Salts, including crystalline salts of 25-hydroxy-cholest-5-ene-3-sulfate, and methods for preparing same

Abstract

Several salts of 25HC3S are provided, including crystalline salts of 25HC3S. Pharmaceutical formulations of the disclosed salts and methods for using them to treat or prevent diseases, such as nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, are also provided. Methods for preparing the disclosed salts are also provided.

Description

[Background technology]

[0001] It has previously been shown that the cholesterol metabolite 5-cholestene-3β-25-diol-3-sulfate ("25HC3S") reduces lipid biosynthesis and increases cholesterol secretion and degradation, and may be useful for the treatment and prevention of one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation.

[0002] Cholesterol is used by the body to make and repair cell membranes and synthesize steroid hormones and vitamin D, and is converted to bile acids in the liver. Cholesterol has both exogenous and endogenous sources. The average American consumes about 450 mg of cholesterol daily and produces an additional 500 mg to 1,000 mg in the liver and other tissues. Another source is biliary cholesterol, with 500 mg to 1,000 mg secreted into the intestine each day, and about 50 percent is reabsorbed (enterohepatic circulation).

[0003] High serum lipid levels (hypercholesterolemia and hypertriglyceridemia) are associated with the accumulation of cholesterol in arterial walls and can lead to NAFLD and atherosclerosis. The plaques that characterize atherosclerosis restrict blood flow, promote clot formation, and can ultimately cause death or severe disability from heart attack and / or stroke. Several therapeutic agents have been developed for the treatment of hyperlipidemia and are widely prescribed by physicians. Unfortunately, only about 35% of patients respond to currently available therapies.

[0004] Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in the United States. This condition is associated with obesity, type 2 adult-onset diabetes, a sedentary lifestyle, and a high-fat diet. If appropriate treatment steps are taken, the earlier stage of NAFLD, fatty liver, is potentially reversible. However, if left unchecked, it can progress to inflammation of liver cells (nonalcoholic steatohepatitis, or NASH), which is much more difficult to treat. If untreated, NASH can lead to irreversible scarring of liver tissue (fat necrosis), potentially leading to cirrhosis, liver failure, and liver cancer.

[0005] 25HC3S has been disclosed as a pharmaceutically acceptable salt, e.g., the sodium salt (e.g., U.S. Pat. No. 10,144,759 and Ogawa et al., Steroids, 74, pp. 81-87 (2009)). Crystalline solids tend to be more convenient for processing, storage, and stability than amorphous solids. However, energetically, the immediate formation of a suitable crystalline solid is not always favored, and polymorphism can make it impractical to produce a stable crystalline solid of a particular active pharmaceutical ingredient. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 10,144,759 [Non-patent literature]

[0007] [Non-Patent Document 1] Ogawa et al., Steroids, 74, pp. 81-87 (2009) Summary of the Invention [Means for solving the problem]

[0008] Also provided herein are 15 salts, including crystalline salts, of 25HC3S, and methods of preparing and using said salts.

[0009] In some embodiments of the present disclosure, crystalline salts of 25HC3S other than crystalline sodium 25HC3S are provided.

[0010] In another embodiment of the present disclosure, a substantially pure crystalline salt of 25HC3S other than sodium is provided.

[0011] In another embodiment of the present disclosure, a method for preparing a crystalline salt of 25HC3S other than sodium is provided.

[0012] In some embodiments of the present disclosure, metal salts of 25HC3S other than sodium are provided.

[0013] In some embodiments of the present disclosure, a salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt is provided.

[0014] In a further aspect of the present disclosure, there is provided a salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt prepared by the method of the present disclosure.

[0015] In an additional embodiment of the present disclosure, there are provided crystalline salts of 25HC3S other than sodium prepared by the methods of the present disclosure.

[0016] In some embodiments of the present disclosure, crystalline salts of 25HC3S other than crystalline 25HC3S sodium and crystalline 25HC3S choline are provided.

[0017] In some embodiments of the present disclosure, substantially pure crystalline salts of 25HC3S other than crystalline 25HC3S sodium and crystalline 25HC3S choline are provided.

[0018] In a further aspect of the present disclosure, a method for preparing crystalline salts of 25HC3S other than sodium and choline is provided.

[0019] In yet a further embodiment of the present disclosure, there is provided a salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt other than choline.

[0020] In a further aspect of the present disclosure, there is provided a method for preparing a crystalline salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt other than choline.

[0021] In an additional embodiment of the present disclosure, there are provided crystalline salts of 25HC3S other than sodium and choline prepared by the methods of the present disclosure.

[0022] In yet a further aspect of the present disclosure, there is provided a pharmaceutical composition comprising a crystalline salt of 25HC3S other than sodium and a pharmaceutically acceptable excipient.

[0023] In yet a further aspect of the present disclosure, there is provided a pharmaceutical composition comprising a salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt, and a pharmaceutically acceptable excipient.

[0024] In a further aspect of the present disclosure, there is provided a pharmaceutical composition comprising a crystalline salt of 25HC3S other than sodium and choline and a pharmaceutically acceptable excipient.

[0025] In yet a further aspect of the present disclosure, there is provided a pharmaceutical composition comprising a salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt other than choline.

[0026] In a further aspect of the present disclosure, there is provided a method for treating or preventing one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, comprising administering to a patient in need thereof an effective amount of a compound of a crystalline salt of 25HC3S other than sodium.

[0027] In a further aspect of the present disclosure, there is provided a method of treating or preventing one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, comprising administering to a patient in need thereof an effective amount of a compound of a salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt.

[0028] In a further aspect of the present disclosure, there is provided a method for treating or preventing one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, comprising administering to a patient in need thereof an effective amount of a compound of a crystalline salt of 25HC3S other than sodium or choline.

[0029] In a further aspect of the present disclosure, there is provided a method for treating or preventing one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, comprising administering to a patient in need thereof an effective amount of a compound of (i) an alkali metal salt or (ii) an ammonium salt other than choline. [Brief explanation of the drawings]

[0030] [Figure 1] 1 is an X-ray powder diffraction pattern of crystalline potassium 25HC3S. [Figure 1A] 1 is a magnified x-ray powder diffraction pattern of crystalline potassium 25HC3S. [Figure 2] 1 is an X-ray powder diffraction pattern of selected peaks of crystalline potassium 25HC3S. [Figure 2A] 1 is an expanded x-ray powder diffraction pattern of selected peaks of crystalline potassium 25HC3S. [Figure 3]1 is an X-ray powder diffraction pattern of crystalline calcium 25HC3S. [Figure 3A] 1 is a magnified X-ray powder diffraction pattern of crystalline calcium 25HC3S. [Figure 4] 1 is an X-ray powder diffraction pattern of selected peaks of crystalline calcium 25HC3S. [Figure 4A] 1 is an enlarged x-ray powder diffraction pattern of selected peaks of crystalline calcium 25HC3S. [Figure 5] 1 is an X-ray powder diffraction pattern of crystalline magnesium 25HC3S. [Figure 5A] 1 is a magnified X-ray powder diffraction pattern of crystalline magnesium 25HC3S. [Figure 6] 1 is an X-ray powder diffraction pattern of selected peaks of crystalline magnesium 25HC3S. [Figure 6A] 1 is an expanded x-ray powder diffraction pattern of selected peaks of crystalline magnesium 25HC3S. [Figure 7] 1 is an X-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium. [Figure 7A] 1 is a magnified X-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium. [Figure 8] 1 is an X-ray powder diffraction pattern of selected peaks of crystalline 25HC3S hydroxyethylammonium. [Figure 8A] 1 is an enlarged X-ray powder diffraction pattern of selected peaks of crystalline 25HC3S hydroxyethylammonium. [Figure 9] This is the X-ray powder diffraction pattern of crystalline 25HC3 hydroxyethylpyrrolidinium. [Figure 10] 1 is an X-ray powder diffraction pattern of selected peaks of crystalline 25HC3S hydroxyethylpyrrolidinium. [Figure 11] 1 is an X-ray powder diffraction pattern of crystalline 25HC3S meglumine. [Figure 11A] 1 is a magnified x-ray powder diffraction pattern of crystalline 25HC3S meglumine. [Figure 12] 1 is an x-ray powder diffraction pattern of selected peaks of crystalline 25HC3S meglumine. [Figure 12A] 1 is an expanded x-ray powder diffraction pattern of selected peaks of crystalline 25HC3S meglumine. [Figure 13] 1 is an X-ray powder diffraction pattern of crystalline 25HC3S tromethammonium. [Figure 13A] This is an enlarged X-ray powder diffraction pattern of crystalline 25HC3S trometaammonium. [Figure 14] 1 is an X-ray powder diffraction pattern of selected peaks of crystalline 25HC3S trometaammonium. [Figure 14A] 1 is an expanded X-ray powder diffraction pattern of selected peaks of crystalline 25HC3S trometaammonium. [Figure 15] 1 is an X-ray powder diffraction pattern of crystalline 25HC3S diethanolamine. [Figure 16] 1 is an X-ray powder diffraction pattern of selected peaks of crystalline 25HC3S diethanolamine. [Figure 17] 1 is an X-ray powder diffraction pattern of crystalline 25HC3S diethylammonium. [Figure 18] 1 is an X-ray powder diffraction pattern of selected peaks of crystalline 25HC3S diethylammonium. [Figure 19] This is the X-ray powder diffraction pattern of crystalline 25HC3 t-butylammonium. [Figure 20] 1 is an X-ray powder diffraction pattern of selected peaks of crystalline 25HC3S t-butylammonium. [Figure 21] 1 is an X-ray powder diffraction pattern of crystalline 25HC3S benzathine. [Figure 22] 1 is an X-ray powder diffraction pattern of selected peaks of crystalline 25HC3S benzathine. [Figure 23] 1 is an X-ray powder diffraction pattern of crystalline 25HC3S lysine. [Figure 23A] 1 is a magnified x-ray powder diffraction pattern of crystalline 25HC3S lysine. [Figure 24] 1 is an X-ray powder diffraction pattern of selected peaks of crystalline 25HC3S lysine. [Figure 24A] 1 is an expanded x-ray powder diffraction pattern of selected peaks of crystalline 25HC3S lysine. [Figure 25] 1 shows a TGA thermogram and a DSC thermogram of crystalline potassium 25HC3S. [Figure 26] 1 is a DVS isotherm of crystalline potassium 25HC3S. [Figure 27] Overlay of X-ray powder diffraction patterns of crystalline potassium 25HC3S before and after DVS. [Figure 28] This is the 1H-NMR spectrum of potassium 25HC3S. [Figure 29] 1 shows a TGA thermogram and a DSC thermogram of crystalline calcium 25HC3S. [Figure 30] 1 is a DVS isotherm of crystalline calcium 25HC3S. [Figure 31] Overlay of X-ray powder diffraction patterns of crystalline calcium 25HC3S before and after DVS. [Figure 32] This is the 1H-NMR spectrum of calcium 25HC3S. [Figure 33] 1 shows a TGA thermogram and a DSC thermogram of crystalline magnesium 25HC3S. [Figure 34] 1 is a DVS isotherm of crystalline magnesium 25HC3S. [Figure 35] Overlay of X-ray powder diffraction patterns of crystalline 25HC3S magnesium before and after DVS. [Figure 36] This is the 1H-NMR spectrum of 25HC3S magnesium. [Figure 37] 1 is a TGA thermogram and a DSC thermogram of crystalline hydroxyethylammonium 25HC3S. [Figure 38] 1 is a DVS isotherm of crystalline hydroxyethylammonium 25HC3S. [Figure 39]Overlay of X-ray powder diffraction patterns of crystalline 25HC3S hydroxyethylammonium before and after DVS. [Figure 40] This is the 1H-NMR spectrum of 25HC3S hydroxyethylammonium. [Figure 41] 1 shows a TGA thermogram and a DSC thermogram of crystalline 25HC3S hydroxyethylpyrrolidinium. [Figure 42] 1 is a DVS isotherm of crystalline 25HC3S hydroxyethylpyrrolidinium. [Figure 43] Overlay of X-ray powder diffraction patterns of crystalline 25HC3S hydroxyethylpyrrolidinium before and after DVS. [Figure 44] This is the 1H-NMR spectrum of 25HC3S hydroxyethylpyrrolidinium. [Figure 45] 1 shows the indexing results of crystalline 25HC3S hydroxyethylpyrrolidinium. [Figure 46] 1 is a TGA thermogram and a DSC thermogram of crystalline 25HC3S meglumine. [Figure 47] 1 is a DVS isotherm of crystalline 25HC3S meglumine. [Figure 48] Overlay of x-ray powder diffraction patterns of crystalline 25HC3S meglumine before and after DVS. [Figure 49] 1H-NMR spectrum of 25HC3S meglumine. [Figure 50] 1 shows the TGA and DSC thermograms of crystalline 25HC3S trometaammonium. [Figure 51] This is the DVS isotherm of crystalline 25HC3S trometaammonium. [Figure 52] Overlay of X-ray powder diffraction patterns of crystalline 25HC3S tromethammonium before and after DVS. [Figure 53] This is the 1H-NMR spectrum of 25HC3S trometaammonium. [Figure 54]1 is a TGA thermogram and a DSC thermogram of crystalline 25HC3S diethanolamine. [Figure 55] 1 is a DVS isotherm of crystalline 25HC3S diethanolamine. [Figure 56] Overlay of X-ray powder diffraction patterns of crystalline 25HC3S diethanolamine before and after DVS. [Figure 57] 1H-NMR spectrum of 25HC3S diethanolamine. [Figure 58] 1 is an indexing result for crystalline 25HC3S diethanolamine. [Figure 59] 1 is a TGA thermogram and a DSC thermogram of crystalline diethylammonium 25HC3S. [Figure 60] 1 is a DVS isotherm of crystalline diethylammonium 25HC3S. [Figure 61] Overlay of X-ray powder diffraction patterns of crystalline diethylammonium 25HC3S before and after DVS. [Figure 62] This is the 1H-NMR spectrum of diethylammonium 25HC3S. [Figure 63] 2 shows the indexing results for crystalline 25HC3S diethylammonium. [Figure 64] 1 is a TGA thermogram and a DSC thermogram of crystalline 25HC3S t-butylammonium. [Figure 65] 1 is a DVS isotherm of crystalline 25HC3S t-butylammonium. [Figure 66] This is the 1H-NMR spectrum of 25HC3S t-butylammonium. [Figure 67] Indexing results for crystalline 25HC3S t-butylammonium. [Figure 68] 1 is a TGA thermogram and a DSC thermogram of crystalline 25HC3S benzathine. [Figure 69] 1 is a DVS isotherm of crystalline 25HC3S benzathine. [Figure 70]Overlay of X-ray powder diffraction patterns of crystalline 25HC3S benzathine before and after DVS. [Figure 71] 1H-NMR spectrum of 25HC3S benzathine. [Figure 72] 1 shows the indexing results for crystalline 25HC3S benzathine. [Figure 73] 1 is a TGA thermogram and a DSC thermogram of crystalline 25HC3S lysine. [Figure 74] 1 is a DVS isotherm of crystalline 25HC3S lysine. [Figure 75] 1H-NMR spectrum of 25HC3S lysine. [Figure 76] 1 is an X-ray powder diffraction pattern of crystalline 25HC3S choline. [Figure 77] 1 is an x-ray powder diffraction pattern of selected peaks of crystalline 25HC3S choline. [Figure 78] Overlay of x-ray powder diffraction patterns of crystalline 25HC3S choline before and after DVS. [Figure 79] 1 shows the indexing results for crystalline 25HC3S choline. [Figure 80] 1 is a DSC (bottom) and TGA (top) thermogram of crystalline 25HC3S choline. [Figure 81] 1H-NMR spectrum of 25HC3S choline in solution. [Figure 82] 1 is a DVS isotherm of crystalline 25HC3S choline. [Figure 83] 1 is an X-ray powder diffraction pattern of crystalline zinc 25HC3S. [Figure 84] 1 is an X-ray powder diffraction pattern of crystalline zinc 25HC3S. [Figure 85] 1 is an x-ray powder diffraction pattern of selected peaks of crystalline zinc 25HC3S. [Figure 86] 1 is an x-ray powder diffraction pattern of selected peaks of crystalline zinc 25HC3S. [Figure 87]Overlay of x-ray powder diffraction patterns of crystalline zinc 25HC3S before and after DVS. [Figure 88] 1 shows a DSC thermogram and a TGA thermogram of crystalline zinc 25HC3S. [Figure 89] This is the 1H-NMR spectrum of zinc 25HC3S. [Figure 90] This is a DVS experiment of crystalline zinc 25HC3S. DETAILED DESCRIPTION OF THE INVENTION

[0031] The compound 25-hydroxy-3β-cholesten-5-ene-3-sulfate (25HC3S) refers to [(3S,10R,13R,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]sulfate, a compound of formula I: [ka]

[0032] Fifteen salts of 25HC3S are described herein. The salts were prepared as described in Examples 27-41. All salts herein may be prepared as crystalline solids. Salts of the present disclosure include inorganic salts and organic salts. Inorganic salts include metal salts, and organic salts include amine salts.

[0033] The metal salts of the present disclosure include dicationic salts, including monocationic alkali metal salts and alkaline earth metal salts. Metal salts of 25HC3S, including crystalline salts other than sodium, are disclosed herein. The metal salts include alkali metals other than sodium. Indeed, the alkali metal salts of the present disclosure include potassium 25HC3S, including crystalline potassium 25HC3S. Other metal salts of 25HC3S include alkaline earth metal salts of 25HC3S, including crystalline alkaline earth metal salts of 25HC3S. Alkaline earth metal salts include (a) calcium 25HC3S, including crystalline calcium 25HC3S, and (b) magnesium 25HC3S, including crystalline magnesium 25HC3S. The present disclosure further includes other metal salts of 25HC3S, including crystalline salts thereof. Other metals within the present disclosure include transition metals, such as dicationic transition metals, including crystalline transition metal salts of 25HC3S, e.g., crystalline dicationic transition metal salts. An exemplary dicationic transition metal is zinc.

[0034] Disclosed herein are organic salts of 25HC3S, including crystalline organic salts of 25HC3S. Examples of such organic salts include amine salts, including crystalline amine salts of 25HC3S. The amines can be aliphatic, cyclic, aromatic, or combinations thereof. The amines can be primary, secondary, tertiary, or quaternary amines. The amines can be further substituted or unsubstituted. Typical substitutions are with one or more alcohol groups. The alcohols can be primary, secondary, or tertiary alcohols.

[0035] The amine functionality of the amine group may be linked to an aliphatic chain of carbons, for example, containing between 1 and 6 carbons. Such linkers may be substituted or unsubstituted. Substitutions include alkyl, alcohol, acid, aryl, and amine groups. Similarly, amino acid salts of the present disclosure are included within the scope of the salts of the present disclosure. Such amino acid salts may be of natural or unnatural amino acids. An exemplary amino acid salt of the present disclosure is the lysine salt of 25HC3S. In many instances herein, the amine salts are amine alcohol salts of 25HC3S, including choline, hydroxyethylammonium, hydroxyethylpyrrolidinium, meglumine, tromethammonium, and diethanolamine. The amine alcohol salts of the present disclosure may contain a single alcohol or multiple alcohol functional groups, such as diethanolamine (two hydroxy groups), tromethammonium (three hydroxy groups), or meglumine (five hydroxy groups). The amine alcohol may be a primary, secondary, tertiary, or quaternary amine alcohol salt. Such amine alcohol salts may include cyclic amines, where the amine group may be linked to the alcohol via an alkyl linker, for example, of from 1 to 6 carbons.

[0036] In many instances herein, the amine salts are amine alkyl salts of 25HS3C, including diethylammonium, t-butylammonium, and benzathine. The alkylamine salts of the present disclosure include primary, secondary, and tertiary amines, and the alkyl chain may contain one or more carbon atoms. In many embodiments, there are two carbon atoms in the linker. The alkylamine salt may also be a diamine. In some instances, the alkylamine salt also contains an aromatic group, such as a phenyl group or a benzyl group.

[0037] In many embodiments, the salts of the present disclosure are crystalline salts of 25HC3S other than (i) alkali metal salts or (ii) ammonium salts. In other embodiments, the salts of the present disclosure are crystalline salts of 25HC3S other than sodium salts or choline salts.

[0038] Further disclosed are substantially pure and crystalline salts of 25HC3S of the present disclosure. "Substantially pure," as used herein, generally refers to forms herein present in significant amounts other than possibly trace levels of other forms of 25HC3S. Examples of trace levels include a total of about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less, based on the total amount (by weight) of the salts of 25HC3S of the present disclosure.

[0039] Methods for preparing salts of 25HC3S of the present disclosure are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt of 25HC3S, for example, as described in Example 41. The triethylammonium salt can then be used to produce other salts of 25HC3S of the present disclosure.

[0040] The present disclosure also relates to pharmaceutical compositions containing the disclosed salts of 25HC3S. Such pharmaceutical compositions comprise one or more pharmaceutically acceptable excipients and salts and crystalline salts of 25HC3S. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, for example, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral preparations.

[0041] In many embodiments, the pharmaceutical compositions of the present disclosure contain a salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt, with the proviso that in some embodiments, a choline salt is included within the scope of such pharmaceutical compositions. In some embodiments, the pharmaceutical compositions comprise a crystalline salt of 25HC3S other than sodium and choline and a pharmaceutically acceptable excipient.

[0042] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, using an effective amount of the salts and crystalline salts of 25HC3S of the present disclosure and / or pharmaceutical compositions comprising such salts and crystalline salts of 25HC3S of the present disclosure. As used throughout this disclosure, examples of "diabetic conditions" that can be treated include one or more of insulin resistance, insulin deficiency, diabetes, and prediabetes. As used throughout this disclosure, examples of "autoimmune conditions" that may be treated include one or more of hepatitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis. As used throughout this disclosure, examples of "inflammatory conditions" that may be treated include one or more of pulpitis, periodontal disease, skin inflammation, psoriasis, ulcerative colitis, osteoarthritis, inflammatory bowel disease (IBD), Crohn's disease, irritable bowel syndrome (IBS), Alzheimer's disease, Parkinson's disease, pancreatitis (acute and / or chronic), hepatitis (viral and / or non-viral), atherosclerosis, myocarditis, idiopathic pulmonary disorder (IPD), chronic obstructive pulmonary disease (COPD), pneumonia, chronic inflammatory lung disease, bronchitis, asthma, chronic kidney disease (CKD), nephritis, sepsis, ankylosing spondylitis, diverticulitis, and fibromyalgia. As used throughout this disclosure, examples of "neurological conditions" that may be treated include one or more of depression, neurodegenerative diseases, multiple sclerosis, Parkinson's disease, spinocerebellar degeneration, Friedreich's ataxia, ataxia-telangiectasia, progressive supranuclear palsy, Huntington's disease, striatonigral degeneration, olivopontocerebellar atrophy, Shy-Drager syndrome, schizophrenia, schizoaffective disorder, manic-depressive (bipolar) disorder, derangements or abnormalities in circadian rhythm entrainment, childhood Alice in Wonderland syndrome, childhood acute cerebellar ataxia, and Alzheimer's disease.

[0043] In many embodiments, the present disclosure further includes methods and uses for treating and / or preventing one or more diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of salts and crystalline salts of 25HC3S of the present disclosure and / or pharmaceutical compositions comprising such salts and crystalline salts of 25HC3S of the present disclosure, wherein the 25HC3S is other than (i) an alkali metal salt or (ii) an ammonium salt, with the proviso that in some embodiments, a choline salt is included. In some other embodiments, the 25HC3S choline used in such methods is a crystalline salt of 25HC3S other than sodium and choline.

[0044] Crystalline 25HC3S choline salts are easily identified by x-ray powder diffraction. An x-ray powder diffraction pattern is an xy graph with °2θ (diffraction angle) on the x-axis and intensity on the y-axis. The x-axis can also be in the form of d-spacing, which is related to the diffraction angle by Bragg's law: 2d sin θ = nλ, where d is the d-spacing and λ is the wavelength of the incident x-ray wave. The pattern contains peaks that can be used to characterize crystalline salts of 25HC3S. Unless otherwise specified, peaks are referred to by their position on the x-axis rather than their y-axis intensity. It is also possible that, due to sample orientation, peaks present in one sample on one instrument may not be present in another sample obtained on a different instrument, due to the orientation of the sample relative to the instrument.

[0045] Data from X-ray powder diffraction may be used in several ways to characterize crystalline forms. For example, the entire X-ray powder diffraction pattern output from a diffractometer may be used to characterize a crystalline salt of 25HC3S. However, smaller subsets of such data may also be, and usually are, suitable for characterizing a crystalline salt of 25HC3S. For example, a collection of one or more peaks from such a pattern may be used to characterize a crystalline salt of 25HC3S. In this application, all reported peak values ​​are in °2θ using CuKα radiation, as described in Examples 24 and 25. In fact, even a single X-ray powder diffraction peak can often be used to characterize such a crystalline form. When a crystalline salt of 25HC3S is characterized herein by "one or more peaks" in an X-ray powder diffraction pattern and such peaks are listed, it generally means that any combination of the listed peaks can be used to characterize the crystalline salt of 25HC3S. Furthermore, the fact that other peaks are present in an X-ray powder diffraction pattern does not generally negate or otherwise limit its characterization.

[0046] In addition to variability in peak intensity, there may also be variability in the position of the peaks along the x-axis. However, this variability can usually be accounted for when reporting peak positions for characterization purposes. Such variability in peak positions along the x-axis can result from several causes (e.g., sample preparation, particle size, water content, solvent content, instrument parameters, data analysis software, and sample orientation). For example, samples of the same crystalline material prepared under different conditions may give slightly different diffractograms, and different X-ray instruments may be operated using different parameters, which may result in slightly different diffraction patterns for the same crystalline solid.

