed primarily bythe abundance of free BAs, which in SPF mice are predominantly derived from deconjugation of BA-taurine and BA-glycine by the intestinal microbiota, indicated that the intestine may be involved in B A-MCY production. Therefore, we additionally profiled BA-MCY levels in the small intestine and caecum, which revealed that BA-MCY conjugates are much more abundant in intestinal tissues than in the liver (Fig. 3 A and Figs. 4K-4P).

[0343] Moreover, the ratio of BA-MCY conjugates relative to their oxidation products BA-MCYO and BA-MCY02 was higher in intestinal tissues than in the liver, serum and feces (Fig. 3A), indicating that BA-MCY conjugates are produced from free BAs following their reuptake in the intestine. A model in which intestinal production of BA-MCYs is dependent on reuptake of free BAs was further consistent with our observation that, across different diets and conditions, abundances of free BAs and BA-MCYs are correlated (Figs. 3M-3O).

[0344] EXAMPLE 4 MCY biosynthesis depends on host VNN1

[0345] As BA-MCY conjugates could plausibly be derived from pantetheine breakdown, we sought to test whether BA-MCY biosynthesis may proceed via the pantetheinase VNN1, which is highly expressed in intestinal tissues, where BA-MCY concentrations were highest. The primary function of VNN1 is to hydrolyze pantetheine into cysteamine and pantothenic acid (Fig. 3B), as part of CoA recycling, and recent studies have shown that VNN1 has important roles in the regulation of metabolism, inflammation and associated diseases.

[0346] Although VNN1 could be a plausible source for the cysteamine moiety required for BA-MCY production, we hypothesized that, in addition to hydrolyzing pantetheine, VNN1 may also be capable of hydrolyzing BA-pantetheine or BA-CoA conjugates. The resulting S-linked BA-cysteamine derivatives would then rearrange to the N-linked isomer, which following S-methylation would produce the BA-MCY conjugates (Fig. 3B). To investigate this hypothesis, we first tested whether recombinant VNN1 could hydrolyze a synthetic cholic acid-pantetheine conjugate (CA-pant). We found that VNN1 breaks down CA-pant as efficiently as pantetheine (Figs. 3C and 4Q) and further showed that the resulting CA-cysteamine conjugate rearranges to the corresponding cholic acid-cysteamide (CA-CY), a plausible precursor of CA-MCY that we had also detected in Baat- / - mice (Fig. 4R). To assess whether VNN1 contributes to BA-MCY biosynthesis in vivo, we compared BA profiles of wild-type (WT) and Vnnl- / - mice in various tissues (Fig. 3D). We found that BA-MCY levels are dramatically reduced in the small intestine, liver and serum, and, to a lesser extent, in the feces of Vnnl - / --mutant mice (Fig. 3D). These results indicate that in vivo BA-MCY biosynthesis largely depends on the host enzyme VNN1. Furthermore, wefound that the predicted precursor of CA-CY, CA-pant, accumulates in the small intestine and feces of Vnnl- / - mice, whereas this compound was absent in the corresponding WT samples and also could not be detected in the Vnnl- / - liver and serum, indicating that BA-pantetheine conjugates are direct precursors for BA-MCY biosynthesis in the intestine (Figs.

[0347] 4S and 4T).

[0348] EXAMPLE 5 Microbial and host metabolism of BA-MCYs

[0349] 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 germ-free compared with SPF mice (Fig. 3G, 6A). Considering the possibility that the gut microbiota may also have a role in the deconjugation of BA-MCYs, we noted that, even though BA-MCY levels are reduced in germ -free mice (Figs, 2O-2Q and 6B), the ratio of BA-MCY conjugates to free BAs was greatly increased in germ-free compared with SPF fecal samples (Fig. 3G). In fact, BA-MCY levels were similar to or exceeded levels of free BAs in feces of germ-free mice (Fig. 3H).

[0350] To determine whether BA-MCYs can indeed be deconjugated by the microbiota, we analyzed faecal and liver samples from SPF and germ-free mice supplemented with stable-isotope-labelled CDCA- d5-MCY for the presence of free labelled CDCA and other labelled 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 (Helf, et al., Nat. Commun. 13:782 (2022)). Targeted analysis of fecal samples from CDCA-d5-MCY-supplemented SPF mice revealed CDCA-d5-MCY as well as d4-labelled and d5-labelled free CDCA (Figs. 31, 6C, 6D, 6K, and 6L), indicating that BA-MCYs can be deconjugated in the gut. In addition, we detected labelled versions of other free BAs that can be derived from CDCA (Fig. 6C and 6L), whereas CA, DCA and other CA-derived BAs remained unlabeled (Fig. 6L), consistent with their separate biosynthetic pathway (Pandak, et al., Liver Res. 3:88-98 (2019); Al-Dury, et al., J. Hepatol. 64:S436 (2016)). Analysis of fecal and liver samples from CDCA-d5-MCY-supplemented SPF mice further indicated that labelled free BAs derived from supplemented CDCA-d5-MCY are partly reconjugated with taurine (Figs. 6E-6J). 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-MCYcould be detected in the liver of supplemented mice, indicating that supplemented CDCA-d5-MCY is quickly oxidized to CDCA-d4 / 5-MCYO(2) (Figs. 6H, 6J).

