Prodrugs of cysteamine and / or cystamine

Abstract

The present disclosure provides cysteamine prodrugs, pharmaceutical compositions made therefrom, and methods, including the treatment of any disease or disorder in a subject that would benefit from one or more of the bioprotective effects of cysteamine, including, but not limited to, binding cystine, reducing oxidative stress, increasing adiponectin levels, and / or increasing brain-derived neurotrophic factor. Examples of such diseases and disorders include, but are not limited to, cystinosis and fatty liver disease, including non-alcoholic steatohepatitis (NASH).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 294,335, filed December 28, 2021, the entire disclosure of which is incorporated herein by reference. FIELD OF THE INVENTION

[0002] Disclosed herein are cysteamine prodrugs, pharmaceutical compositions prepared therefrom, and methods, including the treatment of any disease or disorder in a subject that can benefit from one or more of cysteamine's bioprotective effects, including, but not limited to, cystine binding, reduction of oxidative stress, increased adiponectin levels, and / or increased brain-derived neurotrophic factor. Examples of such diseases and disorders include, but are not limited to, cystinosis and fatty liver disease, including nonalcoholic steatohepatitis (NASH). BACKGROUND

[0003] Cysteine ​​is a commonly occurring amino acid present in the human body primarily in its oxidized form, cystine. Cysteine ​​is essential for the production of glutathione, a potent antioxidant. Unfortunately, the cellular uptake of cysteine ​​(primarily as cystine) is rate-limited. Many conditions are associated with oxidative stress, including nonalcoholic steatohepatitis (NASH) and several neurodegenerative disorders. Cystamine is the dimeric oxidized form of cysteamine. Cysteamine has been shown to reverse liver inflammation associated with NASH. Abnormal sialylsomal cystine deposition occurs in nephropathic cystinosis, leading to organ failure. Cysteamine reduces cystine accumulation and thus improves prognosis in these patients. However, this drug is associated with side effects, such as gastrointestinal symptoms, bad breath, and body odor, which are more likely to occur when plasma concentrations (Cmax) of cysteamine are high. Overview

[0004] Cysteamine is a well-known treatment for nephropathic cystinosis, but it is associated with side effects. It is commercially available as cysteamine bitartrate and is administered as an immediate-release formulation every six hours (Cystagon) and a delayed-release formulation every 12 hours (Procysbi®). Both of these formulations are associated with side effects, although Procysbi® is less so. The latter requires the daily ingestion of a large number of tablets. There are no approved, commercially available medications for the treatment of NASH. Cystamine is not commercially available for the treatment of any medical condition.

[0005] In some embodiments, the compounds of the present disclosure are prodrugs of cysteamine linked to cysteine. Cysteamine-cysteine ​​can be considered a "mutual prodrug," with each moiety (cysteamine or cysteine) possessing biological activity. Cysteamine is recognized as an antioxidant but is not commercially approved for conditions associated with increased oxidative stress. Cysteamine's side effects are associated with a higher Cmax (i.e., higher plasma concentration). The compounds of the present disclosure deliver cysteamine and cysteine ​​intracellularly, where the prodrug is reduced to cysteamine within the cell. Cysteamine is an antioxidant, and cysteine ​​is essential for the production of glutathione, the body's most important antioxidant. Cysteine ​​uptake into cells is rate-limited, which typically controls the availability of cysteine. The compounds of the present disclosure enter cells via different transport pathways, including passive diffusion and / or sulfide, disulfide, and mixed disulfide pathways. In the presence of cystamine, each cystamine compound generates two molecules of cysteamine, allowing delivery of relatively small amounts of the compound compared to cysteamine bitartrate. In addition to delivering cysteamine, certain compounds described in this disclosure will, upon prodrug activation, deliver a molecule of cysteine ​​or a related compound into the cell, promoting the production of glutathione.

[0006] In certain embodiments, the present disclosure provides compounds of formula I: JPEG2025502611000002.jpg61165, or a pharmaceutically acceptable salt or solvate thereof, wherein Y 1 ~Y 8 are each independently selected from H or D, and R is selected from H or an acetyl group. 1 ~Y 8 At least one of Y independently has a deuterium enrichment of about 10% or greater. 1 ~Y 8 At least one of Y is independently deuterium enriched to about 50% or greater. 1 ~Y 8 At least one of Y is independently deuterium enriched to about 90% or greater. 1 ~Y 8 In certain embodiments, at least one of the compounds has a deuterium enrichment of about 98% or greater. JPEG2025502611000003.jpg54136, or a pharmaceutically acceptable salt or solvate thereof, wherein Y 1 ~Y 8 are each independently selected from H or D. In another embodiment, the compound is JPEG2025502611000004.jpg213151JPEG2025502611000005.jpg216154JPEG2025502611000006.jpg145169, or a pharmaceutically acceptable salt or solvate of any one of the foregoing. JPEG2025502611000007.jpg44147, or a pharmaceutically acceptable salt, solvate, or prodrug thereof. In yet another embodiment, the pharmaceutically acceptable salt has the structure of Formula I(b): JPEG2025502611000008.jpg69164, wherein Y 1 ~Y 8are each independently selected from H or D; and X is a pharmaceutically acceptable counterion. In yet another embodiment, the pharmaceutically acceptable counterion is bitartrate or chloride. In another embodiment, the compound is In yet another embodiment, the compound has the structure of Formula I(c): JPEG2025502611000010.jpg52159, or a pharmaceutically acceptable salt or solvate of any one thereof, wherein Y 1 ~Y 8 are each independently selected from H or D; and Ac refers to an acetyl group. In a further embodiment, the compound is JPEG2025502611000011.jpg214153JPEG2025502611000012.jpg212158JPEG2025502611000013.jpg139151, or a pharmaceutically acceptable salt or prodrug thereof. In one embodiment, the pharmaceutically acceptable salt has a structural formula selected from Formula I(d): JPEG2025502611000014.jpg53156, wherein Y 1 ~Y 8 are each independently selected from H or D; Ac is an acetyl group; and X is a pharmaceutically acceptable counterion. In another embodiment, the pharmaceutically acceptable counterion is bitartrate or chloride. In yet another embodiment, the compound is JPEG2025502611000015.jpg52111 or In a further embodiment, the compound has the structure of Formula III(a): JPEG2025502611000017.jpg52136, or a pharmaceutically acceptable salt or solvate thereof, wherein Y 1 ~Y 4 are each independently selected from H or D; and R3 is selected from (C1-C6) alkyl. In a further embodiment, the compound is JPEG2025502611000018.jpg88129, or a pharmaceutically acceptable salt or solvate of any one of the foregoing, wherein R 3 is (C1-C6) alkyl. In another embodiment, the pharmaceutically acceptable salt has formula III(b): JPEG2025502611000019.jpg60137, wherein Y 1 ~Y 4 are each independently selected from H or D; R 3 is (C1-C6) alkyl; and X is a pharmaceutically acceptable counterion. In a further embodiment, the compound has formula III(c): JPEG2025502611000020.jpg61136, or a pharmaceutically acceptable salt or solvate thereof, wherein Y 1 ~Y 4 are each independently selected from H or D; and R 3 is (C1-C6) alkyl. In yet another embodiment, the compound is JPEG2025502611000021.jpg115155, or a pharmaceutically acceptable salt or prodrug thereof, wherein R 3 is (C1-C6) alkyl. In another embodiment, the pharmaceutically acceptable salt has formula III(d): JPEG2025502611000022.jpg65141, wherein Y 1 ~Y 4 are each independently selected from H or D; R 3 is (C1-C6) alkyl; and X is a pharmaceutically acceptable counterion. In one embodiment, the compound has Formula IV: JPEG2025502611000023.jpg63136, or a pharmaceutically acceptable salt or solvate thereof, wherein Y 1 ~Y 4and Y 9 ~Y 16 are each independently selected from H or D; and R 1 is selected from H or an acetyl group. In another embodiment, the compound has formula V: JPEG2025502611000024.jpg52128, or a pharmaceutically acceptable salt or solvate thereof, wherein Y 1 ~Y 4 and Y 9 ~Y 16 are each independently selected from H or D; R 1 is selected from H or an acetyl group; and R 5 is (C1-C6) alkyl.

[0007] The present disclosure also provides a compound of formula VI: JPEG2025502611000025.jpg5681, or a pharmaceutically acceptable salt or solvate thereof, wherein Y 1 ~Y 4 are each independently selected from H or D; and R is a linear or branched aliphatic group (saturated or unsaturated) or aromatic group (substituted or unsubstituted) having 1 to 20 carbon atoms. 1 ~Y 4 At least one of Y independently has a deuterium enrichment of about 10% or greater. 1 ~Y 4 At least one of Y is independently deuterium enriched to about 50% or greater. 1 ~Y 4 At least one of Y is independently deuterium enriched to about 90% or greater. 1 ~Y 4 In one embodiment, the compound has formula VI(a): JPEG2025502611000026.jpg52146 or a pharmaceutically acceptable salt or solvate thereof, wherein Y 1 ~Y 8are each independently selected from H or D; and n is 2 to 6 (e.g., 2, 3, 4, 5, or 6). In another embodiment, Y 1 ~Y 8 At least one of Y independently has a deuterium enrichment of about 10% or greater. 1 ~Y 8 At least one of Y is independently deuterium enriched to about 50% or greater. 1 ~Y 8 At least one of Y is independently deuterium enriched to about 90% or greater. 1 ~Y 8 In yet another embodiment, the compound of formula VI is Selected from JPEG2025502611000027.jpg57161.

[0008] In certain embodiments, the present disclosure also provides a pharmaceutical composition comprising a compound disclosed herein and a pharmaceutically acceptable carrier, diluent, and / or binder. In further embodiments, the pharmaceutical composition is formulated for oral delivery. In yet other embodiments, the composition is in the form of granules, tablets, capsules, or caplets. In other embodiments, the pharmaceutical composition is formulated for delayed release. In yet other embodiments, the pharmaceutical composition comprises an enteric coating.

[0009] In certain embodiments, the present disclosure further provides a method of treating a subject suffering from a disease or disorder selected from the group consisting of cystinosis, fatty liver disease, cirrhosis, eosinophilic diseases or disorders, and Huntington's disease, the method comprising administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition of the present disclosure. In a further embodiment, the subject suffers from cystinosis. In yet another embodiment, the disease or disorder is fatty liver disease. In another embodiment, the fatty liver disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatitis-related fatty liver disease, obesity-related fatty liver disease, diabetes-related fatty liver disease, insulin resistance-related fatty liver disease, hypertriglyceridemia-related fatty liver disease, abetalipoproteinemia, glycogen storage disease, Weber-Christian disease, Wolman disease, acute fatty liver of pregnancy, and lipoatrophy. In certain embodiments, the fatty liver disease is non-alcoholic steatohepatitis (NASH). In another embodiment, the method further comprises measuring one or more liver function markers selected from the group consisting of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and triglycerides. In yet another embodiment, an ALT level of about 60-150 units / liter is indicative of fatty liver disease, and the compound improves the ALT level. In yet another embodiment, an ALP level of about 150-250 units / liter is indicative of fatty liver disease, and the compound improves the ALP level. In yet another embodiment, an AST level of about 40-100 units / liter is indicative of fatty liver disease, and the compound improves the AST level. In yet another embodiment, a GGT level of 50-100 units / liter is indicative of fatty liver disease, and the compound improves the GGT level. In one embodiment, the liver disease or disorder is selected from the group consisting of pediatric type 2 NAFLD, autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cholangitis, chronic drug toxicity, biliary atresia, and idiopathic neonatal hepatitis syndrome.

[0010] In certain embodiments, the present disclosure provides a method for reducing fibrosis or fat content or fat accumulation in the liver associated with non-alcoholic fatty liver disease (NAFLD), the method comprising administering the compound disclosed herein or the pharmaceutical composition of the present disclosure.In further embodiments, the NALFD comprises NASH. [Brief explanation of the drawings]

[0011] FIG. 1 provides a synthetic scheme for producing exemplary compounds of the present disclosure.

[0012] Figure 2 provides the results of a cystine depletion study with constant compound exposure. As shown, the depletion kinetics were nearly identical between the experimental compound tested (BL-0856) and cystamine.

[0013] Figure 3 shows the results of a washout experiment after 3 hours of incubation with the experimental compound tested (BL-0856), which resulted in a rate of cystine re-accumulation comparable to that of cystamine.

[0014] Figure 4 shows the amount of cysteamine or cystamine and the experimental compound (BL-0856) measured over time after administration to cells. As expected, cystamine levels were higher in the cystamine-treated cells, and some cystamine was still present in the compound-treated cells. Cysteamine levels were nearly identical in both treatments, indicating that in both cases, cysteamine is rapidly reduced from its disulfide precursor.

[0015] Figure 5 shows the amount of glutathione (GSH) and oxidized glutathione (GSSG), as well as the GSH / GSSG ratio, measured over time after administration of cystamine or an experimental compound (BL-0856) to cells. There is no clear difference in glutathione between the two drug forms. Interestingly, with both drugs, oxidized glutathione (GSSG) is elevated at the first time point, and the GSH / GSSG ratio is lower across all time points compared to the 0-minute control.

[0016] Figure 6 shows the amount of cysteamine measured over time after cells were treated with cystamine or an experimental compound (BL-0856). As expected, cysteine ​​was higher in BL-0856-treated cells, indicating that cysteine ​​was being released from its precursor form.

[0017] FIG. 7 shows intracellular D2-cysteamine levels after continuous drug exposure (in culture medium) to cystinotic fibroblasts.

[0018] FIG. 8 shows cystine levels after treatment of cystinotic fibroblasts with D4-cystamine, compound 0940, and compound 0948.

[0019] FIG. 9 shows the time course of cysteamine production by D4-cystamine, compound 0940, and compound 0948.

[0020] Figures 10A-E show (A) experimental description; (B) body weight (BW) to liver weight (LW) ratios of mice in each treatment group tested; (C) ALT activity for each drug group; (D) H&E and Sirius Red staining of liver sections for each drug group; and (E) col1 gene expression for each group.

[0021] FIG. 11 shows graphs of BW / LW and ALT activity for the control, drug 2 and drug 4 groups.

[0022] 12A-B show (A) staining of liver sections for inflammatory markers for each drug group; and (B) changes in inflammatory cytokines in each test group.

[0023] 13A-C show (A) aSMA staining for each drug test group; (B) expression levels of inflammatory markers, including matrix metalloproteinases; and (C) aSMA protein staining levels.

[0024] FIG. 14 shows inflammatory marker measurements of the prodrug in a mouse model of NASH.

[0025] FIG. 15 shows fibrosis marker measurements of the prodrug in a mouse model of NASH.

[0026] Figure 16 shows markers of tissue remodeling in a mouse model of NASH undergoing prodrug therapy.

[0027] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, a reference to "the subject" includes a reference to one or more subjects, and so forth.

[0028] Also, the use of "or" means "and / or" unless stated otherwise. Similarly, "include," "including," "including," "including," and "including" are interchangeable and are not intended to be limiting.

[0029] Furthermore, it should be understood that when the term "comprising" is used in describing various embodiments, those skilled in the art will understand that in some specific instances, the embodiments can alternatively be described using the terms "consisting essentially of" or "consisting of."

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods, devices, and materials are described herein.

[0031] The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

[0032] As used herein, the term "about" can allow for a degree of variation in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or the limits of a stated range. When a range or list of continuous values ​​is given, unless otherwise specified, any value within that range or between the given continuous values ​​is also disclosed.

[0033] The terms "active ingredient," "active compound," and "active substance" refer to a compound that is administered to a subject alone, or in combination with one or more pharmaceutically acceptable excipients or carriers, to treat, prevent, or ameliorate one or more symptoms of a disease.