[0047] Because of these sources of variability, it is common to describe x-ray diffraction peaks using the word "about" before the peak value in °2θ. For purposes of data reported herein, that value is generally ±0.2 °2θ. This generally means that with well-maintained equipment, variability in peak measurements of ±0.2 °2θ is expected. Unless otherwise specified, x-ray powder diffraction peaks cited herein are generally reported with this variability of ±0.2 °2θ, and as disclosed herein, are generally intended to be reported with such variability whether or not the word "about" is present; however, variability may in some cases be ±0.2 °2θ or even greater depending on instrument conditions. In many of the crystalline salts of the present disclosure, some of the x-ray powder diffraction peaks, and in many cases peaks at low angles, e.g., intensity less than 5 °2θ, fall closely together, e.g., within 0.2 °2θ, which is the same variability with which the peaks are reported. Because of such variability, in some measurements, the two peaks may coincide, making it difficult to distinguish between them. However, to characterize a crystalline salt of 25HC3S of the present disclosure, and such characterization identifies only a single peak, that peak may then be used to characterize or assist in characterizing such crystalline salt of 25HC3S, as the case may be, so long as the single peak is within a specified variability range of ±0.2°2θ. Furthermore, in additional embodiments of the present invention, the variability of the quoted peak values ​​or groupings of the quoted peaks in °2θ units is ±0.1°2θ, or even ±0.05°2θ, rather than ±0.2°2θ.

[0048] In addition to x-ray powder diffraction, several salts of 25HC3S were observed by polarized optical microscopy. Some x-ray powder diffraction patterns were also indexed. "Indexing," as used herein, generally refers to the process of determining the size and shape of a crystallographic unit cell given peak positions in a diffraction pattern. The term derives its name from the assignment of Miller index labels to individual peaks. For example, if all peaks in a pattern are indexed by a single unit cell, this can be strong evidence that the sample contains a single crystalline phase. With the indexing solution, the unit cell volume can be directly calculated, which can be useful in determining their solvation state. Indexing can also be a description of crystalline morphology, providing a summary of all available peak positions in terms of a specific thermodynamic state for that phase.

[0049] Differential scanning calorimetry (DSC) is a thermal analysis technique that measures the difference in the amount of heat required to raise the temperature between a sample and a reference as a function of temperature. This technique can be used to measure phase transitions of a sample, such as melting points. Decomposition as a dehydration event can also be observed by DSC. In the context of DSC measurements, there is variability, and the term "about" means ±1°C, and such variability should be understood whether the DSC measurement is preceded by "about" or not, unless otherwise specified. An exemplary method for collecting DSC data is described in Example 21. Thermogravimetric analysis, also known as thermal gravimetric analysis (TGA), is a thermal method of thermal analysis in which the mass of a sample is measured as a function of time as the sample temperature is changed. For example, if a sample disclosed herein is heated and a decrease in mass before decomposition is measured, this is likely an indication of the loss of water from the sample and may indicate that the heated sample is a hydrate. An exemplary method for collecting a TGA is described in Example 23.

[0050] Dynamic vapor sorption (DVS) measures the uptake of vapor (usually water) by a sample when exposed to changes in relative humidity. Water loss may also be measured as the relative humidity decreases. Hygroscopic materials tend to absorb water more readily than non-hygroscopic materials. In some cases, hydrates absorb water to form higher hydrates (e.g., a monohydrate converts to a dihydrate when exposed to increasingly humid conditions). In some salts of the present disclosure, moisture is retained upon exposure in DVS experiments, and little or no weight loss is observed upon heating from room temperature. In such experiments, the hygroscopicity of the salt under consideration may indicate the formation of a hydrate from the anhydrate. In other salts of the present disclosure, weight loss is observed upon heating, suggesting that the salt form at the start of such a thermal experiment is a hydrate. An exemplary method for collecting DVS data is described in Example 26.

[0051] Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) is an analytical technique used to detect chemical elements. This spectroscopic technique can be used to determine the relative stoichiometry of compounds, and was used herein to identify metal salts of 25HC3S.

[0052] Turning to inorganic salts of 25HC3S, an exemplary inorganic salt of the present disclosure is a metal salt of 25HC3S and potassium. Crystalline potassium 25HC3S can be prepared as described in Example 27. The x-ray powder diffraction pattern of crystalline potassium 25HC3S can be seen in Figure 1, with Figure 1A being a magnified version of the pattern. The x-ray powder diffraction pattern of selected peaks can be seen in Figure 2, with Figure 2A being a magnified version of the pattern. Table 1 lists selected peaks from Figure 2.

[0053] [Table 1] TIFF2026501266000004.tif15161

[0054] Crystalline 25HC3S potassium can be characterized by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S potassium, or a portion thereof, can be used to identify the crystalline 25HC3S potassium. Crystalline 25HC3S potassium contains various x-ray powder diffraction peaks, which, alone or together, can help identify the presence of crystalline 25HC3S potassium.

[0055] In some cases, crystalline potassium-25HC3S may be characterized by an x-ray powder diffraction pattern having one or more peaks in Figure 2. The two most intense peaks in Figure 2 are at about 2.2° 2θ and about 2.3° 2θ. Although these peaks appear very close together in Figure 2 due to their individual intensities, the XRPD instrument is able to distinguish them. However, due to variability associated with these XRPD peaks, they may appear "underneath" each other when analyzed as a single peak. Because such a single-appearing peak is possible in many cases herein, crystalline potassium-25HC3S may be characterized, at least in part, as having an x-ray powder diffraction pattern comprising two peaks between about 2.2° 2θ and about 2.3° 2θ that (a) do not overlap, (b) partially overlap, or (c) overlap so as to appear as a single peak, for example.

[0056] The ICP-OES results are consistent with a 1:1 stoichiometry of potassium ions to 25HC3S ions. For example, a peak at about 2.2°2θ can be used to characterize crystalline potassium-25HC3S. In these and other cases, the crystalline potassium-25HC3S may be further characterized by one or more peaks at about 2.3°2θ, about 8.8°2θ, about 9.3°2θ, and about 15.3°2θ.

[0057] In some instances, the crystalline potassium-25HC3S may be characterized by an x-ray powder diffraction pattern having a peak at about 2.3°2θ. In these and other instances, the crystalline potassium-25HC3S may be further characterized by one or more peaks at about 2.2°2θ, about 8.8°2θ, about 9.3°2θ, and about 15.3°2θ.

[0058] In some instances, the crystalline potassium-25HC3S may be characterized by an x-ray powder diffraction pattern having a peak at about 8.8°2θ. In these and other instances, the crystalline potassium-25HC3S may be further characterized by one or more peaks at about 2.2°2θ, about 2.3°2θ, about 9.3°2θ, and about 15.3°2θ.

[0059] In some instances, the crystalline potassium-25HC3S may be characterized by an x-ray powder diffraction pattern having a peak at about 9.3°2θ. In these and other instances, the crystalline potassium-25HC3S may be further characterized by one or more peaks at about 2.2°2θ, about 2.3°2θ, about 8.8°2θ, and about 15.3°2θ.

[0060] In some instances, the crystalline potassium-25HC3S may be characterized by an x-ray powder diffraction pattern having a peak at about 15.3°2θ. In these and other instances, the crystalline potassium-25HC3S may be further characterized by one or more peaks at about 2.2°2θ, about 2.3°2θ, about 8.8°2θ, and about 9.3°2θ.

[0061] In some cases, crystalline potassium 25HC3S may be further characterized by one or more peaks at about 4.6°2θ, 14.7°2θ, 14.9°2θ, and about 16.1°2θ. For example, crystalline potassium 25HC3S may be further characterized by a peak at about 4.6°2θ. Alternatively or additionally, crystalline potassium 25HC3S may be further characterized by a peak at about 14.7°2θ. Alternatively or additionally, crystalline potassium 25HC3S may be further characterized by a peak at about 14.9°2θ. Alternatively or additionally, crystalline potassium 25HC3S may be further characterized by a peak at about 16.1°2θ.

[0062] In some cases, the crystalline potassium-25HC3S can be characterized by an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern set forth in Figure 2 or Figure 2A.

[0063] Crystalline potassium 25HC3S has limited hygroscopicity according to DVS, gaining only 2% water as the relative humidity approaches 95% RH (Figure 26), and by TGA, it exhibits a weight loss of about 0.9% up to about 100°C, and a further weight loss of 1.7% between 100°C and 180°C (Figure 25). These data suggest that crystalline potassium 25HC3S is anhydrous. Figure 27 is an overlay showing the XRPD diffractograms of crystalline potassium 25HC3S before and after DVS. There is no significant change in the pattern. Figure 28 shows the XRPD diffractogram of a solution of potassium 25HC3S. 1 The H-NMR spectrum is consistent with the chemical structure. The DSC thermogram in Figure 25 also shows a small endotherm near 100°C, then again at 139°C. Additional endotherms at 198°C and 244°C are observed, suggesting decomposition at 198°C.

[0064] Further disclosed is substantially pure crystalline potassium 25HC3S. "Substantially pure," as used herein, generally refers to forms herein present in significant amounts other than possibly trace levels of other forms of potassium 25HC3S. Examples of trace levels include about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less in total, based on the total amount (by weight) of potassium 25HC3S present.

[0065] Methods for preparing potassium 25HC3S are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce potassium 25HC3S, as described in Example 27.

[0066] The present disclosure also relates to pharmaceutical compositions containing 25HC3S potassium, including the crystalline 25HC3S potassium disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S potassium, including the crystalline 25HC3S potassium. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, for example, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral preparations.

[0067] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans), such as one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3 potassium, including the crystalline 25HC3 potassium of the present disclosure, and / or an effective amount of a pharmaceutical composition comprising the crystalline 25HC3 potassium of the present disclosure.

[0068] Another exemplary salt of the present disclosure is a metal salt of 25HC3S and calcium. Crystalline calcium 25HC3S can be prepared as described in Example 28. The x-ray powder diffraction pattern of crystalline calcium 25HC3S can be seen in Figure 3, with Figure 3A being a magnified version. The x-ray powder diffraction pattern of selected peaks can be seen in Figure 4, with Figure 4A being a magnified version. Table 2 lists selected peaks from Figure 4. The calcium salt of 25HC3S contains two ions of 25HC3S for every ion of calcium, as determined by ICP-OES. Therefore, the calcium salt can also be referred to as a hemicalcium salt.

[0069] [Table 2] TIFF2026501266000006.tif146161

[0070] Crystalline 25HC3S calcium can be characterized by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S calcium, or a portion thereof, can be used to identify the crystalline 25HC3S calcium. Crystalline 25HC3S calcium contains various x-ray powder diffraction peaks, which, alone or together, can help identify the presence of crystalline 25HC3S calcium.

[0071] Two of the most intense peaks in Figure 4 are those at about 15.0°2θ and about 15.1°2θ. While these peaks appear very close together in Figure 4 due to their individual intensities, the XRPD instrument is able to distinguish them. However, due to variability associated with these XRPD peaks, they may appear "underneath" each other when analyzed as a single peak. Because such single-appearing peaks are possible in many instances herein, crystalline 25HC3S calcium is characterized, at least in part, as having an x-ray powder diffraction pattern that includes two peaks between about 15.0°2θ and about 15.1°2θ that are (a) non-overlapping, (b) partially overlapping, or (c) overlapping, e.g., to appear as a single peak. In some instances, crystalline 25HC3S calcium may be characterized by an x-ray powder diffraction pattern with one or more peaks in Figure 4. For example, a peak at about 2.2°2θ can be used to characterize crystalline 25HC3S calcium. In these and other cases, the crystalline calcium 25HC3S may be further characterized by one or more peaks at about 4.5°2θ, about 9.0°2θ, about 10.0°2θ, about 15.0°2θ, about 15.1°2θ, and about 15.7°2θ.

[0072] In some cases, the crystalline 25HC3S calcium may be characterized by an x-ray powder diffraction pattern having a peak at about 4.5°2θ. In these and other cases, the crystalline 25HC3S calcium may be further characterized by one or more peaks at about 2.2°2θ, about 9.0°2θ, about 10.0°2θ, about 15.0°2θ, about 15.1°2θ, and about 15.7°2θ.

[0073] In some instances, the crystalline 25HC3S calcium may be characterized by an x-ray powder diffraction pattern having a peak at about 9.0°2θ. In these and other instances, the crystalline 25HC3S calcium may be further characterized by one or more peaks at about 2.2°2θ, about 4.5°2θ, about 10.0°2θ, about 15.0°2θ, about 15.1°2θ, and about 15.7°2θ.

[0074] In some instances, the crystalline 25HC3S calcium may be characterized by an x-ray powder diffraction pattern having a peak at about 10.0°2θ. In these and other instances, the crystalline 25HC3S calcium may be further characterized by one or more peaks at about 2.2°2θ, about 4.5°2θ, about 9.0°2θ, about 15.0°2θ, about 15.1°2θ, and about 15.7°2θ.

[0075] In some instances, the crystalline 25HC3S calcium may be characterized by an x-ray powder diffraction pattern having a peak at about 15.0°2θ. In these and other instances, the crystalline 25HC3S calcium may be further characterized by one or more peaks at about 2.2°2θ, about 4.5°2θ, about 9.0°2θ, about 10.0°2θ, about 15.1°2θ, and about 15.7°2θ.

[0076] In some instances, the crystalline 25HC3S calcium may be characterized by an x-ray powder diffraction pattern having a peak at about 15.1°2θ. In these and other instances, the crystalline 25HC3S calcium may be further characterized by one or more peaks at about 2.2°2θ, about 4.5°2θ, about 9.0°2θ, about 10.0°2θ, about 15.0°2θ, and about 15.7°2θ.

[0077] In some instances, the crystalline 25HC3S calcium may be characterized by an x-ray powder diffraction pattern having a peak at about 15.7°2θ. In these and other instances, the crystalline 25HC3S calcium may be further characterized by one or more peaks at about 2.2°2θ, about 4.5°2θ, about 9.0°2θ, about 10.0°2θ, about 15.0°2θ, and about 15.1°2θ.

[0078] In some cases, the crystalline 25HC3S calcium may be further characterized by one or more peaks at about 15.4°2θ, 16.5°2θ, 18.0°2θ, 18.1°2θ, 18.4°2θ, and about 19.2°2θ. For example, the crystalline 25HC3S calcium may be further characterized by a peak at about 15.4°2θ. Alternatively or additionally, the crystalline 25HC3S calcium may be further characterized by a peak at about 16.5°2θ. Alternatively or additionally, the crystalline 25HC3S calcium may be further characterized by a peak at about 18.0°2θ. Alternatively or additionally, the crystalline 25HC3S calcium may be further characterized by a peak at about 18.1°2θ. Alternatively or additionally, the crystalline 25HC3S calcium may be further characterized by a peak at about 18.4°2θ. Alternatively, or in addition, the crystalline calcium 25HC3S may be further characterized by a peak at about 19.2° 2θ.

[0079] In some cases, the crystalline 25HC3S calcium may be characterized by an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern in Figure 4 or Figure 4A.

[0080] Crystalline 25HC3S calcium can occur as a hydrate. A sample of crystalline 25HC3S calcium lost approximately 0.9% of its weight between room temperature and about 93°C, and an additional 3.1% between about 112°C and about 130°C, as shown in the TGA thermogram in Figure 29. The TGA data was calculated based on a 25HC3S salt with one 25HC3S ion and 0.5 calcium ions. The weight loss is estimated to be due to water at these temperatures. Therefore, for the calcium salt, a 3-4% water loss is associated with approximately one mole of water per mole of 25HC3S ion (and thus 0.5 moles of calcium dication), suggesting a monohydrate. According to the DVS data in Figure 30, higher water uptake can occur when exposed to higher relative humidity, which can result in a dihydrate, as seen by the plateau in the DVS isotherm. Thus, this data indicates that crystalline calcium 25HC3S can exist in several hydration states, including monohydrate or dihydrate, when referring to a single ion of 25HC3S and half ions of the calcium dication per salt molecule. In Example 28, for example, the crystalline 25HC3S hemicalcium salt prepared contained two equivalents of water.

[0081] The DSC thermogram of crystalline 25HC3S calcium shows a small endothermic peak at about 70.3°C and a larger endothermic peak at about 130°C, with decomposition associated with the higher value (Figure 29). Figure 31 is an overlay showing the XRPD diffractograms of crystalline 25HC3S calcium before and after DVS. There is no significant change in the pattern. Figure 32 shows the XRPD diffractogram of a solution of 25HC3S calcium. 1 H-NMR spectrum, which is consistent with the chemical structure.

[0082] Further disclosed is a substantially pure crystalline 25HC3S calcium. "Substantially pure," as used herein, generally refers to forms herein present in significant amounts other than possibly trace levels of other forms of 25HC3S calcium. Examples of trace levels include a total of about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less, based on the total amount (by weight) of 25HC3S calcium present.

[0083] Methods for preparing calcium 25HC3S are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce calcium 25HC3S, as described in Example 28.

[0084] The present disclosure also relates to pharmaceutical compositions containing 25HC3S calcium, including the crystalline 25HC3S calcium disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S calcium, including crystalline 25HC3S calcium. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, for example, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral preparations.

[0085] The present disclosure further includes methods and uses for treating and / or preventing one or more of diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3S calcium comprising the crystalline 25HC3S calcium of the present disclosure, and / or an effective amount of a pharmaceutical composition comprising the crystalline 25HC3S calcium of the present disclosure.

[0086] Another exemplary inorganic salt of the present disclosure is a metal salt of 25HC3S and zinc. Crystalline zinc 25HC3S can be prepared as described in Example 29. The x-ray powder diffraction pattern of crystalline zinc 25HC3S can be seen in Figure 83, with a magnified view in Figure 84. The x-ray powder diffraction pattern of selected peaks can be seen in Figure 85, with a magnified view in Figure 86. Table 3 shows selected peaks from Figure 85. The zinc salt of 25HC3S was confirmed by ICP-OES to contain two 25HC3S ions for each zinc ion, confirming the presence of 5.72% zinc, which is consistent with the calculated value of 5.98% zinc at 0.5 mol / mol. Therefore, the zinc salt of 25HC3S can also be referred to as a hemi-zinc salt, as in the case of calcium. The zinc salt of 25HC3S contains two 25HC3S ions for each zinc ion, confirming the presence of 5.72% zinc, which is consistent with the calculated value of 5.98% zinc at 0.5 mol / mol. Therefore, the zinc salt of 25HC3S can also be referred to as a hemi-zinc salt, as in the case of calcium. 1 The 1 H-NMR spectrum is consistent with the chemical structure shown in Figure 89.

[0087] [Table 3] TIFF2026501266000008.tif109160

[0088] The two most intense peaks in Figure 85 are those at about 2.1° 2θ and about 2.3° 2θ. In Figure 85, these peaks appear very close together due to their individual intensities, but the XRPD instrument is able to distinguish them. However, due to the variability associated with these XRPD peaks, they may appear "underneath" each other when analyzed as a single peak. Because such a single peak is possible in many instances herein, crystalline zinc-25HC3S is characterized, at least in part, as having an x-ray powder diffraction pattern that includes two peaks between about 2.1° 2θ and about 2.3° 2θ that (a) do not overlap, (b) partially overlap, or (c) overlap so as to appear, for example, as a single peak. To help illustrate other peaks in the pattern, a magnified diffraction pattern can be seen in Figure 86.

[0089] In some cases, the crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 2.1°2θ. In these and other cases, the crystalline zinc-25HC3S can be further characterized by one or more peaks at about 2.3°2θ, about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0090] In some cases, the crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 2.3°2θ. In these and other cases, the crystalline zinc-25HC3S can be further characterized by one or more peaks at about 2.1°2θ, about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0091] In some cases, the crystalline zinc-25HC3S may be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1°2θ and about 2.3°2θ. In these and other cases, the crystalline zinc-25HC3S may be characterized by one or more peaks at about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0092] In some cases, crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1°2θ and about 2.3°2θ, and one peak at about 18.3°2θ. In these and other cases, crystalline zinc-25HC3S can be characterized by one or more peaks at about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, and about 18.8°2θ.

[0093] In some cases, the crystalline zinc-25HC3S may be characterized by an x-ray powder diffraction pattern having a peak at about 6.0°2θ. In these and other cases, the crystalline zinc-25HC3S may be further characterized by one or more peaks at about 2.1°2θ, about 2.3°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0094] In some cases, the crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 8.6°2θ. In these and other cases, the crystalline zinc-25HC3S can be further characterized by one or more peaks at about 2.1°2θ, about 2.3°2θ, about 6.0°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0095] In some instances, the crystalline zinc-25HC3S may be characterized by an x-ray powder diffraction pattern having a peak at about 8.9°2θ. In these and other instances, the crystalline zinc-25HC3S may be further characterized by one or more peaks at about 2.1°2θ, about 2.3°2θ, about 6.0°2θ, about 8.6°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0096] In some cases, the crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 9.3°2θ. In these and other cases, the crystalline zinc-25HC3S can be further characterized by one or more peaks at about 2.1°2θ, about 2.3°2θ, about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0097] In some instances, the crystalline zinc-25HC3S may be characterized by an x-ray powder diffraction pattern having a peak at about 15.1°2θ. In these and other instances, the crystalline zinc-25HC3S may be further characterized by one or more peaks at about 2.1°2θ, about 2.3°2θ, about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 18.3°2θ, and about 18.8°2θ.

[0098] In some instances, the crystalline zinc-25HC3S may be characterized by an x-ray powder diffraction pattern having a peak at about 18.3°2θ. In these and other instances, the crystalline zinc-25HC3S may be further characterized by one or more peaks at about 2.1°2θ, about 2.3°2θ, about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, and about 18.8°2θ.

[0099] In some cases, the crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 18.8°2θ. In these and other cases, the crystalline zinc-25HC3S can be further characterized by one or more peaks at about 2.1°2θ, about 2.3°2θ, about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, and about 18.3°2θ.

[0100] In some cases, the crystalline zinc-25HC3S may be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1°2θ and about 2.3°2θ and one peak at about 6.0°2θ. In these and other cases, the crystalline zinc-25HC3S may be further characterized by one or more peaks at about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0101] In some cases, the crystalline zinc-25HC3S may be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1°2θ and about 2.3°2θ, one peak at about 6.0°2θ, and one peak at about 8.6°2θ. In these and other cases, the crystalline zinc-25HC3S may be further characterized by one or more peaks at about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0102] In some cases, the crystalline zinc-25HC3S may be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1°2θ and about 2.3°2θ, one peak at about 6.0°2θ, one peak at about 8.6°2θ, and one peak at about 9.3°2θ. In these and other cases, the crystalline zinc-25HC3S may be further characterized by one or more peaks at about 8.9°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0103] In some cases, crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1°2θ and about 2.3°2θ, as well as one peak at about 6.0°2θ, one peak at about 8.6°2θ, one peak at about 9.3°2θ, and one peak at about 15.1°2θ. In these and other cases, crystalline zinc-25HC3S can be further characterized by one or more peaks at 8.9°2θ, about 18.3°2θ, and about 18.8°2θ.

[0104] In some cases, the crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1°2θ and about 2.3°2θ, one peak at about 6.0°2θ, one peak at about 8.6°2θ, one peak at about 9.3°2θ, one peak at about 15.1°2θ, and one peak at about 18.3°2θ. In these and other cases, the crystalline zinc-25HC3S can be further characterized by one or more peaks at about 8.9°2θ and about 18.8°2θ.

[0105] In some cases, the crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1°2θ and about 2.3°2θ, one peak at about 6.0°2θ, one peak at about 8.6°2θ, one peak at about 9.3°2θ, one peak at about 15.1°2θ, one peak at about 18.3°2θ, and one peak at about 18.8°2θ. In these and other cases, the crystalline zinc-25HC3S can be further characterized by a peak at about 8.9°2θ.

[0106] In some cases, the crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 2.1°2θ, about 2.3°2θ, about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0107] In some cases, the crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1°2θ and about 2.3°2θ, as well as peaks at about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0108] In some cases, the crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0109] In some cases, the crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0110] In some cases, the crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0111] In some cases, crystalline zinc-25HC3S can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 9.3° 2θ, about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0112] In some cases, crystalline zinc-25HC3S may be characterized by an x-ray powder diffraction pattern having one or more peaks at about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0113] In some cases, crystalline zinc-25HC3S may be characterized by an x-ray powder diffraction pattern having one or more peaks at about 18.3° 2θ and about 18.8° 2θ.

[0114] In some cases, crystalline zinc-25HC3S may be characterized by an x-ray powder diffraction pattern with a peak at about 18.8° 2θ.

[0115] In some cases, crystalline zinc 25HC3S may be characterized by an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern shown in Figure 85 or Figure 86.

[0116] Crystalline zinc 25HC3S may exhibit a differential scanning calorimetry (DSC) thermogram with one or more small endothermic peaks at about 68°C, about 86°C, and about 102°C, as can be seen in Figure 88. Thermal decomposition of crystalline zinc 25HC3S is believed to occur at about 119°C, which can be observed as the large endothermic peak in Figure 88.

[0117] Crystalline zinc 25HC3S can be hydrated. In such cases, the water of hydration can be part of the crystalline structure, thus making the crystalline 25HC3S a hydrate. A sample of crystalline zinc 25HC3S lost approximately 5.1% weight between room temperature and about 130°C, as shown in the TGA thermogram in Figure 88. The weight loss is estimated to be due to water at these temperatures. For the zinc salt, the 5.1% water content is associated with approximately 1.5 moles of water per mole of 25HC3S. The TGA data was calculated based on a zinc 25HC3S salt having one ion of 25HC3S and one half-ion of zinc.