[0351] In contrast to SPF mice, deconjugation-derived, labelled CDCA or other labelled free BAs were not detected in germ-free mice supplemented with CDCA-d5-MCY (Fig. 3E, 6M, and 6N), indicating that deconjugation of CDCA-MCY is dependent on microbiota.

[0352] Similarly, suppression of microbiota in SPF mice treated with antibiotics (ABX) resulted in significantly reduced deconjugation of supplemented CDCA-d5-MCY compared with SPF mice (Figs. 3E, 6C, 6D, 6G, 6H). In ABX and germ-free mice, supplemented CDCA-d5-MCY was instead primarily converted into the corresponding oxidation products, CDCA-d4 / 5-MCYO and CDCA-d4 / 5-MCYO2 (Figs. 6D, 6H, 6J).

[0353] Next, we demonstrated that BA-MCY conjugates are deconjugated by fecal suspensions obtained from SPF mice and individual gut bacteria known to have the gene encoding BSH42 (Figs. 7A and 7B). To determine whether BSH is required for BA-MCY deconjugation, we tested gnotobiotic mice colonized 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) (Arifuzzaman, et al. Nature doi.org / 10.1038 / s41586-022-05380-y (2022); Yao, et al., eLife doi.org / 10.7554 / eLife.37182 (2018)). 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. 3F), where the supplemented CDCA-d5-MCY was exclusively converted to the oxidized CDCA-d4 / 5-MCYO(2), as in germ-free mice (Fig. 7C). These results indicate that BSH of gut microbiota can deconjugate BA-MCY conjugates, albeit less efficiently than the corresponding taurine conjugates (Fig.

[0354] 3F), 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. 3G).

[0355] EXAMPLE 6 BA-MCYs act as FXR antagonists in vitro

[0356] 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, for example, the broadly conserved CDCA, CA and DCA14, as well as amino acid conjugates of CA43, function as potent FXR agonists that negatively regulate BA production. By contrast, it is unclear whether there are any conserved endogenous FXR antagonists that would promote BA production.

[0357] To test for potential FXR agonist or antagonist activity of the identified BA-MCY conjugates, we selected four derivatives, CA-MCY, CA-MCYO, PMCA-MCY and CDCA-MCY based on their relative abundance in SPF mouse serum samples and considering FXR agonist activity of the corresponding free BAsl4. 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 co-activator peptide (SRCP)-derived nuclear fusion protein44. Whereas none of the tested conjugates showed agonist activity at any of the tested concentrations (Fig. 4 and 7D), 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 FXR45 (Fig. 4). By 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 (Figs. 7E-7I). In parallel, we also tested TpMCA, which is a weak, murine-specific FXR antagonist (Sayin, et al., Cell Metab. 17, 225-235 (2013)).

[0358] However, TpMCA was inactive at the tested range of concentrations in this assay (Fig. 7J). These results indicate that BA-MCY conjugates function as endogenous FXR antagonists that complement the role of free BAs as FXR agonists.

[0359] EXAMPLE 6a BA-aminoethyl thioesters regulate FXR signaling in vivo

[0360] In a related experiment, the Bile Acid aminoethyl thioesters shown in Tables 1, 2, 3, and 4 are synthesized and, using the method described in Example 6 are evaluated for their activity as FXR agonists or antagonists.

[0361] EXAMPLE 7 BA-MCYs regulate FXR signaling in vivo

[0362] BA biosynthesis in the liver is controlled by a complex signaling network regulated by hepatic and intestinal FXR via distinct pathways (Fig. 5 A). In the liver, FXR agonists promote expression of short heterodimer partner (SHP), which in turn antagonizes expression of Cyp7al, encoding a cytochrome P450 enzyme required for the first and rate-limiting step in BA synthesis (Fig. 5 A). 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 BAs that are 12a-hydroxylated (such as CA) and BAs that are not 12a-hydroxylated (such as CDCA). By contrast, intestinal FXR activation promotes production of the ileal hormone fibroblast growth factor 15 (FGF15; FGF19 in humans), a signaling peptide that travels to the liver via the enterohepatic circulation to suppress expression of Cyp7al. Conversely, FXR antagonistspromote BA synthesis by relieving repression of Cyp7al and Cyp8bl by suppressing SHP and FGF15 / FGF19 expression (Fig. 5A).