[0034] To represent a structural position range, "R x From R xx "Up" or "R x ~R xx " may be used, where X and XX represent numbers. Thus, unless otherwise specified, this notation is intended to encompass all numbered positions enclosed by X and XX, not just the numbers represented by X and XX themselves. For example, "R 1 From R 4 "Up" or "R 1 ~R 4 " means R unless otherwise specified. 1 , R 2 , R 3 , and R 4 is equivalent to

[0035] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic disorder described in this disclosure. Such administration includes substantially simultaneous administration of these agents, such as in a single capsule having a fixed ratio of active ingredients, or in multiple separate capsules for each active ingredient. Such administration also includes sequential use of each type of therapeutic agent. In either case, the treatment regimen will provide the beneficial effects of the drug combination in treating the disease described herein.

[0036] The term "deuterium enrichment" refers to the percentage of deuterium incorporated into a molecule at a particular position instead of hydrogen. For example, 1% deuterium enrichment at a particular position means that 1% of the molecules in a particular sample contain deuterium at that particular position. Because the distribution of naturally occurring deuterium is approximately 0.0156%, a compound synthesized using non-enriched starting materials will have a deuterium enrichment of approximately 0.0156% at any position. Deuterium enrichment can be measured using conventional analytical methods known to those skilled in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy.

[0037] The term "deuterated," when used to describe a particular position within a molecule, such as RR or the symbol "D," or when used to represent a particular position within a molecular structure diagram, means that deuterium is enriched beyond the naturally occurring distribution of deuterium at the specified position. In one embodiment, the deuterium enrichment is at least about 1% of deuterium at the specified position, at least about 5% of deuterium, at least about 10% of deuterium, at least about 20% of deuterium, at least about 50% of deuterium, at least about 70% of deuterium, at least about 80% of deuterium, at least about 90% of deuterium, or at least about 98% of deuterium.

[0038] As used herein, the term "disorder" is generally intended to be synonymous with, and is used interchangeably with, the terms "disease," "syndrome," and "condition" (in the context of medical conditions), all of which reflect an abnormal condition of the human or animal body, or part thereof, that impairs normal functioning and is usually manifested by distinguishable signs or symptoms.

[0039] The terms "drug" and "therapeutic agent" refer to a chemical compound or pharmaceutical composition thereof that is administered to a subject to treat, prevent, or ameliorate one or more symptoms of a disease or disorder.

[0040] The term "non-deuterated," when used to describe a compound, refers to a compound that has not been prepared to increase the level of deuteration beyond that which occurs naturally without an active deuteration process. In some cases, a non-deuterated molecule lacks a deuterated atom.

[0041] The term "isotopic enrichment" refers to the rate at which a less common isotope of an element is incorpo- rated at a particular position in a molecule in place of a more common isotope of that element.

[0042] The term "non-isotopically enriched" refers to a molecule in which the proportions of various isotopes are substantially the same as those found in nature.

[0043] The term "non-release controlling excipient" refers to an excipient whose primary function does not include altering the duration or location of release of an active agent from a dosage form compared to conventional immediate release dosage forms.

[0044] The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" refer to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each component must be "pharmaceutically acceptable" in the sense of being compatible with the other components of the drug. It must also be suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio. See Remington, Pharmacy and Practice, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Pharmaceutical Excipients Handbook, 5th ed.; Rowe et al., Eds. Pharmaceutical Journal and American Pharmaceutical Association: 2005; Pharmaceutical Excipients Handbook, 3rd ed.; Ash and Ash Eds. Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, Fla., 2004).

[0045] The compounds disclosed herein can and do exist as therapeutically acceptable salts. As used herein, the term "pharmaceutically acceptable salt" refers to a salt or zwitterionic form of a compound disclosed herein that is therapeutically acceptable as defined herein. Such salts can be prepared during the final isolation and purification of the compound, or separately by reacting the appropriate compound with a suitable acid or base. Therapeutically acceptable salts include acid and base addition salts. For a more complete discussion of salt preparation and selection, see "Handbook of Pharmaceutical Salts, Properties and Uses," Stah and Wermuth, Ed. (Wiley-VCH and VHCA, Zurich, 2002) and Berge et al., J. Pharm. Sci. 1977, 66, 1-19.

[0046] Acids suitable for preparing pharmaceutically acceptable salts include, but are not limited to, acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, boric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, C -oxo-glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, (+)-L-lactic acid, (t)-DL-lactic acid, lactobionic acid, lauric acid, maleic acid, (-)-L-malic acid, malonic acid, (+)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, perchloric acid, phosphoric acid, L-pyroglutamic acid, saccharic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid.

[0047] Bases applicable to the preparation of pharmaceutically acceptable salts include, but are not limited to, inorganic bases such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, or sodium hydroxide; and organic bases such as primary, secondary, tertiary, and quaternary, aliphatic amines, and aromatic amines, for example, L-arginine, benzathine, benzathine, choline, denol, diethanolamine, diethylamine, dimethylamine, dipropylamine, diisopropylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine, methyl ... amine, isopropylamine, N-methylglucamine, hydrabamine, 1H-imidazole, L-lysine, morpholine, 4-(2-hydroxyethyl)-morpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline, isoquinoline, secondary amines, triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine, 2-amino-2-(hydroxymethyl)-1,3-propanediol, and tromethamine.

[0048] The terms "prevent", "preventing", and "prevention" refer to a method of delaying or arresting the onset of a disease and / or its associated symptoms, preventing a subject from developing a disease, or reducing a subject's risk of developing a disease.

[0049] The term "prodrug" refers to a compound disclosed herein that is readily convertible into the parent compound in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for example, be bioavailable by oral administration, whereas the parent compound is not. A prodrug may also have improved solubility in pharmaceutical compositions over the parent compound. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, Advances in Pharmaceutical Research 1962, 4, 221-294; Morozowich et al., "Designing Biopharmaceutical Properties with Prodrugs and Analogues," Roche Ed., APHA Acad. Pharm. Sci. 1977; "Bioreversible Drug Carriers, Theory and Applications in Drug Design," Roche Ed., APHA Acad. Pharm. Sci. 1987; "Design of Prodrugs," Bundgaard, Elsevier, 1985; Wang et al., Curr. Pharm. Design 1999, 5, 265-287; Pauletti et al., Adv. Drug. Delivery Rev. 1997, 27, 235-256; Mizen et al., Pharm. Biotech. 1998, 11, 345-365; Gaignault et al., Pract. Med. Chem. 1996, 671-696; Asgharnejad "Transport Processes in Pharmaceutical Systems" Amidon et al., Ed., Marcell Dekker, 185-218, 2000; Balant et al., Eur. J. Drug Metab. Pharmacokinet. 1990, 15, 143-53; Balimane and Sinko, Adv. Drug Delivery Rev. 1999, 39, 183-209: Browne, Clin. Neuropharmacol. 1997, 20, 1-12; Bundgaard, Arch. Pharm. Chem. 1979, 86, 1-39: Bundgaard, Controlled Drug Delivery 1987, 17, 179-96: Bundgaard, Adv.Drug Delivery Rev. 1992, 8, 1-38; Fleisher et al., Adv. Drug Delivery Rev. 1996, 19, 115-130; Fleisher et al., Methods Enzymol. 1985, 112, 360-381; Farquhar et al., J. Pharm. Sci. 1983, 72, 324-325; Freeman et al., J. Chem. Soc., Chem. Commun. 1991, 875-877; Friis and Bundgaard, Eur. J. Pharm. Sci. 1996, 4, 49-59; Gangwar et al., Des. Biopharm. Prop. Prodrugs Analogs, 1977, 409-421; Nathwani and Wood, Drugs 1993, 45, 866-94; Sinhababu and Thakker, Adv. Drug Delivery Rev. 1996, 19, 241-273; Stella et al., Drugs 1985, 29, 455-73; Tan et al., Adv. Drug Delivery Rev. 1999, 39, 117-151; Taylor, Adv. Drug Delivery Rev. 1996, 19, 131-148; Valentino and Borchardt, Drug Discovery Today 1997, 2, 148-155; Wiebe and Knaus, Adv. Drug Delivery Rev. 1999, 39, 63-80; Waller et al., Br. J. Clin. Pharmac. 1989, 28, 497-507.

[0050] The term "controlled-release excipient" refers to an excipient whose primary function is to modify the duration or location of release of an active agent from a dosage form as compared to conventional immediate-release dosage forms.

[0051] The term "subject" refers to an animal, including, but not limited to, primates (e.g., humans, monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, etc.), lagomorphs, swine (e.g., pigs, minipigs), horses, dogs, cats, etc. The terms "subject" and "patient" are used interchangeably herein to refer to a mammalian subject, such as a human patient.

[0052] As used herein, the term "substantially" refers to a majority or major portion, such as at least about 51%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

[0053] The term "therapeutically acceptable" refers to compounds (or salts, prodrugs, tautomers, zwitterionic forms, etc.) that are suitable for use in contact with the tissues of a patient without excessive toxicity, irritation, allergic response, or immunogenicity, commensurate with a reasonable benefit / risk ratio, and that are effective in their intended use.

[0054] The term "therapeutically effective amount" refers to that amount of a compound that, when administered, is sufficient to prevent or alleviate to some extent one or more of the symptoms of the disease being treated. The term "therapeutically effective amount" also refers to that amount of a compound that is sufficient to elicit the biological or medical response in a cell, tissue, system, animal, or human that is desired by a researcher, veterinarian, physician, or clinician.

[0055] The terms "treat," "treating," and "treatment" are meant to include alleviating or suppressing a disease, or one or more symptoms associated with a disease, or reducing or eradicating the cause of the disease itself. As used herein, reference to "treatment of a disease" is intended to include prophylaxis.

[0056] Cysteamine is a small aminothiol molecule that is easily transported across cell membranes. Cysteamine significantly reduces intralysosomal cysteine ​​accumulation and is currently approved as a treatment for cystinosis. Cysteamine can increase cellular thiol and free thiol tripeptide glutathione pools, thereby regulating the scavenging of reactive oxygen species (ROS) and reducing lipid peroxidation and glutathione peroxidase activity. Furthermore, cysteamine also increases adiponectin levels.

[0057] Cysteamine reacts with cystine to produce cysteine, which can be further metabolized to glutathione, a potent endogenous antioxidant. Therefore, cysteamine is an attractive candidate for the treatment of fatty liver disease, including NASH. Cysteamine is a precursor to the protein glutathione (GSH) precursor and is currently FDA-approved for use in the treatment of cystinosis, a lysosomal cystine storage disorder. In cystinosis, cysteamine acts by converting cystine to cysteine ​​and cysteine-cysteamine mixed disulfide, both of which can then leave the lysosome via cysteine ​​and lysine transporters, respectively (Gahl et al., N Engl J Med 2002;347(2):111-21). Within the cytosol, the mixed disulfide is reduced by reaction with glutathione, and the released cysteine ​​can be used for further GSH synthesis. The synthesis of GSH from cysteine ​​is catalyzed by two enzymes: gamma-glutamylcysteine ​​synthetase and GSH synthase. This pathway occurs in nearly all cell types, with the liver being the primary producer and exporter of GSH. Reduced cysteine-cysteamine mixed disulfide also releases cysteamine, which can theoretically re-enter lysosomes, bind to more cystine, and repeat the process (Dohil et al., J Pediatr 2006;148(6):764-9). A recent study in children with cystinosis showed that enteral administration of cysteamine resulted in increased cysteamine absorption, which subsequently had a long-lasting effect in reducing leukocyte cystine levels (Dohil et al., J Pediatr 2006;148(6):764-9). This may be due to "recycling" of cysteamine when sufficient amounts of the drug reach the lysosomes. When cysteamine acts in this way, it can also significantly stimulate GSH production.

[0058] Cysteamine is a potent gastric acid secretagogue that has been used to induce duodenal ulcer formation in experimental animals. Human and animal studies have shown that cysteamine-induced gastric acid hypersecretion is most likely mediated by hypergastrinemia. Previous studies in children with cystinosis who suffered from regular upper gastrointestinal symptoms have shown that a single oral dose of cysteamine (11–23 mg / kg) caused hypergastrinemia, a two- to three-fold increase in gastric acid hypersecretion, and a 50% increase in serum gastrin levels. Symptoms experienced by these individuals included abdominal pain, heartburn, nausea, vomiting, and loss of appetite. U.S. Patent Application No. 11 / 990,869 and published International Publication No. WO 2007 / 089670 (both claiming priority to U.S. Provisional Patent Application No. 60 / 762,715, filed January 26, 2006, all of which are incorporated herein by reference in their entireties) demonstrated that cysteamine-induced hypergastrinemia occurs, in part, as a local effect on the gastric antrum-predominant G cells in susceptible humans. The data also suggest that this is a systemic effect of cysteamine-induced gastrin release. For both routes of administration, plasma gastrin levels typically peak within 30 minutes after intragastric delivery, while plasma cysteamine levels peak thereafter.

[0059] Sulfhydryl (SH) compounds, such as cysteamine, cystamine, and glutathione, are among the most important and active intracellular antioxidants. Cysteamine protects animals from bone marrow and gastrointestinal radiation syndrome. The rationale for the importance of SH compounds is further supported by observations in mitotic cells. These cells are most sensitive to radiation damage in terms of cell reproductive death and have been noted to have the lowest levels of SH compounds. Conversely, using the same criteria, S-phase cells, which are most resistant to radiation damage, exhibit the highest levels of intrinsic SH compounds. Furthermore, treatment of mitotic cells with cysteamine significantly enhanced their resistance to radiation. Cysteamine has also been noted to directly protect cells from induced mutations. This protection is thought to result from the scavenging of free radicals, either directly or via the release of protein-bound GSH. An enzyme that liberates cysteamine from coenzyme A has been reported in avian liver and porcine kidney. Recently published studies have demonstrated the protective effect of cysteamine against the hepatotoxic substances acetaminophen, bromobenzene, and phalloidin.

[0060] In addition to its role as a radioprotector, cystamine has been found to reduce tremors and extend lifespan in mice with a genetic mutation for Huntington's disease (HD). The drug may act by increasing the activity of proteins that protect nerve cells or neurons from degeneration. Cystamine inactivates an enzyme called transglutaminase, resulting in a decrease in huntingtin protein (Nature Medicine 8, 143-149, 2002). Cystamine has also been found to increase the levels of certain neuroprotective proteins. However, current cystamine delivery methods and formulations require excessive administration due to insufficient degradation and uptake.

[0061] Currently, cysteamine is only FDA-approved for the treatment of cystinosis. Patients with cystinosis typically require oral cysteamine every six hours or enteric-coated cysteamine (PROCYSBI®) every 12 hours. Subjects with cystinosis require oral cysteamine (CYSTAGON®) every six hours or enteric-coated cysteamine (PROCYSBI®) every 12 hours, day and night. Regular cysteamine intake can deplete intracellular cystine (measured in circulating white blood cells) by up to 90%, slow the rate of progression to renal failure / transplant, and prevent the need for thyroid replacement therapy. Because CYSTAGON® is difficult to administer, reducing the required dose improves compliance with the treatment plan. International Publication No. WO 2007 / 089670 demonstrates that delivery of cysteamine to the small intestine reduces gastric distress and ulcer formation, increases Cmax, and increases AUC. Delivery of cysteamine to the small intestine is beneficial due to improved absorption rate from the small intestine and / or reduced hepatic first-pass excretion of cysteamine upon absorption through the small intestine. Reductions in leukocyte cystine were observed within 1 hour of treatment.

[0062] In a pilot study by Dohil et al., 11 children with biopsy-confirmed nonalcoholic fatty liver disease (NAFLD) were given enteric-coated (EC) cysteamine bitartrate orally for 24 weeks. Treatment resulted in statistically significant decreases in mean serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total adiponectin, leptin, and cytokeratin 18 fragment, but there was no associated decrease in body mass index. Seven of the 11 subjects achieved the primary endpoint (at least a 50% reduction in ALT). Mean ALT and AST levels continued to decrease 16 weeks after the end of treatment.