[0118] The DVS data, referring to a single ion of 25HC3S and half ion of the zinc dication per salt molecule, indicates that water uptake may again stabilize at about 2.8% (mass change) between 25% and 45% relative humidity, suggesting a hydrate or monohydrate consisting of 0.75 moles of water per mole of 25HC3S, and at relative humidities higher than 45%, suggesting a sesquihydrate or dihydrate. Thus, in many cases, the level of hydration may average between 0.75 and 2 water molecules (inclusive) per ion of 25HC3S in the crystalline 25HC3S zinc. A weight loss of 11.5% in TGA was also observed between about 131°C and about 190°C, which may be due to decomposition. The XRPD patterns of crystalline zinc before and after DVS are shown in Figure 87. Figure 89 shows the XRPD patterns of crystalline zinc before and after DVS. 1 H-NMR spectrum, which is consistent with the chemical structure.

[0119] Further disclosed is substantially pure crystalline zinc 25HC3S. "Substantially pure," as used herein, generally refers to forms herein present in significant amounts other than possibly trace levels of other forms of zinc 25HC3S. Examples of trace levels include about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less in total, based on the total amount (by weight) of zinc 25HC3S present.

[0120] Methods for preparing zinc 25HC3S are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce zinc 25HC3S, as described in Example 29.

[0121] The triethylammonium salt of 25HC3S can be prepared, for example, by passing a mixture of triethylammonium chloride and triethylamine through a column and treating with a solvent, such as alcohol, until a neutral pH is reached. Separately, crystalline sodium 25HC3S can be dissolved in a solvent, such as alcohol. This solution can then be passed through the same column previously exposed to triethylamine and combined with the triethylammonium solution. The resulting solid can then be isolated, for example, under vacuum or by drying, to yield crystalline triethylammonium salt of 25HC3S, which can be homogenized, for example, with a mortar and pestle. Suitable alcohols for this method include methanol.

[0122] Crystalline zinc-25HC3S, including zinc-25HC3S, may be prepared by starting with sodium-25HC3S, converting it to a second salt of 25HC3S, such as the triethylammonium salt, and then converting the second salt of 25HC3S to zinc-25HC3S, including zinc-25HC3S. Crystalline zinc-25HC3S may be prepared by preparing a suspension of the triethylammonium salt of 25HC3S in a suitable solvent, such as acetonitrile, and treating it with a zinc source, such as zinc chloride in water, to form crystalline zinc-25HC3S. The zinc-25HC3S may be purified, for example, by rinsing with a suitable solvent, such as acetonitrile. Further treatment with a zinc source, such as zinc chloride in water, may be used to improve the yield. Further processing may include stirring and / or further processing with a suitable solvent, such as acetonitrile. Additional processing, such as drying under vacuum, or other methods, may also be performed to aid in the preparation of crystalline zinc-25HC3S. The present disclosure further includes crystalline zinc 25HC3S made by the methods described herein.

[0123] The present disclosure also relates to pharmaceutical compositions containing zinc 25HC3S, including the crystalline zinc 25HC3S disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and zinc 25HC3S, including crystalline zinc 25HC3S. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, such as parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral formulations.

[0124] As discussed elsewhere herein, the 25HC3S zinc of the present disclosure advantageously provides supplemental zinc to patients suffering from conditions targeted by 25HC3S. As discussed elsewhere herein, zinc deficiency may contribute to these conditions, and it may be advantageous to provide zinc simultaneously with 25HC3S during the course of treatment. Thus, 25HC3S zinc surprisingly and advantageously combines advantageous salt / crystal form properties with the inherent ability to advantageously provide zinc supplementation during the course of treatment.

[0125] The present disclosure further includes methods and uses for treating and / or preventing one or more of diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3S zinc, including the crystalline 25HC3S zinc of the present disclosure, and / or an effective amount of a pharmaceutical composition, including the crystalline 25HC3S zinc of the present disclosure.

[0126] Another exemplary inorganic salt of the present disclosure is a metal salt of 25HC3S and magnesium. Crystalline 25HC3S magnesium can be prepared as described in Example 30. The x-ray powder diffraction pattern of crystalline 25HC3S magnesium can be seen in Figure 5, with Figure 5A being a magnified version. The x-ray powder diffraction pattern of selected peaks can be seen in Figure 6, with Figure 6A being a magnified version. Table 4 lists selected peaks from Figure 6. The magnesium salt contains two ions of 25HC3S for each magnesium ion, as confirmed by ICP-EOS. Therefore, the magnesium salt can also be referred to as a hemimagnesium salt.

[0127] [Table 4]

[0128] Crystalline 25HC3S magnesium can be characterized by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S magnesium, or a portion thereof, can be used to identify the crystalline 25HC3S magnesium. Crystalline 25HC3S magnesium contains various x-ray powder diffraction peaks, which, alone or together, can help identify the presence of crystalline 25HC3S magnesium.

[0129] In some cases, crystalline 25HC3S magnesium may be characterized by an x-ray powder diffraction pattern having one or more peaks in Figure 6. For example, a peak at about 2.2°2θ can be used to characterize crystalline 25HC3S magnesium. In these and other cases, crystalline 25HC3S magnesium may be further characterized by one or more peaks at about 4.4°2θ, about 6.6°2θ, about 8.9°2θ, and about 15.1°2θ. In some cases, crystalline 25HC3S magnesium may be characterized by an x-ray powder diffraction pattern having a peak at about 4.4°2θ. In these and other cases, crystalline 25HC3S calcium may be further characterized by one or more peaks at about 2.2°2θ, about 6.6°2θ, about 8.9°2θ, and about 15.1°2θ.

[0130] In some cases, crystalline magnesium-25HC3S may be characterized by an x-ray powder diffraction pattern having a peak at about 6.6°2θ. In these and other cases, crystalline calcium-25HC3S may be further characterized by one or more peaks at about 2.2°2θ, about 4.4°2θ, about 8.9°2θ, and about 15.1°2θ.

[0131] In some cases, crystalline magnesium-25HC3S may be characterized by an x-ray powder diffraction pattern having a peak at about 8.9°2θ. In these and other cases, crystalline calcium-25HC3S may be further characterized by one or more peaks at about 2.2°2θ, about 4.4°2θ, about 6.6°2θ, and about 15.1°2θ.

[0132] In some cases, crystalline magnesium-25HC3S may be characterized by an x-ray powder diffraction pattern having a peak at about 15.1°2θ. In these and other cases, crystalline calcium-25HC3S may be further characterized by one or more peaks at about 2.2°2θ, about 4.4°2θ, about 6.6°2θ, and about 8.9°2θ.

[0133] In some cases, crystalline 25HC3S magnesium may be further characterized by one or more peaks at about 15.6°2θ, 16.4°2θ, 17.6°2θ, and about 17.8°2θ. For example, crystalline 25HC3S magnesium may be further characterized by a peak at about 15.6°2θ. Alternatively or additionally, crystalline 25HC3S magnesium may be further characterized by a peak at about 16.4°2θ. Alternatively or additionally, crystalline 25HC3S magnesium may be further characterized by a peak at about 17.6°2θ. Alternatively or additionally, crystalline 25HC3S magnesium may be further characterized by a peak at about 17.8°2θ.

[0134] Crystalline 25HC3S magnesium may be characterized by an x-ray powder diffraction pattern having substantially the same diffraction pattern as Figure 6 or Figure 6A.

[0135] Crystalline magnesium-25HC3S can occur as a hydrate. The weight of a sample of crystalline magnesium-25HC3S lost approximately 1.5% by 127°C, as shown in the TGA thermogram in Figure 33. The weight loss is estimated to be due to water at these temperatures. For magnesium salts, a 1.5% water loss corresponds to approximately 0.5 moles of water per mole of 25HC3S. The plateau-like region in the DVS of Figure 34 for crystalline magnesium-25HC3S at approximately 5% to 7% mass change suggests that higher hydrates, such as sesquihydrates or dihydrates, of 1.5 to 2 moles of water per mole of 25HC3S are available. Thus, this data indicates that crystalline magnesium-25HC3S can exist as a hydrate, such as a hemihydrate, sesquihydrate, or dihydrate, when referring to a single ion of 25HC3S and a half ion of the magnesium dication per salt molecule. The x-ray powder diffraction patterns before and after DVS are shown in Figure 35. The H-NMR spectrum in Figure 36 is consistent with the chemical structure of 25HC3S magnesium. The DSC thermogram shows an endothermic peak at approximately 139 °C, which is believed to be due to decomposition.

[0136] Further disclosed is substantially pure crystalline magnesium 25HC3S. "Substantially pure," as used herein, generally refers to forms herein present in significant amounts other than possibly trace levels of other forms of magnesium 25HC3S. Examples of trace levels include about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less in total, based on the total amount (by weight) of magnesium 25HC3S present.

[0137] Methods for preparing magnesium 25HC3S are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce magnesium 25HC3S, as described in Example 30.

[0138] The present disclosure also relates to pharmaceutical compositions containing 25HC3S magnesium, including the crystalline 25HC3S magnesium disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S magnesium, including crystalline 25HC3S magnesium. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, for example, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral formulations.

[0139] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans), such as one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3 magnesium, including the crystalline 25HC3 magnesium of the present disclosure, and / or an effective amount of a pharmaceutical composition comprising the crystalline 25HC3 magnesium of the present disclosure.

[0140] Another exemplary organic salt of 25HC3S of the present disclosure is an organic amine hydroxyethylammonium salt of 25HC3S. The preparation of crystalline hydroxyethylammonium salt of 25HC3S is found in Example 31. The x-ray powder diffraction pattern of crystalline hydroxyethylammonium 25HC3S can be seen in Figure 7, with Figure 7A being a magnified version of the pattern. The x-ray powder diffraction pattern of selected peaks can be seen in Figure 8, with Figure 8A being a magnified version of the pattern. Table 5 lists selected peaks from Figure 8.

[0141] [Table 5]

[0142] Crystalline 25HC3S hydroxyethylammonium can be characterized by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium, or a portion thereof, can be used to identify the crystalline 25HC3S hydroxyethylammonium. Crystalline 25HC3S hydroxyethylammonium contains various x-ray powder diffraction peaks, which, alone or together, can help identify the presence of crystalline 25HC3S hydroxyethylammonium.

[0143] In some cases, crystalline 25HC3S hydroxyethylammonium may be characterized by an x-ray powder diffraction pattern having one or more peaks in Figure 8. For example, a peak at about 2.1 degrees 2θ may be used to characterize crystalline 25HC3S hydroxyethylammonium. In these and other cases, crystalline 25HC3S hydroxyethylammonium may be further characterized by a peak at about 8.6 degrees 2θ.

[0144] Crystalline 25HC3S hydroxyethylammonium is hygroscopic as determined by DVS in Figure 38. Crystalline 25HC3S hydroxyethylammonium exhibits a weight loss of about 2.7% from ambient temperature to 134°C by TGA in Figure 37. 1The H-NMR spectrum shows isopropyl alcohol in solution, consistent with the salt preparation according to Example 31, in which the salt was prepared with isopropyl alcohol. Therefore, without being bound by theory, it is believed that the crystalline 25HC3S hydroxyethylammonium salt may exist as an isopropyl alcohol solvate. The x-ray analysis after DVS in Figure 39 also shows the appearance of additional x-ray powder diffraction peaks, which may indicate further physical transformation. The DSC of crystalline 25HC3S hydroxyethylammonium can be seen in Figure 37 and shows multiple endotherms.

[0145] Further disclosed is a substantially pure crystalline 25HC3S hydroxyethylammonium. "Substantially pure," as used herein, generally refers to forms herein present in significant amounts other than possibly trace levels of other forms of 25HC3S hydroxyethylammonium. Examples of trace levels include a total of about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less, based on the total amount (by weight) of 25HC3S hydroxyethylammonium present.

[0146] Methods for preparing hydroxyethylammonium 25HC3S are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce hydroxyethylammonium 25HC3S, as described in Example 31.

[0147] The present disclosure also relates to pharmaceutical compositions containing 25HC3S hydroxyethylammonium, including the crystalline 25HC3S hydroxyethylammonium disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S hydroxyethylammonium, including the crystalline 25HC3S hydroxyethylammonium. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, for example, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral formulations.

[0148] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans), such as one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3S hydroxyethylammonium, including the crystalline 25HC3S hydroxyethylammonium of the present disclosure, and / or an effective amount of a pharmaceutical composition comprising the crystalline 25HC3S hydroxyethylammonium of the present disclosure.

[0149] An exemplary organic salt of 25HC3S of the present disclosure is the organic amine tromethammonium salt of 25HC3S. The preparation of crystalline tromethammonium salt of 25HC3S is found in Example 32. The x-ray powder diffraction pattern of crystalline 25HC3S tromethammonium can be seen in Figure 13, with Figure 13A being a magnified version of the pattern. The x-ray powder diffraction pattern of selected peaks can be seen in Figure 14, with Figure 14A being a magnified version of the pattern. Table 6 lists selected peaks from Figure 14.

[0150] [Table 6] TIFF2026501266000012.tif116160

[0151] Crystalline 25HC3S trometaammonium can be characterized by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S trometaammonium, or a portion thereof, can be used to identify crystalline 25HC3S trometaammonium. Crystalline 25HC3S trometaammonium contains various x-ray powder diffraction peaks, which, alone or together, can assist in identifying the presence of crystalline 25HC3S trometaammonium.

[0152] In some cases, crystalline 25HC3S trometaammonium may be characterized by an x-ray powder diffraction pattern having one or more peaks in Figure 14 or Figure 14A. For example, a peak at about 1.9°2θ may be used to characterize crystalline 25HC3S trometaammonium. In these and other cases, crystalline 25HC3S trometaammonium may be further characterized by a peak at about 2.1°2θ or about 3.8°2θ.

[0153] The two most intense peaks in Figure 14 are those at about 1.9°2θ and about 2.1°2θ. In Figure 14, these peaks appear very close together due to their individual intensities, but the XRPD instrument is able to distinguish them. However, due to the variability associated with these XRPD peaks, they may appear "underneath" each other when analyzed as a single peak. Because such a single-appearing peak is possible in many instances herein, crystalline 25HC3S trometaammonium is characterized, at least in part, as having an x-ray powder diffraction pattern that includes two peaks between about 1.9°2θ and about 2.1°2θ that are (a) non-overlapping, (b) partially overlapping, or (c) overlapping, e.g., to appear as a single peak. In some instances, crystalline 25HC3S trometaammonium may be characterized by an x-ray powder diffraction pattern with a peak at about 2.1°2θ. In these and other cases, the crystalline calcium 25HC3S may be further characterized by one or more peaks at about 1.9° 2θ and about 3.8° 2θ.

[0154] In some cases, crystalline 25HC3S trometaammonium may be characterized by an x-ray powder diffraction pattern having a peak at about 3.8° 2θ. In these and other cases, crystalline 25HC3S calcium may be further characterized by one or more peaks at about 1.9° 2θ and about 2.1° 2θ.

[0155] In some cases, crystalline 25HC3S trometaammonium may be further characterized by one or more peaks at about 4.2°2θ and about 15.4°2θ. For example, crystalline 25HC3S trometaammonium may be further characterized by a peak at about 4.2°2θ. Alternatively, or in addition, crystalline 25HC3S trometaammonium may be further characterized by a peak at about 15.4°2θ.

[0156] In some cases, crystalline 25HC3S trometaammonium can be characterized by an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of Figure 14 or Figure 14A.

[0157] Crystalline 25HC3S trometaammonium is hygroscopic, as seen by DVS in Figure 51. In fact, crystalline 25HC3S trometaammonium takes up more than 20% water by weight when exposed to approximately 95% relative humidity. By TGA in Figure 50, only a weight loss of approximately 0.3% was observed up to approximately 80°C, suggesting an anhydrous form under ambient conditions. Furthermore, the x-ray powder diffraction patterns before and after DVS are presented in Figure 52, showing some potential difference that may be due to its hygroscopic nature. The DSC of crystalline 25HC3S trometaammonium can be seen in Figure 50, showing several small endotherms at approximately 54°C, approximately 114°C, and approximately 158°C, followed by decomposition at approximately 184°C. In Figure 53, 1 The H-NMR structure is consistent with the chemical structure.

[0158] Further disclosed is a substantially pure crystalline 25HC3S trometaammonium. "Substantially pure," as used herein, generally refers to forms herein present in significant amounts other than possibly trace levels of other forms of 25HC3S trometaammonium. Examples of trace levels include a total of about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less, based on the total amount (by weight) of 25HC3S trometaammonium present.

[0159] Methods for preparing 25HC3S trometaammonium salt are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce 25HC3S trometaammonium salt, as described in Example 32.

[0160] The present disclosure also relates to pharmaceutical compositions containing 25HC3S trometaammonium, including the crystalline 25HC3S trometaammonium disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S trometaammonium, including the crystalline 25HC3S trometaammonium. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, for example, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral formulations.

[0161] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans), such as one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3S trometaammonium, including the crystalline 25HC3S trometaammonium of the present disclosure, and / or an effective amount of a pharmaceutical composition, including the crystalline 25HC3S trometaammonium of the present disclosure.

[0162] An exemplary organic salt of 25HC3S of the present disclosure is the organic amino acid lysine salt of 25HC3S. Preparation of the crystalline l-lysine salt of 25HC3S is found in Example 33. The x-ray powder diffraction pattern of crystalline 25HC3S lysine can be seen in Figure 23, with Figure 23A being a magnified version of the pattern. The x-ray powder diffraction pattern of selected peaks can be seen in Figure 24, with Figure 24A being a magnified version of the pattern. Table 7 lists selected peaks from Figure 24.

[0163] [Table 7] TIFF2026501266000014.tif54161

[0164] Crystalline 25HC3S lysine can be characterized by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S lysine, or a portion thereof, can be used to identify the crystalline 25HC3S lysine. Crystalline 25HC3S lysine contains various x-ray powder diffraction peaks, which, alone or together, can help identify the presence of crystalline 25HC3S lysine. In some cases, crystalline 25HC3S lysine can be characterized by an x-ray powder diffraction pattern having one or more peaks in Figure 24. For example, a peak at about 1.5° 2θ can be used to characterize crystalline 25HC3S lysine. In these and other cases, crystalline 25HC3S lysine can be further characterized by one or more peaks at about 7.0° 2θ, about 10.7° 2θ, about 11.8° 2θ, and about 16.8° 2θ.

[0165] In some cases, the crystalline 25HC3S lysine may be characterized by an x-ray powder diffraction pattern having a peak at about 7.0°2θ. In these and other cases, the crystalline 25HC3S lysine may be further characterized by one or more peaks at about 1.5°2θ, about 10.7°2θ, about 11.8°2θ, and about 16.8°2θ.

[0166] In some cases, the crystalline 25HC3S lysine may be characterized by an x-ray powder diffraction pattern having a peak at about 10.7°2θ. In these and other cases, the crystalline 25HC3S lysine may be further characterized by one or more peaks at about 1.5°2θ, about 7.0°2θ, about 11.8°2θ, and about 16.8°2θ.

[0167] In some cases, the crystalline 25HC3S lysine may be characterized by an x-ray powder diffraction pattern having a peak at about 11.8°2θ. In these and other cases, the crystalline 25HC3S lysine may be further characterized by one or more peaks at about 1.5°2θ, about 7.0°2θ, about 10.7°2θ, and about 16.8°2θ.

[0168] In some cases, the crystalline 25HC3S lysine may be characterized by an x-ray powder diffraction pattern having a peak at about 16.8°2θ. In these and other cases, the crystalline 25HC3S lysine may be further characterized by one or more peaks at about 1.5°2θ, about 7.0°2θ, about 10.7°2θ, and about 11.8°2θ.

[0169] In some cases, crystalline 25HC3S lysine may be further characterized by one or more peaks at about 3.2°2θ, about 10.0°2θ, about 12.2°2θ, and about 15.2°2θ. For example, crystalline 25HC3S lysine may be further characterized by a peak at about 3.2°2θ. Alternatively or additionally, crystalline 25HC3S lysine may be further characterized by a peak at about 10.0°2θ. Alternatively or additionally, crystalline 25HC3S lysine may be further characterized by a peak at about 12.2°2θ. Alternatively or additionally, crystalline 25HC3S lysine may be further characterized by a peak at about 15.2°2θ.

[0170] In some cases, the crystalline 25HC3S lysine may be characterized by an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of FIG.

[0171] Crystalline 25HC3S lysine is hygroscopic at relative humidities above 75%, as seen by the DVS in Figure 74. As can be seen in Figure 73, the TGA shows a weight loss of about 1.5% up to 100°C, and a further weight loss of about 8.8% between 100°C and 235°C, some of which may be due to water loss and some to decomposition. Therefore, it is believed that crystalline 25HC3S lysine exists as a hydrate. The DSC of crystalline 25HC3S lysine can be seen in Figure 73, and shows a weak endotherm at about 96°C. The endotherm at about 186°C is believed to indicate decomposition. In Figure 75 1 The H-NMR spectrum is consistent with the chemical structure.

[0172] Further disclosed is a substantially pure crystalline 25HC3S lysine. "Substantially pure," as used herein, generally refers to a form herein present in a form that is free of significant amounts, possibly other than trace levels of other forms of 25HC3S lysine. Examples of trace levels include a total of about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less, based on the total amount of 25HC3S lysine present (by weight).

[0173] Methods for preparing 25HC3S lysine are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce 25HC3S lysine, as described in Example 33.

[0174] The present disclosure also relates to pharmaceutical compositions containing 25HC3S lysine, including the crystalline 25HC3S lysine disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S lysine, including crystalline 25HC3S lysine. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, for example, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral formulations.

[0175] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans), such as one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3S lysine, including the crystalline 25HC3S lysine of the present disclosure, and / or an effective amount of a pharmaceutical composition comprising the crystalline 25HC3S lysine of the present disclosure.

[0176] An exemplary organic salt of 25HC3S of the present disclosure is the organic amine meglumine salt of 25HC3S. Preparation of the crystalline meglumine salt of 25HC3S is found in Example 34. The x-ray powder diffraction pattern of crystalline 25HC3S meglumine can be seen in Figure 11, with Figure 11A being a magnified version of the pattern. The x-ray powder diffraction pattern of selected peaks can be seen in Figure 12, with Figure 12A being a magnified version of the pattern. Table 8 lists selected peaks from Figure 12.

[0177] [Table 8] TIFF2026501266000016.tif29161

[0178] Crystalline 25HC3S meglumine can be characterized by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S meglumine, or a portion thereof, can be used to identify crystalline 25HC3S meglumine. Crystalline 25HC3S meglumine contains various x-ray powder diffraction peaks, which, alone or together, can help identify the presence of crystalline 25HC3S meglumine.

[0179] In some instances, crystalline 25HC3S meglumine may be characterized by an x-ray powder diffraction pattern having one or more peaks in Figure 12. For example, a peak at about 1.7°2θ can be used to characterize crystalline 25HC3S meglumine. In these and other instances, crystalline 25HC3S meglumine may be further characterized by one or more peaks at about 3.5°2θ, about 5.2°2θ, about 14.9°2θ, and about 24.2°2θ.

[0180] In some instances, crystalline 25HC3S meglumine may be characterized by an x-ray powder diffraction pattern having a peak at about 3.5°2θ. In these and other instances, crystalline 25HC3S meglumine may be further characterized by one or more peaks at about 1.7°2θ, about 5.2°2θ, about 14.9°2θ, and about 24.2°2θ.

[0181] In some instances, crystalline 25HC3S meglumine may be characterized by an x-ray powder diffraction pattern having a peak at about 5.2°2θ. In these and other instances, crystalline 25HC3S meglumine may be further characterized by one or more peaks at about 1.7°2θ, about 3.5°2θ, about 14.9°2θ, and about 24.2°2θ.

[0182] In some instances, crystalline 25HC3S meglumine may be characterized by an x-ray powder diffraction pattern having a peak at about 14.9°2θ. In these and other instances, crystalline 25HC3S meglumine may be further characterized by one or more peaks at about 1.7°2θ, about 3.5°2θ, about 5.2°2θ, and about 24.2°2θ.

[0183] In some instances, crystalline 25HC3S meglumine may be characterized by an x-ray powder diffraction pattern having a peak at about 24.2°2θ. In these and other instances, crystalline 25HC3S meglumine may be further characterized by one or more peaks at about 1.7°2θ, about 3.5°2θ, about 5.2°2θ, and about 14.9°2θ.

[0184] In some cases, crystalline 25HC3S meglumine may be further characterized by one or more peaks at about 8.6°2θ, about 14.5°2θ, about 15.1°2θ, about 17.5°2θ, and about 18.2°2θ. For example, crystalline 25HC3S meglumine may be further characterized by a peak at about 8.6°2θ. Alternatively or additionally, crystalline 25HC3S meglumine may be further characterized by a peak at about 14.5°2θ. Alternatively or additionally, crystalline 25HC3S meglumine may be further characterized by a peak at about 15.1°2θ. Alternatively or additionally, crystalline 25HC3S meglumine may be further characterized by a peak at about 17.5°2θ. Alternatively or additionally, crystalline 25HC3S meglumine may be further characterized by a peak at about 18.2°2θ.

[0185] In some cases, the crystalline 25HC3S meglumine can be characterized by an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of Figure 12 or Figure 12A.