[0363] 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 CDCA-MCY (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 FXR-FGF15 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 BA-MCY production may be dependent on reabsorption of free BAs from the ileum, we additionally tested whether CDCA-MCY supplementation affects expression of Slcl0a2, the transporter mediating BA reuptake from the gut; however, Slcl0a2 expression was unchanged (Fig. 5E).

[0364] EXAMPLE 7a BA-MCYs regulate BA production in vivo

[0365] To measure the effects of CDCA-MCY on BA production in vivo, we conducted additional supplementation studies using stable isotope-labelled CDCA-d5-MCY. The use of labelled 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, labelled 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 and 8A). Similarly, levels of CA-derived BAs were increased in fecal samples of animals supplemented with CDCA-d5-MCY or CDCA- MCY (Fig. 5G and 8B). Faucal BA levels were also increased by CDCA-d5-MCY supplementation in ABX mice (Fig. 6H). Given that levels of unlabeled BAs in the liver and serum of supplemented mice were not significantly changed (Figs. 8C-8F), the large increase in fecal excretion of both CA-family 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).Next, we tested whether upregulation of BA synthesis by CDCA-MCY supplementation is in fact dependent on FXR. We found that CDCA- MCY supplementation increased fecal BA abundance in WT mice but not in FXR-deficient (Nrlh4- / -) mice (Fig.

[0366] 51). BA levels in the liver and serum of WT and Nrlh4- / - mice were not significantly affected by CDCA supplementation (Figs. 8G, 8H), consistent with results from our initial supplementation study (Figs. 8C-8F). These data demonstrate that CDCA-MCY supplementation increases BA biosynthesis in an FXR-dependent manner.

[0367] EXAMPLE 7b BA-MCYs regulate lipid metabolism in vivo

[0368] 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 O staining revealed greatly decreased hepatic lipid accumulation in CDCA-MCY-supplemented HCD-fed mice compared with untreated HCD-fed mice (Fig. 51, 5J), consistent with studies of synthetic compounds acting as intestinal FXR antagonists. We observed similar effects when CDCA-MCY was supplied at a tenfold lower dose (Fig. 81, 8J).

[0369] 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. 5K).

[0370] EXAMPLE 7c BA-aminoethyl thioesters regulate FXR signaling in vivo

[0371] In a related experiment, the Bile Acid aminoethyl thioesters shown in Tables 1, 2, 3, and 4 are synthesized and using the method described in Example 7 are evaluated for their ability to decrease hepatic liver accumulation.

[0372] EXAMPLE 8 Chemical Synthesis of CA-pant

[0373] (a) Synthesis of D-pantetheine

[0374]

[0375] D-Pantethine (Sigma, 138 mg, 0.25 mmol) and tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 79 mg, 0.28 mmol, 1.1 equiv.) were dissolved in 4 mL of 10 mMtri s(hydroxymethyl)aminom ethane hydrochloride (pH 7.0). The reaction mixture was incubated at room temperature for 2 hr and extracted three times with 7:3 dichloromethane / isopropanol. Combined organics were dried with sodium sulfate, filtered, and concentrated in vacuo to afford D-pantetheine (108 mg, 78 %).

[0376] D-pantetheine HRMS (ESI) m / z [M+H]+ calcd for C11H23N2O4S+ 279.1373; found 279.1368.

[0377] D-pantetheine 1H NMR, 600 MHz, methanol -t / 4: 6 (ppm) 3.88 (s, 1H), 3.55-3.32 (m, 6H), 2.59 (t, J = 6.8 Hz, 2H), 2.43 (t, J = 6.7 Hz, 2H), 0.91 (s, 6H).

[0378] (b) Synthesis of CA-pant (21)

[0379]

[0380] To a stirred solution of solution of cholic acid (la, Sigma, 41 mg, 0.1 mmol), 4-dimethylaminopyridine (Sigma, 12 mg, 0.1 mmol, 1.0 equiv.), and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (TCI, 38 mg, 0.2 mmol, 2.0 equiv.) in 1 mL of dichloromethane was added D-pantetheine (56 mg, 0.2 mmol, 2.0 equiv.). The reaction mixture was stirred at room temperature for 24 hr and concentrated in vacuo.

[0381] Purification by flash chromatography on silica using a gradient of 0-30 % methanol in dichloromethane afforded CA-pant (21, 49 mg, 73 %).

[0382] CA-pant (21) HRMS (ESI) m / z [M+H]+ calcd for C35H61N2O5S+ 669.41431; found 669.4140.

[0383] See Figures 9-12 for NMR spectra.