[0063] In certain embodiments, the present disclosure provides compounds of formula I: JPEG2025502611000028.jpg63127, or a pharmaceutically acceptable salt or solvate thereof, wherein: R 1 is selected from H or an acetyl group; R 2 teeth, JPEG2025502611000029.jpg54150 and Selected from JPEG2025502611000030.jpg51115; R 3 is selected from optionally substituted (C1-C6) alkyl, optionally substituted cycloalkyl, optionally substituted benzyl, or optionally substituted aryl; R 4 teeth, JPEG2025502611000031.jpg2134 or Selected from JPEG2025502611000032.jpg4165; R 5 is selected from optionally substituted (C1-C6) alkyl, optionally substituted cycloalkyl, optionally substituted benzyl, or optionally substituted aryl; and Y 1 ~Y 16 are each independently selected from H or D.

[0064] In another embodiment, the present disclosure provides a compound of formula II: JPEG2025502611000033.jpg61164, or a pharmaceutically acceptable salt or solvate thereof, wherein: Y 1 ~Y 8 are each independently selected from H or D; and R 1 is selected from H or an acetyl group.

[0065] In another embodiment, the present disclosure provides a compound of formula II(a): JPEG2025502611000034.jpg64167, or a pharmaceutically acceptable salt or solvate thereof, wherein Y 1 ~Y 8 are each independently selected from H or D.

[0066] In another embodiment, the present disclosure provides: JPEG2025502611000035.jpg216149JPEG2025502611000036.jpg220151JPEG2025502611000037.jpg145162, or a pharmaceutically acceptable salt or solvate of any one of the above.

[0067] In a further embodiment, the pharmaceutically acceptable salt of Formula I has Formula II(b): JPEG2025502611000038.jpg63170, wherein: Y 1 ~Y 8 are each independently selected from H or D; and X is a pharmaceutically acceptable counterion.

[0068] In another embodiment, the present disclosure provides a compound of formula II(c): JPEG2025502611000039.jpg53163, or a pharmaceutically acceptable salt or solvate thereof, wherein: Y 1 ~Y 8 are each independently selected from H or D.

[0069] In another embodiment, the present disclosure provides: JPEG2025502611000040.jpg211154JPEG2025502611000041.jpg213153JPEG2025502611000042.jpg143153, or a pharmaceutically acceptable salt or prodrug thereof.

[0070] In a further embodiment, the pharmaceutically acceptable salt of Formula I has Formula II(d): JPEG2025502611000043.jpg36115, wherein: Y 1 ~Y 8 are each independently selected from H or D; and X is a pharmaceutically acceptable counterion.

[0071] In another embodiment, the present disclosure provides a compound of formula III: JPEG2025502611000044.jpg63135, or a pharmaceutically acceptable salt or solvate thereof, wherein: Y 1 ~Y 4 are each independently selected from H or D; R 1 is selected from H or an acetyl group; and R 3 is (C1-C6) alkyl.

[0072] In another embodiment, the present disclosure provides a compound of formula III(a): JPEG2025502611000045.jpg61139, or a pharmaceutically acceptable salt or solvate thereof, wherein: Y 1 ~Y 4 are each independently selected from H or D; and R 3 is selected from (C1-C6) alkyl.

[0073] In another embodiment, the present disclosure provides: JPEG2025502611000046.jpg117163, or a pharmaceutically acceptable salt or solvate of any one of the foregoing, wherein R 3 is (C1-C6) alkyl.

[0074] In a further embodiment, the pharmaceutically acceptable salt of Formula I has Formula III(b): JPEG2025502611000047.jpg63137, wherein: Y 1 ~Y 4 are each independently selected from H or D; R 3 is (C1-C6) alkyl; and X is a pharmaceutically acceptable counterion.

[0075] In another embodiment, the present disclosure provides a compound of formula III(c): JPEG2025502611000048.jpg56132, or a pharmaceutically acceptable salt or solvate thereof, wherein: Y 1 ~Y 4 are each independently selected from H or D; and R 3 is (C1-C6) alkyl.

[0076] In another embodiment, the present disclosure provides: JPEG2025502611000049.jpg115151, or a pharmaceutically acceptable salt or solvate thereof, wherein R 3 is (C1-C6) alkyl.

[0077] In a further embodiment, the pharmaceutically acceptable salt of Formula I has Formula III(d): JPEG2025502611000050.jpg60136, wherein: Y 1 ~Y 4 are each independently selected from H or D; R 3 is (C1-C6) alkyl; and X is a pharmaceutically acceptable counterion.

[0078] In another embodiment, the present disclosure provides a compound of formula IV: JPEG2025502611000051.jpg65134, or a pharmaceutically acceptable salt or solvate thereof, wherein: Y 1 ~Y 4 and Y 9 ~Y 16 are each independently selected from H or D; and R 1 is selected from H or an acetyl group.

[0079] In another embodiment, the present disclosure provides a compound of formula V: JPEG2025502611000052.jpg53134, or a pharmaceutically acceptable salt or solvate thereof, wherein: Y 1 ~Y 4 and Y 9 ~Y 16 are each independently selected from H or D; R 1 is selected from H or an acetyl group; and R 5 is (C1-C6) alkyl.

[0080] In another embodiment, the present disclosure provides a compound of formula VI: JPEG2025502611000053.jpg5286, or a pharmaceutically acceptable salt or solvate thereof, wherein Y 1 ~Y 4 are each independently selected from H or D; and R is a linear or branched aliphatic group (saturated or unsaturated) or aromatic group (substituted or unsubstituted) having 1 to 20 carbon atoms. 1 ~Y 4 At least one of Y independently has a deuterium enrichment of about 10% or greater. 1 ~Y 4 At least one of Y is independently deuterium enriched to about 50% or greater. 1 ~Y 4At least one of Y is independently deuterium enriched to about 90% or greater. 1 ~Y 4 At least one of the following independently has a deuterium enrichment of about 98% or greater.

[0081] In another embodiment, the present disclosure provides a compound of formula VI(a): JPEG2025502611000054.jpg53147, or a pharmaceutically acceptable salt or solvate thereof, wherein Y 1 ~Y 8 are each independently selected from H or D; and n is 2 to 6 (e.g., 2, 3, 4, 5, or 6). 1 ~Y 8 At least one of Y independently has a deuterium enrichment of about 10% or greater. 1 ~Y 8 At least one of Y is independently deuterium enriched to about 50% or greater. 1 ~Y 8 At least one of Y is independently deuterium enriched to about 90% or greater. 1 ~Y 8 At least one of the following independently has a deuterium enrichment of about 98% or greater.

[0082] In another embodiment, the present disclosure provides: The present invention provides a compound having a structure selected from the group consisting of: JPEG2025502611000055.jpg60159.

[0083] Animal bodies use cytochrome P to eliminate foreign substances such as therapeutic drugs. 450They express a variety of enzymes, including CYPs (cytochrome P450), ester hydrolases, proteases, reductases, dehydrogenases, and monoamine oxidases, to react with these xenobiotics and convert them into more polar intermediates or metabolites for renal excretion. Such metabolic reactions often involve the oxidation of carbon-hydrogen (CH) bonds to either carbon-oxygen (CO) or carbon-carbon (CC) JU bonds. The resulting metabolites may be stable or unstable under physiological conditions and may have substantially different pharmacokinetic, pharmacodynamic, acute, and long-term toxicity profiles from the parent compound. For most drugs, such oxidation is generally rapid, ultimately resulting in multiple daily or large doses.

[0084] The relationship between activation energy and reaction rate is given by the Arrhenius equation The Arrhenius equation states that the rate of a chemical reaction depends on the activation energy (E a ) is exponentially dependent on

[0085] A transition state in a reaction is a short-lived state along the reaction pathway where the original bonds are stretched to their limits. The activation energy E of the reaction a By definition, is the energy required to reach the transition state of that reaction. Once the transition state is reached, the molecules can either revert to the original reactants or form new bonds to produce reaction products. Catalysts accelerate the reaction process by lowering the activation energy leading to the transition state. Enzymes are examples of biological catalysts.

[0086] The strength of a carbon-hydrogen bond is directly proportional to the absolute value of the ground-state vibrational energy of the bond. This vibrational energy depends on the mass of the atoms forming the bond and increases as the mass of one or both of the atoms forming the bond increases. Because deuterium (D) has twice the mass of protium (H), a C-D bond is stronger than the corresponding C-H bond. If a C-H bond is broken during the rate-determining step of a chemical reaction (i.e., the step with the highest transition state energy), replacing the protium with deuterium reduces the reaction rate. This phenomenon is known as the deuterium kinetic isotope effect (DKIE). The magnitude of the DKIE can be expressed as the ratio of the rate of a particular reaction in which a C-H bond is broken to the rate of the same reaction in which protium is replaced by deuterium. The DKIE can range from approximately 1 (no isotope effect) to very large numbers, e.g., 50 or greater. Substitution of hydrogen with tritium creates an even stronger bond than deuterium, resulting in a numerically larger isotope effect.

[0087] Deuterium (D) is a stable, non-radioactive isotope of hydrogen, with approximately twice the mass of the most common hydrogen isotope, protium (H). Deuterium oxide (DO), or deuterium dioxide (DO), or "heavy water," looks and tastes similar to H2O but has different physical properties. When pure DO is given to rodents, it is readily absorbed. The amount of deuterium required to cause toxicity is extremely high. When approximately 0-15% of the body water has been replaced by heavy water, animals are healthy but do not gain weight as quickly as untreated controls. When approximately 15-20% of the body water has been replaced by heavy water, animals become agitated. When approximately 20-25% of the body water has been replaced by heavy water, animals become highly agitated and frequently convulse when stimulated. Skin lesions, ulcers on the paws and muzzle, and necrosis of the tail appear. Animals also become highly aggressive. When approximately 30% of an animal's body's water is replaced by heavy water, it will refuse to eat, become lethargic, lose weight rapidly, and experience a much slower metabolic rate than normal. When approximately 30-35% of the animal's body's water is replaced by heavy water, it will die. This effect is reversible as long as the animal does not lose more than 30% of its previous body weight to heavy water. Research has also shown that the use of heavy water can slow the growth of cancer cells and enhance the cytotoxicity of certain antitumor agents.

[0088] Deuteration of pharmaceuticals to improve pharmacokinetics (PK), pharmacodynamics (PD), and toxicity profiles has previously been demonstrated for several drug classes. For example, DKIE was used to reduce the hepatotoxicity of halothane, presumably by limiting the production of reactive species such as trifluoroacetyl chloride. However, this method is not applicable to all drug classes. For example, the introduction of deuterium can induce metabolic switching. Metabolic switching occurs when heterologous substances sequestered by phase I enzymes temporarily bind and recombine in various conformations before chemical reactions (e.g., oxidation). Metabolic switching is made possible by the relatively large binding pockets of many phase I enzymes and the random nature of many metabolic reactions. Metabolic switching can result not only in different ratios of known metabolites but also in entirely new metabolites. This new metabolic profile may confer greater or lesser toxicity. Such pitfalls are not obvious and cannot be predicted in advance for any drug class.

[0089] Compounds containing deuterium atoms as described herein are expected to prevent or delay their metabolism. Other sites on the molecule may also undergo transformation, leading to metabolites whose pharmacology / toxicology is yet unknown. Limiting the production of such metabolites may reduce the risks of administering such drugs and allow for increased doses and therefore improved efficacy. All of these transformations, among other potential transformations, may occur through polymorphically expressed enzymes, leading to patient-to-patient variability. Furthermore, diseases ameliorated by the compositions and methods of the present disclosure, such as NAFLD, NASH, or cystinosis, are very typically best administered around the clock for extended periods of time to alleviate symptoms.

[0090] For all of the above reasons, drugs with longer half-lives can provide greater efficacy and cost savings. Various deuteration patterns can be used to (a) reduce or eliminate undesirable metabolites, (b) extend the half-life of the parent drug, (c) reduce the number of doses required to achieve the desired effect, (d) reduce the dose required to achieve the desired effect, (e) increase the formation of active metabolites, if they are formed, (f) reduce the production of harmful metabolites in specific tissues, and / or (g) create more effective and / or safer drugs for polypharmacy, whether or not polypharmacy is intended. Compounds of the present disclosure containing deuterium atoms can slow metabolism and / or selectively shunt the metabolism of the compound to more preferred enzymatic pathways. For example, it is expected that compounds containing deuterium atoms as described herein can potentially prevent or reduce the production of odorous cysteamine metabolites, which can lead to patient non-compliance.

[0091] The present disclosure provides bioprotective compounds and pharmaceutical compositions, as well as methods of synthesis of the compounds and methods of use, including methods of treating liver diseases and disorders in patients by administering the disclosed compounds.

[0092] The present disclosure is not limited with respect to a particular salt form of Formula I (e.g., the present disclosure is not limited to any particular pharmaceutically acceptable salt). Furthermore, pharmaceutical compositions of the present disclosure can contain compounds of the present disclosure individually or in combination with compounds of the present disclosure, in which one or both compounds are deuterated. The active ingredients in the compositions, i.e., compounds of the present disclosure, can be administered in the form of their pharmacologically acceptable salts or solvates. Salts and solvates of the compounds are known to those skilled in the art of organic synthetic chemistry and may be prepared using standard procedures, for example, as described in "Advanced Organic Chemistry: Reactions, Mechanisms, and Structures," 4th Edition, by J. March (New York: Wiley-Interscience, 1992). For example, base addition salts are prepared from neutral drugs using conventional methods involving the reaction of one or more free hydroxyl groups of the active ingredient with a suitable base. Generally, the neutral form of the drug is dissolved in a polar organic solvent, such as methanol or ethanol, and a base is added thereto. The resulting salt may precipitate or be removed from solution by adding a less polar solvent. Suitable bases for the formation of base addition salts include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, and trimethylamine.

[0093] The compounds disclosed herein include, for example, 13 C or 14 C, sulfur 33 S, 34 S, or 36 S, nitrogen 15 N and oxygen 17 O or 18 It may also contain lower isotopes of other elements, including O.

[0094] In certain embodiments, assuming that all CD bonds in the compounds disclosed herein are metabolized and released as DO or DHO, the compounds disclosed herein may expose a patient to a maximum of about 0.000005% DO or about 0.00001% DHO. In certain embodiments, levels of DO that have been shown to cause toxicity in animals are far greater than the maximum exposure caused by administration of the deuterium-enriched compounds disclosed herein. Thus, in certain embodiments, the deuterium-enriched compounds disclosed herein should not cause additional toxicity due to the formation of DO or DHO during drug metabolism.

[0095] In a further embodiment, the compounds of the present disclosure exhibit a reduced rate of metabolism by at least one polymorphically expressed cytochrome P450 isotope per dosage unit in a subject compared to non-isotopically enriched cysteamine and cystamine. Examples of polymorphically expressed cytochrome P450 isotopes include, but are not limited to, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. In another embodiment, the compounds of the present disclosure exhibit a reduced rate of metabolism by at least one cytochrome P450 isotope or monoamine oxidase isotope per dosage unit in a subject compared to non-isotopically enriched cysteamine and cystamine. Examples of cytochrome P450 isotopes and monoamine oxidase isotopes include, but are not limited to, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2G1, CYP2J2, CYP2R1, CYP2S1, CYP3A4, CY P3A5, CYP3A5P1, CYP3A5P2, CYP3A7, CYP4A11, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, These include CYP4X1, CYP4Z1, CYP5A1, CYP7A1, CYP7B1, CYP8A1, CYP8B1, CYP11A1, CYP11B1, CYP11B2, CYP17, CYP19, CYP21, CYP24, CYP26A1, CYP26B1, CYP27A1, CYP27B1, CYP39, CYP46, CYP51, MAOA, and MAOB.

[0096] In certain embodiments, the compounds of the present disclosure exhibit improved diagnostic hepatobiliary function endpoints compared to the corresponding non-isotopically enriched cysteamine and cystamine. Examples of diagnostic hepatobiliary function endpoints include, but are not limited to, alanine aminotransferase (ALT), serum glutamate pyruvate aminotransferase (SGPT), aspartate aminotransferase (AST, SGOT), ALT / AST ratio, serum aldolase, alkaline phosphatase (ALP), ammonia level, bilirubin, gamma glutamyl transpeptidase (GGTP, γ-GTP, GGT), leucine aminopeptidase (LAP), liver biopsy, liver ultrasound, hepatic nuclear scan, 5'-nucleotidase, and blood proteins.