[0186] Crystalline 25HC3S meglumine takes up water in a DVS experiment, as can be seen in Figure 47, showing approximately 3% water uptake, with a stable plateau at about that level of water uptake at relative humidities above 35%. This level of water uptake is consistent with the formation of a monohydrate. TGA shows a loss of approximately 3.2% water loss starting from ambient, which is also consistent with a monohydrate. X-ray powder diffraction patterns before and after DVS are attached as Figure 48. The DSC of crystalline 25HC3S meglumine can be seen in Figure 46, showing several weak endotherms between 50°C and 90°C. The solution state of this salt 1 The H-NMR spectrum shows several additional peaks between 4.3 and 5.5 ppm, others consistent with the chemical structure.

[0187] Further disclosed is a substantially pure crystalline 25HC3S meglumine. "Substantially pure," as used herein, generally refers to forms herein that are present in significant amounts other than possibly trace levels of other forms of 25HC3S meglumine. Examples of trace levels include less than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less in total, based on the total amount of 25HC3S meglumine present (by weight).

[0188] Methods for preparing 25HC3S meglumine are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce 25HC3S meglumine, as described in Example 34.

[0189] The present disclosure also relates to pharmaceutical compositions containing 25HC3S meglumine, including the crystalline 25HC3S meglumine disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S meglumine, including crystalline 25HC3S meglumine. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, such as parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral formulations.

[0190] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans), such as one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3S meglumine, including the crystalline 25HC3S meglumine of the present disclosure, and / or an effective amount of a pharmaceutical composition, including the crystalline 25HC3S meglumine of the present disclosure.

[0191] An exemplary organic salt of 25HC3S of the present disclosure is the organic cyclic amine hydroxyethylpyrrolidinium salt of 25HC3S. The preparation of the crystalline hydroxyethylpyrrolidinium salt of 25HC3S is found in Example 35. The x-ray powder diffraction pattern of crystalline hydroxyethylpyrrolidinium 25HC3S can be seen in Figure 9. The x-ray powder diffraction pattern of selected peaks can be seen in Figure 10. Table 9 lists selected peaks from Figure 10.

[0192] [Table 9] TIFF2026501266000018.tif139162

[0193] Crystalline 25HC3S hydroxyethylpyrrolidinium can be characterized by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylpyrrolidinium, or a portion thereof, can be used to identify the crystalline 25HC3S hydroxyethylpyrrolidinium. Crystalline 25HC3S hydroxyethylpyrrolidinium contains various x-ray powder diffraction peaks, which, alone or together, can help identify the presence of crystalline 25HC3S hydroxyethylpyrrolidinium.

[0194] In some instances, crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction pattern having one or more peaks in Figure 10. For example, a peak at about 3.8°2θ can be used to characterize crystalline 25HC3S hydroxyethylpyrrolidinium. In these and other instances, crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by one or more peaks at about 7.5°2θ, about 7.6°2θ, about 8.2°2θ, about 8.6°2θ, about 12.4°2θ, about 13.3°2θ, and about 15.0°2θ.

[0195] Two of the most intense peaks in Figure 10 are those at about 7.5°2θ and about 7.6°2θ. In Figure 10, these peaks appear very close together due to their individual intensities, but the XRPD instrument is able to distinguish them. However, due to variability associated with these XRPD peaks, they may appear "underneath" each other when analyzed as a single peak. Because such a single-appearing peak is possible in many instances herein, crystalline 25HC3S hydroxyethylpyrrolidinium is characterized, at least in part, as having an x-ray powder diffraction pattern comprising two peaks between about 7.5°2θ and about 7.6°2θ that are (a) non-overlapping, (b) partially overlapping, or (c) overlapping so as to appear, for example, as a single peak.

[0196] Two of the most intense peaks in Figure 10 are those at about 8.2°2θ and about 8.6°2θ. In Figure 10, these peaks appear very close together due to their individual intensities, but the XRPD instrument is able to distinguish them. However, due to variability associated with these XRPD peaks, they may appear "underneath" each other when analyzed as a single peak. Because such a single-appearing peak is possible in many instances herein, crystalline 25HC3S hydroxyethylpyrrolidinium is characterized, at least in part, as having an x-ray powder diffraction pattern comprising two peaks between about 8.2°2θ and about 8.6°2θ that are (a) non-overlapping, (b) partially overlapping, or (c) overlapping so as to appear, for example, as a single peak.

[0197] In some instances, the crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction pattern having a peak at about 7.5°2θ. In these and other instances, the crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by one or more peaks at about 3.8°2θ, about 7.6°2θ, about 8.2°2θ, about 8.6°2θ, about 12.4°2θ, about 13.3°2θ, and about 15.0°2θ.

[0198] In some instances, the crystalline 25HC3S hydroxyethylpyrrolidinium can be characterized by an x-ray powder diffraction pattern having a peak at about 7.6°2θ. In these and other instances, the crystalline 25HC3S hydroxyethylpyrrolidinium can be further characterized by one or more peaks at about 3.8°2θ, about 7.5°2θ, about 8.2°2θ, about 8.6°2θ, about 12.4°2θ, about 13.3°2θ, and about 15.0°2θ.

[0199] In some instances, the crystalline 25HC3S hydroxyethylpyrrolidinium can be characterized by an x-ray powder diffraction pattern having a peak at about 8.2°2θ. In these and other instances, the crystalline 25HC3S hydroxyethylpyrrolidinium can be further characterized by one or more peaks at about 3.8°2θ, about 7.5°2θ, about 7.6°2θ, about 8.6°2θ, about 12.4°2θ, about 13.3°2θ, and about 15.0°2θ.

[0200] In some instances, the crystalline 25HC3S hydroxyethylpyrrolidinium can be characterized by an x-ray powder diffraction pattern having a peak at about 8.6°2θ. In these and other instances, the crystalline 25HC3S hydroxyethylpyrrolidinium can be further characterized by one or more peaks at about 3.8°2θ, about 7.5°2θ, about 7.6°2θ, about 8.2°2θ, about 12.4°2θ, about 13.3°2θ, and about 15.0°2θ.

[0201] In some instances, the crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction pattern having a peak at 12.4°2θ. In these and other instances, the crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by one or more peaks at about 3.8°2θ, about 7.5°2θ, about 7.6°2θ, about 8.2°2θ, about 8.6°2θ, about 13.3°2θ, and about 15.0°2θ.

[0202] In some instances, the crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction pattern having a peak at 13.3°2θ. In these and other instances, the crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by one or more peaks at about 3.8°2θ, about 7.5°2θ, about 7.6°2θ, about 8.2°2θ, about 8.6°2θ, about 12.4°2θ, and about 15.0°2θ.

[0203] In some instances, the crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction pattern having a peak at 15.0°2θ. In these and other instances, the crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by one or more peaks at about 3.8°2θ, about 7.5°2θ, about 7.6°2θ, about 8.2°2θ, about 8.6°2θ, about 12.4°2θ, and about 13.3°2θ. In some instances, the crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of FIG. 10.

[0204] In some cases, crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by one or more peaks at about 10.5°2θ, about 15.3°2θ, about 15.6°2θ, about 16.3°2θ, about 16.7°2θ, and about 20.9°2θ. For example, crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by a peak at about 10.5°2θ. Alternatively or additionally, crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by a peak at about 15.3°2θ. Alternatively or additionally, crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by a peak at about 15.6°2θ. Alternatively or additionally, crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by a peak at about 16.3°2θ. Alternatively or additionally, the crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by a peak at about 16.7° 2θ. Alternatively or additionally, the crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by a peak at about 20.9° 2θ.

[0205] Crystalline 25HC3S hydroxyethylpyrrolidinium takes up and retains water, as seen in DVS experiments. The stable plateau in DVS in Figure 42 indicates a water content between about 2.5% and about 6% between about 25% relative humidity and about 75% relative humidity. These data suggest that crystalline 25HC3S hydroxyethylpyrrolidinium may convert to a hydrate at such relative humidities, including levels that would be consistent with a monohydrate. Upon heating to 181°C, weight loss was limited to about 0.1%, as seen by TGA in Figure 41, suggesting that this salt is anhydrous at ambient conditions and therefore converts to a hydrate upon exposure to the appropriate relative humidity by DVS. After the DVS experiment in Figure 42, the XRPD pattern seen in Figure 43 differs from the previous one, which is also consistent with a form change, for example, from anhydrous to hydrate. The solution in Figure 44 1 H-NMR reveals the presence of an extra proton at 5.3 ppm that is not accounted for in the structure, but otherwise the spectrum is consistent with the chemical structure. Therefore, without being bound by theory, it is believed that crystalline 25HC3S hydroxyethylpyrrolidinium may exist as both an anhydrate and a hydrate, e.g., a monohydrate, at the appropriate relative humidity. The DSC of crystalline 25HC3S hydroxyethylpyrrolidinium can be seen in Figure 41, and shows a weak endotherm at about 40°C and another endotherm at about 140°C, followed presumably by decomposition at about 181°C.

[0206] The x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylpyrrolidinium was successfully indexed, showing that the pattern represents a single crystalline phase with the results shown in Figure 45. The indexing results reveal that the crystalline 25HC3S hydroxyethylpyrrolidinium has a triclinic lattice with a lattice volume consistent with the anhydrous form and the proposed salt stoichiometry. The formula volume is 3416.8 Å. 3 / lattice (±5%), which can accommodate 1 mole of hydroxyethylpyrrolidinium. Some lattice parameters are shown in Table 10 below.

[0207] [Table 10]

[0208] Further disclosed is a substantially pure crystalline 25HC3S hydroxyethylpyrrolidinium. "Substantially pure," as used herein, generally refers to a form herein present in a form that is present in a significant amount other than possibly trace levels of other forms of 25HC3S hydroxyethylpyrrolidinium. Examples of trace levels include a total of about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less, based on the total amount (by weight) of 25HC3S hydroxyethylpyrrolidinium present.

[0209] Methods for preparing 25HC3S hydroxyethylpyrrolidinium are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce 25HC3S hydroxyethylpyrrolidinium, as described in Example 35.

[0210] The present disclosure also relates to pharmaceutical compositions containing 25HC3S hydroxyethylpyrrolidinium, including the crystalline 25HC3S hydroxyethylpyrrolidinium disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S hydroxyethylpyrrolidinium, including crystalline 25HC3S hydroxyethylpyrrolidinium. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, such as parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral formulations.

[0211] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans), such as one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3S hydroxyethylpyrrolidinium, including the crystalline 25HC3S hydroxyethylpyrrolidinium of the present disclosure, and / or an effective amount of a pharmaceutical composition, including the crystalline 25HC3S hydroxyethylpyrrolidinium of the present disclosure.

[0212] An exemplary organic salt of 25HC3S of the present disclosure is the organic aliphatic amine diethylammonium salt of 25HC3S. The preparation of the crystalline diethylammonium salt of 25HC3S can be found in Example 36. The x-ray powder diffraction pattern of crystalline diethylammonium 25HC3S can be found in Figure 17. The x-ray powder diffraction pattern of selected peaks can be found in Figure 18. Table 11 shows selected peaks from Figure 18.

[0213] [Table 11] TIFF2026501266000021.tif146160

[0214] Crystalline 25HC3S diethylammonium can be characterized by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S diethylammonium, or a portion thereof, can be used to identify the crystalline 25HC3S diethylammonium. Crystalline 25HC3S diethylammonium contains various x-ray powder diffraction peaks, which, alone or together, can help identify the presence of crystalline 25HC3S diethylammonium.

[0215] In some cases, crystalline 25HC3S diethylammonium may be characterized by an x-ray powder diffraction pattern having one or more peaks in Figure 18. For example, a peak at about 3.8°2θ may be used to characterize crystalline 25HC3S diethylammonium. In these and other cases, crystalline 25HC3S diethylammonium may be further characterized by one or more peaks at about 7.9°2θ, about 8.6°2θ, about 9.6°2θ, about 10.9°2θ, about 12.3°2θ, about 15.4°2θ, and about 17.2°2θ.

[0216] In some instances, the crystalline 25HC3S diethylammonium may be characterized by an x-ray powder diffraction pattern having a peak at about 7.9°2θ. In these and other instances, the crystalline 25HC3S diethylammonium may be further characterized by one or more peaks at about 3.8°2θ, about 8.6°2θ, about 9.6°2θ, about 10.9°2θ, about 12.3°2θ, about 15.4°2θ, and about 17.2°2θ.

[0217] In some instances, the crystalline 25HC3S diethylammonium may be characterized by an x-ray powder diffraction pattern having a peak at about 8.6°2θ. In these and other instances, the crystalline 25HC3S diethylammonium may be further characterized by one or more peaks at about 3.8°2θ, about 7.9°2θ, about 8.6°2θ, about 9.6°2θ, about 12.3°2θ, about 15.4°2θ, and about 17.2°2θ.

[0218] In some instances, the crystalline 25HC3S diethylammonium may be characterized by an x-ray powder diffraction pattern having a peak at about 9.6°2θ. In these and other instances, the crystalline 25HC3S diethylammonium may be further characterized by one or more peaks at about 3.8°2θ, about 7.9°2θ, about 8.6°2θ, about 10.9°2θ, about 12.3°2θ, about 15.4°2θ, and about 17.2°2θ.

[0219] In some instances, the crystalline 25HC3S diethylammonium may be characterized by an x-ray powder diffraction pattern having a peak at about 10.9°2θ. In these and other instances, the crystalline 25HC3S diethylammonium may be further characterized by one or more peaks at about 3.8°2θ, about 7.9°2θ, about 8.6°2θ, about 9.6°2θ, about 12.3°2θ, about 15.4°2θ, and about 17.2°2θ.

[0220] In some cases, the crystalline 25HC3S diethylammonium may be characterized by an x-ray powder diffraction pattern having a peak at 12.3°2θ. In these and other cases, the crystalline 25HC3S diethylammonium may be further characterized by one or more peaks at about 3.8°2θ, about 7.9°2θ, about 8.6°2θ, about 9.6°2θ, about 10.9°2θ, about 12.3°2θ, and about 15.4°2θ, and about 17.2°2θ.

[0221] In some instances, the crystalline 25HC3S diethylammonium may be characterized by an x-ray powder diffraction pattern having a peak at 15.4°2θ. In these and other instances, the crystalline 25HC3S diethylammonium may be further characterized by one or more peaks at about 3.8°2θ, about 7.9°2θ, about 8.6°2θ, about 9.6°2θ, about 10.9°2θ, about 12.3°2θ, and about 17.2°2θ.

[0222] In some instances, the crystalline 25HC3S diethylammonium may be characterized by an x-ray powder diffraction pattern having a peak at 17.2°2θ. In these and other instances, the crystalline 25HC3S diethylammonium may be further characterized by one or more peaks at about 3.8°2θ, about 7.9°2θ, about 8.6°2θ, about 9.6°2θ, about 10.9°2θ, about 12.3°2θ, and about 15.4°2θ.

[0223] In some cases, crystalline diethylammonium 25HC3S can be characterized by an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of FIG.

[0224] Crystalline 25HC3S diethylammonium takes up water in a DVS experiment, as can be seen in Figure 60, indicating that it is hygroscopic at relative humidities above 85%. As can be seen by TGA in Figure 59, upon heating to 204°C, weight loss is limited to about 0.1%, suggesting an anhydrous structure at ambient conditions. The DSC of crystalline 25HC3S diethylammonium can be seen in Figure 59 and shows an endotherm at about 134°C and an endotherm at about 215°C. The x-ray powder diffraction patterns before and after DVS are shown in Figure 61 and in Figure 62. 1 The H-NMR spectrum is consistent with the chemical structure.

[0225] The x-ray powder diffraction pattern of crystalline 25HC3S diethylammonium was successfully indexed, showing that the pattern represents a single crystalline phase with the results shown in Figure 63. The indexing results show that crystalline 25HC3S diethylammonium has an average diffraction index of 3293.3 Å, consistent with the anhydrous form and the proposed salt stoichiometry. 3 It is found to have an orthorhombic lattice with a lattice volume of (±5%). Some lattice parameters are shown in Table 12 below.

[0226] [Table 12]

[0227] Further disclosed is a substantially pure crystalline 25HC3S diethylammonium. "Substantially pure," as used herein, generally refers to forms herein present in significant amounts other than possibly trace levels of other forms of 25HC3S diethylammonium. Examples of trace levels include a total of about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less, based on the total amount (by weight) of 25HC3S diethylammonium present.

[0228] Methods for preparing diethylammonium 25HC3S are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce diethylammonium 25HC3S, as described in Example 36.

[0229] The present disclosure also relates to pharmaceutical compositions containing 25HC3S diethylammonium, including the crystalline 25HC3S diethylammonium disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S diethylammonium, including the crystalline 25HC3S diethylammonium. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, for example, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral formulations.

[0230] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans), such as one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3S diethylammonium, including the crystalline 25HC3S diethylammonium of the present disclosure, and / or an effective amount of a pharmaceutical composition comprising the crystalline 25HC3S diethylammonium of the present disclosure.

[0231] An exemplary organic salt of 25HC3S of the present disclosure is the organic amine, diethanolamine, salt of 25HC3S. Preparation of the crystalline diethanolamine salt of 25HC3S can be found in Example 37. The x-ray powder diffraction pattern of crystalline 25HC3S diethanolamine can be found in Figure 15. The x-ray powder diffraction pattern of selected peaks can be found in Figure 16. Table 13 lists selected peaks from Figure 16.

[0232] [Table 13] TIFF2026501266000024.tif195161

[0233] Crystalline 25HC3S diethanolamine can be characterized by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S diethanolamine, or a portion thereof, can be used to identify the crystalline 25HC3S diethanolamine. Crystalline 25HC3S diethanolamine contains various x-ray powder diffraction peaks, which, alone or together, can help identify the presence of crystalline 25HC3S diethanolamine.

[0234] In some cases, crystalline 25HC3S diethanolamine may be characterized by an x-ray powder diffraction pattern having one or more peaks in Figure 16. For example, a peak at about 3.8°2θ can be used to characterize crystalline 25HC3S diethanolamine. In these and other cases, crystalline 25HC3S diethanolamine may be further characterized by one or more peaks at about 7.7°2θ, about 8.1°2θ, about 8.8°2θ, about 14.6°2θ, and about 15.2°2θ.

[0235] Two of the most intense peaks in Figure 16 are those at about 7.7°2θ and about 8.1°2θ. In Figure 16, these peaks appear very close together due to their individual intensities, but the XRPD instrument is able to distinguish them. However, due to the variability associated with these XRPD peaks, they may appear "under" each other when analyzed as a single peak. Because such a single-appearing peak is possible in many instances herein, crystalline 25HC3S diethanolamine is characterized, at least in part, as having an x-ray powder diffraction pattern that includes two peaks between about 7.7°2θ and about 8.1°2θ that are (a) non-overlapping, (b) partially overlapping, or (c) overlapping so as to appear, for example, as a single peak.

[0236] In some cases, the crystalline 25HC3S diethanolamine may be characterized by an x-ray powder diffraction pattern having a peak at about 7.7°2θ. In these and other cases, the crystalline 25HC3S diethanolamine may be further characterized by one or more peaks at about 3.8°2θ, about 8.1°2θ, about 8.8°2θ, about 14.6°2θ, and about 15.2°2θ.

[0237] In some cases, the crystalline 25HC3S diethanolamine may be characterized by an x-ray powder diffraction pattern having a peak at about 8.1°2θ. In these and other cases, the crystalline 25HC3S diethanolamine may be further characterized by one or more peaks at about 3.8°2θ, about 7.5°2θ, about 8.2°2θ, about 8.6°2θ, about 12.4°2θ, about 13.3°2θ, and about 15.0°2θ.

[0238] In some instances, the crystalline 25HC3S diethanolamine may be characterized by an x-ray powder diffraction pattern having a peak at about 8.2°2θ. In these and other instances, the crystalline 25HC3S diethanolamine may be further characterized by one or more peaks at about 3.8°2θ, about 7.7°2θ, about 8.8°2θ, about 14.6°2θ, and about 15.2°2θ.

[0239] In some instances, the crystalline 25HC3S diethanolamine may be characterized by an x-ray powder diffraction pattern having a peak at about 8.8°2θ. In these and other instances, the crystalline 25HC3S diethanolamine may be further characterized by one or more peaks at about 3.8°2θ, about 7.7°2θ, about 8.1°2θ, about 14.6°2θ, and about 15.2°2θ.

[0240] In some cases, the crystalline 25HC3S diethanolamine may be characterized by an x-ray powder diffraction pattern having a peak at 14.6°2θ. In these and other cases, the crystalline 25HC3S diethanolamine may be further characterized by one or more peaks at about 3.8°2θ, about 7.7°2θ, about 8.1°2θ, about 14.6°2θ, and about 15.2°2θ.

[0241] In some cases, crystalline 25HC3S diethanolamine may be characterized by an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of FIG.

[0242] Crystalline 25HC3S diethanolamine takes up water in the DVS experiment in Figure 55, demonstrating that it is hygroscopic at relative humidities above about 75%. As can be seen in Figure 54, negligible weight loss is observed upon heating to 175°C, suggesting an anhydrous structure at ambient conditions. The solution in Figure 57 1 H-NMR reveals the presence of an extra proton at 5.3 ppm that is not explained by the structure, but is otherwise consistent with the chemical structure. The DSC of crystalline 25HC3S diethanolamine can be seen in Figure 54 and shows a single isotherm at about 181°C. The x-ray powder diffraction patterns both before and after DVS are shown in Figure 56.

[0243] The x-ray powder diffraction pattern of crystalline 25HC3S diethanolamine was successfully indexed, showing that the pattern represents a single crystalline phase with the results shown in Figure 58. The indexing results show an average diffraction angle of 3294.1 Å, consistent with the crystalline 25HC3S diethanolamine, anhydrous form, and proposed salt stoichiometry.3 It is found to have a monoclinic lattice with a lattice volume of (±5%). Some lattice parameters are shown in Table 14 below.

[0244] [Table 14]

[0245] Further disclosed is a substantially pure crystalline 25HC3S diethanolamine. "Substantially pure," as used herein, generally refers to forms herein present in significant amounts other than possibly trace levels of other forms of 25HC3S diethanolamine. Examples of trace levels include a total of about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less, based on the total amount (by weight) of 25HC3S diethanolamine present.

[0246] Methods for preparing 25HC3S diethanolamine are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce 25HC3S diethanolamine, as described in Example 37.

[0247] The present disclosure also relates to pharmaceutical compositions containing 25HC3S diethanolamine, including the crystalline 25HC3S diethanolamine disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S diethanolamine, including the crystalline 25HC3S diethanolamine. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, such as parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral formulations.

[0248] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans), such as one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3S diethanolamine, including the crystalline 25HC3S diethanolamine of the present disclosure, and / or an effective amount of a pharmaceutical composition, including the crystalline 25HC3S diethanolamine of the present disclosure.

[0249] An exemplary organic salt of 25HC3S of the present disclosure is the organic amine t-butylammonium salt of 25HC3S. The preparation of the crystalline t-butylammonium salt of 25HC3S can be found in Example 38. The x-ray powder diffraction pattern of crystalline 25HC3S t-butylammonium can be found in Figure 19. The x-ray powder diffraction pattern of selected peaks can be found in Figure 20. Table 15 lists selected peaks from Figure 20.

[0250] [Table 15] TIFF2026501266000027.tif80158

[0251] Crystalline 25HC3S t-butylammonium can be characterized by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S t-butylammonium, or a portion thereof, can be used to identify the crystalline 25HC3S t-butylammonium. Crystalline 25HC3S t-butylammonium contains various x-ray powder diffraction peaks, which, alone or together, can help identify the presence of crystalline 25HC3S t-butylammonium.

[0252] In some cases, crystalline 25HC3S t-butylammonium may be characterized by an x-ray powder diffraction pattern having one or more peaks in Figure 19. For example, a peak at about 4.0°2θ may be used to characterize crystalline 25HC3S t-butylammonium. In these and other cases, crystalline 25HC3S t-butylammonium may be further characterized by one or more peaks at about 8.0°2θ, about 11.5°2θ, about 12.2°2θ, about 14.4°2θ, about 15.4°2θ, and about 16.3°2θ.

[0253] In some instances, the crystalline 25HC3S t-butylammonium may be characterized by an x-ray powder diffraction pattern having a peak at about 8.0°2θ. In these and other instances, the crystalline 25HC3S t-butylammonium may be further characterized by one or more peaks at about 4.0°2θ, about 11.5°2θ, about 12.2°2θ, about 14.4°2θ, about 15.4°2θ, and about 16.3°2θ.

[0254] In some instances, the crystalline 25HC3S t-butylammonium may be characterized by an x-ray powder diffraction pattern having a peak at about 11.5°2θ. In these and other instances, the crystalline 25HC3S t-butylammonium may be further characterized by one or more peaks at about 4.0°2θ, about 8.0°2θ, about 12.2°2θ, about 14.4°2θ, about 15.4°2θ, and about 16.3°2θ.

[0255] In some instances, the crystalline 25HC3S t-butylammonium may be characterized by an x-ray powder diffraction pattern having a peak at about 12.2°2θ. In these and other instances, the crystalline 25HC3S t-butylammonium may be further characterized by one or more peaks at about 4.0°2θ, about 8.0°2θ, about 11.5°2θ, about 14.4°2θ, about 15.4°2θ, and about 16.3°2θ.

[0256] In some instances, the crystalline 25HC3S t-butylammonium may be characterized by an x-ray powder diffraction pattern having a peak at about 14.4°2θ. In these and other instances, the crystalline 25HC3S t-butylammonium may be further characterized by one or more peaks at about 4.0°2θ, about 8.0°2θ, about 11.5°2θ, about 12.2°2θ, about 15.4°2θ, and about 16.3°2θ.