[0384] ’II (600 MHz) and13C (151 MHz) NMR spectroscopic data for CA-pant (21) in methanol-

[0385] Chemical shifts were referenced to 6(CHD2OD) = 3.31 and 5(13CHD2OD) = 49.0.13C chemical determined from the acquired1H or dqfCOSY spectra. HMBC correlations are from the proton(s) stated to the indicated13C atom.

[0386]

[0387]

[0388]

[0389] 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.

[0390] 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 2-aminoethylthioester of a carboxylic acid.

2. The method of claim 1, wherein the 2-aminoethylthioester of a carboxylic acid comprises a compound of Formula I:wherein:A is an optionally substituted C1-39 aliphatic group; andeach of R1and R2is independently selected from -H, an optionally substituted C1-40 aliphatic group, and a linker moiety attached to the nitrogen atom of one or more additional molecules of formula I, where R1and R2may also be taken together to form a an optionally unsaturated ring.

3. The method of claim 2, wherein the moiety A is derived from, or is identical to, an endogenous carboxylic acid.

4. The method of claim 3, wherein the endogenous carboxylic acid comprises a shortchain fatty acid.

5. The method of claim 3, wherein the endogenous carboxylic acid comprises a bile acid.

6. The method of claim 4, wherein the short chain fatty acid is selected from propionic acid, butyric acid, and valeric acid.

7. The method of claim 4, wherein the short chain fatty acid comprises butyric acid.

8. The method of any one of claims 1 through 7, wherein R1is -H.

9. The method of any one of claims 1 through 7, wherein R2is other than -H.

10. The method of claim 9, wherein R2is an optionally substituted moiety selected from the group consisting of: 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, and a methyl group.

11. The method of claim 9, wherein R2is a C1-30 acyl moiety.

12. The method of claim 9, wherein R2is an amino acid or a peptide.

13. The method of claim 9, wherein R2is an acyl moiety that is labile under physiological conditions.

14. The method of claim 9, wherein R2, wherein each of R3and R4are independently selected from the group consisting of -H, a C1-32 optionally substituted aliphatic group, a C1-32 acyl group, and a hydroxyl protecting group.

15. The method of claim 9, wherein R2is16. The method of any claim above, wherein treatment with the 2-aminoethylthioester of a carboxylic acid, results in rearrangement of the amino thioester or a metabolite thereof to a cysteamide of the carboxylic acid.

17. The method of claim 16, wherein the treatment further results in methylation of the cysteamide.

18. The method of claim 16 or 17, wherein the treatment further results in S-oxidation of the cysteamide.

19. A method for improving the health of an animal, the method comprising the step of contacting the animal with a compound of the formula:wherein:A is an optionally substituted C1-39 aliphatic group; andR2is selected from -H, an optionally substituted C1-40 aliphatic group, and a linker moiety attached to the nitrogen atom of one or more additional molecules of the formula.

20. The method of claim 19, wherein the moiety A is derived from, or is identical to, an endogenous carboxylic acid.

21. The method of claim 20, wherein the endogenous carboxylic acid comprises a shortchain fatty acid.

22. The method of claim 20, wherein the endogenous carboxylic acid comprises a bile acid.

23. The method of claim 21, wherein the short chain fatty acid is selected from propionic acid, butyric acid, and valeric acid.

24. The method of claim 21, wherein the short chain fatty acid comprises butyric acid.

25. The method of any one of claims 19 through 24, wherein R2is -H.

26. The method of any one of claims 19-25, wherein the compound rearranges to a cysteamide.

27. The method of claim 26, wherein the cysteamide is A'-methylated after rearrangement.

28. The method of claim 26 or 27, wherein the cysteamide is 5-oxidized after rearrangement.

29. The method of any one of claims 19 through 24, wherein R2is other than -H.

30. The method of claim 29, wherein R2is an optionally substituted moiety selected from the group consisting of: 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, and a methyl group.

31. The method of claim 29, wherein R2is a C1-30 acyl moiety.

32. The method of claim 29, wherein R2is an amino acid or a peptide.

33. The method of claim 29, wherein R2is an acyl moiety that is labile under physiological conditions.

34. The method of claim 29, wherein R2is, wherein each of R3and R4are independently selected from the group consisting of -H, a C1-32 optionally substituted aliphatic group, a C1-32 acyl group, and a hydroxyl protecting group.

35. The method of claim 29, wherein R is36. A compound of Formula I:wherein:A is an optionally substituted C1-39 aliphatic group; andeach of R1and R2is independently selected from -H, an optionally substituted C1-40 aliphatic group, and a linker moiety attached to the nitrogen atom of one or more additional molecules of formula I, where R1and R2may also be taken together to form a an optionally unsaturated ring.

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