[0097] As shown in the results herein, the compounds of the present disclosure exhibited nearly identical cystine depletion kinetics to cystamine. Furthermore, with the compounds of the present disclosure, cystine reaccumulated after washout at a rate similar to that described for cystamine. This suggests that both drugs acted similarly in depleting intracellular cystine.

[0098] In further experiments presented herein, the intracellular reduction of the compounds disclosed herein to cysteamine is very rapid, similar to that of cystamine.In addition, the intracellular level of cystamine is higher after cystamine administration than that of the compounds disclosed herein, because the compound does not need to be reduced before detection.The release of cysteine ​​is greater with the compounds disclosed herein than with cystamine.Interestingly, this does not affect the level of cystine depletion during continuous drug exposure or the degree of cystine accumulation after drug washout.

[0099] Based on these studies, it is clear that the compounds disclosed herein make more cysteine ​​available in cells than cysteamine or cystamine, and glutathione was induced at the same level as when cystamine or the compounds disclosed herein were administered.

[0100] In another embodiment, methods for preparing the compounds disclosed herein, or other pharmaceutically acceptable derivatives (e.g., salts, solvates, or prodrugs), as antioxidants and for the treatment of cystinosis and fatty liver diseases such as NAFLD and NASH.

[0101] Although the compounds of the present disclosure can be administered as raw chemicals, they can also be provided as pharmaceutical compositions. Thus, provided herein are pharmaceutical compositions comprising one or more specific deuterated compounds disclosed herein, or one or more pharmaceutically acceptable salts, prodrugs, or solvates thereof, and one or more pharmaceutically acceptable carriers thereof, and optionally one or more other therapeutic ingredients. Appropriate formulations vary depending on the selected route of administration. Any of the well-known techniques, carriers, and excipients can be used as appropriate, as understood in the art (e.g., Remington's Pharmaceuticals). The pharmaceutical compositions disclosed herein may be manufactured by any method known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping, or compressing processes.

[0102] The pharmaceutical compositions can also be formulated as modified-release dosage forms, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-, and gastroretentive-release dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art (see Remington, Pharmacy and Practice, supra; Modified-Release Drug Delivery Technologies, edited by Rathbone et al., Drugs and Pharmaceutical Sciences, Marcel Dekker, Inc.: New York, NY, 2002; Vol. 126). For example, in one embodiment, deuterated cysteamine and / or cystamine can be enterically coated (e.g., enterically coated beads or capsules). As noted above and elsewhere herein, non-deuterated enteric-coated formulations of cysteamine bitartrate have been shown to improve compliance, reduce dosing frequency, and provide long-term reductions in cystine levels in patients with cystinosis. Enteric coated formulations comprising deuterated compounds of Formula I and / or II provide improved administration and longer biological activity due to the longer half-life of the deuterated forms of the compounds of the present disclosure. In some embodiments, enteric coated formulations of compounds of Formula I and / or II can be administered at lower doses and / or less frequently than non-deuterated enteric formulations.

[0103] The compositions include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal, and topical (including cutaneous, buccal, sublingual, and ocular) administration. The optimal route of administration will depend on various factors, including patient-to-patient variability and the type of disease, and therefore the present disclosure is not limited to a single form of administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts. Typically, these methods include the step of bringing into association the compound of the present disclosure or a pharmaceutical salt, prodrug, or solvate thereof (the "active ingredient") with the carrier, which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation.

[0104] Formulations of the compounds disclosed herein suitable for oral administration can be presented as discrete units, for example, as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules (including enteric-coated granules); as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion. The active ingredient can also be presented as a bolus, electuary, or paste.

[0105] Orally usable pharmaceutical formulations include tablets, push-fit capsules made of gelatin, and soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form, such as a powder or granules, optionally mixed with a binder, inert diluent, or lubricant, surfactant, or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated to provide slow or controlled release of the active ingredient therein. All formulations intended for oral administration should be in dosages suitable for such administration. The push-fit capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active compound can be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Stabilizers may also be added. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used. These may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0106] The compounds can be formulated for parenteral administration by injection, e.g., bolus injection or continuous infusion. Injectable formulations may be presented in unit dosage form (e.g., in ampoules) or in multi-dose containers with added preservatives. The compositions may take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulating agents such as suspending, stabilizing, and / or dispersing agents. The formulations may be presented in unit-dose or multi-dose containers, e.g., sealed ampoules and vials, and may be stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, such as saline or sterile pyrogen-free water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0107] Formulations for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compound, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the subject's blood; and aqueous and non-aqueous sterile suspensions may contain suspending agents and thickening agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound, allowing for the preparation of highly concentrated solutions.

[0108] In addition to the above-mentioned formulations, the compound can also be formulated as depot preparations.Such long-acting preparations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.Thus, for example, the compound can be formulated with suitable polymer or hydrophobic material (for example, as an emulsion in acceptable oil) or ion exchange resin, or as sparingly soluble derivatives, for example, as sparingly soluble salts.

[0109] For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, troches, or gels formulated in a conventional manner. Such compositions may comprise the active ingredient in a flavored base such as sucrose and acacia or tragacanth.

[0110] Certain compounds disclosed herein may be administered topically, i.e., by non-systemic administration. This includes applying the compounds disclosed herein externally to the epidermis or buccal cavity, and instilling such compounds into the ears, eyes, and nose so that the compounds do not enter the bloodstream as much. In contrast, systemic administration refers to oral, intravenous, intraperitoneal, and intramuscular administration.

[0111] Formulations suitable for topical administration include liquid or semi-liquid formulations suitable for penetration through the skin to the site of inflammation, such as gels, salves, lotions, creams, ointments or pastes, and drops suitable for administration to the eyes, ears or nose.

[0112] For administration by inhalation, the compound may be delivered from an inhaler, a nebulizer pressurized pack, or other convenient means for delivering a propellant spray. The pressurized pack may contain a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized propellant, the dosage unit may be determined by providing a valve to deliver a metered amount. Alternatively, for administration by inhalation or insufflation, the compound according to the present disclosure may be in the form of a dry powder composition, for example, a powder mix of the compound and a suitable powder base, such as lactose or starch. The powder composition may be presented in unit dosage form, such as capsules, cartridges, gelatin, or blister packs from which the powder can be administered with the aid of an inhaler or insufflator. Typical unit-dose formulations contain an effective dose, or an appropriate fraction thereof, of the active ingredient, as described herein below.

[0113] Tablets or other presentation forms provided as discrete units may conveniently contain an amount of one or more compounds, said amount being effective in such doses or multiples thereof, e.g., 1 mg to 1000 mg of a compound disclosed herein, typically a unit containing about 100 mg to 500 mg of said compound.

[0114] The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

[0115] The compound can be administered in various ways, for example, orally, topically, or by injection. The exact amount of compound administered to a patient is the responsibility of the attending physician. The specific dose level for any particular patient will depend on various factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, route of administration, excretion rate, drug combination, the exact disease being treated, and the severity of the disease being treated. The route of administration may also vary depending on the disease and its severity.

[0116] In this disclosure, oral administration is considered to be a typical delivery method. Formulations for such delivery include enteric-coated and non-enteric-coated formulations containing at least one deuterated form of cystamine and / or cysteamine. As shown below, the deuterated forms include pharmacokinetic and pharmacodynamic changes relative to the non-deuterated forms. Furthermore, conventional formulations containing enteric-coated cysteamine and / or cystamine have also shown improved pharmacokinetic and pharmacodynamic data compared to non-enteric-coated formulations. Therefore, the combination of enteric-coated and deuterated forms of cystamine and / or cysteamine is expected to further modulate the pharmacokinetics and pharmacodynamics of cysteamine and / or cysteamine delivery, including, for example, AUC, C max and / or T max This includes both delayed and sustained release of the active ingredient as reflected by the regulation of

[0117] If the patient's condition does not improve, administration of the compound may, at the physician's discretion, be administered long-term, i.e., throughout the patient's life, to ameliorate or otherwise control or limit the symptoms of the patient's disease.

[0118] If the patient's condition improves, administration of the compound may be continued or temporarily discontinued for a period of time (i.e., a "drug period"), at the discretion of the physician. Once the patient's condition has improved, a maintenance dose is administered, if necessary. Thereafter, depending on the symptoms, the dosage or frequency of administration, or both, may be reduced to a level at which the improved condition is maintained. However, if symptoms recur, the patient may require intermittent treatment on a long-term basis.

[0119] The present disclosure identifies patient populations, particularly young patients, that may benefit from the compounds disclosed herein. The present disclosure provides compositions of the compounds disclosed herein that can be used to treat a variety of diseases, including cystinosis, Huntington's disease, and NAFLD (including NASH).

[0120] Cystinosis is a rare disease usually diagnosed by the age of two. It is a genetic metabolic disorder that causes the amino acid cystine to accumulate in various organs of the body. Cystine crystals accumulate in the kidneys, eyes, liver, muscles, pancreas, brain, and white blood cells. Without specific treatment, children with cystinosis develop end-stage renal failure by approximately age nine. Cystinosis also causes complications in other organs of the body. These complications include muscle weakness, difficulty swallowing, diabetes, and hypothyroidism. Cystinosis is estimated to affect at least 2,000 people worldwide, but the exact number is difficult to determine because the disease is often undiagnosed and / or misdiagnosed. There are three forms of cystinosis. Infantile nephrotic cystinosis is the most severe form of the disease. Children with cystinosis appear normal at birth but are significantly shorter than children of the same age by 10 months of age. They have frequent urination, excessive thirst, and often appear to be picky eaters. By 12 months of age, they are unable to walk and weigh very little. One of the major complications of cystinosis is renal tubular Fanconi syndrome, a condition in which the kidneys are unable to reabsorb nutrients and minerals. Minerals are lost in the urine, and these losses must be replaced. They typically have a picky eater, a salt craving, and very slow growth. If left untreated, the disease can lead to renal failure by age 10. In patients with intermediate or juvenile (adolescent) cystinosis, kidney and eye symptoms usually appear in the teenage years or early adulthood. In benign or adult cystinosis, cystine accumulates primarily in the cornea of ​​the eye. Cystinosis is treated symptomatically. Renal tubular dysfunction requires a high intake of fluids and electrolytes to prevent excessive water loss (dehydration) from the body. Sodium bicarbonate, sodium citrate, and potassium citrate may be administered to maintain normal electrolyte balance. Phosphate and vitamin D are also needed to correct impaired renal phosphate uptake and prevent rickets. Carnitine may help compensate for muscle carnitine deficiency.

[0121] Cysteamine (Cystagon®) is approved by the Food and Drug Administration (FDA) as the standard treatment for cystinosis. Cysteamine is a cystine-depleting agent that reduces intracellular cystine levels. Cysteamine has proven effective in delaying or preventing renal failure. Cysteamine also improves growth in children with cystinosis. Given the deleterious effects of chronic cystine accumulation and the lack of evidence of efficacy of cysteamine therapy in various tissues and organ systems, patients with post-transplant cystinosis should use oral cysteamine. Procysbi® (cysteamine bitartrate delayed-release capsules) was approved by the FDA in May 2013. Cystaran (cysteamine ophthalmic solution) 0.44% is an FDA-approved eye drop for the treatment of corneal cystine crystal accumulation in patients with cystinosis.

[0122] In one embodiment, a subject suffering from cystinosis is administered a compound of the present disclosure or a pharmaceutically acceptable salt thereof to achieve a plasma level of the compound of about 10-200 μmol (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or any value therebetween). In one embodiment, the dose is up to about 10-95 mg / kg. In another embodiment, the dose is about 100 mg-1 g administered two to four times daily. In a further embodiment, the compound of the present disclosure is administered at a dose of 2.0 g / m 2 The compound is administered in multiple doses not exceeding 95 mg / kg / day or 95 mg / kg / day. If the compound is well tolerated, the goal of treatment is to maintain leukocyte cystine levels at 1 nmol / kg for 5-6 hours after administration of the compound of the present disclosure. 1 / 2 The goal is to maintain leukocyte cystine levels below 2 nmol / mg protein. Patients with poor tolerance should have leukocyte cystine levels below 2 nmol / mg protein. 1 / 2 Less than cystine / mg protein still provides significant benefit. To achieve this level, doses of the compounds disclosed herein can be increased to a maximum of 2.0 g / m 2 / day.

[0123] Patients aged 12 years or older weighing 110 pounds or more should receive a starting maintenance dose of 2.0 g per day in 4 divided doses. This dose should be reached after 4 to 6 weeks of titration as described above. If leukocyte cystine levels are >2 nmol / 1 / 2 If cystine / mg protein remains, the dose should be increased.

[0124] For new patients after reaching the maintenance dose, a leukocyte cystine measurement performed 5 to 6 hours after dose administration is recommended. Patients switching from a solution containing the compound to capsules should have their leukocyte cystine levels measured within 2 weeks and then every 3 months to assess the optimal dose, as described above.

[0125] If the compounds of the present disclosure are initially poorly tolerated due to gastrointestinal symptoms or transient skin rash, treatment should be temporarily discontinued and then resumed at a lower dose and gradually increased to an appropriate dose.

[0126] The compositions and methods of the present disclosure can also be used to treat NAFLD and NASH and liver fibrosis.Non-alcoholic fatty liver disease (NAFLD) represents a range of diseases that occur in the absence of alcohol abuse.It is characterized by the presence of fatty liver (fat in the liver) and can represent the liver symptoms of metabolic syndrome (including obesity, diabetes and hypertriglyceridemia).NAFLD is associated with insulin resistance and can cause liver disease in adults and children, eventually leading to cirrhosis (Skelly et al., J Hepatol 2001;35:195-9; Chitturi et al., Hepatology 2002;35(2):373-9). The severity of NAFLD ranges from relatively benign isolated macrovesicular steatosis (i.e., nonalcoholic fatty liver or NAFL) to nonalcoholic steatohepatitis (NASH) (Angulo et al., J Gastroenterol Hepatol 2002;17 Suppl:S186-90). NASH is characterized by the histological presence of fatty liver, cytological ballooning, diffuse inflammation, and pericellular fibrosis (Contos et al., Adv Anat Pathol 2002;9:37-51). Liver fibrosis caused by NASH can progress to cirrhosis or liver failure and, in some cases, hepatocellular carcinoma.

[0127] The degree of insulin resistance (and hyperinsulinemia) correlates with the severity of NAFLD and is more pronounced in patients with NASH than in those with simple fatty liver (Sanyal et al., Gastroenterology 2001;120(5):1183-92). This results in the suppression of insulin-mediated lipolysis and elevated levels of circulating fatty acids. Two factors associated with NASH include insulin resistance and increased delivery of free fatty acids to the liver. Insulin blocks mitochondrial fatty acid oxidation. Increased hepatic production of free fatty acids for re-esterification and oxidation leads to the accumulation of fat in the liver and increases the liver's vulnerability to secondary injury.

[0128] Glutathione (γ-glutamyl-cysteinyl-glycine; GSH) is a major endogenous antioxidant, and its depletion has been implicated in the development of hepatocellular injury (Wu et al., J Nutr 2004;134(3):489-92). One such injury is acetaminophen poisoning, in which reduced GSH levels are depleted in an attempt to bind and inactivate the drug's hepatotoxic metabolites. After ingesting a toxic dose of acetaminophen, excess metabolite (N-acetyl-benzoquinoneimine) covalently binds to hepatic proteins and enzymes, causing liver injury (Wu et al., J Nutr 2004;134(3):489-92; Prescott et al., Annu Rev Pharmacol Toxicol 1983;23:87-101). Therefore, elevated glutathione levels may have some protective effect through the reduction of ROS. Glutathione itself does not readily enter cells, even in large amounts. However, glutathione precursors do enter cells, and some GSH precursors, such as N-acetylcysteine, have been shown to be effective in treating conditions such as acetaminophen toxicity by slowing or preventing GSH depletion (Prescott et al., Annu Rev Pharmacol Toxicol 1983;23:87-101). Examples of GSH precursors include cysteine, N-acetylcysteine, methionine, and other sulfur-containing compounds such as cysteamine (Prescott et al., J Int Med Res 1976;4(4 Suppl):112-7).