[0257] In some instances, the crystalline 25HC3S t-butylammonium may be characterized by an x-ray powder diffraction pattern having a peak at 15.4°2θ. In these and other instances, the crystalline 25HC3S t-butylammonium may be further characterized by one or more peaks at about 4.0°2θ, about 8.0°2θ, about 11.5°2θ, about 12.2°2θ, about 14.4°2θ, and about 16.3°2θ.

[0258] In some instances, the crystalline 25HC3S t-butylammonium may be characterized by an x-ray powder diffraction pattern having a peak at 16.3°2θ. In these and other instances, the crystalline 25HC3S t-butylammonium may be further characterized by one or more peaks at about 4.0°2θ, about 8.0°2θ, about 11.5°2θ, about 12.2°2θ, about 14.4°2θ, and about 15.4°2θ.

[0259] In some cases, crystalline 25HC3S t-butylammonium may be further characterized by one or more peaks at about 10.3°2θ, about 10.5°2θ, about 13.8°2θ, about 16.8°2θ, about 17.0°2θ, and about 17.4°2θ. For example, crystalline 25HC3S t-butylammonium may be further characterized by a peak at about 10.3°2θ. Alternatively or additionally, crystalline 25HC3S t-butylammonium may be further characterized by a peak at about 10.5°2θ. Alternatively or additionally, crystalline 25HC3S t-butylammonium may be further characterized by a peak at about 13.8°2θ. Alternatively or additionally, crystalline 25HC3S t-butylammonium may be further characterized by a peak at about 16.8°2θ. Alternatively or additionally, the crystalline 25HC3S t-butylammonium may be further characterized by a peak at about 17.0° 2θ. Alternatively or additionally, the crystalline 25HC3S t-butylammonium may be further characterized by a peak at about 17.4° 2θ.

[0260] In some cases, the crystalline 25HC3S t-butylammonium may be characterized by an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of FIG.

[0261] Crystalline 25HC3S t-butylammonium has low hygroscopicity between 5% and 95% relative humidity, as seen by DVS in Figure 65. By TGA in Figure 64, a weight loss of 0.2% was measured upon heating to 200°C, suggesting an anhydrous structure at ambient conditions. The DSC of crystalline 25HC3S t-butylammonium can be seen in Figure 64, showing isotherms near 205°C and 218°C. 1 The H-NMR spectrum is consistent with the chemical structure.

[0262] The x-ray powder diffraction pattern of crystalline 25HC3S t-butylammonium was successfully indexed, showing that the pattern represents a single crystalline phase with the results shown in Figure 67. The indexing results show that the crystalline 25HC3S t-butylammonium has an average diffraction index of 3333.3 Å, consistent with the anhydrous form and the proposed salt stoichiometry. 3 It is found to have a monoclinic lattice with a lattice volume of (±5%). Some lattice parameters are shown in Table 16 below.

[0263] [Table 16]

[0264] Further disclosed is a substantially pure crystalline 25HC3S t-butylammonium. "Substantially pure," as used herein, generally refers to forms herein present in significant amounts other than possibly trace levels of other forms of 25HC3S t-butylammonium. Examples of trace levels include a total of about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less, based on the total amount (by weight) of 25HC3S t-butylammonium present.

[0265] Methods for preparing 25HC3S t-butylammonium are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce 25HC3S t-butylammonium, as described in Example 38.

[0266] The present disclosure also relates to pharmaceutical compositions containing 25HC3S t-butylammonium, including the crystalline 25HC3S t-butylammonium disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S t-butylammonium, including the crystalline 25HC3S t-butylammonium. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, for example, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral formulations.

[0267] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans), such as one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3S t-butylammonium, including the crystalline 25HC3S t-butylammonium of the present disclosure, and / or an effective amount of a pharmaceutical composition comprising the crystalline 25HC3S t-butylammonium of the present disclosure.

[0268] An exemplary organic salt of 25HC3S of the present disclosure is the benzathine salt, an organic amine of 25HC3S. The preparation of crystalline benzathine salt of 25HC3S is found in Example 39. The x-ray powder diffraction pattern of crystalline 25HC3S benzathine can be seen in Figure 21. The x-ray powder diffraction pattern of selected peaks can be seen in Figure 22. Table 17 shows selected peaks from Figure 22. The benzathine salt of 25HC3S contains two ions of 25HC3S for each ion of benzathine, as determined by ICP-OES.

[0269] [Table 17] TIFF2026501266000030.tif121158

[0270] Crystalline 25HC3S benzathine can be characterized by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S benzathine, or a portion thereof, can be used to identify crystalline 25HC3S benzathine. Crystalline 25HC3S benzathine contains various x-ray powder diffraction peaks, which, alone or together, can help identify the presence of crystalline 25HC3S benzathine.

[0271] In some cases, crystalline 25HC3S benzathine may be characterized by an x-ray powder diffraction pattern having one or more peaks in Figure 22. For example, a peak at about 4.1°2θ can be used to characterize crystalline 25HC3S benzathine. In these and other cases, crystalline 25HC3S benzathine may be further characterized by one or more peaks at about 6.9°2θ, about 8.2°2θ, about 12.3°2θ, and about 16.2°2θ.

[0272] In some cases, crystalline 25HC3S benzathine may be characterized by an x-ray powder diffraction pattern having a peak at about 6.9°2θ. In these and other cases, crystalline 25HC3S benzathine may be further characterized by one or more peaks at about 4.1°2θ, about 8.2°2θ, about 12.3°2θ, and about 16.2°2θ.

[0273] In some cases, crystalline 25HC3S benzathine may be characterized by an x-ray powder diffraction pattern having a peak at about 8.2°2θ. In these and other cases, crystalline 25HC3S benzathine may be further characterized by one or more peaks at about 4.1°2θ, about 6.9°2θ, about 12.3°2θ, and about 16.2°2θ.

[0274] In some cases, crystalline 25HC3S benzathine may be characterized by an x-ray powder diffraction pattern having a peak at about 12.3°2θ. In these and other cases, crystalline 25HC3S benzathine may be further characterized by one or more peaks at about 4.1°2θ, about 6.9°2θ, and about 16.2°2θ.

[0275] In some cases, crystalline 25HC3S benzathine may be characterized by an x-ray powder diffraction pattern having a peak at about 16.2°2θ. In these and other cases, crystalline 25HC3S benzathine may be further characterized by one or more peaks at about 4.1°2θ, about 6.9°2θ, and about 12.3°2θ.

[0276] In some cases, crystalline 25HC3S benzathine may be further characterized by one or more peaks at about 7.3°2θ, about 15.1°2θ, about 16.4°2θ, about 17.8°2θ, and about 20.4°2θ. For example, crystalline 25HC3S benzathine may be further characterized by a peak at about 7.3°2θ. Alternatively or additionally, crystalline 25HC3S benzathine may be further characterized by a peak at about 15.1°2θ. Alternatively or additionally, crystalline 25HC3S benzathine may be further characterized by a peak at about 16.4°2θ. Alternatively or additionally, crystalline 25HC3S benzathine may be further characterized by a peak at about 17.8°2θ. Alternatively or additionally, crystalline 25HC3S benzathine may be further characterized by a peak at about 20.4°2θ.

[0277] In some cases, crystalline 25HC3S benzathine has an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of FIG.

[0278] Crystalline 25HC3S benzathine has low hygroscopicity at 5% to 95% relative humidity. As seen in Figure 68, a weight loss of 0.4% was measured by heating to 200°C as seen by TGA, suggesting that crystalline 25HC3S benzathine is anhydrous at ambient conditions. The DSC of crystalline 25HC3S benzathine can be seen in Figure 68, showing isotherms near 216°C and 223°C. The DVS isotherm in Figure 69 indicates low hygroscopicity. The x-ray powder diffraction patterns before and after DVS are shown in Figure 70. 1 The H-NMR spectrum is consistent with the chemical structure.

[0279] The x-ray powder diffraction pattern of crystalline 25HC3S benzathine was successfully indexed, showing that the pattern represents a single crystalline phase with the results shown in Figure 72. The indexing results show that crystalline 25HC3S benzathine has an average diffraction index of 3394.0 Å, consistent with the anhydrous form and the proposed salt stoichiometry. 3 It appears to have a monoclinic lattice with a lattice volume of (±5%). Some lattice parameters are shown in Table 18 below.

[0280] [Table 18]

[0281] Further disclosed is a substantially pure crystalline 25HC3S benzathine. "Substantially pure," as used herein, generally refers to forms herein present in significant amounts other than possibly trace levels of other forms of 25HC3S benzathine. Examples of trace levels include a total of about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less, based on the total amount (by weight) of 25HC3S benzathine present.

[0282] Methods for preparing 25HC3S benzathine are further described herein. In some cases, the sodium salt of 25HC3S can be first prepared. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce 25HC3S benzathine, as described in Example 39.

[0283] The present disclosure also relates to pharmaceutical compositions containing 25HC3S benzathine, including the crystalline 25HC3S benzathine disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S benzathine, including crystalline 25HC3S benzathine. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, for example, parenterally, topically, nasally, ophthalmically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral formulations.

[0284] The present disclosure further includes methods and uses for treating and / or preventing one or more diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, with an effective amount of 25HC3S benzathine, including the crystalline 25HC3S benzathine of the present disclosure, and / or an effective amount of a pharmaceutical composition, including the crystalline 25HC3S benzathine of the present disclosure.

[0285] An exemplary organic salt of 25HC3S of the present disclosure is the organic amine choline salt of 25HC3S. Preparation of the crystalline choline salt of 25HC3S can be found in Example 40. The x-ray powder diffraction pattern of crystalline 25HC3S choline can be found in Figure 76. The x-ray powder diffraction pattern of selected peaks can be found in Figure 77. Table 19 lists selected peaks from Figure 77.

[0286] [Table 19] TIFF2026501266000033.tif145159

[0287] Crystalline 25HC3S choline can be identified by various analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S choline, or a portion thereof, can be used to identify crystalline 25HC3S choline. Crystalline 25HC3S choline includes various x-ray powder diffraction peaks, which, alone or together, can help identify the presence of crystalline 25HC3S choline.

[0288] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 3.9°2θ. In these and other cases, the crystalline 25HC3S choline can be further characterized by one or more peaks at about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0289] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 7.8°2θ. In these and other cases, the crystalline 25HC3S choline can be further characterized by one or more peaks at about 3.9°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0290] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 9.5°2θ. In these and other cases, the crystalline 25HC3S choline can be further characterized by one or more peaks at about 3.9°2θ, about 7.8°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0291] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 10.1°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by one or more peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0292] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 11.0°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0293] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 12.2°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0294] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 13.7°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0295] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 14.7°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0296] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 15.1°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0297] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 15.8°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 16.3°2θ, and about 19.1°2θ.

[0298] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 16.3°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, and about 19.1°2θ.

[0299] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 19.1°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, and about 16.3°2θ.

[0300] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9°2θ and about 7.8°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0301] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9°2θ, about 7.8°2θ, and about 9.5°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0302] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, and about 10.1°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0303] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, and about 11.0°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0304] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, and about 12.2°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0305] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, and about 13.7°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0306] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, and about 14.7°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0307] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, and about 15.1°2θ. In these and other cases, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0308] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, and about 15.8°2θ. In these and other cases, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having one or more peaks at about 16.3°2θ and about 19.1°2θ.

[0309] In some instances, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, and about 16.3°2θ. In these and other instances, the crystalline 25HC3S choline can be further characterized by an x-ray powder diffraction pattern having a peak at about 19.1°2θ.

[0310] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0311] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0312] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0313] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0314] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0315] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0316] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0317] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0318] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 15.1° 2θ, about 15.8° 2θ, about 16.3° 2θ, and about 19.1° 2θ.

[0319] In some cases, crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 15.8° 2θ, about 16.3° 2θ, and about 19.1° 2θ.

[0320] In some cases, crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at 16.3° 2θ and about 19.1° 2θ.

[0321] In some cases, crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern with a peak at about 19.1° 2θ.

[0322] In some cases, crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having substantially the same pattern as that seen in Figure 77.

[0323] The x-ray powder diffraction pattern of crystalline 25HC3S choline was successfully indexed, showing that the pattern represents a single crystalline phase with the results shown in Figure 79. The indexing results show that crystalline 25HC3S choline has an average peak size of 3371.5 Å, consistent with the anhydrous form. 3 It is found to have an orthorhombic lattice with a lattice volume of (±5%). Some lattice parameters are shown in Table 20 below.

[0324] [Table 20]

[0325] The DSC thermogram of crystalline 25HC3S choline in Figure 80 shows endothermic peaks at about 198° C. and about 220° C. The TGA thermogram in Figure 80 shows that there is negligible weight loss up to 198° C.

[0326] Without being bound by theory, crystalline 25HC3S choline is believed to be anhydrous, meaning that there are no water crystals in the unit cell. This does not exclude the possibility of the presence of additional water in solids containing crystalline 25HC3S choline. Furthermore, crystalline 25HC3S choline exhibits no appreciable hygroscopicity up to approximately 95% relative humidity, with a weight gain of only approximately 0.5% up to this relative humidity, as evidenced by dynamic vapor sorption experiments according to Example 26, the results of which are shown in Figure 82. Furthermore, the x-ray powder diffraction pattern of crystalline 25HC3S choline does not change significantly after DVS, as shown in Figure 78. Only a 0.5% weight gain was observed from 5% to 95% relative humidity, and a 0.5% weight loss was observed when the relative humidity returned to 5%, showing no hysteresis. Such low hygroscopicity indicates good stability under such stresses, which may be suitable for pharmaceutical processing, as further discussed elsewhere herein. Thus, in practice, the present disclosure further includes stable crystalline 25HC3S choline, where such stability includes crystalline 25HC3S choline that is sufficiently stable to be formulated for delivery to a patient. 1 The H-NMR spectrum is consistent with the structure seen in Figure 81, except for a peak at 5.3 ppm. There was no evidence of residual solvent. The methylene overlap from the chlorine at approximately 3.8 ppm appears to be 1 mole per mole of choline.

[0327] Choline salts have additional advantages over sodium salts of 25HC3S and some other salts in that the choline counterion has additional advantageous properties. For example, choline is an essential nutrient, and choline deficiency has been shown to cause fat and cholesterol accumulation in the liver. Furthermore, 25HC3S choline forms crystals of superior quality and diffraction to those of prior art. Finally, crystalline 25HC3S choline is less hygroscopic and therefore more physically stable than, for example, crystalline sodium 25HC3S. Crystalline sodium 25HC3S is stable as a hydrate when exposed to humid conditions. In particular, monohydrate, dihydrate, and various hydrates of crystalline sodium 25HC3S have been prepared. The hydrate, Form I, has been found to be hygroscopic and can form liquid crystals at high water activities (e.g., greater than 0.73). Another hydrate, Form II, is stable at relative humidities between about 21% and about 30%. By comparison, only about 0.5% water by weight is absorbed under conditions up to about 95% relative humidity, indicating that crystalline 25HC3S choline is stable as an anhydrate.

[0328] Further disclosed is a substantially pure crystalline 25HC3S choline. As used herein, "substantially pure" generally refers to a form that is not present in any significant amount, except potentially trace levels of other forms of 25HC3S choline. Examples of trace levels include a total of about 10% or less, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less by weight of the total amount of 25HC3S choline present.

[0329] Methods for preparing 25HC3S choline are further described herein. In some cases, the sodium salt of 25HC3S can be first prepared. Examples of such preparations are described herein. The sodium salt of 25HC3S, which can be crystalline, can be converted to the triethylammonium salt, for example, as described in Example 41. The triethylammonium salt can then be used to produce 25HC3S choline, as described in Example 40.

[0330] The present disclosure also relates to pharmaceutical compositions containing 25HC3S choline, including the crystalline 25HC3S choline disclosed herein. Such pharmaceutical compositions consist of one or more pharmaceutically acceptable excipients and 25HC3S choline, including crystalline 25HC3S choline. Such pharmaceutical compositions may be administered orally or may be configured to be delivered in any effective conventional dosage form, such as parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, etc., including immediate-release, sustained-release, delayed-release, and extended-release oral preparations.

[0331] As discussed elsewhere herein and demonstrated in the Examples, the 25HC3S choline of the present disclosure has surprisingly low hygroscopicity, including compared to other salt forms of 25HC3S. Accordingly, 25HC3S choline can be advantageously manufactured and utilized in the preparation of pharmaceutical formulations, particularly dosage forms for oral administration (e.g., solid dosage forms such as tablets, capsules (each of which includes immediate-release, sustained-release, or extended-release formulations), pills, powders, or granules).

[0332] Furthermore, the 25HC3S choline of the present disclosure advantageously provides supplemental choline to patients suffering from conditions targeted by 25HC3S.As discussed elsewhere herein, choline deficiency may contribute to these conditions, and it may be advantageous to provide choline simultaneously with 25HC3S during the course of treatment.Thus, 25HC3S choline surprisingly and advantageously combines advantageous salt form properties, contributing to the preparation of oral dosage forms that are particularly well suited to treating certain conditions, with the inherent ability to advantageously provide choline supplementation during, for example, the course of treatment using said oral dosage form.

[0333] The present disclosure further includes methods and uses for treating and / or preventing one or more diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, using an effective amount of 25HC3S choline, including the crystalline 25HC3S choline of the present disclosure, and / or an effective amount of a pharmaceutical composition, including the crystalline 25HC3S choline of the present disclosure.

[0334] An exemplary organic salt of 25HC3S of the present disclosure is the organic amine triethylammonium salt of 25HC3S. The preparation of the triethylammonium salt of 25HC3S is found in Example 41.

[0335] The triethylammonium salt of 25HC3S can be prepared, for example, by passing a mixture of triethylammonium chloride and triethylamine through a column and treating with a solvent, such as alcohol, until a neutral pH is reached. Alternatively, crystalline sodium 25HC3S can be dissolved in a solvent, such as alcohol. This solution can then be passed through the same column previously exposed to triethylamine and combined with the triethylammonium solution. The resulting solid can then be isolated, for example, under vacuum or by drying, to yield crystalline triethylammonium salt of 25HC3S, which can be homogenized, for example, with a mortar and pestle. Suitable alcohols for this method include methanol.

[0336] 25HC3S choline, including crystalline 25HC3S choline, may be prepared by starting with 25HC3S sodium, converting it to a second salt of 25HC3S, such as the triethylammonium salt, and then converting the second salt of 25HC3S to 25HC3S choline, including crystalline 25HC3S choline. Crystalline 25HC3S choline may be prepared by preparing a suspension of the triethylammonium salt of 25HC3S in a suitable solvent, such as acetonitrile, and treating it with a choline source, such as aqueous choline hydroxide, to form 25HC3S choline, including crystalline 25HC3S choline. 25HC3S choline may be purified, for example, by rinsing with a suitable solvent. Additional processing, such as drying under vacuum or other methods, may also be performed. The present disclosure further includes crystalline 25HC3S choline produced by the methods described herein.

[0337] Method for preparing 25-hydroxy-cholesten-5-ene-3-sulfate (25HC3S) Methods for preparing 25-hydroxycholesten-5-ene-3-sulfate, e.g., 25-hydroxy-3β-cholesten-5-ene-3-sulfate (25HC3S), are described herein. While much of the teaching herein encompasses sulfates at the 3β position, the teachings of the present disclosure are also generally applicable to sulfates at the 3α position. The components used in each step of the subject method for preparing 25-hydroxy-3β-cholesten-5-ene-3-sulfate described herein may be purified or crude compositions, as desired. The term "purified" is used in its conventional sense to refer to a composition that has undergone at least some isolation or purification process, such as filtration of a reaction mixture or aqueous workup. In some cases, purification includes at least one of liquid chromatography, recrystallization, distillation (e.g., azeotropic distillation), and other types of compound purification. For example, the compounds described herein may be purified by chromatographic means, such as high-performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), thin-layer chromatography, flash column chromatography, and ion exchange chromatography. In addition to ionic resins, any suitable stationary phase, including normal phase and reverse phase, may be used. The mobile phase can be selected from polar and non-polar solvents. In some cases, the mobile phase comprises a polar solvent. In some cases, the polar solvent is selected from chloroform, dichloromethane, tetrahydrofuran, dichloroethane, acetone, dioxane, ethyl acetate, dimethyl sulfoxide, aniline, diethylamine, nitromethane, acetonitrile, pyridine, isopropanol, ethanol, methanol, ethylene glycol, acetic acid, and water. In some cases, the mobile phase comprises a non-polar solvent. In some cases, the non-polar solvent is selected from diethyl ether, toluene, benzene, pentane, hexane, cyclohexane, petroleum ether, and carbon tetrachloride.See, for example, Introduction to Modern Liquid Chromatography, 2nd Edition, ed. LR Snyder and JJ Kirkland, John Wiley and Sons, 1979, and Thin Layer Chromatography, ed. E. Stahl, Springer-Verlag, New York, 1969.

[0338] In some cases, the reaction mixture is used as a crude mixture in subsequent steps of the methods described herein without purification or other work-up of the reaction mixture. In some cases, the crude mixture contains the compound of interest of sufficient purity, for example, where the reaction mixture is purified by chromatography (e.g., HPLC or SFC), nuclear magnetic resonance spectroscopy (e.g., 1 H NMR or 13The crude reaction mixture (separate from the solvent, if present) comprises the compound of interest in a purity including 70% or more, such as 75% or more, for example 80% or more, for example 85% or more, such as 90% or more, for example 95% or more, such as 97% or more, for example 99% or more, such as 99.5% or more, for example 99.9% or more, for example 99.99% or more, and 99.999% or more of the crude reaction mixture (separate from the solvent, if present) as determined by C NMR or a combination thereof. In some cases, the compound of interest is present in the reaction mixture in an amount which may range from 5% to 99.999% by weight, such as 30% to 99.99% by weight, such as 40% to 99.9% by weight, such as 50% to 99% by weight, such as 60% to 99% by weight, such as 70% to 95% by weight, for example 75% to 99% by weight, such as 80% to 99% by weight, for example 95% to 99% by weight, such as 99.5% to 99.9% by weight, for example 99.99% by weight, and such as 99.999% by weight or more of the crude reaction mixture (separate from solvent, if present). In some cases, the compound of interest is present in the reaction mixture comprising 30 mol% or more, such as 40 mol% or more, for example 50 mol% or more, for example 60 mol% or more, for example 70 mol% or more, for example 75 mol% or more, such as 80 mol% or more, for example 85 mol% or more, for example 90 mol% or more, for example 95 mol% or more, such as 97 mol% or more, for example 99 mol% or more, for example 99.5 mol% or more, for example 99.9 mol% or more, for example 99.99 mol% or more, and 99.999 mol% or more, relative to the crude reaction mixture (separate from the solvent, if present), and may be in the range of 30 mol% to 99.999 mol%, for example 50 mol% to 99 mol%, 70 mol% to 95 mol%, 75 mol% to 90 mol%, 80 mol% to 99 mol% or 80 mol% to 95 mol%.

[0339] The method for preparing the metal salt of 25-hydroxy-3β-cholesten-5-ene-3-sulfate ([(3S,10R,13R,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3-yl]sulfate metal salt) according to the present disclosure comprises: The method includes contacting hydroxy-(3β)-cholest-5-en-3-ol with a sulfating agent to produce 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt, and contacting 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt with at least one metal salt to produce 5-cholesten-3β,25-diol 3-sulfate metal salt (Scheme Ia).

[0340] [ka]

[0341] [ka]

[0342] 25-Hydroxy-(3β)-cholest-5-en-3-ol may be sulfated by contacting it with a sulfating agent (Scheme IA1). In some cases, the sulfating reagent is selected from sulfur trioxide complexes, sulfate compounds, sulfonic acid compounds, and sulfonate compounds. In some cases, the sulfating reagent is selected from sulfur trioxide dimethylformamide, sulfur trioxide triethylamine, and sulfur trioxide trimethylamine. In some cases, the sulfating reagent comprises sulfuric acid, acetic anhydride, and pyridine. In some cases, the sulfating reagent comprises sulfur trioxide triethylamine and pyridine. In some cases, the sulfating reagent is selected from 1) chlorosulfonic acid and pyridine, and 2) chlorosulfonic acid and 2,6-lutidine. In some cases, the sulfating reagent is ethyl chloride sulfate.

[0343] 25-Hydroxy-(3β)-cholest-5-en-3-ol may be sulfated at a temperature in the range of, and including, from −10° C. to 50° C., such as from −5° C. to 45° C., for example from −4° C. to 40° C., for example from −3° C. to 35° C., for example from −2° C. to 30° C., for example from −1° C. to 25° C. The reaction may be carried out for a duration in the range of, and including, from 0.1 hours to 72 hours, for example from 0.2 hours to 48 hours, for example from 0.3 hours to 24 hours, for example from 0.4 hours to 21 hours, for example from 0.5 hours to 20 hours, for example from 0.6 hours to 19 hours, for example from 0.7 hours to 18 hours, for example from 0.8 hours to 17 hours, for example from 0.9 hours to 16 hours, for example from 1 hour to 15 hours. The amount of sulfating agent used relative to 25-hydroxy-(3β)-cholest-5-en-3-ol may vary and may be 0.001 equivalents or more, such as 0.01 equivalents or more, for example 0.1 equivalents or more, such as 0.2 equivalents or more, for example 0.3 equivalents or more, such as 0.4 equivalents or more, for example 0.5 equivalents or more, such as 0.6 equivalents or more, for example 0.7 equivalents or more, such as 0.8 equivalents or more, for example 0.9 equivalents or more, such as 1 equivalent or more, for example 1.1 equivalents or more, such as 1.2 equivalents or more, for example 1.3 equivalents or more, such as 1.4 equivalents or more, for example 1.5 equivalents or more, such as 1.6 equivalents or more, for example 1.7 equivalents or more, such as 1.8 equivalents or more, for example 1.9 equivalents or more, such as 2 equivalents or more, for example 3 equivalents or more, for example It may be 4 equivalents or more, for example, 5 equivalents or more, including 10 equivalents or more, and is 0.001 to 10 equivalents relative to 25-hydroxy-(3β)-cholest-5-en-3-ol, for example, 0.1 to 10 equivalents, 0.1 to 8 equivalents, 0.1 to 5 equivalents, 0.5 to 10 equivalents, 0.5 to 8 equivalents, 0.5 to 5 equivalents, 0.9 to 10 equivalents The amount may be in the range of 0.9 to 8 equivalents, 0.9 to 5 equivalents, 1.3 to 10 equivalents, 1.3 to 8 equivalents, 1.3 to 5 equivalents, 1.5 to 10 equivalents, 1.5 to 8 equivalents, 1.5 to 5 equivalents, 2 to 10 equivalents, 2 to 8 equivalents, 2 to 5 equivalents, 1 to 2 equivalents, 1 to 1.5 equivalents, or 1.1 to 1.2 equivalents.