[0129] Cysteine ​​is the major limiting factor for GSH synthesis. Factors that stimulate cysteine ​​uptake by cells (e.g., insulin and growth factors) generally result in elevated intracellular GSH levels (Lyons et al., Proc Natl Acad Sci USA 2000;97(10):5071-6; Lu SC. Curr Top Cell Regul 2000;36:95-11).

[0130] N-acetylcysteine ​​has been administered to patients with NASH. A report from Turkey showed that obese individuals with NASH treated with N-acetylcysteine ​​for 4 to 12 weeks showed improvements in aminotransferase and γ-GT levels, although no changes in body mass index were reported (Pamuk et al., J Gastroenterol Hepatol 2003;18(10):1220-1).

[0131] Studies in mice and humans have shown that cysteamine is effective in preventing acetaminophen-induced hepatocellular injury (Prescott et al., Lancet 1972;2(7778):652; Prescott et al., Br Med J 1978;1(6116):856-7; Mitchell et al., Clin Pharmacol Ther 1974;16(4):676-84). Cystamine and cysteine ​​have been reported to reduce hepatocellular necrosis induced by several hepatotoxins (Toxicol Appl Pharmacol. 1979 Apr;48(2):221-8). Cystamine has been shown to ameliorate carbon tetrachloride-induced liver fibrosis through the inhibition of tissue transglutaminase (Qiu et al., World J Gastroenterol. 13:4328-32, 2007).

[0132] The prevalence of NAFLD in children is unknown because liver histology is required to confirm the diagnosis (Schwimmer et al., Pediatrics 2006;118(4):1388-93). However, prevalence estimates can be inferred from childhood obesity data using liver ultrasound and elevated serum aminotransferase levels, as well as the knowledge that 85% of children with NAFLD are obese. Data from the National Health and Nutrition Examination Survey reveal a three-fold increase in childhood and adolescent obesity rates over the past 35 years. Data from 2000 onward suggest that 14-16% of children aged 6-19 years are obese, with a BMI >95% (Fishbein et al., J Pediatr Gastroenterol Nutr 2003;36(1):54-61). Furthermore, 85% of children with NAFLD are obese.

[0133] The exact mechanism by which NAFLD develops into NASH remains unclear. Because insulin resistance is associated with both NAFLD and NASH, it is hypothesized that additional factors are also required for the development of NASH. This is known as the "two-hit" hypothesis (Day CP. Best Pract Res Clin Gastroenterol 2002;16(5):663-78), which involves the accumulation of fat in the liver and the presence of a large amount of free radicals accompanied by increased oxidative stress. Macrovesicular steatosis represents the accumulation of triglycerides in the liver, which is due to an imbalance between the delivery and utilization of free fatty acids to the liver. When caloric intake increases, triglycerides accumulate and serve as a reserve energy source. When dietary calories are insufficient, stored triglycerides (in adipose tissue) undergo lipolysis, releasing fatty acids into the circulation and for uptake by the liver. Fatty acids are oxidized to provide energy for utilization. Currently, treatment of NASH focuses on reducing two major pathogenic factors: hepatic fat accumulation and the excessive accumulation of free radicals that cause oxidative stress. Fat accumulation can be reduced by reducing fat intake and increasing calorie consumption. One treatment is sustained, steady weight loss. Although not conclusively proven, several cases have shown that a weight loss of 10% or more reduces hepatic fat accumulation, normalizes hepatic aminotransferases, and improves liver inflammation and fibrosis (Ueno et al., J Hepatol 1997, 27(1):103-7; Vajro et al., J Pediatr 1994;125(2):239-41; Franzese et al., Dig Dis Sci 1997, 42(7):1428-32).

[0134] Reducing oxidative stress through treatment with antioxidants has also been shown to be effective in several studies. For example, obese children with fatty liver were treated with vitamin E (400–1000 IU / day) for 4–10 months (Lavine, J Pediatr 2000, 136(6):734–8). Despite significant changes in BMI, mean alanine aminotransferase (ALT) levels decreased from 175 ± 106 IU / L to 40 ± 26 IU / L (P<0.01), and mean aspartate aminotransferase (AST) levels decreased from 104 ± 61 IU / L to 33 ± 11 IU / L (P<0.002). Patients who chose to discontinue vitamin E therapy experienced increases in liver aminotransferases. A study in adults using vitamin E for one year demonstrated similar reductions in liver aminotransferase and fibrosis marker TGFβ levels (Hasegawa et al., Aliment Pharmacol Ther 2001, 15(10):1667-72).

[0135] Fatty liver can also develop into steatohepatitis through oxidative stress caused by reactive oxygen species (ROS) and a decline in antioxidant defenses (Sanyal et al., Gastroenterology 2001, 120(5):1183-92). ROS are generated in the liver through several pathways, including mitochondria, peroxisomes, cytochrome P450, NADPH oxidase, and lipoxygenase (Sanyal et al., Nat Clin Pract Gastroenterol Hepatol, 2005; 2(1):46-53). Insulin resistance and hyperinsulinism have been shown to increase hepatic oxidative stress and lipid peroxidation through increased hepatic CYP2EI activity (Robertson et al., Am J Physiol Gastrointest Liver Physiol, 2001 281(5):G 1135-9; Leclercq et al., J Clin Invest 2000, 105(8):1067-75).

[0136] Currently, much of our understanding of the pathogenesis of NAFLD comes from animal studies. Numerous mouse models exhibiting fatty liver / steatohepatitis exist, including genetically engineered leptin-deficient (ob / ob) or leptin-resistant (db / db) models and dietary methionine / choline deficiency (MCD) models. Studies have compared various strains of female and male rats (Wistar, Sprague-Dawley, and Long-Evans) with mouse strains (C57BL / 6) as models of NASH. These animals were fed an MCD diet for 4 weeks. While elevated ALT and hepatic steatosis were more pronounced in Wistar rats, the overall histological changes in the livers of mice were more consistent with NASH. More recently, the use of super-nutritional diets in animals has resulted in NAFLD models that physiologically resemble the human phenotype. The pathologies most commonly associated with NAFLD are obesity, type 2 diabetes, and dyslipidemia. These symptoms can be induced in mice and rats by feeding a high-fat or sucrose diet. Rats fed a diet rich in >70% fat for 3 weeks developed pancytopenia, patchy inflammation, increased oxidative stress, and increased plasma insulin levels suggestive of insulin resistance. NASH mice were induced by intragastric overfeeding. Mice were fed up to 85% above their standard intake for 9 weeks. The mice became obese, with a final weight gain of 71%, and exhibited increased white adipose tissue, hyperglycemia, hyperinsulinemia, hyperleptinemia, glucose intolerance, and insulin resistance. 46% of these mice developed elevated ALT (121 = / - 27 vs. 13 + / - 1 U / L) and histological features suggestive of NASH. The livers of overfed mice were approximately twice the expected size. This was consistent with microscopic evidence of lipid droplets, cytoplasmic vacuoles, and clusters of inflammation.

[0137] Mouse models of NASH can be used to study various treatments. Mouse models are generated by a specific diet (methionine choline deficiency, MCD) or intragastric hyperphagia. These mice develop serological and histological characteristics of NASH. NASH mice are useful for screening and measuring the effects of cysteamine on NASH-related diseases and disorders. For example, the effects of the treatment can be measured by dividing the NASH mice into a control group that continues to receive only the MCD diet and three other treatment groups that receive the MCD diet and antioxidant therapy. For example, the three treatment groups can receive 50 mg / kg / day of cysteamine, 100 mg / kg / day of sAME, and sAME.

[0138] As mentioned above, NASH is a disease subset within the category of NAFLD, and various biomarkers and histological tests have characterized two types of NASH, including type 1 and type 2. These have some distinct biomarker and histological characteristics, but there are also other features that overlap between the two types. These two types, type 1 and type 2 NASH, are typically identified in younger patients.

[0139] Type 1 NASH is characterized by hepatic steatosis, lobular inflammation, balloon degeneration, and perisinusoidal fibrosis. Type 2 NASH is characterized by hepatic steatosis, portal venous inflammation, and portal vein fibrosis. Schwimmer et al. described various criteria and biomarkers used to distinguish type 1 NASH from type 2 NASH (Hepatology, 42(3):641-649, 2005; incorporated herein by reference). In particular, Schwimmer et al. disclosed that subjects with type 1 NASH have higher levels of AST, ALT, and triglycerides compared with patients with type 2 NASH. The most compelling difference between the two types of NASH is best found on histological examination. As noted above, type 1 NASH exhibits widespread lobular inflammation in the liver, in contrast to the widespread portal venous inflammation in type 2 NASH. Thus, the present disclosure contemplates that one of the important discriminators that can be used in the methods disclosed herein is distinguishing the presence of Type 1 versus Type 2 NASH by histological examination.

[0140] A diagnosis of fatty liver is usually made when lipid deposition is present in 5% or more of hepatocytes. NASH is diagnosed when both inflammatory infiltrates, ballooning, and hepatocellular injury are present in addition to hepatic steatosis. The NAFLD Activity Score (NAS) was developed to provide a numerical score for patients most likely to have NASH. The NAS is the sum of separate scores for hepatic steatosis (0-3), hepatocyte ballooning (0-2), and lobular inflammation (0-3). The majority of NASH patients have an NAS score of 5 or higher (Kleiner DE, Brunt EM, Van Natta M, et al., Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41(6), 1313-1321 (2005)).

[0141] Various studies have also shown that cytokeratin 18, released from apoptotic hepatocytes, is a useful indicator of inflammation in NASH. Normal cytokeratin 18 levels are typically characterized by levels below 200 units per liter. In contrast, subjects with liver diseases, including NALFD and NASH, have statistically significant elevations of cytokeratin 18 (e.g., 200-300 U / L). Furthermore, cytokeratin 18 levels can be used as a marker to determine whether a treatment is effective. For example, a reduction in cytokeratin 18 levels of more than 10% (e.g., 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 90-100%) indicates a beneficial effect of treatment. Other markers include, for example, commonly used tests of liver function including measurement of one or more of serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and gamma-glutamyl transpeptidase (GGT).

[0142] The effectiveness of the disclosed methods or compositions can be assessed by measuring fatty acid content and metabolism in the liver. Dosage adjustments and treatments can be performed by a specialist, for example, depending on the severity of NAFL.

[0143] In patients with histologically proven NAFLD, serum liver aminotransferase levels, particularly alanine aminotransferase (ALT), are elevated 10-fold above the upper limit of normal (Schwimmer et al., J Pediatr 2003, 143(4):500-5; Rashid et al., J Pediatr Gastroenterol Nutr 2000, 30(1):48-53). The ALT / AST (aspartate aminotransferase) ratio is >1 (range 1.5-1.7), which differs from alcoholic steatohepatitis, where the ALT / AST ratio is typically <1. Other abnormal serological tests that may be abnormally elevated in NASH include γ-glutamyltransferase (γ-GT) and fasting plasma insulin, cholesterol, and triglyceride levels.

[0144] ALT levels have been shown to indicate liver function. For example, normal ALT levels, approximately 7-55 units per liter (e.g., 10-40 units per liter), have been shown to be associated with normal liver function. This value varies somewhat during childhood and adolescence. Thus, in some cases, an ALT level of less than 25 units per liter is considered "normal" during childhood and adolescence. Elevated ALT levels have been shown to be associated with liver disease and injury. For example, subjects with NAFLD and NASH typically exhibit ALT levels of 60-150 (e.g., 60-145, 70-140, 80-135, 90-130, 105-125, 110-120, or any number between any two of these values). In some embodiments, ALT levels greater than 25 units per liter may be indicative of NASH or NAFLD, particularly during childhood and adolescence. When determining whether a subject has NAFLD or NASH or is susceptible to treatment with the compounds of the present disclosure, ALT can be measured alone, but preferably, this measurement should be performed in combination with one or more other markers of liver function or dysfunction.For example, a subject with an ALT level greater than about 80 indicates liver disease or dysfunction.

[0145] AST levels have been shown to indicate liver function. For example, AST levels of approximately 8-48 units per liter (e.g., 10-40 units) have been shown to be associated with normal liver function. Elevated AST levels have been shown to be associated with liver disease and dysfunction. For example, subjects with NAFLD and NASH typically exhibit AST levels of 40-100 (e.g., 45-95, 55-90, 65-85, 70-80, or any number between any two of these values). While AST can be measured alone when determining whether a subject has NAFLD or NASH or would be susceptible to treatment with a deuterated cysteamine or cystamine composition, it is preferable to measure AST in combination with one or more other markers of liver function or dysfunction. For example, a subject with an AST level greater than approximately 50 indicates liver disease or dysfunction.

[0146] ALP levels have been shown to indicate liver function. For example, ALP levels of approximately 45-115 units per liter (e.g., 50-110 units) have been shown to be associated with normal liver function. Elevated ALP levels have been shown to be associated with liver disease and injury. For example, subjects with NAFLD and NASH typically exhibit ALP levels of 150-250 (e.g., 155-245, 160-240, 165-235, 170-230, 175-225, 180-220, 185-215, 190-210, 195-200, or any number between any two values). While ALP can be measured alone when determining whether a subject has NAFLD or NASH or is susceptible to treatment with a deuterated cysteamine or cystamine composition, it is preferable to measure ALP in combination with one or more other markers of liver function or dysfunction. For example, a subject with an ALP level above about 150 indicates liver disease or dysfunction.

[0147] GGT levels have been shown to indicate liver function. For example, GGT levels of approximately 9-48 units per liter (e.g., 10-40 units per liter) have been shown to be associated with normal liver function. Elevated GGT levels have been shown to be associated with liver disease and dysfunction. For example, subjects with NAFLD and NASH typically exhibit GGT levels of 50-100 (e.g., 55-95, 60-90, 65-85, 70-80, or any number between any two of these values). While GGT can be measured alone when determining whether a subject has NAFLD or NASH or would be susceptible to treatment with a deuterated cysteamine or cystamine composition, it is preferable to measure it in combination with one or more other markers of liver function or dysfunction. For example, a subject with a GGT level greater than approximately 50 indicates liver disease or dysfunction.

[0148] Triglyceride levels have been shown to indicate liver function. For example, triglyceride levels below about 150 mg / dL (e.g., 100-150 mg / dL) have been shown to be associated with normal liver function. Elevated triglyceride levels have been shown to be associated with liver disease and dysfunction. For example, subjects with NAFLD and NASH typically exhibit triglyceride levels of 150-200 (e.g., 155-195, 160-190, 165-185, 170-180, or any number between any two of these values). While triglycerides can be measured alone when determining whether a subject has NAFLD or NASH or is susceptible to treatment with a deuterated cysteamine or cystamine composition, this measurement should preferably be performed in combination with one or more other markers of liver function or dysfunction. For example, a subject with a triglyceride level above about 150 mg / dL indicates liver disease or dysfunction.

[0149] High triglyceride levels are known to be a major cause of various forms of inflammation. Triglycerides are the form in which fat travels through the bloodstream. Triglycerides are metabolized by various organs, including the liver, to form phospholipids (LDL and HDL), cholesterol, and oxidized forms. Oxidized phospholipids (OxPL), including oxLDL, are known as inflammatory mediators and are strongly associated with cardiovascular disease. For example, Bieghs et al. reported that the use of antibodies against oxLDL reduced liver inflammation (Hepatology, 65(3):894-903, 2012).

[0150] Increased adipose tissue mass is associated with decreased adiponectin production. Data from mouse and human studies increasingly suggest that adiponectin deficiency is a major factor in the development of fatty liver and steatohepatitis. Adiponectin circulates in serum as trimers (low molecular weight adiponectin), hexamers (medium molecular weight adiponectin), and higher-order multimers (high molecular weight adiponectin), and isotope-specific effects have been demonstrated. Epidemiological studies have shown that decreased adiponectin levels are associated with NASH. Furthermore, adiponectin is thought to have hepatoprotective properties due to its protective effect against oxidative damage. Normal adiponectin levels vary with age and gender. For example, women have higher baseline adiponectin levels compared to men. Normal-weight women typically have adiponectin levels of approximately 8.5–11 μg / ml, while men typically have adiponectin levels of approximately 6–8 μg / ml. In contrast, subjects with fatty liver disease, NASH, and / or obesity have adiponectin levels that are approximately 50-90% of normal levels (e.g., a 10-50% decrease from normal, or any value therebetween) (see, e.g., Merl et al., Int. J. Obes (Lond), 29(8), 998-1001, 2005).

[0151] In contrast, resistin protein is elevated in subjects with NASH compared with normal subjects. Human resistin is a cysteine-rich, 108-amino acid peptide hormone with a molecular weight of 12.5 kDa. In adults, resistin is expressed in the bone marrow. Furthermore, resistin mRNA is barely detectable in adipocytes from subjects with low BMI or healthy subjects. Consistent with this serum resistin concentration, women may have higher resistin concentrations than men. Resistin mRNA expression in human peripheral mononuclear cells is increased by proinflammatory cytokines. Serum resistin is significantly elevated in both NASH and simple steatosis subjects. Hepatic resistin is significantly elevated at both the mRNA and protein levels in NASH patients compared with subjects with simple steatosis and normal control subjects. Because resistin's structure is rich in cysteine, changes in sulfur availability (mainly from cysteine ​​and glutathione) can affect the protein's structure and function. As noted above, cysteamine and cystamine can regulate cysteine ​​and / or glutathione levels in subjects taking cysteamine or cystamine.

[0152] Subjects with NAFLD or NASH tend to be in the higher weight percentiles for their age group (e.g., above the 97th percentile for BMI for their age group). Early treatment of pediatric patients can provide lifelong benefits in liver function and obesity management.

[0153] The compositions and methods of the present disclosure demonstrate that deuterated compositions of the present disclosure reduce liver fibrosis and improve liver function markers in animal models of NAFLD. For example, data demonstrated that animal models treated with a high-fat NASH diet developed nonalcoholic steatohepatitis accompanied by liver fibrosis, and that administration of a deuterated compound during the induction of fatty liver disease reduced the risk of developing NASH and its markers. In further studies, the present disclosure demonstrated that administration of a deuterated compound of the present disclosure following treatment to induce fatty liver disease resulted in an improvement in ALT markers of liver function and a reduction in inflammatory infiltration, liver fibrosis, and fibrosis markers such as collagen 1 and TIMPs. Thus, the present disclosure demonstrates that deuterated cystamine and / or cysteamine can be used to prevent and / or treat NAFLD, NASH, and liver fibrosis resulting from these diseases.

[0154] The present disclosure provides a population of subjects with NASH likely to respond to treatment with deuterated cysteamine or cystamine compositions. The present disclosure also provides a method of treating a subject suffering from a fatty liver disease, such as NASH, comprising administering a therapeutically effective amount of a compound of the present disclosure. In one embodiment, the fatty liver disease is selected from the group consisting of nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), fatty liver disease due to hepatitis, fatty liver disease due to obesity, fatty liver disease due to diabetes, fatty liver disease due to insulin resistance, fatty liver disease due to hypertriglyceridemia, abetalipoproteinemia, glycogen storage disease, Weber-Christian disease, Wolman disease, acute fatty liver of pregnancy, and lipodystrophy.

[0155] In certain embodiments, pediatric and adolescent patients are treated with deuterated compounds of the present disclosure. In one embodiment, a subject suffering from NASH, biliary cholangitis, biliary atresia, etc., is orally administered a formulation containing a deuterated compound and / or prodrug of the present disclosure in an amount sufficient to achieve about 10-200 μmol in plasma (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or any value therebetween). In a further embodiment, the formulation is a delayed-release oral formulation. In one embodiment, the dose is about 10-95 mg / kg. In another embodiment, the dose is about 100 mg-1 g administered two to four times daily. Typically, the dose is adjusted over time to achieve the subject's maximum tolerated dose, usually about 30-200 μmol of compound in plasma. For example, an initial dose can provide a circulating level of about 10 μmol of compound, which is then adjusted to the maximum tolerated dose. In certain embodiments of any of the foregoing, subjects under 15 years of age (e.g., under 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year of age) and with a body mass index (BMI) above the 97th percentile for their age are treated with a compound of the present disclosure. In some embodiments, the subjects have a BMI above the 97th percentile for their age and weigh less than 65 kg. In some embodiments, these same subjects have high triglyceride levels, low LDH, and low or low normal adiponectin levels. In yet another embodiment, the subjects have high or high normal resistin levels. In various embodiments of any of the foregoing, the patient weighs less than 65 kg. In various embodiments, the patient weighs about 35-65 kg, or about 40-60 kg, or about 45-55 kg, or about 35, 40, 45, 50, 55, 60, or 65 kg. In various embodiments, a patient weighing less than 65 kg is administered 600-1200 mg / day of a deuterated compound of the disclosure, or an amount that results in a circulating plasma level of about 10-200 μmol of the compound (usually about 30-80 μmol, more usually about 40 μmol).In any of the foregoing embodiments, the subject has Type 1 NASH or Type 1 histological pattern of NASH. In yet another embodiment of the foregoing, the subject has lobular inflammation of the liver. In a further embodiment of any of the foregoing, the subject has low adiponectin and high triglycerides, which are characteristic of NASH. In yet another embodiment of any of the foregoing, the subject has levels of markers consistent with NASH (e.g., AST, ALT, GGT, or other liver markers) described herein.

[0156] In various embodiments, the patient weighs between 65 and 80 kg and may be administered 500 to about 2000 mg / day of a compound of the present disclosure, or an amount that results in a circulating plasma level of the compound of between about 10 and 200 μmol (usually about 30 to 80 μmol, more typically about 40 μmol). In any of the foregoing embodiments, the subject is suffering from Type 1 NASH. In yet another embodiment of the above, the subject is suffering from liver lobular inflammation. In any further embodiment of the above, the subject has low adiponectin and elevated triglycerides, which are characteristic of NASH.

[0157] In various embodiments, the patient weighs more than 65 kg and is administered 900 to about 2000 mg / day of a compound of the present disclosure, or an amount that results in a circulating plasma level of about 10 to 200 μmol of deuterated compound (usually about 30 to 50 μmol, more typically about 40 μmol). In any of the foregoing embodiments, the subject is suffering from Type 1 NASH. In yet another embodiment of the above, the subject is suffering from liver lobular inflammation. In any further embodiment of the above, the subject has low adiponectin and elevated triglycerides, which are characteristic of NASH.

[0158] The subject can be an adult, an adolescent, or a child. In various embodiments, the patient is 2-7 years old, 8-11 years old, 9-12 years old, or 13-18 years old. In various embodiments, the adolescent is 10-19 years old, as defined by the National Institutes of Health.

[0159] In various embodiments, the administration results in a reduction of 2 or more points in the NAFLD activity score, no worsening of fibrosis or an improvement in fibrosis, a reduction in serum aminotransferases and gamma-glutamyl transpeptidase (GGT); a reduction in liver fat fraction measured by MRI; a change in markers of oxidative and antioxidant status; a change in fasting insulin and glucose; an increase in circulating adiponectin levels; a reduction in circulating resistin levels; a reduction in triglyceride levels; a reduction in oxidized phospholipids; a change in weight, height, body mass index (BMI), and waist circumference; a change in pediatric quality of life score; a change in any symptoms the patient may be experiencing; the rate of change from histologically diagnosed definite NASH or indeterminate NASH to non-NASH at the end of treatment; individual histological features at the end of treatment compared to baseline, such as steatosis (fatty liver), lobular inflammation, portal vein chronic inflammation, ballooning, fibrosis score, and stage 1a and 1b fibrosis; and a change in mean NAS.

[0160] In various embodiments of the present disclosure, the deuterated compounds of the present disclosure are administered at a daily dose ranging from about 10 mg / kg to about 2.5 mg / kg, or from about 100 mg / kg to about 250 mg / kg, or from about 60 mg / kg to about 100 mg / kg, or from about 50 mg / kg to about 90 mg / kg, or from about 30 mg / kg to about 80 mg / kg, or from about 20 mg / kg to about 60 mg / kg, or from about 10 mg / kg to about 50 mg / kg. Additionally, the effective doses are 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, 250 mg / kg, 275 mg / kg, 300 mg / kg, 325 mg / kg, 350 mg / kg, 375 mg / kg, 400 mg / kg, 425 mg / kg, 450 mg / kg, 475 mg / kg, 500 mg / kg, 525 mg / kg, 550 In some embodiments, the deuterated compounds of the present disclosure may be at or below about 0.25 mg / kg, 575 mg / kg, 600 mg / kg, 625 mg / kg, 650 mg / kg, 675 mg / kg, 700 mg / kg, 725 mg / kg, 750 mg / kg, 775 mg / kg, 800 mg / kg, 825 mg / kg, 850 mg / kg, 875 mg / kg, 900 mg / kg, 925 mg / kg, 950 mg / kg, 975 mg / kg, or 1000 mg / kg, or a range between any two of the foregoing values. In some embodiments, the deuterated compounds of the present disclosure may be at or below about 0.25 mg / kg 2 ~4.0 g / m 2 Body surface area, approximately 0.5~2.0 g / m 2 body surface area, or 1–1.5 g / m 2 body surface area, or 1–1.95 g / m 2 body surface area, or 0.5–1 g / m 2 body surface area, or approximately 0.7–0.8 g / m 2body surface area, or approximately 1.35 g / m 2 Body surface area, or approximately 1.3 to 1.95 g / m 2 / day, or about 0.5 to about 1.5 g / m 2 / day, or about 0.5 to about 1.0 g / m 2 / day, e.g., at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 g / m 2 , or up to approximately 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 3.25, 3.5, or 3.75 g / m 2 or a total daily dose ranging between any two of these values.

[0161] In some embodiments, the delayed-release and sustained-release formulations comprise an enteric coating that releases the compounds disclosed herein when they reach a region of a subject's small intestine or gastrointestinal tract where the pH is above 4.5. In various embodiments, the formulations release at about pH 4.5-6.5, 4.5-5.5, 5.5-6.5, or at about pH 4.5, 5.0, 5.5, 6.0, or 6.5.

[0162] In certain embodiments, the compounds of the present disclosure or their pharmaceutically acceptable salts, prodrugs, or solvates are formulated for oral administration (e.g., as capsules, tablets, caplets, solutions, etc.). In further embodiments, the present disclosure provides capsules, tablets, or caplets containing 50-200 mg of the compounds of the present disclosure or their pharmaceutically acceptable salts (e.g., bitartrates), prodrugs, or solvates. In yet other embodiments, the capsules, tablets, or caplets further comprise inactive ingredients such as colloidal silicon dioxide, croscarmellose sodium, D&C Yellow No. 10 Aluminum Lake, FD&C Blue No. 1 Aluminum Lake, FD&C Blue No. 2 Aluminum Lake, FD&C Red No. 40 Aluminum Lake, gelatin, magnesium stearate, microcrystalline cellulose, pharmaceutical glaze, pregelatinized starch, silicon dioxide, sodium lauryl sulfate, synthetic black iron oxide, and / or titanium dioxide.

[0163] In yet another embodiment, the compounds disclosed herein are administered no more than four times per day (e.g., once, twice, or three times per day). In various embodiments, the compositions are in delayed- or controlled-release dosage forms that increase delivery of the compounds disclosed herein to the small intestine.

[0164] In one embodiment, a compound of the present disclosure, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, is formulated for oral administration (e.g., as a capsule, tablet, caplet, solution, etc.) to provide delayed release. In a further embodiment, the present disclosure provides a delayed-release capsule, tablet, or caplet comprising 25-75 mg of a deuterated compound of the present disclosure, or a pharmaceutically acceptable salt (e.g., bitartrate), prodrug, or solvate thereof. In yet another embodiment, the delayed-release capsule, tablet, or caplet further comprises inactive ingredients such as microcrystalline cellulose, Eudragit® L 30 D-55, hypromellose, talc, triethyl citrate, sodium lauryl sulfate, purified water, gelatin, titanium dioxide, blue ink, and / or white ink.

[0165] The delayed or controlled release form may be a dose of C provided by an immediate release dosage form containing the same amount of compound. max at least about 35%, 50%, 75%, or more of the C of the compounds disclosed herein or their biologically active metabolites max In another embodiment, the delayed-release and sustained-release dosage forms provide an improved AUC compared to an immediate-release dosage form of the compound. For example, the AUC is increased compared to an immediate-release formulation. In yet another embodiment, the delayed-release or controlled-release dosage form includes an enteric coating that releases the compound disclosed herein when the compound reaches a region of the subject's small intestine or gastrointestinal tract where the pH exceeds 4.5. In various embodiments, the pH is between 4.5 and 6.5. In one embodiment, the pH is about 5.5 and 6.5. In one embodiment, the compound of the present disclosure is delivered throughout the small intestine and sustained-released within the small intestine.

[0166] The delayed or controlled release form is provided by enteric coated, non-deuterated cystamine and / or cysteamine (e.g., Procysbi®) dosage forms containing the same amount of cysteamine and / or cystamine base. max C of the compounds disclosed herein or their biologically active metabolites at least about 10%, 20%, 30%, or more than maxIn another embodiment, delayed-release and sustained-release dosage forms comprising the compounds disclosed herein provide improved AUC compared to approved cysteamine formulations. For example, the AUC of the disclosed compounds is increased compared to approved cysteamine formulations. In yet another embodiment, delayed-release or controlled-release dosage forms comprising the disclosed compounds include an enteric coating that releases the disclosed compounds when the compounds reach a region of the subject's small intestine or gastrointestinal tract where the pH exceeds 4.5. In various embodiments, the pH is between 4.5 and 6.5. In one embodiment, the pH is about 5.5 and 6.5. In one embodiment, the disclosed compounds are delivered throughout the small intestine and sustained-released within the small intestine.

[0167] In various embodiments, enteric-coated formulations containing the compounds of the present disclosure are granulated, and the granules are compressed into tablets or filled into capsules. In certain embodiments, the granules are enteric-coated before being compressed into tablets or capsules. The capsule material may be either hard or soft and is typically sealed with a gelatin band or the like. Tablets and capsules for oral use will generally contain one or more commonly used excipients, such as those described herein.

[0168] Suitable pH-sensitive polymers are those that dissolve in the intestinal environment at higher pH levels (above pH 4.5), such as in the small intestine, thereby enabling release of the pharmacologically active agent in regions of the small intestine rather than in the upper gastrointestinal tract, such as the stomach.

[0169] In various embodiments, exemplary formulations comprising the deuterated compounds of the present disclosure contemplated for use in the methods of the present invention include those described in International Patent Applications PCT / US2007 / 002325, PCT / US2014 / 042607, and PCT / US2014 / 042616, the disclosures of which are incorporated herein by reference.

[0170] For administration of a dosage form, i.e., a tablet or capsule containing an enterically coated compound of the present disclosure, a total weight of about 50 to 1500 mg is used. In various embodiments, the tablet or capsule contains 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 mg of a compound of the present disclosure as the active ingredient, with multiple tablets or capsules being administered to reach the desired dose. The dosage form is orally administered to a subject in need thereof.