[0344] In some cases, the method includes sulfating 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent in which the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product exhibits low solubility. In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in at least one solvent in which the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product has a solubility of 100 mmol / L or less, such as 90 mmol / L or less, for example 80 mmol / L or less, for example 70 mmol / L or less, such as 60 mmol / L or less, for example 50 mmol / L or less, such as 40 mmol / L or less, for example 30 mmol / L or less, such as 20 mmol / L or less, for example 10 mmol / L or less, and the method comprises sulfating 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent in which the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product has a solubility of 5 mmol / L or less. In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in at least one solvent in which the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product precipitates after formation. In some cases, the at least one solvent is selected from chloroform, methylene chloride, acetone, acetonitrile, toluene, tetrahydrofuran, and methyltetrahydrofuran.

[0345] In some cases, the method includes sulfating 25-hydroxy-(3β)-cholest-5-en-3-ol in a manner sufficient to reduce or eliminate bis-sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated and a bis-sulfate product (i.e., 5-cholesten-3β-25-diol-disulfate, Structure IA) is formed in an amount that is 10% by weight or less, such as 9% by weight or less, for example 8% by weight or less, such as 7% by weight or less, for example 6% by weight or less, such as 5% by weight or less, for example 4% by weight or less, such as 3% by weight or less, for example 2% by weight or less, such as 1% by weight or less, for example 0.5% by weight or less, for example 0.1% by weight or less, such as 0.01% by weight or less, for example 0.001% by weight or less, of the reaction product formed by contacting 25-hydroxy-(3β)-cholest-5-en-3-ol with the sulfating agent, and 10 wt% to 0.001 wt%, for example, 10 wt% to 0.1 wt%, 10 wt% to 1 wt%, 10 wt% to 2 wt%, 8 wt% to 0.001 wt%, 8 wt% to 0.1 wt%, 8 wt% to 1 wt%, 8 wt% to 2 wt%, 6 wt% to 0.001 wt%, for example, 10 wt% to 0.1 wt%, 10 wt% to 1 wt%, 8 wt% to 2 wt%, 6 wt% to 0.001 wt%, for example, 10 wt% to 0.1 wt%, 10 wt% to 1 wt%, 10 wt% to 2 wt%, 8 wt% to 0.001 wt%, 8 wt% to 0.1 wt%, 8 wt% to 1 wt%, 8 wt% to 2 wt%, 6 wt% to 0.001 wt%, for example, 10 wt% to 0.1 wt%, 10 wt% to ... The range may be 1% by weight, 6% to 0.1% by weight, 6% to 1% by weight, 6% to 2% by weight, 4% to 0.001% by weight, 4% to 0.1% by weight, 4% to 1% by weight, 4% to 2% by weight, 3% to 0.001% by weight, 3% to 0.1% by weight, 3% to 1% by weight, 2% to 0.001% by weight, 2% to 0.1% by weight, or 2% to 1% by weight.

[0346] In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to 5-cholesten-3β-25-diol-disulfate formed is 10:1 or more, such as 25:1 or more, for example 50:1 or more, for example 100:1 or more, such as 250:1 or more, for example 500:1 or more, for example 1000:1 or more, for example 2500:1 or more, for example 5000:1 or more, for example 10,000:1 or more, for example 25,000:1 or more, for example 50,000:1 or more, for example 100,000:1 or more, for example 10 6 : 1 or more, e.g. 10 7 : 1 or more, e.g. 10 8 :1 or more, and the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the formed 5-cholesten-3β-25-diol disulfate is 10 9 10:1 weight ratio to 10:1, including cases where the ratio is greater than 10:1 9 :1 weight ratio, for example 10:1 weight ratio ~ 10 6 :1 weight ratio, 10:1 weight ratio ~10 3 :1 weight ratio, 10:1 weight ratio to 100:1 weight ratio, 100:1 weight ratio to 10 9 :1 weight ratio, 100:1 weight ratio ~10 6 :1 weight ratio, 100:1 weight ratio ~10 3 :1 weight ratio, 250:1 weight ratio ~10 9 :1 weight ratio, 250:1 weight ratio ~10 6 :1 weight ratio, 250:1 weight ratio ~10 3 :1 weight ratio, 500:1 weight ratio ~10 9 :1 weight ratio, 500:1 weight ratio ~10 6 :1 weight ratio, 500:1 weight ratio ~10 3 :1 weight ratio, 10 3 :1 weight ratio ~10 9 :1 weight ratio, 10 3 :1 weight ratio ~10 6 :1 weight ratio or 250:1 weight ratio ~10 3 The weight ratio may be in the range of 1:1.

[0347] [ka]

[0348] In some cases, when 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated, the resulting 5-cholestene-3β-25-diol disulfate remains solubilized in at least one solvent. In some cases, 5-cholestene-3β-25-diol disulfate has high solubility in at least one solvent. In some cases, 5-cholestene-3β-25-diol disulfate exhibits a solubility of 500 mmol / L or more in at least one solvent, for example, 600 mmol / L or more, for example, 700 mmol / L or more, for example, 800 mmol / L or more, for example, 900 mmol / L or more, including a solubility of 1 mol / L or more in at least one solvent.

[0349] In certain instances, the method further includes separating the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product from the bisulfate product (i.e., 5-cholesten-3β-25-diol disulfate). In some instances, the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product is separated from the bisulfate product by vacuum filtration. In some instances, the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product is separated from the bisulfate product by recrystallization of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product. In some instances, the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product is separated from the bisulfate product by chromatography (e.g., silica column).

[0350] In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in a reaction mixture having a pH in the range of 5.0 to 8.0, such as pH 5.1 to 7.9, for example pH 5.2 to 7.8, for example pH 5.3 to 7.7, for example pH 5.4 to 7.6, for example pH 5.5 to 7.5, for example pH 5.6 to 7.4, for example pH 5.7 to 7.3, for example pH 5.8 to 7.2, for example pH 5.9 to 7.1, comprising sulfating 25-hydroxy-(3β)-cholest-5-en-3-ol in a reaction mixture having a pH of 6.0 to 7.0.

[0351] In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in the presence of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt. In some cases, the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt is present as particles (e.g., seed crystals of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt produced in a previous reaction or purified reaction batch). In some cases, the step of sulfating 25-hydroxy-(3β)-cholest-5-en-3-ol in the presence of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt (e.g., as particles) is sufficient to reduce the solubility of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt produced by the reaction of the sulfating agent with 25-hydroxy-(3β)-cholest-5-en-3-ol compared to the solubility in the absence of the 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, the solubility of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt formed in the reaction mixture is reduced by 5% or more, such as 10% or more, for example 25% or more, such as 50% or more, for example 75% or more, for example 90% or more, compared to the solubility in the absence of the added 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt, including a 99% or greater reduction in the solubility of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt formed. The size of the particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt added to the reaction mixture may vary and may have dimensions (e.g. length, width or diameter) including 0.01 mm or more, such as 0.025 mm or more, for example 0.05 mm or more, for example 0.075 mm or more, such as 0.1 mm or more, for example 0.25 mm or more, such as 0.5 mm or more, for example 0.75 mm or more, such as 1 mm or more, for example 2 mm or more, such as 3 mm or more, for example 4 mm or more, and 5 mm or more.In some cases, the particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salt are added to the reaction mixture immediately after contacting the sulfating agent with 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, the particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salt are added to the reaction mixture 1 minute or more, for example, 5 minutes or more, for example, 10 minutes or more, for example, 15 minutes or more, for example, 20 minutes or more, for example, 30 minutes or more, for example, 40 minutes or more, for example, 50 minutes or more after contacting the sulfating agent with 25-hydroxy-(3β)-cholest-5-en-3-ol, and the method includes adding the particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salt to the reaction mixture 60 minutes or more after contacting the sulfating agent with 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0352] In certain instances, the sulfating agent is characterized prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol. In some instances, characterizing the sulfating agent includes determining the extent of decomposition of the sulfating agent prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol. In certain instances, determining the extent of decomposition of the sulfating agent includes determining the amount of impurities in the sulfating reagent prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0353] In some cases, the decomposition of sulfating agents was monitored by proton nuclear magnetic resonance spectroscopy ( 1 The proton NMR spectroscopy of the sulfating agent may be performed in at least one deuterated solvent. In some cases, the at least one deuterated solvent is deuterated acetone ((CD3)2CO). In some cases, the at least one deuterated solvent is not deuterated benzene (CD6D6). In some cases, the at least one deuterated solvent is not deuterated acetonitrile (CD3CN). In some cases, the at least one deuterated solvent is not deuterated chloroform (CD3Cl).

[0354] In some cases, the method for determining the degree of degradation includes: 1 In some instances, the method for determining the degree of degradation includes integrating one or more peaks at a chemical shift between 9.2 ppm and 9.3 ppm in the H NMR spectrum, and calculating the sulfating agent impurity level for the integrated peaks. 1 In some cases, the sulfating agent impurity level is below a predetermined threshold, for example, the impurity level is determined by integrating one or more peaks at a chemical shift of about 9.25 ppm in the H NMR spectrum, and calculating the sulfating agent impurity level for the integrated peaks. In some cases, the sulfating agent impurity level is below a predetermined threshold, for example, the impurity level is determined by integrating one or more peaks at a chemical shift of 9.2 ppm to 9.3 ppm in the proton NMR spectrum, and is 25% or less, for example, 24% or less, for example, 23% or less, for example, 22% or less, for example, 21% or less, for example, 20% or less, for example, 19% or less, for example, 18% or less, for example, 17% or less, for example, 16% or less, for example, 15% or less, for example, 14% or less, for example, 13% or less. The sulfating agent is contacted with 25-hydroxy-(3β)-cholest-5-en-3-ol, including when the impurity level is 1% or less as determined by integrating one or more peaks at a chemical shift between 9.2 ppm and 9.3 ppm in a proton NMR spectrum, such as when the impurity level is 12% or less, for example 11% or less, for example 10% or less, such as 9% or less, for example 8% or less, such as 7% or less, for example 6% or less, such as 5% or less, for example 4% or less, such as 3% or less, for example 2% or less. In some cases, the sulfating agent is not contacted with 25-hydroxy-(3β)-cholest-5-en-3-ol if the impurity level exceeds a predetermined threshold, for example, if the impurity level is 25% or more, such as 26% or more, for example 27% or more, such as 28% or more, for example 29% or more, such as 30% or more, for example 31% or more, such as 32% or more, for example 33% or more, for example 34% or more, as determined by integrating one or more peaks at a chemical shift between 9.2 ppm and 9.3 ppm in a proton NMR spectrum, including when the impurity level is 35% or more, as determined by integrating one or more peaks at a chemical shift between 9.2 ppm and 9.3 ppm in a proton NMR spectrum.

[0355] In certain instances, the resulting 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product includes one or more by-products. In some instances, the by-product is 5-cholesten-3β-25-diol disulfate. In some instances, the 5-cholesten-3β-25-diol disulfate by-product is present in a composition produced by sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol at a concentration of 10% by weight or less, such as 9% by weight or less, for example 8% by weight or less, such as 7% by weight or less, for example 6% by weight or less, such as 5% by weight or less, for example 4% by weight or less, for example 3% by weight or less, for example 2% by weight or less, for example 5 ... For example, the sulfation product may be present in an amount of 1 wt% or less, such as 0.5 wt% or less, for example 0.1 wt% or less, for example 0.01 wt% or less, for example 0.001 wt% or less, including where 5-cholesten-3β-25-diol-disulfate by-product is present in an amount of 0.001 wt% or less in compositions produced by sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol, which may be in the range of 0.1 wt% to 50 wt%, for example 0.5 wt% to 20 wt% or 1 wt% to 12 wt%. In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the 5-cholesten-3β-25-diol-disulfate by-product formed is 10:1 or more, such as 25:1 or more, for example 50:1 or more, such as 100:1 or more, for example 250:1 or more, for example 500:1 or more, for example 1000:1 or more, for example 2500:1 or more, for example 5000:1 or more, for example 10,000:1 or more, for example 25,000:1 or more, for example 50,000:1 or more, for example 100,000:1 or more, for example 10 6 : 1 or more, e.g. 10 7 : 1 or more, e.g. 10 8 :1 or more, and the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the formed 5-cholesten-3β-25-diol disulfate is 10 9In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the formed 5-cholesten-3β-25-diol disulfate is 10:1 to 10:1. 9 :1, for example 100:1~10 8 :1, for example 1000:1~10 7 :1 range, 10000:1~10 6 :1 included.

[0356] Embodiments of the present disclosure also include compositions having 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and 5-cholesten-3β-25-diol-disulfate, wherein the 5-cholesten-3β-25-diol-disulfate is present in an amount of 10% by weight or less, such as 9% by weight or less, for example 8% by weight or less, such as 7% by weight or less, for example 6% by weight or less, such as 5% by weight or less, for example 4% by weight or less, such as 3% by weight or less, for example 2% by weight or less, such as 1% by weight or less, for example 0.5% by weight or less, such as 0.1% by weight or less, for example 0.01% by weight or less, for example 0.001% by weight or less, %, including 0.001% or less by weight, and may be in the range of 10% to 0.001% by weight, e.g., 10% to 0.1% by weight, 10% to 1% by weight, 10% to 2% by weight, 8% to 0.001% by weight, 8% to 0.1% by weight, 8% to 1% by weight, 8% to 2% by weight, 6% to 0.001% by weight, 6% to 0.1% by weight, 6% to 1% by weight, 6% to 2% by weight, 4% to 0.001% by weight, 4% to 0.1% by weight, 4% to 1% by weight, 4% to 2% by weight, 3% to 0.001% by weight, 3% to 0.1% by weight, 3% to 1% by weight, 2% to 0.001% by weight, 2% to 0.1% by weight, or 2% to 1% by weight.

[0357] In some cases, the composition may be 10:1 or more, such as 25:1 or more, for example 50:1 or more, such as 100:1 or more, for example 250:1 or more, such as 500:1 or more, for example 1000:1 or more, such as 2500:1 or more, for example 5000:1 or more, such as 10,000:1 or more, for example 25,000:1 or more, such as 50,000:1 or more, for example 100,000:1 or more, for example 10 6 : 1 or more, e.g. 10 7 : 1 or more, e.g. 10 8 :1 or more, and the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to 5-cholesten-3β-25-diol-disulfate in the composition is 10:1 or more. 9 In some cases, the composition may be 10:1 to 10:1. 9 :1, for example 100:1~10 8 :1, for example 1000:1~10 7 :1 range, 10000:1~10 6 :1 by weight of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and 5-cholesten-3β-25-diol-disulfate.

[0358] In some cases, the by-product is sulfated desmosterol (structure IB).

[0359] [ka]

[0360] In some cases, sulfated desmosterol ([(3S,8S,9S,10R,13R,14S,17R)-17-[(1R)-1,5-dimethylhex-4-enyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3-yl]sulfate) is present in an amount of 10% by weight or less, such as 9% by weight or less, for example 8% by weight or less, for example 7% by weight or less, e.g., 25-hydroxy-(3β)-cholest-5-en-3-ol in a composition produced by sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol. For example, it may be present in an amount of 6% by weight or less, such as 5% by weight or less, for example 4% by weight or less, for example 3% by weight or less, such as 2% by weight or less, for example 1% by weight or less, such as 0.5% by weight or less, for example 0.1% by weight or less, for example 0.01% by weight or less, such as 0.001% by weight or less, including when sulfated desmosterol is present in an amount of 0.001% by weight or less relative to 25-hydroxy-(3β)-cholest-5-ene-3-sulfate in a composition produced by sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol, and may be in the range of 0.1% by weight to 10% by weight, for example 0.2% by weight to 5% by weight or 0.3% by weight to 3% by weight. In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the sulfated desmosterol formed is 10:1 or more, such as 25:1 or more, for example 50:1 or more, such as 100:1 or more, for example 250:1 or more, for example 500:1 or more, such as 1000:1 or more, for example 2500:1 or more, for example 5000:1 or more, such as 10,000:1 or more, for example 25,000:1 or more, for example 50,000:1 or more, such as 100,000:1 or more, for example 10 6 : 1 or more, e.g. 10 7 : 1 or more, e.g. 10 8 :1 or more, and the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the sulfated desmosterol formed is 10 9In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the sulfated desmosterol formed is 10:1 to 10:1. 9 :1, for example 100:1~10 8 :1, for example 1000:1~10 7 :1 range, 10000:1~10 6 :1 included.

[0361] Embodiments of the present disclosure also include a composition having 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and sulfated desmosterol, wherein the sulfated desmosterol is present in the composition in an amount of 10% by weight or less, such as 9% by weight or less, for example 8% by weight or less, such as 7% by weight or less, for example 6% by weight or less, such as 5% by weight or less, for example 4% by weight or less, such as 3% by weight or less, for example 2% by weight or less, such as 1% by weight or less, for example 0.5% by weight or less, such as 0.1% by weight or less, for example 0.01% by weight or less, for example 0.001% by weight or less, relative to the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate; -sulfate, and may be in the range of 0.001% w / w or less, 10% to 0.001% w / w, e.g., 10% to 0.1%, 10% to 1%, 10% to 2%, 8% to 0.001%, 8% to 0.1%, 8% to 1%, 8% to 2%, 6% to 0.001%, 6% to 0.1%, 6% to 1%, 6% to 2%, 4% to 0.001%, 4% to 0.1%, 4% to 1%, 4% to 2%, 3% to 0.001%, 3% to 0.1%, 3% to 1%, 2% to 0.001%, 2% to 0.1%, or 2% to 1% by weight.

[0362] In some cases, the composition has a porosity of 10:1 or more, such as 25:1 or more, for example 50:1 or more, such as 100:1 or more, for example 250:1 or more, such as 500:1 or more, for example 1000:1 or more, such as 2500:1 or more, for example 5000:1 or more, such as 10,000:1 or more, for example 25,000:1 or more, such as 50,000:1 or more, for example 100,000:1 or more, for example 10 6 : 1 or more, e.g. 10 7 : 1 or more, e.g. 10 8 :1 or more, and the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to sulfated desmosterol in the composition is 10 9 In some cases, the composition may be 10:1 to 10:1. 9 :1, for example 100:1~10 8 :1, for example 1000:1~10 7 :1 and 10000:1~10 6 The weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and sulfated desmosterol includes a range of 1:1.

[0363] In some cases, the by-product of sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol present in the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate composition is a thermal decomposition product. In some cases, when the components of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate composition are separated by liquid chromatography (e.g., HPLC), the by-product is identified by its relative retention time. In some cases, when the components of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate composition are separated by HPLC using a C8 stationary phase, operated at about 45°C, and using a first mobile phase containing a buffer (e.g., aqueous sodium phosphate buffer) and a second mobile phase containing one or more organic solvents (see, for example, Tables 13 and 14 below), the by-product is sulfated desmosterol, a compound having a retention time of about 18.3 minutes. In some cases, the first mobile phase is an aqueous buffer. In some cases, the first mobile phase includes sodium phosphate. In some cases, the second mobile phase is selected from one or more of methoxypropyl acetate, acetonitrile, and methanol. In some cases, the flow rate of the first mobile phase is about 1.0 mL / min. In some cases, the flow rate of the second mobile phase is about 1.0 mL / min or greater. In some cases, 25-hydroxy-(3β)-cholest-5-ene-3-sulfate has a retention time of about 7.7 minutes under the same HPLC conditions. In some cases, when the components of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate composition are separated by HPLC using a C8 stationary phase, operated at about 45°C, and separating the components of the composition using a first mobile phase containing a buffer (e.g., an aqueous sodium phosphate buffer) and a second mobile phase containing one or more organic solvents (see, for example, Tables 13 and 14 below), the by-product is a compound having a retention time of about 37.7 minutes. Without wishing to be bound by theory, it is believed that the compound having a retention time of about 37.7 minutes is desmosterol. In some cases, the first mobile phase is an aqueous buffer. In some cases, the first mobile phase contains sodium phosphate.In some cases, the second mobile phase is selected from one or more of methoxypropyl acetate, acetonitrile, and methanol. In some cases, the flow rate of the first mobile phase is about 1.0 mL / min. In some cases, the flow rate of the second mobile phase is about 1.0 mL / min or greater. In some cases, 25-hydroxy-(3β)-cholest-5-ene-3-sulfate also has a retention time of about 7.7 minutes under the same HPLC conditions, so that sulfated desmosterol has a relative retention time of about 2.4 (=18.3 / 7.7) and the compound believed to be desmosterol has a relative retention time of about 4.9 (=37.7 / 7.7).

[0364] Embodiments of the present disclosure also include compositions having one or more by-products of the sulfation of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, the one or more by-products are present in the composition in an amount of 10% by weight or less, such as 9% by weight or less, for example 8% by weight or less, for example 7% by weight or less, such as 6% by weight or less, for example 5% by weight or less, such as 4% by weight or less, for example 3% by weight or less, for example 2% by weight or less, such as 1% by weight or less, for example 0.5% by weight or less, such as 0.1% by weight or less, for example 0.01% by weight or less, such as 0.001% by weight or less, including 0.001% by weight or less, and may be in the range of 0.1% by weight to 5% by weight, for example 0.2% by weight to 10% by weight or 0.3% by weight to 15% by weight. In some cases, the composition comprises 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and one or more by-products in an amount ranging from 0.0001% to 10% by weight, such as 0.005% to 9.5% by weight, for example, 0.001% to 9.0% by weight, such as 0.05% to 8.5% by weight, for example, 0.1% to 8.0% by weight, such as 0.5% to 7.5% by weight, for example, 1% to 7% by weight, such as 1.5% to 6.5% by weight, and including 2% to 6% by weight, relative to the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate.

[0365] In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to one or more by-products formed is 10:1 or more, such as 25:1 or more, for example 50:1 or more, for example 100:1 or more, such as 250:1 or more, for example 500:1 or more, for example 1000:1 or more, for example 2500:1 or more, for example 5000:1 or more, for example 10,000:1 or more, such as 25,000:1 or more, for example 50,000:1 or more, for example 100,000:1 or more, for example 10 6 : 1 or more, e.g. 10 7 : 1 or more, e.g. 10 8 :1 or more, and the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to one or more by-products formed is 10 9 In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to one or more by-products formed is 10:1 to 10:1. 9 :1, for example 100:1~10 8 :1, for example 1000:1~10 7 :1 range, 10000:1~10 6 :1 included.

[0366] In some cases, the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt is a 25-hydroxy-(3β)-cholest-5-ene-3-sulfate pyridinium salt (Scheme IA2).

[0367] [ka]

[0368] In certain instances, the sulfating agent is contacted with an anhydride prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol. In some instances, the anhydride is selected from acetic anhydride, trifluoroacetic anhydride, and triflic anhydride. The amount of anhydride relative to 25-hydroxy-(3β)-cholest-5-en-3-ol may vary and may be 0.001 equivalents or more, such as 0.2 equivalents or more, for example 0.3 equivalents or more, for example 0.4 equivalents or more, such as 0.5 equivalents or more, for example 0.6 equivalents or more, such as 0.7 equivalents or more, for example 0.8 equivalents or more, such as 0.9 equivalents or more, for example 1 equivalent or more, such as 1.1 equivalents or more, for example 1.2 equivalents or more, such as 1.3 equivalents or more, for example 1.4 equivalents or more, such as 1.5 equivalents or more, for example 1.6 equivalents or more, such as 1.7 equivalents or more, for example 1.8 equivalents or more, for example 1.9 equivalents or more, such as 2 equivalents or more, for example 3 equivalents or more, such as 4 equivalents or more, for example 5 equivalents or more. It may contain 10 equivalents or more, and is 0.001 equivalents to 10 equivalents, for example, 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 5 equivalents, 0.5 equivalents to 10 equivalents, 0.5 equivalents to 8 equivalents, 0.5 equivalents to 5 equivalents, 0.9 equivalents to 10 equivalents, 0.9 equivalents to The amount may be in the range of 8 equivalents, 0.9 to 5 equivalents, 1.3 to 10 equivalents, 1.3 to 8 equivalents, 1.3 to 5 equivalents, 1.5 to 10 equivalents, 1.5 to 8 equivalents, 1.5 to 5 equivalents, 2 to 10 equivalents, 2 to 8 equivalents, 2 to 5 equivalents, 0.1 to 1.5 equivalents, 0.5 to 1.1 equivalents, or 0.1 to 1 equivalent.