[0171] In one embodiment, the tablet core comprises about 50-500 mg of a compound of the present disclosure encapsulated in an enteric coating material having a thickness of about 60-100 μm (e.g., about 71, 73, 75, 77, or 79 μm, or any value therebetween) and / or about 10-13% by weight of the tablet (e.g., about 10.5, 11.0, 11.2, 11.4, 11.6, 11.8, 12.0, 12.2, 12.4, 12.6, 12.8%, or any value therebetween). In another embodiment, the tablet core comprises about 100-100 mg of a compound of the present disclosure encapsulated in an enteric coating material having a thickness of about 90-130 μm (e.g., about 97, 99, 101, 103, 105, 107, 109, 111, 113 μm, or any value therebetween) and / or about 9-14% by weight of the tablet (e.g., about 9.5, 9.7, 9.9, 10.1, 10.3, 10.5, 11.0, 11.2, 11.4, 11.6, 11.8, 12.0, 12.2, 12.4, 12.6, 12.8, 13.0, 13.2, 13.4, 13.6, 13.8%, or any value therebetween).

[0172] In any of the foregoing embodiments, the enteric coating material can be selected from the group comprising polymerized gelatin, shellac, methacrylic acid copolymer type C NF, cellulose butyrate phthalate, cellulose hydrogen phthalate, cellulose phthalate propionate, polyvinyl acetate phthalate (PVAP), cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate, dioxypropyl methylcellulose succinate, carboxymethylethylcellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and acrylic acid polymers and copolymers typically formed from methyl acrylate, ethyl acrylate, methyl methacrylate, and / or copolymers of ethyl methacrylate with acrylic and methacrylic acid esters.

[0173] The composition can be administered orally or parenterally. In another embodiment, the method results in an improvement in liver fibrosis compared to levels prior to administration of the disclosed compound. In yet another embodiment, the method results in a reduction in liver fat content, a reduction in the incidence or progression of cirrhosis, or a decrease in the incidence of hepatocellular carcinoma. In one embodiment, the method results in a reduction in liver aminotransferase levels compared to levels prior to administration of the disclosed deuterated compound. In a further embodiment, the administration reduces liver aminotransferase levels by about 10% to 70%, e.g., 10, 20, 30, 40, 50, 60, or 70%, compared to pre-treatment levels, or any value therebetween. In yet another embodiment, the administration reduces alanine or aspartate aminotransferase levels in the treated patient by about 50%, 40%, 30%, 20%, or 10% or more below normal ALT levels, or to normal ALT levels. In yet other embodiments, the administration reduces serum ferritin levels compared to levels prior to treatment with a compound of the present disclosure. In various embodiments, the administration reduces the NAS score.

[0174] In any of the foregoing embodiments, formulations for use in the methods described herein can include a pharmaceutically acceptable salt of a compound of the present disclosure, e.g., chloride or bitartrate salt, in place of the free base compound.

[0175] The methods and compositions of the present disclosure can also include administering a second agent in combination with a compound of the present disclosure to treat a disease or disorder. Thus, in another embodiment of any of the above methods or compositions, a subject can be treated with a combination of an active agent for treating cystinosis or fatty liver disease, such as NAFLD and NASH. The combination includes a compound of the present disclosure and one or more of metformin, a statin, an antioxidant, and / or an antibody against oxidized phospholipids. Such combinations may result in unexpected synergistic effects through a multifaceted approach to modulating inflammation and inflammatory mediators. Such combinations may enhance the antioxidant effects of adiponectin by increasing adiponectin levels, reduce triglyceride levels, thereby reducing circulating phospholipids, reducing insulin resistance, and blocking the pro-inflammatory effects of oxidized phospholipids.

[0176] While the present disclosure has been described in connection with specific embodiments thereof, it is to be understood that the foregoing description and the following examples are illustrative, but not limiting, of the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure is directed. [Example]

[0177] Chemical synthesis of novel prodrugs of cysteamine and cysteine. S-(2-((tert-butoxycarbonyl)amino)ethyl)-(S)-2-((tert-butoxycarbonyl)amino)-3-((4-methoxybenzyl)thio)propanethioate JPEG2025502611000057.jpg63139

[0178] To a solution of N-(tert-butoxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine ​​(6.00 g, 1.00 equiv., 17.6 mmol) in THF (65.00 mL) cooled to 0 °C, DCC (3.66 g, 1.01 equiv., 17.7 mmol) and chilled DMAP (0.0215 g, 0.0100 equiv., 0.176 mmol) were added, and the resulting mixture was stirred for 1 h. tert-Butyl (2-mercaptoethyl)carbamate (3.43 g, 1.1 equiv., 19.3 mmol) was then added at room temperature, and stirring was continued for an additional 3 h. The reaction was then filtered and concentrated. Purification by silica gel chromatography (30% EtOAc in hexanes) afforded the title compound (7.02 g, 14.0 mmol, 79.8%) as a white solid. 1 H NMR (600 MHz, chloroform-d) δ 7.21 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 5.30 (d, J = 8.5 Hz, 1H), 4.85 (s, 1H), 4.55-4.44 (m, 1H), 3.78 (s, 3H), 3.67-3.61 (m, 2H), 3.27 (t, J = 6.7 Hz, 2H), 3.00 (t, J = 7.0 Hz, 2H), 2.88-2.74 (m, 2H), 1.46 (s, 9H), 1.41 (s, 9H) ppm. C 23 H 36 N2O6S2[M + Na] + The calculated HRMS (ES+) for was 523.1907 and the observed value was 523.1902. S-(2-((tert-butoxycarbonyl)amino)ethyl)-(S)-2-((tert-butoxycarbonyl)amino)-3-((3-nitropyridin-2-yl)disulfanyl)propanethioate JPEG2025502611000058.jpg64144

[0179] At 0 °C, 3-nitropyridin-2-yl hypochlorothioite (0.476 g, 2.50 equiv., 2.50 mmol) was added to a solution of S-(2-((tert-butoxycarbonyl)amino)ethyl)(S)-2-((tert-butoxycarbonyl)amino)-3-((4-methoxybenzyl)thio)propanethioate (0.500 g, 1.00 equiv., 0.999 mmol) in DCM (20 mL). The resulting mixture was stirred for 2 h. After complete conversion of the starting material was determined by TLC, the solvent was evaporated in vacuo. Purification by silica gel chromatography (DCM, 30% EtOAc in hexanes) afforded the title compound as a yellow solid (0.254 g, 0.475 mmol, 47.6%). 1 H NMR (600 MHz, chloroform-d) δ 8.95 (d, J = 4.6 Hz, 1H), 8.53 (d, J = 8.2 Hz, 1H), 7.40 (dd, J = 8.3, 4.6 Hz, 1H), 7.08 (d, J = 7.9 Hz, 1H), 4.87 (s, 1H), 4.57 (q, J = 6.1 Hz, 1H), 3.57 (dd, J = 14.0, 6.0 Hz, 1H), 3.23 (s, 3H), 3.13 (dd, J = 14.0, 4.6 Hz, 1H), 2.99-2.92 (m, 3H), 1.46 (s, 9H), 1.39 (s, 9H) ppm. C 20 H 30 N4O7S3[M + Na] + The calculated HRMS (ES+) for was 557.1169 and the observed value was 557.1164. S-(2-((tert-butoxycarbonyl)amino)ethyl) (S)-2-((tert-butoxycarbonyl)amino)-3-((2-((tert-butoxycarbonyl)amino)ethyl)disulfanyl)propanethioate JPEG2025502611000059.jpg66151

[0180] To a solution of S-(2-((tert-butoxycarbonyl)amino)ethyl)(S)-2-((tert-butoxycarbonyl)amino)-3-((3-nitropyridin-2-yl)disulfanyl)propanethioate in CAN (0.325 mL) and water (425 mL) acidified to pH 6 with acetic acid was added tert-butyl (2-mercaptoethyl)carbamate (0.040 g, 3 equiv., 0.22 mmol) at room temperature, and the resulting mixture was stirred for 4 h. The reaction mixture was then diluted with water and extracted with EtOAc (3×). The combined organic layers were washed with a saturated aqueous solution of NaCl, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. Purification by silica gel chromatography (30% EtOAc in hexanes) afforded the title compound (0.031 g, 0.056 mmol, 75%) as a white solid. 1 H NMR (600 MHz, chloroform-d) δ 5.43 (d, J = 8.5 Hz, 1H), 5.04 (s, 1H), 4.85 (s, 1H), 4.64 (q, J = 7.4, 6.8 Hz, 1H), 3.44 (d, J = 8.5 Hz, 2H), 3.31 (d, J = 6.6 Hz, 2H), 3.13 (d, J = 6.0 Hz, 2H), 3.04 (tq, J = 13.6, 6.6 Hz, 2H), 2.86-2.77 (m, 2H), 1.49-1.41 (m, 27H) ppm. C 22 H 41 N3O7S3[M + Na] + The calculated HRMS (ES+) for was 578.1999 and the observed value was 578.2005. (S)-3-((2-ammonioethyl)disulfanyl)-1-((2-ammonioethyl)thio)-1-oxopropane-2-aminium chloride (BL-0856) JPEG2025502611000060.jpg54123

[0181] S-(2-((tert-butoxycarbonyl)amino)ethyl)-(S)-2-((tert-butoxycarbonyl)amino)-3-((2-((tert-butoxycarbonyl)amino)ethyl)disulfanyl)propanethioate (25 mg, 1 equiv., 0.045 mmol) was dissolved in MeOH (0.600 mL) and a solution of HCl in dioxane (4 M, 0.300 mL, 27 equiv., 1.2 mmol), and the resulting mixture was stirred at room temperature for 3 h. The solvent was then evaporated in vacuo to give the title compound as a white solid (16 mg, 0.044 mmol, 97%). 1 H NMR (600 MHz, heavy water) δ 3.49 (dd, J = 15.3, 4.8 Hz, 1H), 3.44 (t, J = 6.6 Hz, 2H), 3.39 (q, J = 6.5, 6.0 Hz, 2H), 3.36-3.32 (m, 1H), 3.32-3.21 (m, 2H), 3.15-3.02 (m, 2H) ppm. 13 C NMR (150 MHz, heavy water) δ 196.41, 58.64, 39.24, 38.24, 37.67, 33.86, 26.82 ppm. C7H 18 N3OS3[M + H] + The calculated HRMS (ES+) for was 256.0607 and the found value was 256.0603. JPEG2025502611000061.jpg26115

[0182] To a solution of the thioester (0.110 g, 0.5 mmol, 1 equiv.) in diethyl ether (5 mL) was added dropwise a solution of HCl (0.181 g, 2.49 mL, 4.97 mmol, 2 M, 10 equiv.) in diethyl ether at room temperature. The solution was stirred at this temperature for 16 h and then concentrated. The solid was then triturated with cold diethyl ether and filtered through a sintered glass to give the title compound (0.066 g, 0.42 mmol, 85%) as a white solid. 1H NMR (600 MHz, DO): 3.22 (2H, s), 2.43 (3H, s) ppm. HRMS calculated for C4H7D2NOS: 121.0530, found: 121.0528. JPEG2025502611000062.jpg52147

[0183] To a solution of the thioester (0.378 g, 1.71 mmol, 1 equiv.) in a mixture of MeOH and water (1 / 1, 4 mL) was added K2CO3 (0.472 g, 3.42 mmol, 2 equiv.), and the reaction was stirred at room temperature for 1 hour, then concentrated and extracted twice with EtOAc. The combined organic fractions were washed twice with brine, dried, and concentrated under reduced pressure. This intermediate (0.246 mg, 1.37 mmol, 4 equiv.) was dissolved in dry DCM (4 mL), and triethylamine (0.070 g, 0.096 mL, 0.688 mmol, 2 equiv.) was added. The mixture was cooled to 0 °C, and succinyl dichloride (0.053 g, 0.038 mL, 0.343 mmol, 1 equiv.) was added dropwise. The reaction was stirred at room temperature for 2 h, then concentrated and purified by flash chromatography (hexane / EtOAc: 99 / 1 to 50 / 50) to give the title compound as a clear oil (0.129 g, 0.293 mmol, 65%). 1 H NMR (600 MHz, CDCl3): 4.43 (s, 2H), 3.26 (s, 4H), 2.75 (s, 4H), 1.43 (s, 18H) ppm. JPEG2025502611000063.jpg40156

[0184] At room temperature, a solution of thioester (0.060 g, 0.140 mmol, 1 equiv.) in diethyl ether (4 mL) was added dropwise with a solution of HCl (0.050 g, 0.680 mL, 1.400 mmol, 2 M, 10 equiv.). The solution was stirred at this temperature for 24 hours and concentrated. Then, cold diethyl ether was added to the solid, which was triturated and filtered on a sintered glass to give the title compound as a white solid (0.016 g, 0.051 mmol, 36%). 1H NMR (600 MHz, DO): 3.21 (4H, s), 2.43 (4H, s) ppm. C8H 12 The calculated HRMS for D4N2O2S2 was 240.0904 and the found value was 240.0901. JPEG2025502611000064.jpg42118

[0185] To a solution of the thioester (0.348 g, 1.57 mmol, 1 equiv.) in a mixture of MeOH and water (1 / 1, 4 mL) was added K2CO3 (0.435 g, 3.14 mmol, 2 equiv.), and the reaction was stirred at room temperature for 1 hour, then concentrated and extracted twice with EtOAc. The combined organic fractions were washed twice with brine, dried, and concentrated under reduced pressure. This intermediate (0.307 mg, 1.73 mmol, 2 equiv.) was dissolved in dry DCM (2.5 mL), and triethylamine (0.088 g, 0.121 mL, 0.866 mmol, 1 equiv.) was added. The mixture was cooled to 0 °C, and succinyl dichloride (0.093 g, 0.091 mL, 0.866 mmol, 1 equiv.) was added dropwise. The reaction was stirred at room temperature for 2 h, then concentrated and purified by flash chromatography (hexane / EtOAc: 99 / 1 to 50 / 50) to give the title compound as a clear oil (0.214 g, 0.865 mmol, 99%). 1 H NMR (600 MHz, CDCl3): 4.47 (s, 1H), 3.29 (s, 2H), 2.77-2.72 (m, 1H), 1.43 (s, 9H), 1.19 (d, J = 6 Hz, 6H) ppm. JPEG2025502611000065.jpg24121

[0186] To a solution of the thioester (0.220 g, 0.882 mmol, 1 equiv.) in diethyl ether (4 mL) was added dropwise a solution of HCl (0.161 g, 2.210 mL, 4.410 mmol, 2 M, 5 equiv.) in diethyl ether at room temperature. The solution was stirred at this temperature for 24 h and then concentrated. The solid was then triturated with cold diethyl ether and filtered through a sintered glass to give the title compound (0.075 g, 0.040 mmol, 46%) as a white solid. 1 H NMR (600 MHz, D2O): 3.21 (s, 2H), 2.93-2.96 (m, 1H), 1.19 (d, J = 6 Hz, 6H) ppm. C6H 11 The calculated HRMS for D2NOS was 1490.0843 and the observed value was 149.0842.

[0187] Drug concentration: BL-0856 powder was resuspended in water at a concentration of 25 mM. 40 μL of this solution was added to 10 mL of medium to give a final concentration of 100 μM. When tested one week after resuspension, a portion of the resuspended drug contained cysteamine and cystamine.

[0188] Cystamine was produced by high pH oxidation of 25 mM cysteamine bitartrate powder (w / ammonium hydroxide). During the experiment, approximately 25% of the drug remained as cysteamine, and approximately 75% was oxidized to cystamine. This was considered sufficient for the purpose, as it was only used at some control time points. The drug was also administered to cells at a final concentration of 100 μM.

[0189] Based on the LC-MS profile, the purity of BL-0856 when used in this experiment was likely greater than 80-90% (unhydrolyzed). Methods for the reduced intermediates were also developed, but none existed in neat standards.