[0369] In some cases, the method includes quenching (i.e., deactivating) the unreacted sulfating agent after producing the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt. In some cases, quenching the sulfating agent includes adding water to the reaction mixture. The amount of water added to the reaction mixture relative to the amount of sulfating agent contacted with 25-hydroxy-(3β)-cholest-5-en-3-ol can vary and can be 1 equivalent or more, for example, 2 equivalents or more, for example, 3 equivalents or more, for example, 4 equivalents or more, for example, 5 equivalents or more, for example, 6 equivalents or more, for example, 7 equivalents or more, for example, 8 equivalents or more, for example, 9 equivalents or more, for example, 10 equivalents or more, for example, 15 equivalents or more, for example, 20 equivalents or more, including 25 equivalents or more.

[0370] In some cases, quenching the reactivity of the unreacted sulfating agent involves adding water to the reaction mixture, followed by adding at least one base. In some cases, the at least one base is a trialkylamine, such as trimethylamine or triethylamine. In some cases, the at least one base is 2,6-lutidine. In some cases, the at least one base is pyridine. Pyridine may be added to the reaction mixture 1 minute or more, for example, 5 minutes or more, for example, 10 minutes or more, for example, 15 minutes or more, for example, 30 minutes or more, for example, 45 minutes or more, for example, 60 minutes or more, for example, 90 minutes or more, for example, 120 minutes or more, for example, 150 minutes or more, for example, 180 minutes or more, for example, 210 minutes or more, including 240 minutes or more after adding water to the reaction mixture. In some cases, pyridine is added to the reaction mixture 60 minutes after adding water. The amount of pyridine added to the reaction mixture may vary relative to the amount of sulfating agent and may be 0.001 equivalents or more, such as 0.005 equivalents or more, for example 0.01 equivalents or more, such as 0.05 equivalents or more, for example 0.1 equivalents or more, such as 0.5 equivalents or more, for example 1 equivalent or more, such as 2 equivalents or more, for example 3 equivalents or more, such as 4 equivalents or more, for example 5 equivalents or more, such as 6 equivalents or more, including 10 equivalents or more.

[0371] In some cases, the unreacted sulfating agent in the reaction mixture is quenched with slow agitation. In some cases, quenching the unreacted sulfating agent with slow agitation includes stirring the reaction mixture in a manner sufficient to maintain aggregates of the unreacted sulfating agent in the reaction mixture. In some cases, slow agitation of the reaction mixture is sufficient to reduce the size of the aggregates of the unreacted sulfating agent by 10% or less, for example, 9% or less, for example, 8% or less, for example, 7% or less, for example, 6% or less, for example, 5% or less, for example, 4% or less, for example, 3% or less, for example, 2% or less, for example, 1% or less during the quench, including when the reaction mixture is slowly agitated to reduce the size of the aggregates of the unreacted sulfating agent by 0.1% or less during the quench. In some cases, slow agitation of the reaction mixture is sufficient to maintain the aggregates of the unreacted sulfating agent at the bottom of the reaction flask during the quench. In some cases, gentle agitation of the reaction mixture is sufficient so that little to no aggregates of unreacted sulfating agent are present in the agitating vortex of the agitated reaction mixture.

[0372] In some cases, the method includes purifying the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt before contacting the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt with at least one metal salt. In some cases, the purified 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt has a purity of 97% or greater, such as a purity of 98% or greater, for example, a purity of 99% or greater, for example, a purity of 99.5% or greater, for example, a purity of 99.7% or greater, for example, a purity of 99.9% or greater, including a purity of 99.99% or greater. In some cases, the purified 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt has one or more by-products of sulfation (e.g., by-products from the sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol), wherein the one or more by-products are 5% w / w or less, such as 4% w / w or less, for example 3% w / w or less, such as 2% w / w or less, for example 1% w / w or less, of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt. , such as 0.9% w / w or less, for example 0.8% w / w or less, such as 0.7% w / w or less, for example 0.6% w / w or less, such as 0.5% w / w or less, for example 0.4% w / w or less, such as 0.3% w / w or less, for example 0.2% w / w or less, such as 0.1% w / w or less, for example 0.05% w / w or less, for example 0.01% w / w or less, including being present in an amount of 0.001% w / w or less relative to the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt.In some cases, the bis-sulfated product (i.e., 5-cholesten-3β-25-diol disulfate) is present in the purified 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt composition in an amount of 1% w / w or less, for example, 0.9% w / w or less, for example, 0.8% w / w or less, for example, 0.7% w / w or less, relative to the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt. % w / w or less, such as 0.6% w / w or less, for example 0.5% w / w or less, such as 0.4% w / w or less, for example 0.3% w / w or less, such as 0.2% w / w or less, for example 0.1% w / w or less, such as 0.05% w / w or less, for example 0.01% w / w or less, including being present in an amount of 0.001% w / w or less relative to the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt.

[0373] In some cases, 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salts are purified by liquid chromatography. In some cases, purification of 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salts involves liquid chromatography using a silica gel stationary phase (e.g., a silica gel plug column, 5 mass equivalents or more). In some cases, 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salts are purified using a silica gel stationary phase and a mobile phase comprising pyridine. In some cases, the mobile phase comprises methylene chloride, methanol, and pyridine. In some cases, the mobile phase comprises a mixture of methylene chloride-methanol (85:15) and pyridine (1%).

[0374] In some cases, one or more fractions collected from the stationary phase may be combined. In some cases, the combined fractions may be concentrated. In some cases, the combined fractions are concentrated by distillation. In some cases, the combined fractions are concentrated under vacuum. In some cases, the combined fractions are concentrated by distillation under vacuum.

[0375] In some cases, the combined fractions are contacted with one or more particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt (e.g., particles from a previously purified sample of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt). In some cases, contacting the particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt with the combined fractions is sufficient to precipitate the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt in the combined fractions. In some cases, contacting the particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt with the combined fractions comprises adding the particles during distillation of the combined fractions. In some cases, the particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt are added to the combined fractions before distilling the combined fractions. In some cases, the particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salt are added to the combined fractions during distillation, for example, 1 minute or more, for example, 5 minutes or more, for example, 10 minutes or more, for example, 15 minutes or more, for example, 20 minutes or more, for example, 30 minutes or more, for example, 40 minutes or more, for example, 50 minutes or more, and the particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salt are added to the combined fractions at least 60 minutes after the start of distillation of the combined fractions. In some cases, the combined fractions are distilled under constant pressure, for example, where the pressure varies by 10% or less, for example, 9% or less, for example, 8% or less, for example, 7% or less, for example, 6% or less, for example, 5% or less, for example, 4% or less, for example, 3% or less, for example, 2% or less, for example, 1% or less, and including 0.1% or less. In some cases the pressure during distillation varies, including 10 inHg or less, such as 9 inHg or less, for example 8 inHg or less, for example 7 inHg or less, such as 6 inHg or less, for example 5 inHg or less, such as 4 inHg or less, for example 3 inHg or less, such as 2 inHg or less, for example 1 inHg or less, such as 0.5 inHg or less, for example 0.1 inHg or less, such as 0.05 inHg or less, 0.01 inHg or less.In some cases, the combined fractions are distilled under reduced pressure, with the pressure maintained between 15 inHg and 30 inHg, such as 17.5 inHg and 27.5 inHg, for example, 20 inHg and 25 inHg, for example, 21 inHg and 24 inHg, including maintaining a pressure between 22 inHg and 23 inHg.

[0376] In some cases, the combined fractions are concentrated under vacuum, and the concentrated combined fractions are contacted with a composition comprising particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salt. In some cases, the concentrated combined fractions are contacted with a composition comprising particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salt and at least one solvent. In some cases, the at least one solvent is selected from tetrahydrofuran, such as 2-methyltetrahydrofuran. The concentrated combined fraction can be contacted with a composition comprising particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salt for a duration of 0.001 minutes or more, for example, 0.005 minutes or more, for example, 0.01 minutes or more, for example, 0.05 minutes or more, for example, 0.1 minutes or more, for example, 0.5 minutes or more, for example, 1 minute or more, for example, 2 minutes or more, for example, 3 minutes or more, for example, 4 minutes or more, for example, 5 minutes or more, for example, 10 minutes or more, for example, 15 minutes or more, for example, 30 minutes or more, for example, 45 minutes or more, and including 60 minutes or more.In some cases, the combined fraction is added dropwise to a composition comprising 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salt in 2-methyltetrahydrofuran.

[0377] In some cases, 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt is contacted with a metal salt to produce 25-hydroxy-(3β)-cholest-5-ene-3-sulfate metal salt (Scheme IB1).

[0378] [ka]

[0379] In some cases, a method for producing 25-hydroxy-(3β)-cholest-5-ene-3-sulfate metal salt includes contacting a 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt with at least one sodium salt. In some cases, the at least one sodium salt is selected from sodium acetate, sodium iodide, sodium chloride, sodium hydroxide, and sodium methoxide. The 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt may be contacted with the metal salt at a temperature ranging from -10°C to 75°C, for example, from -5°C to 70°C, for example, from -4°C to 65°C, for example, from -3°C to 60°C, for example, from -2°C to 55°C, for example, from -1°C to 50°C, for example, from 0°C to 45°C, for example, from 5°C to 40°C, including 10°C to 35°C.

[0380] The reaction may be carried out for a duration ranging from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, for example, from 0.3 hours to 24 hours, for example, from 0.4 hours to 21 hours, for example, from 0.5 hours to 20 hours, for example, from 0.6 hours to 19 hours, for example, from 0.7 hours to 18 hours, for example, from 0.8 hours to 17 hours, for example, from 0.9 hours to 16 hours, and inclusive of from 1 hour to 15 hours. The amount of metal salt used may vary relative to the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt and may be 0.0001 equivalents or more, such as 0.001 equivalents or more, for example 0.01 equivalents or more, for example 0.1 equivalents or more, such as 0.2 equivalents or more, for example 0.3 equivalents or more, for example 0.4 equivalents or more, such as 0.5 equivalents or more, for example 0.6 equivalents or more, such as 0.7 equivalents or more, for example 0.8 equivalents or more, for example 0.9 equivalents or more, such as 1 equivalent or more, for example 1.1 equivalents or more, such as 1.2 equivalents or more, for example 1.3 equivalents or more, for example 1.4 equivalents or more, for example 1.5 equivalents or more, such as 1.6 equivalents or more, for example 1.7 equivalents or more, for example 1.8 equivalents or more. It may be 9 equivalents or more, for example, 2 equivalents or more, for example, 3 equivalents or more, for example, 4 equivalents or more, for example, 5 equivalents or more, including 10 equivalents or more, such as 0.001 equivalents to 10 equivalents, for example, 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 6 equivalents, 0.1 equivalents to 4 equivalents, 0.1 equivalents to 3 equivalents, 1 equivalent to 10 equivalents, 1 equivalent to 8 equivalents, 1 equivalent to 6 equivalents The amount may be in the range of 1 to 4 equivalents, 1 to 3 equivalents, 1.5 to 10 equivalents, 1.5 to 8 equivalents, 1.5 to 6 equivalents, 1.5 to 4 equivalents, 1.5 to 3 equivalents, 2 to 10 equivalents, 2 to 8 equivalents, 2 to 6 equivalents, 2 to 4 equivalents, or 2 to 3 equivalents, 1 to 100 equivalents, 1 to 5 equivalents, or 1 to 2 equivalents.

[0381] In some cases, the method includes contacting 25-hydroxy-(3β)-cholest-5-ene-3-sulfate pyridinium salt with sodium iodide to produce 25-hydroxy-(3β)-cholest-5-ene-3-sulfate sodium salt (Scheme IB2).

[0382] [ka]

[0383] In some cases, a method for preparing 25-hydroxy-3β-cholesten-5-ene-3-sulfate includes contacting 25-hydroxy-(3β)-cholest-5-en-3-ol with sulfur trioxide-pyridine complex to produce 25-hydroxy-(3β)-cholest-5-ene-3-sulfate pyridinium salt, and contacting 25-hydroxy-(3β)-cholest-5-ene-3-sulfate pyridinium salt with sodium salt to produce 5-cholesten-3β,25-diol 3-sulfate sodium salt (Scheme Ib).

[0384] [ka]

[0385] In some cases, the method for preparing 25-hydroxy-3β-cholesten-5-ene-3-sulfate includes contacting (3β)-cholest-5-en-3-ol with a sulfating agent to produce a first (3β)-cholest-5-ene-3-sulfate organic cation salt; contacting the first (3β)-cholest-5-ene-3-sulfate organic cation salt with an organic base to produce a second (3β)-cholest-5-ene-3-sulfate organic cation salt; oxidizing the second (3β)-cholest-5-ene-3-sulfate organic cation salt in the presence of at least one surfactant. generating 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt from the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cation salt by deoxygenation; and contacting the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt with at least one metal salt to produce 5-cholestene-3β,25-diol 3-sulfate metal salt (Scheme IIa).

[0386] [ka]

[0387] In some cases, cholesterol is sulfated using a sulfating agent (Scheme IIA1). In some cases, the sulfating agent is selected from sulfur trioxide complexes, sulfate compounds, sulfonic acid compounds, and sulfonate compounds. In some cases, the sulfating agent is a sulfur trioxide-pyridine complex. In some cases, the sulfating agent is selected from sulfur trioxide dimethylformamide, sulfur trioxide triethylamine, and sulfur trioxide trimethylamine. In some cases, the sulfating agent is sulfuric acid, acetic anhydride, and pyridine. In some cases, the sulfating agent is selected from chlorosulfonic acid and pyridine. In some cases, the sulfating agent is selected from chlorosulfonic acid and 2,6-lutidine. In some cases, the sulfating agent is selected from ethyl chloridosulfate.

[0388] Cholesterol may be sulfated at a temperature ranging from 0° C. to 100° C., for example, 5° C. to 95° C., for example, 10° C. to 90° C., for example, 15° C. to 85° C., for example, 20° C. to 80° C., for example, 25° C. to 75° C., and including 30° C. to 70° C. The reaction may be carried out for a duration ranging from 0.1 hours to 72 hours, for example, 0.2 hours to 48 hours, for example, 0.3 hours to 24 hours, for example, 0.4 hours to 21 hours, for example, 0.5 hours to 20 hours, for example, 0.6 hours to 19 hours, and including 0.7 hours to 18 hours. The amount of sulfating agent used may vary relative to cholesterol and may be 0.0001 equivalents or more, such as 0.001 equivalents or more, for example 0.01 equivalents or more, such as 0.1 equivalents or more, for example 0.2 equivalents or more, such as 0.3 equivalents or more, for example 0.4 equivalents or more, such as 0.5 equivalents or more, for example 0.6 equivalents or more, such as 0.7 equivalents or more, for example 0.8 equivalents or more, such as 0.9 equivalents or more, for example 1 equivalent or more, such as 1.1 equivalents or more, for example 1.2 equivalents or more, such as 1.3 equivalents or more, for example 1.4 equivalents or more, such as 1.5 equivalents or more, for example 1.6 equivalents or more, such as 1.7 equivalents or more, for example 1.8 equivalents or more, such as 1.9 equivalents or more, for example 2 equivalents or more. For example, it may be 3 equivalents or more, for example, 4 equivalents or more, for example, 5 equivalents or more, including 10 equivalents or more, such as 0.001 equivalents to 10 equivalents, for example, 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 6 equivalents, 0.1 equivalents to 4 equivalents, 0.1 equivalents to 3 equivalents, 1 equivalent to 10 equivalents, 1 equivalent to 8 equivalents, 1 equivalent to 6 equivalents, 1 equivalent The amount may be in the range of 1 to 4 equivalents, 1 to 3 equivalents, 1.5 to 10 equivalents, 1.5 to 8 equivalents, 1.5 to 6 equivalents, 1.5 to 4 equivalents, 1.5 to 3 equivalents, 2 to 10 equivalents, 2 to 8 equivalents, 2 to 6 equivalents, 2 to 4 equivalents, 2 to 3 equivalents, 1 to 30 equivalents, 1 to 5 equivalents, or 1 to 2 equivalents.

[0389] [ka]

[0390] In some cases, the first (3β)-cholest-5-ene-3-sulfate organic cation salt is a (3β)-cholest-5-ene-3-sulfate pyridinium salt (Scheme IIA2).

[0391] [ka]

[0392] In some cases, a first (3β)-cholest-5-ene-3-sulfate organic cation salt (structure IIA) is contacted with an organic base to produce a second (3β)-cholest-5-ene-3-sulfate organic cation salt (structure IIB) (Scheme IIB1).

[0393] [ka]

[0394] In some cases, the organic base contacted with the first (3β)-cholest-5-ene-3-sulfuric acid organic cation salt is selected from hydroxide bases. In some cases, the hydroxide base is selected from tetraethylammonium hydroxide, tetrabutylammonium hydroxide, tetrapropylammonium hydroxide, and tetramethylammonium hydroxide. In some cases, the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt is selected from tetraethylammonium cation salts, tetrabutylammonium cation salts, tetrapropylammonium cation salts, and tetramethylammonium cation salts. In some cases, the organic base is contacted with the first (3β)-cholest-5-ene-3-sulfuric acid organic cation salt at a temperature ranging from -10°C to 75°C, for example, from -5°C to 70°C, for example, from -4°C to 65°C, for example, from -3°C to 60°C, for example, from -2°C to 55°C, for example, from -1°C to 50°C, including 0°C to 15°C. The reaction may be carried out for a duration ranging from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, for example, from 0.3 hours to 24 hours, for example, from 0.4 hours to 21 hours, for example, from 0.5 hours to 20 hours, for example, from 0.6 hours to 19 hours, for example, from 0.7 hours to 18 hours, for example, from 0.8 hours to 17 hours, for example, from 0.9 hours to 16 hours, and inclusive of from 1 hour to 15 hours.The amount of organic base used may vary relative to the first (3β)-cholest-5-ene-3-sulfate organic cation salt and may be 0.0001 equivalents or more, such as 0.001 equivalents or more, for example 0.01 equivalents or more, for example 0.1 equivalents or more, such as 0.2 equivalents or more, for example 0.3 equivalents or more, for example 0.4 equivalents or more, such as 0.5 equivalents or more, for example 0.6 equivalents or more, such as 0.7 equivalents or more, for example 0.8 equivalents or more, for example 0.9 equivalents or more, such as 1 equivalent or more, for example 1.1 equivalents or more, such as 1.2 equivalents or more, for example 1.3 equivalents or more, for example 1.4 equivalents or more, such as 1.5 equivalents or more, for example 1.6 equivalents or more, for example 1.7 equivalents or more, for example 1.8 equivalents or more, such as 1.9 equivalents The amount may be 10 or more, for example, 2 or more, for example, 3 or more, for example, 4 or more, for example, 5 or more, including 10 or more, such as 0.001 to 10 equivalents, for example, 0.1 to 10 equivalents, 0.1 to 8 equivalents, 0.1 to 6 equivalents, 0.1 to 4 equivalents, 0.1 to 3 equivalents, 1 to 10 equivalents, 1 to 8 equivalents, 1 to 6 equivalents, The amount may be in the range of 1 to 4 equivalents, 1 to 3 equivalents, 1.5 to 10 equivalents, 1.5 to 8 equivalents, 1.5 to 6 equivalents, 1.5 to 4 equivalents, 1.5 to 3 equivalents, 2 to 10 equivalents, 2 to 8 equivalents, 2 to 6 equivalents, 2 to 4 equivalents, 2 to 3 equivalents, 1 to 10 equivalents, 1 to 5 equivalents, or 1 to 2 equivalents.

[0395] In certain instances, the method includes contacting a first (3β)-cholest-5-ene-3-sulfate organic cation salt with tetrabutylammonium hydroxide to generate (3β)-cholest-5-ene-3-sulfate tetrabutylammonium cation salt (Structure IIB1) (Scheme IIB2).

[0396] [ka]

[0397] In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is oxidized to produce 25-hydroxy-(3β)-cholesta-(5,6-epoxy)-3-sulfate organic cation salt (Structure IIC) (Scheme IIC1).

[0398] [ka]

[0399] In some cases, oxidizing the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt includes contacting the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt with a composition having an oxidizing agent and at least one surfactant.

[0400] In some cases, the at least one surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, and zwitterionic surfactants. The nonionic surfactant may be selected from polyoxyethylene glycol ethers (e.g., polyoxyethylene glycol octylphenol ether), polyoxyethylene glycol sorbitan alkyl esters, sorbitan alkyl esters, block copolymers of polyethylene glycol and polypropylene glycol, among other nonionic surfactants. The anionic surfactant may be selected from surfactants having an anionic functional head group, such as surfactants containing a sulfonate, phosphate, sulfate, or carboxylate head group. For example, the anionic surfactant may be selected from alkyl sulfates, such as ammonium lauryl sulfate, dioctyl sodium sulfosuccinate, perfluorooctanesulfonate, perfluorononanoate, perfluorooctanoate, linear alkylbenzenesulfonate, alkylaryletherphosphate, sodium lauryl ether sulfate, lignosulfonate, or sodium stearate, among other anionic surfactants. The cationic surfactant may be selected from surfactants having a cationic functional head group, such as a pyridinium or quaternary ammonium head group. For example, the cationic surfactant may be selected from cetyltrimethylammonium hydrogen sulfate, tetrabutylammonium hydrogen sulfate, cetyltrimethylammonium bromide, tetrabutylammonium bromide, tetrabutylammonium iodide, tetrabutylphosphonium bromide, tetraoctylammonium bromide, tetraoctylammonium iodide, benzyltriethylammonium chloride, benzyltriethylammonium bromide, benzylcetyldimethylammonium chloride, or benzylcetyldimethylammonium bromide. The zwitterionic surfactant contains both a cationic center and an anionic center, such as a sultaine (e.g., 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or a betaine (e.g., cocamidopropyl betaine).In some cases, the surfactant is Extran laboratory soap, La Parisienne soap, or DL-α-tocopherol methoxypolyethylene glycol succinate (e.g., TPGS-750-M2).

[0401] The amount of surfactant used may vary relative to the second (3β)-cholest-5-ene-3-sulfate organic cation salt, and in some cases may be 0.0001 equivalents or more, such as 0.001 equivalents or more, for example 0.01 equivalents or more, for example 0.1 equivalents or more, such as 0.2 equivalents or more, for example 0.3 equivalents or more, for example 0.4 equivalents or more, such as 0.5 equivalents or more, for example 0.6 equivalents or more, such as 0.7 equivalents or more, for example 0.8 equivalents or more, for example 0.9 equivalents or more, such as 1 equivalent or more, for example 1.1 equivalents or more, such as 1.2 equivalents or more, for example 1.3 equivalents or more, for example 1.4 equivalents or more, such as 1.5 equivalents or more, for example 1.6 equivalents or more, for example 1.7 equivalents or more, for example 1.8 equivalents or more, such as 1.9 equivalents or more. For example, 2 equivalents or more, for example 3 equivalents or more, for example 4 equivalents or more, for example 5 equivalents or more of a surfactant is used, including 10 equivalents or more of a surfactant, 0.001 equivalents to 10 equivalents, for example 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 6 equivalents, 0.1 equivalents to 4 equivalents, 0.1 equivalents to 3 equivalents, 1 equivalent to 10 equivalents, 1 equivalent to 8 equivalents, 1 equivalent to 6 equivalents The amount may be in the range of 1 equivalent to 4 equivalents, 1 equivalent to 3 equivalents, 1.5 equivalents to 10 equivalents, 1.5 equivalents to 8 equivalents, 1.5 equivalents to 6 equivalents, 1.5 equivalents to 4 equivalents, 1.5 equivalents to 3 equivalents, 2 equivalents to 10 equivalents, 2 equivalents to 8 equivalents, 2 equivalents to 6 equivalents, 2 equivalents to 4 equivalents, 2 equivalents to 3 equivalents, 0.1 equivalents to 5 equivalents, 0.15 equivalents to 1 equivalent, or 0.2 equivalents to 0.3 equivalents.

[0402] In some cases, oxidizing the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt includes contacting the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt with an oxidizing agent and at least one ketone in the presence of at least one surfactant.

[0403] In some cases, the at least one ketone is selected from tetrahydrothiopyran-4-one 1,1-dioxide and a halogenated ketone. In some cases, the halogenated ketone is selected from 1,1,1-trifluoro-2-butanone, 4,4-difluorocyclohexanone, 2-2-2-4'-tetrafluoroacetophenone, and 1,1,1-trifluoroacetone. In some cases, the at least one ketone is 1,1,1-trifluoro-2-butanone. The amount of ketone used may vary relative to the oxidizing agent in the subject reaction and may be 1 equivalent or more, such as 2 equivalents or more, for example 3 equivalents or more, for example 4 equivalents or more, such as 5 equivalents or more, for example 6 equivalents or more, such as 7 equivalents or more, for example 8 equivalents or more, such as 9 equivalents or more, for example 10 equivalents or more, such as 15 equivalents or more, for example 20 equivalents or more, such as 25 equivalents or more, for example 30 equivalents or more, for example 35 equivalents or more, including 50 equivalents or more of ketone, from 1 equivalent to 50 equivalents, for example The amount may be in the range of 1 equivalent to 35 equivalents, 1 equivalent to 25 equivalents, 1 equivalent to 15 equivalents, 1 equivalent to 10 equivalents, 1 equivalent to 8 equivalents, 1 equivalent to 5 equivalents, 2 equivalents to 50 equivalents, 2 equivalents to 35 equivalents, 2 equivalents to 25 equivalents, 2 equivalents to 15 equivalents, 2 equivalents to 10 equivalents, 2 equivalents to 8 equivalents, 2 equivalents to 5 equivalents, 4 equivalents to 50 equivalents, 4 equivalents to 35 equivalents, 4 equivalents to 25 equivalents, 4 equivalents to 15 equivalents, 4 equivalents to 10 equivalents, 4 equivalents to 8 equivalents, 1 equivalent to 50 equivalents, 2 equivalents to 25 equivalents, or 5 equivalents to 10 equivalents.