[0190] In vitro experiments using prodrugs in cystinotic fibroblasts. Fibroblasts from an anonymized cystinosis patient were split from two plates to 12 plates (triplicates). The following protocol was used to test the experimental compound BL-0856. For comparison, several time points for cystamine were also performed. The time series of constant incubation for the novel compound was 0, 15 min, 30 min, 1 h, 3 h, and 5 h. The time points for cystamine were 0, 30 min, 1 h, and 5 h. A washout experiment was also performed for two time points for the experimental compound. In this experiment, after 3 h of constant incubation, the medium was replaced without compound and incubated for 1 or 3 h. The cells on the plate were harvested as follows: To capture free thiol groups, the cells were washed twice with PBS containing 5 mM N-ethylmaleimide, followed by addition of d4-NEM-cysteamine, d4-cystine, d8-cystamine, and d8-cystamine as internal standards. 13 C 15 N NEM-glutathione, and 13 C. 15 Plates were scraped with an acidic organic solvent extract containing N-GSSG. All samples were run on an API 4500 triple quadrupole LC-MS / MS with selective reaction monitoring (SRM / MRM) for the following compounds of interest: cystine, cysteine, cystamine, reduced glutathione, and oxidized glutathione (GSSG). BL-0856 and thiol reduction intermediates were scanned but were not found at levels high enough to be identified at any time point. This is likely due to the drug's near-instantaneous reduction and / or hydrolysis within the cells. Except where noted, all data fit well to a single exponential curve (decay or reversion).

[0191] Measurement of cystine during constant cystamine or BL-0856 exposure: Fibroblast cultures were exposed to 100 μM cystamine or 100 μM BL-0856 at 0, 10, 30, 1, 3, and 5 h. After incubation, cells were washed twice in PBS containing N-ethylmaleimide. After removing the wash solution, cells were harvested on ice using 80% acetonitrile / 1% formic acid containing a stable isotope internal standard for cystine (d4-cystine). As shown in Figure 2, the difference in cystine depletion rates was similar between cystamine and BL-0856.

[0192] Measurement of cystine after washout: Fibroblast cultures were exposed to 100 μM BL-0856 for 3 hours. After incubation, cells were washed with PBS and then incubated with regular medium (containing FBS) at 0 minutes, 1 hour, and 3 hours. Cells were harvested as described above. The rate of cystine reaccumulation with BL-0856 (see Figure 3) was comparable to that of cystamine (data not shown).

[0193] Measurement of cysteamine and cystamine during constant cystamine or BL-0856 exposure: Fibroblast cultures were exposed to 100 μM cystamine or 100 μM BL-0856 at 0, 10, 30, 1, 3, and 5 h. After incubation, cells were washed twice in PBS containing N-ethylmaleimide. After removing the wash solution, cells were harvested on ice using 80% acetonitrile / 1% formic acid containing a stable isotope internal standard of cystamine or cysteamine. As shown in Figure 4, cystamine levels were higher in the cystamine-treated cells, although some cystamine was still present in the BL-0856-treated cells. Cysteamine levels were nearly identical in both treatments, indicating that the depletion of cysteamine from its disulfide precursor is rapid in both treatments.

[0194] Measurement of glutathione and oxidized glutathione during constant cystamine or BL-0856 exposure: Fibroblast cultures were exposed to 100 μM cystamine or 100 μM BL-0856 at 0, 10, 30, 1, 3, and 5 h. After incubation, cells were washed twice in PBS containing N-ethylmaleimide. After removing the wash solution, cells were harvested on ice using 80% acetonitrile / 1% formic acid containing stable isotope internal standards for glutathione and oxidized glutathione. As shown in Figure 5, there was no apparent difference in glutathione between the two drug forms. Interestingly, both drugs elevated oxidized glutathione (GSSG) at the initial time point, and the GSH / GSSG ratio was lower at all time points compared to the 0-min control.

[0195] Measurement of cysteine ​​during constant cystamine or BL-0856 exposure: Fibroblast cultures were exposed to 100 μM cystamine or 100 μM BL-0856 at 0, 10, 30, 1, 3, and 5 hours. After incubation, cells were washed twice in PBS containing N-ethylmaleimide. After removing the wash solution, cells were harvested on ice using 80% acetonitrile / 1% formic acid containing stable isotope internal standards for glutathione and oxidized glutathione. As shown in Figure 6, cysteine ​​was elevated in BL-0856-treated cells, indicating that cysteine ​​was released from its precursor form.

[0196] Results: BL-0856 is degraded early in cells, as evidenced by the early release of both cysteamine and cysteine. The depletion and reaccumulation kinetics of cystine appear to be similar to that of cystamine. There is no evidence of increased glutathione accumulation, but the levels of glutathione reduction / oxidation decreased after administration of both drugs.

[0197] Cystine depletion test. Dermal cystinotic fibroblasts were cultured in medium containing BL0948, BL0940, or D4-cystamine at the same 100 μM concentration. JPEG2025502611000066.jpg31154

[0198] BL0984 and BL0940 generate one molecule of D2-cysteamine, while D4-cystamine generates two molecules of D2-cysteamine. The fact that BL-940 is less effective than D4-cystamine is expected based on the following: when administered at the same mg / kg dose as D4-cystamine, this molecule would have half the D2-cysteamine in cells treated with BL-940 compared to cells treated with D4-cystamine, assuming complete prodrug activation. BL-948 produced a significant effect, which was unexpected considering that it spontaneously rearranges most of the prodrug into the corresponding, more stable, N-acetylated isomer and therefore is not completely converted to D2-cysteamine. This is interesting because it indicates that N-acetyl-D2-cysteamine is more potent than D2-cysteamine at depleting cystine.

[0199] In the assay, the cells are incubated in 100 μM drug so that sufficient drug is available in both free cysteamine and rearranged isomeric forms.

[0200] BL-0940 D2-cysteamine is a mutual prodrug that binds to a stable N-acetylcysteine. This undeuterated compound may be known.

[0201] This prodrug analog, BL0940, is highly stable both in culture medium and inside cells.

[0202] Intracellular D2-cysteamine levels are measured after continuous drug exposure (in culture medium) to cystinotic fibroblasts (Figure 7).

[0203] Depletion of intracellular cystine over time. The reduction in cystine levels after exposure to D4-cystamine and BL0948 is rapid and pronounced. This likely represents rapid exposure to D2-cysteamine after cellular uptake. D4-cystamine most likely decreases after crossing the plasma membrane (maximum levels seen at 45 min). BL0948 is most likely reduced to D2-cysteamine and its rearranged isomer in the medium before crossing the plasma membrane. After extracellular reduction, the ratio of D2-cysteamine to the rearranged analog can be 10-20:1. This explains why relatively high levels of intracellular D2-cysteamine are observed at baseline, and why a dramatic reduction in cystine levels is observed with BL0948.

[0204] BL0940 may provide a slower but longer-lasting effect due to a potential reduction in the rate of disulfide bond formation in this molecule. Notably, after continuous exposure to BL0940, the intracellular levels of BL0940 and D2-cysteamine increased over time (e.g., at 240 min, Figures 8-9).

[0205] A study using a mouse model of choline-deficient, L-amino acid-defined, and high-fat diet (CDAHFD)-induced nonalcoholic steatohepatitis (NASH).

[0206] Study 1 - NASH / mouse. Mice were fed a CDAHFD diet for at least 8 weeks before the start of the study and continued on this diet throughout the following study phases. Four mice (C57BL / 6, male) were used in each group: A control group (c1) received water via gavage once daily for 2 weeks; group receiving once-daily gavage therapy with 200 mg / kg of drug 0 for 2 weeks; a group receiving once-daily gavage therapy with 200 mg / kg of drug 2 for 2 weeks; A group received 200 mg / kg of drug 4 once daily through a gastric tube for two weeks. Figure 10A shows the study summary. JPEG2025502611000067.jpg70161

[0207] Figures 10B-E show the results of one liver tissue study after 10 weeks of continuous CDAHFD diet, showing red staining of intrahepatic fibrosis. The treatment group also showed a significant reduction in liver aminotransferase ALT compared to the control group (Figure 11). Significant reduction in intrahepatic fibrosis was observed with D2 and D4 compared to the control group. Figures 12A-B further demonstrate a significant reduction in inflammatory markers when comparing D2-cystamine and D4-cystamine compared to the control group. Significant reductions in inflammatory (F4 / 80) and fibrosis (ASMA) markers were observed when comparing D2-cystamine and D4-cystamine compared to the control group (Figures 13A-C).

[0208] CDAHFD NASH Test in Mice 2-Comparison of Two Prodrug Analogues, BL0940 and BL0948

[0209] Mice were fed the CDAHDF diet for at least 8 weeks before the start of the study and continued to receive this diet throughout the following study phases. Eight mice (C57BL / 6, male) were used in each group. Control group Group receiving once-daily gastric tube therapy with HD BL0940 (high dose) for 4 weeks A group that received gastric tube therapy with LD BL0940 (low dose) once a day for 4 weeks A group that received BL0948 once-daily gastric tube therapy for 4 weeks

[0210] BL0940 was administered at a higher dose than BL0948 due to its higher molecular weight, i.e., HD BL0940 and BL0948 had the capacity to release the same number of D2-cysteamine molecules.

[0211] Figures 14-16 show the results of prodrug testing, demonstrating reductions in various markers of inflammation and disease. In studies of NASH mice (using the CDAHFD model), high doses of BL0940 and the same molar doses of BL0948 and D4 appeared to have significant effects on markers of inflammation, remodeling, and fibrosis. However, not surprisingly, low doses of BL0940 (which would release less D2 cysteamine) did not have the same effect in studies of NASH mice.

[0212] It will be understood that various modifications can be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. Formula I: “During the ceremony, R 1 is selected from H or an acetyl group; R 2 teeth, and Selected from: R 3 is arbitrarily substituted (C 1 ~C 6 ) alkyl, optionally substituted cycloalkyl, optionally substituted benzyl, or optionally substituted aryl; R 4 teeth, or Selected from: R 5 is arbitrarily substituted (C 1 ~C 6 ) alkyl, optionally substituted cycloalkyl, optionally substituted benzyl, or optionally substituted aryl; and Y 1 ~Y 16 are each independently selected from H or D, or a pharmaceutically acceptable salt or solvate thereof.

2. Y 1 ~Y 16 2. The compound of claim 1, wherein at least one of the following independently has a deuterium enrichment of about 10% or greater.

3. The compound has the formula II(a): “During the ceremony, Y. 1 ~Y 16 are each independently selected from H or D, or a pharmaceutically acceptable salt or solvate thereof.

4. The compound is 4. The compound of claim 3, which is a compound selected from: or a pharmaceutically acceptable salt or solvate thereof.

5. The compound is 2. The compound of claim 1, which is a compound having the structure:

6. The pharmaceutically acceptable salt is of formula II(b): “During the ceremony, Y 1 ~Y 8 are each independently selected from H or D; and The compound according to claim 3, wherein X is a pharmaceutically acceptable counter ion.

7. 7. The compound of claim 6, wherein the pharmaceutically acceptable counterion is bitartrate or chloride.

8. The compound of claim 6 having the structure:

9. The compound has the formula II(c): “During the ceremony, Y 1 ~Y 8 are each independently selected from H or D; and 2. The compound according to claim 1, wherein Ac represents an acetyl group, or a pharmaceutically acceptable salt or solvate thereof.

10. The compound is 10. The compound of claim 9, which is a compound selected from: or a pharmaceutically acceptable salt or solvate thereof.

11. The pharmaceutically acceptable salt has the formula II(d): “During the ceremony, Y 1 ~Y 8 are each independently selected from H or D; Ac is an acetyl group; and 10. The compound of claim 9, having the structure:

12. 12. The compound of claim 11, wherein the pharmaceutically acceptable counterion is bitartrate or chloride.

13. The compound is 12. The compound of claim 11 having the structure:

14. The compound has formula III(a): “During the ceremony, Y 1 ~Y 4 are each independently selected from H or D; and R 3 is (C 1 ~C 6 2. The compound of claim 1, wherein the compound is selected from the group consisting of:

15. The compound is wherein R 3 is (C 1 ~C 6 15. The compound of claim 14, wherein R is 1 or 2; R is 2 or 3; R is 3 or 4; R is 4 or 5; R is 5 or 6; R is 6 or 7;

16. The pharmaceutically acceptable salt is of formula III(b): “During the ceremony, Y 1 ~Y 4 are each independently selected from H or D; R 3 is (C 1 ~C 6 ) alkyl; and 15. The compound of claim 14, having the structure:

17. The compound has formula III(c): “During the ceremony, Y 1 ~Y 4 are each independently selected from H or D; and R 3 is (C 1 ~C 6 2. The compound according to claim 1, which is a compound represented by the formula:

18. The compound is wherein R 3 is (C 1 ~C 6 20. The compound of claim 17, wherein: R is 1 or 2; R is 2 or 3; R is 3 or 4; R is 4 or 5; R is 5 or 6; R is 6 or 7;

19. The pharmaceutically acceptable salt has formula III(d): “During the ceremony, Y 1 ~Y 4 are each independently selected from H or D; R 3 is (C 1 ~C 6 ) alkyl; and 18. The compound of claim 17, having the structure:

20. The compound has formula IV: “During the ceremony, Y 1 ~Y 4 are each independently selected from H or D; and R 1 The compound according to claim 1, wherein R is a substituted or unsubstituted aryl group, or a pharmaceutically acceptable salt or solvate thereof.

21. The compound has formula V: “During the ceremony, Y 1 ~Y 4 and Y 9 ~Y 16 are each independently selected from H or D; R 1 is selected from H or an acetyl group; and R 5 is (C 1 ~C 6 2. The compound according to claim 1, which is a compound represented by the formula: "having a structure in which R is 1 or 2; R is 2 or 3; R is 3 or 4; R is 4 or 5; R is 5 or 6; R is 6 or 7; R is 7 or 8; R is 8 or 9; R is 9 or 10; R is 10 or 11; R is 11 or 12; R is 13 or 14; R is 15 or 16; R is 17 or 18; R is 19 or 20; R is 19 or 21; R is 19 or 22; R is 19 or 23; R is 19 or 24; R is 19 or 25; R is 19 or 26; R is 19 or 26;

22. Formula VI: “During the ceremony, Y 1 ~Y 4 are each independently selected from H or D; and R has a structure that is a linear or branched aliphatic group (saturated or unsaturated) or aromatic group (substituted or unsubstituted) having 1 to 20 carbon atoms, or a pharmaceutically acceptable salt or solvate thereof.

23. The compound has formula VI(a): “During the ceremony, Y 1 ~Y 8 are each independently selected from H or D; and The compound according to claim 22, which is a compound represented by the formula: "having a structure in which n is 2 to 6," or a pharmaceutically acceptable salt or solvate thereof.

24. The compound is 23. The compound of claim 22, selected from the group consisting of:

25. 10. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier, diluent, and / or binder.

26. 10. A pharmaceutical composition for treating a disease or disorder selected from the group consisting of cystinosis, fatty liver disease, cirrhosis, eosinophilic diseases or disorders, and Huntington's disease, comprising a therapeutically effective amount of a compound of claim 1.

27. 27. The pharmaceutical composition of claim 26, wherein the fatty liver disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fatty liver disease due to hepatitis, fatty liver disease due to obesity, fatty liver disease due to diabetes, fatty liver disease due to insulin resistance, fatty liver disease due to hypertriglyceridemia, abetalipoproteinemia, glycogen storage disease, Weber-Christian disease, Wolman disease, acute fatty liver of pregnancy, and lipoatrophy.

28. 10. A pharmaceutical composition for reducing fibrosis, fat content, or fat accumulation in the liver associated with non-alcoholic fatty liver disease (NAFLD), comprising the compound of claim 1.

29. 29. The pharmaceutical composition of claim 28, wherein the NALFD comprises NASH.

30. A pharmaceutical composition comprising the compound of claim 22 and a pharmaceutically acceptable carrier, diluent, and / or binder.

31. A pharmaceutical composition for treating a disease or disorder selected from the group consisting of cystinosis, fatty liver disease, cirrhosis, eosinophilic diseases or disorders, and Huntington's disease, comprising a therapeutically effective amount of the compound described in claim 22.

32. A pharmaceutical composition for reducing liver fibrosis or fat content or fat accumulation associated with non-alcoholic fatty liver disease (NAFLD), comprising a therapeutically effective amount of a compound according to claim 22.

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