[0404] In some cases, the ketone is further purified before use. For example, the ketone may be purified by distillation before use. In some cases, the reactivity of the ketone is tested to determine if purification may be necessary (e.g., 1 Tested for impurities by HNMR).

[0405] In certain instances, oxidizing the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt includes contacting the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt with an oxidizing agent and at least one ketone in the presence of at least one surfactant and water. The amount of water present may vary from 0.0000001% w / v or more, such as 0.000001% w / v or more, for example 0.00001% w / v or more, such as 0.00001% w / v or more, for example 0.0001% w / v or more, such as 0.0001% w / v or more, for example 0.01% w / v or more, such as 0.1% w / v, for example 0.05% w / v or more, such as 0.1% w / v or more, for example 0.5% w / v or more, such as 1% w / v or more, for example 5% w / v or more, such as 10% w / v or more, for example 15% w / v or more, of the reaction mixture, including 25% w / v or more of the reaction mixture, 0.0000001%w / v~5%w / v, 0.0000001%w / v~1%w / v, 0.001%w / v~25%w / v, 0.001%w / v~15%w / v, 0 .001%w / v~10%w / v, 0.001%w / v~5%w / v, 0.001%w / v~1%w / v, 0.1%w / v~25%w / v, 0.1%w / v~15%w / The range may be 0.1% w / v to 10% w / v, 0.1% w / v to 5% w / v, 0.1% w / v to 1% w / v, 1% w / v to 25% w / v, 1% w / v to 15% w / v, 1% w / v to 10% w / v, 1% w / v to 5% w / v, 0.1% w / v to 50% w / v, 0.1% w / v to 10% w / v, or 0.5% w / v to 1% w / v.

[0406] The second (3β)-cholest-5-ene-3-sulfate organic cation salt may be oxidized at a temperature ranging from -25°C to 50°C, for example, from -20°C to 45°C, for example, from -15°C to 40°C, for example, from -10°C to 35°C, for example, from -5°C to 30°C, for example, from -1°C to 25°C, including from 0°C to 15°C. In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is oxidized at a temperature ranging from 0°C to 5°C. When the reaction mixture contains a certain amount of water, the reaction may be carried out at a temperature ranging from -10°C to 50°C, for example, from -5°C to 45°C, for example, from 0°C to 40°C, for example, from 0°C to 35°C, for example, from 0°C to 30°C, for example, from 0°C to 25°C, for example, from 0°C to 20°C, including from 0°C to 15°C.

[0407] The second (3β)-cholest-5-ene-3-sulfate organic cation salt may be oxidized at a pH in the range of 5 to 7.5, for example, a pH of 5.5 to 7.0, including a pH of 5.5 to 6.5. In some cases, when the reaction mixture includes water (e.g., in a two-phase solvent system), the pH is in the range of 5.0 to 6.0, for example, pH 5.0 to 5.9, for example, pH 5.0 to 5.8, for example, pH 5.0 to 5.7, for example, pH 5.0 to 5.6, including pH 5.0 to 5.5.

[0408] The reaction may be carried out for a duration ranging from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, for example, from 0.3 hours to 24 hours, for example, from 0.4 hours to 21 hours, for example, from 0.5 hours to 20 hours, for example, from 0.6 hours to 19 hours, for example, from 0.7 hours to 18 hours, for example, from 0.8 hours to 17 hours, for example, from 0.9 hours to 16 hours, and inclusive of from 1 hour to 15 hours.

[0409] In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is contacted in situ with a composition having potassium peroxymonosulfate and at least one ketone in the presence of at least one surfactant. In some cases, the method includes contacting potassium peroxymonosulfate with at least one ketone in the presence of at least one surfactant to form a separate oxidized reactive mixture, and adding the oxidized reactive mixture to the second (3β)-cholest-5-ene-3-sulfate organic cation salt. In these cases, the potassium peroxymonosulfate is contacted with the at least one ketone in the presence of the at least one surfactant for a duration including 0.1 minutes or more, e.g., 1 minute or more, e.g., 2 minutes or more, e.g., 3 minutes or more, e.g., 5 minutes or more, and 10 minutes or more, before contacting the oxidized reactive mixture with the second (3β)-cholest-5-ene-3-sulfate organic cation salt, which may range from 2 minutes to 180 minutes, e.g., 3 minutes to 120 minutes, or 4 minutes to 60 minutes. In some cases, potassium peroxymonosulfate may be contacted with at least one ketone in the presence of at least one surfactant to form a separate oxidative reactive mixture, and the oxidative reactive mixture may then be immediately contacted with a second (3β)-cholest-5-ene-3-sulfate organic cation salt. The oxidative reactive mixture may be formed at a temperature ranging from -10°C to 50°C, for example, from -5°C to 45°C, for example, from -4°C to 40°C, for example, from -3°C to 35°C, for example, from -2°C to 30°C, for example, from -1°C to 25°C, and inclusive, from 0°C to 15°C. If the oxidative reactive mixture is not immediately contacted with the second (3β)-cholest-5-ene-3-sulfate organic cation salt, the oxidative reactive mixture may be maintained at a temperature ranging from -10°C to 50°C, for example, from -5°C to 45°C, for example, from -4°C to 40°C, for example, from -3°C to 35°C, for example, from -2°C to 30°C, for example, from -1°C to 25°C, and inclusive, from 0°C to 15°C.

[0410] In some cases, the method further includes adding the oxidation reactive mixture to a second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt. In some cases, the method includes adding the oxidation reactive mixture dropwise to the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt. In some cases, the oxidation reactive mixture is added to a measured amount of the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt. The measured amount may be added continuously or at predetermined time intervals (e.g., every 30 seconds, every minute, every 2 minutes, every 3 minutes, every 5 minutes, or some other interval). In some cases, the oxidation reactive mixture is added to the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt by controlled addition, for example, using a mechanically or computer-controlled pump, such as a syringe pump. In some cases, the method includes generating an oxidation reactive mixture and adding a composition containing a second (3β)-cholest-5-ene-3-sulfate organic cation salt to the oxidation reactive mixture. In some cases, the method includes adding the second (3β)-cholest-5-ene-3-sulfate organic cation salt dropwise to the oxidation reactive mixture. In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is added to the oxidation reactive mixture in a measured amount. The measured amount may be added continuously or at predetermined time intervals (e.g., every 30 seconds, every minute, every 2 minutes, every 3 minutes, every 5 minutes, or some other interval). In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is added to the oxidation reactive mixture by controlled addition, for example, using a mechanically or computer-controlled pump, such as a syringe pump.

[0411] In certain cases, oxidizing the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt comprises contacting the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt with at least one oxidizing species. In some cases, the at least one oxidizing species is selected from a dioxirane. In some cases, the dioxirane is generated in situ in the composition containing the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt. In some cases, the dioxirane is generated separately (e.g., in a separate reaction vessel, e.g., a flask) and added to the composition containing the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt.

[0412] In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is oxidized in the presence of at least one base. In some cases, the at least one base is selected from weak bases. In some cases, the at least one base is selected from potassium bicarbonate, sodium bicarbonate, potassium phenoxide, sodium citrate buffer, sodium phosphate buffer, potassium formate, and potassium acetate. In some cases, the at least one base is potassium bicarbonate. In some cases, the at least one base may be added to the reaction mixture over time, for example, in a measured amount, where the base is added at predetermined time intervals (e.g., every 30 seconds, every minute, every 2 minutes, every 3 minutes, every 5 minutes, or some other interval). In some cases, the at least one base may be a composition with water. The base may be present in the composition at 0.0000001% w / v or more, such as 0.000001% w / v or more, for example 0.00001% w / v or more, for example 0.0001% w / v or more, such as 0.001% w / v or more, for example 0.01% w / v or more, such as 0.05% w / v or more, for example 0.1% w / v or more, such as 0.5% w / v or more, for example 1% w / v or more, such as 5% w / v or more, for example 10% w / v or more, such as 15% w / v or more, including 25% w / v or more of the composition, such as 0.0000001% w / v to 25% w / v, for example 0.0000001% w / v to 15% w / v, 0.0000001% w / v to 10% w / v, 0.00000 01%w / v~5%w / v, 0.0000001%w / v~1%w / v, 0.001%w / v~25%w / v, 0.001%w / v~15%w / v, 0.001%w / v~10%w / v, 0.001%w / v~5%w / v, 0.001%w / v~1%w / v, 0.1%w / v~25%w / v, 0.1%w / v~15%w / v, 0. The range may be between 1% w / v and 10% w / v, 0.1% w / v and 5% w / v, 0.1% w / v and 1% w / v, 1% w / v and 25% w / v, 1% w / v and 15% w / v, 1% w / v and 10% w / v, 1% w / v and 5% w / v, 0.1% w / v and 20% w / v, 0.2% w / v and 15% w / v, or 0.3% w / v and 10% w / v. In some cases, the at least one base may be an aqueous potassium bicarbonate composition.

[0413] In one instance, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is oxidized by contacting it with Oxone in the presence of cetyltrimethylammonium hydrogen sulfate (CTAHS), followed by the addition of trifluorobutanone and potassium hydrogen sulfate to form 25-hydroxy-(3β)-cholesta-(5,6-epoxy)-3-sulfate organic cation salt (Scheme IIC2).

[0414] [ka]

[0415] In some instances, the method includes forming an oxidizing species in situ using a second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt, for example, by contacting potassium peroxymonosulfate and trifluorobutanone with the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt in a reaction mixture in the presence of cetyltrimethylammonium hydrogen sulfate (CTAHS). In some instances, forming an oxidizing species in situ using the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt includes forming a dioxirane in situ using the second (3β)-cholest-5-ene-3-sulfuric acid organic cation salt.

[0416] In some cases, the method includes forming a dioxirane in a separate reaction and adding the dioxirane to a second (3β)-cholest-5-ene-3-sulfate organic cation salt. In these cases, potassium peroxymonosulfate may be contacted with trifluorobutanone in the presence of cetyltrimethylammonium hydrogen sulfate (CTAHS) for a duration including 0.1 minutes or more, such as 1 minute or more, for example 2 minutes or more, for example 3 minutes or more, for example 5 minutes or more, and 10 minutes or more, prior to contacting the reactive composition with the second (3β)-cholest-5-ene-3-sulfate organic cation salt, the duration may range from 0.01 minutes to 120 minutes, for example 0.1 minutes to 90 minutes, or 0.5 minutes to 60 minutes. In some cases, potassium peroxymonosulfate may be contacted with trifluorobutanone in the presence of cetyltrimethylammonium hydrogen sulfate (CTAHS) to form an oxidation-reactive composition, which is immediately contacted with a second (3β)-cholest-5-ene-3-sulfate organic cation salt.

[0417] 25-Hydroxy-(3β)-cholesta-(5,6-epoxy)-3-sulfuric acid organic cation salt is deoxygenated to produce 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salt (Structure IID) (Scheme IID1).

[0418] [ka]

[0419] In some cases, generating 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salt from 25-hydroxy-(3β)-cholesta-(5,6-epoxy)-3-sulfuric acid organic cation salt includes deoxygenating the 25-hydroxy-(3β)-cholesta-(5,6-epoxy)-3-sulfuric acid organic cation salt by contacting it with zinc. In some cases, the 25-hydroxy-(3β)-cholesta-(5,6-epoxy)-3-sulfuric acid organic cation salt is contacted with zinc in the presence of at least one halide and at least one acid. In some cases, the at least one halide is selected from iodine and a metal halide. In some cases, the metal halide is selected from sodium iodide and lithium iodide. In some cases, the at least one acid is selected from a weak acid. In some cases, the at least one acid is selected from acetic acid, hydrochloric acid, citric acid, paratoluenesulfonic acid, formic acid, and methanesulfonic acid.

[0420] The amount of reagent used to deoxygenate the 25-hydroxy-(3β)-cholesta-(5,6-epoxy)-3-sulfonic acid organic cation salt may vary, and in some cases may be 0.0001 equivalents or more, such as 0.001 equivalents or more, for example 0.01 equivalents or more, for example 0.1 equivalents or more, such as 0.2 equivalents or more, for example 0.3 equivalents or more, such as 0.4 equivalents or more, for example 0.5 equivalents or more, such as 0.6 equivalents or more, for example 0.7 equivalents or more, such as 0.8 equivalents or more, for example 0.9 equivalents or more, such as 1 equivalent or more, for example 1.1 equivalents or more, for example 1.2 equivalents or more, such as 1.3 equivalents or more, for example 1.4 equivalents or more, for example 1.5 equivalents or more, for example 1.6 equivalents A reagent containing 10 or more equivalents, for example 1.7 equivalents or more, for example 1.8 equivalents or more, for example 1.9 equivalents or more, for example 2 equivalents or more, for example 3 equivalents or more, for example 4 equivalents or more, for example 5 equivalents or more, is used, and 0.001 equivalents to 10 equivalents, for example 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 6 equivalents, 0.1 equivalents to 4 equivalents, 0.1 equivalents to 3 equivalents, 1 equivalent to 10 equivalents A reagent in the range of 1 equivalent to 8 equivalents, 1 equivalent to 6 equivalents, 1 equivalent to 4 equivalents, 1 equivalent to 3 equivalents, 1.5 equivalents to 10 equivalents, 1.5 equivalents to 8 equivalents, 1.5 equivalents to 6 equivalents, 1.5 equivalents to 4 equivalents, 1.5 equivalents to 3 equivalents, 2 equivalents to 10 equivalents, 2 equivalents to 8 equivalents, 2 equivalents to 6 equivalents, 2 equivalents to 4 equivalents, 2 equivalents to 3 equivalents, 1 equivalent to 20 equivalents, 1 equivalent to 10 equivalents, or 4 equivalents to 6 equivalents is used.

[0421] The 25-hydroxy-(3β)-cholesta-(5,6-epoxy)-3-sulfuric acid organic cation salt may be deoxygenated at a temperature ranging from −10° C. to 75° C., for example, −5° C. to 70° C., for example, −4° C. to 65° C., for example, −3° C. to 60° C., for example, −2° C. to 55° C., for example, −1° C. to 50° C., including 0° C. to 25° C. The reaction may be carried out for a duration ranging from 0.1 hours to 72 hours, for example, 0.2 hours to 48 hours, for example, 0.3 hours to 24 hours, for example, 0.4 hours to 21 hours, for example, 0.5 hours to 20 hours, for example, 0.6 hours to 19 hours, for example, 0.7 hours to 18 hours, for example, 0.8 hours to 17 hours, for example, 0.9 hours to 16 hours, including 1 hour to 15 hours.

[0422] In certain instances, the method includes contacting 25-hydroxy-(3β)-cholesta-(5,6-epoxy)-3-sulfuric acid organic cation salt with zinc in the presence of iodine and acetic acid to generate 25-hydroxy-(3β)-cholest-5-ene-3-sulfuric acid organic cation salt (Scheme IID2).

[0423] [ka]

[0424] In some cases, 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt (structure IID) is contacted with a metal salt to produce 25-hydroxy-(3β)-cholest-5-ene-3-sulfate metal salt (structure IIE) (Scheme IIE1).

[0425] [ka]

[0426] In some cases, a method for producing 25-hydroxy-(3β)-cholest-5-ene-3-sulfate metal salt includes contacting a 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt with at least one sodium salt. In some cases, the at least one sodium salt is selected from sodium acetate, sodium iodide, sodium chloride, sodium hydroxide, and sodium methoxide. The 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt may be contacted with the metal salt at a temperature ranging from -10°C to 75°C, such as from -5°C to 70°C, for example, from -4°C to 65°C, for example, from -3°C to 60°C, for example, from -2°C to 55°C, for example, from -1°C to 50°C, for example, from 0°C to 45°C, for example, from 5°C to 40°C, and including 10°C to 35°C.

[0427] The reaction may be carried out for a duration ranging from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, for example, from 0.3 hours to 24 hours, for example, from 0.4 hours to 21 hours, for example, from 0.5 hours to 20 hours, for example, from 0.6 hours to 19 hours, for example, from 0.7 hours to 18 hours, for example, from 0.8 hours to 17 hours, for example, from 0.9 hours to 16 hours, and inclusive of from 1 hour to 15 hours. The amount of metal salt used may vary relative to the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt and may be 0.0001 equivalents or more, such as 0.001 equivalents or more, for example 0.01 equivalents or more, such as 0.1 equivalents or more, for example 0.2 equivalents or more, such as 0.3 equivalents or more, for example 0.4 equivalents or more, for example 0.5 equivalents or more, such as 0.6 equivalents or more, for example 0.7 equivalents or more, for example 0.8 equivalents or more, such as 0.9 equivalents or more, for example 1 equivalent or more, such as 1.1 equivalents or more, for example 1.2 equivalents or more, for example 1.3 equivalents or more, such as 1.4 equivalents or more, for example 1.5 equivalents or more, for example 1.6 equivalents or more, for example 1.7 equivalents or more, for example 1.8 equivalents or more, e.g. For example, it may be 1.9 equivalents or more, for example, 2 equivalents or more, for example, 3 equivalents or more, for example, 4 equivalents or more, for example, 5 equivalents or more, including 10 equivalents or more, such as 0.001 equivalents to 10 equivalents, for example, 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 6 equivalents, 0.1 equivalents to 4 equivalents, 0.1 equivalents to 3 equivalents, 1 equivalent to 10 equivalents, 1 equivalent to 8 equivalents, 1 equivalent The amount may be in the range of 1 to 6 equivalents, 1 to 4 equivalents, 1 to 3 equivalents, 1.5 to 10 equivalents, 1.5 to 8 equivalents, 1.5 to 6 equivalents, 1.5 to 4 equivalents, 1.5 to 3 equivalents, 2 to 10 equivalents, 2 to 8 equivalents, 2 to 6 equivalents, 2 to 4 equivalents, 2 to 3 equivalents, 1 to 20 equivalents, 1 to 10 equivalents, or 1 to 7 equivalents.

[0428] In some cases, the method includes contacting 25-hydroxy-(3β)-cholest-5-ene-3-sulfate pyridinium salt with sodium iodide to produce 25-hydroxy-(3β)-cholest-5-ene-3-sulfate sodium salt (Scheme IIE2).

[0429] [ka]

[0430] In some embodiments, salts of 25HC3S, including crystalline salts of 25HC3S, have relatively high solubility, which is useful, for example, for preparing concentrated solutions for IV administration. For example, salts of 25HC3S that have relatively high solubility in water, saline, dextrose, and / or ethanol may be useful. Exemplary embodiments of such salts include crystalline diethylammonium 25HC3S, which has relatively high solubility in water and saline, crystalline hydroxyethylpyrrolidinium 25HC3S, which has relatively high solubility in dextrose, and crystalline zinc 25HC3S, which has relatively high solubility in ethanol.

[0431] In some embodiments, salts of 25HC3S have relatively high solubility, which is useful, for example, for making concentrated solutions for topical administration. For example, crystalline zinc 25HC3S has relatively high solubility in ethanol, a well-known topical penetration enhancer, and can be used in transdermal delivery systems.

[0432] In some embodiments, salts of 25HC3S have relatively high solubility, which is useful, for example, for making concentrated solutions for oral administration. For example, crystalline zinc 25HC3S has relatively high solubility in ethanol.

[0433] In some embodiments, salts of 25HC3S have relatively low solubility, which may be useful, for example, in controlled-release formulations, such as injectable or oral controlled-release formulations. As shown in the examples, the following salts of 25HC3S may be useful in controlled-release formulations due to their low solubility in at least saline: crystalline 25HC3S potassium, crystalline 25HC3S calcium, crystalline 25HC3S zinc, crystalline 25HC3S choline, crystalline 25HC3S meglumine, crystalline 25HC3S tromethammonium, crystalline 25HC3S benzathine, crystalline 25HC3S diethanolamine, and crystalline 25HC3S magnesium. The following salts of 25HC3S may be useful in controlled-release formulations due to their low solubility in at least fasted-state simulated gastric fluid (FaSSGF): crystalline 25HC3S potassium, crystalline 25HC3S calcium, crystalline 25HC3S zinc, crystalline 25HC3S choline, crystalline 25HC3S hydroxyethylammonium, crystalline 25HC3S benzathine, crystalline 25HC3S magnesium, and crystalline 25HC3S lysine. The benzathine salt of 25HC3S may be useful in controlled-release formulations due to its low solubility in fasted-state simulated intestinal fluid (FaSSIF) and fed-state simulated intestinal fluid (FeSSIF).

[0434] In some embodiments, salts of 25HC3S may be orally bioavailable. For example, salts of 25HC3S that are highly soluble in fasted-state simulated gastric fluid (FaSSGF) may be orally bioavailable. As shown in the examples, crystalline 25HC3S meglumine has relatively high solubility in FaSSGF. As another example, salts of 25HC3S that are highly soluble in fasted-state simulated intestinal fluid (FaSSIF) may be orally bioavailable. The following salts of 25HC3S have relatively high solubility in FaSSIF: crystalline 25HC3S calcium, crystalline 25HC3S choline, crystalline 25HC3S hydroxyethylpyrrolidinium, crystalline 25HC3S diethanolamine, crystalline 25HC3S diethylammonium, and crystalline 25HC3S t-butylammonium. Salts of 25HC3S that are highly soluble in fed-state simulated intestinal fluid (FeSSIF) may also be orally bioavailable. The following salts of 25HC3S have relatively high solubility in FeSSIF: crystalline 25HC3S choline, crystalline 25HC3S hydroxyethylpyrrolidinium, crystalline 25HC3S diethanolamine, and crystalline 25HC3S diethylammonium.

[0435] In some embodiments, salts of 25HC3S have high solubility in solvents that can be used during synthesis. For example, as shown in the examples, crystalline 25HC3S diethylammonium has relatively high solubility in isopropyl alcohol (IPA). Crystalline 25HC3S diethylammonium and crystalline 25HC3S magnesium have relatively high solubility in methanol, which can be used in synthesis and spray-dried drug-polymer dispersions for amorphous preparations of drug substances to increase oral bioavailability. Crystalline 25HC3S hydroxyethylpyrrolidinium has relatively high solubility in acetonitrile (ACN).

[0436] In some embodiments, 25HC3S salts are non-hygroscopic, which facilitates handling of the drug substance at ambient conditions and avoids the need for special precautions, such as handling in low-humidity conditions, handling in a dry environment, or maintaining in a tightly closed container. Weighing these drug substance salts at ambient conditions is not a problem because there is no concern that moisture uptake will change the weight on the balance. Similarly, containers of these salts can be opened and closed multiple times at ambient conditions without concern that moisture absorption will change the powder composition. The non-hygroscopic nature of these salts also enables the preparation of wet granulations for oral tablet and capsule products, minimizing the possibility of conversion to polymorphs or other solid forms, such as hydrate formation. For example, as shown in the Examples, the crystalline 25HC3S t-butylammonium salt, crystalline 25HC3S benzathine salt, and crystalline 25HC3S choline salt of 25HC3S gain less than 0.5% water at 95% relative humidity. Furthermore, as shown by the DVS isotherms, if the crystalline 25HC3S t-butylammonium, crystalline 25HC3S benzathine, and crystalline 25HC3S choline salts of 25HC3S gain a small amount of water when the relative humidity increases to 95%, they reversibly lose all of that water when the relative humidity decreases to 5%.

[0437] In some embodiments, salts of 25HC3S are highly crystalline, which can be advantageous, for example, from a processing standpoint. Crystalline 25HC3S hydroxyethylpyrrolidinium, crystalline 25HC3S diethylammonium, crystalline 25HC3S diethanolamine, crystalline 25HC3S t-butylammonium, crystalline 25HC3S benzathine, and crystalline 25HC3S choline salts are highly crystalline. XRPD patterns have been successfully indexed with a single unit cell, providing a strong description of the crystalline form through tentative crystallographic unit cell parameters. Formula unit volumes from the indexing results are consistent with all of the anhydrous forms and predicted salt stoichiometries.

[0438] In some embodiments, salts of 25HC3S have a relatively high DSC (differential scanning calorimetry) endothermic transition (indicative of thermal decomposition or solid-state transformation). Without wishing to be bound by theory, this property may enable dry heat sterilization of the drug substance (e.g., 160°C for 2 hours) and facilitate the preparation of sterile dosage forms. For example, as shown in the Examples, the first significant endothermic transitions for potassium, lysine, diethanolamine, t-butylammonium, benzathine, and choline are near 198°C, 186°C, 181°C, 201°C, 211°C, and 198°C, respectively, indicating that these salts of 25HC3S can be sterilized by dry heat processing.

[0439] In some embodiments, powders of 25HC3S salts have relatively good flowability, which can be useful during manufacturing. For example, as shown in the examples, crystalline 25HC3S hydroxyethylammonium and crystalline 25HC3S lysine salts have relatively good flowability.

[0440] In some embodiments, salts of 25HC3S have good temperature stability. As shown in the examples, the following salts of 25HC3S have good stress stability at 80°C: crystalline 25HC3S diethylammonium, crystalline 25HC3S t-butylammonium, crystalline 25HC3S choline, crystalline 25HC3S diethanolamine, crystalline 25HC3S tromethammonium, and crystalline 25HC3S lysine.

[0441] In some embodiments, the counterion of 25HC3S can have beneficial effects in vivo. For example, choline salts can be advantageous because choline deficiency is also associated with conditions related to fat accumulation and inflammation, and choline supplementation has been proposed to be potentially desirable for the treatment and / or management of such conditions (see, e.g., Zeisel et al., Nutr Rev. 2009 Nov; 67(11): 615-623; Corbin et al., Curr Opin Gastroenterol. 2012 Mar; 28(2): 159-165). Because zinc is an essential trace element required for cell proliferation, development, and differentiation, zinc salts of 25HC3S can be beneficial. Zinc deficiency is observed in many pathological conditions, including those for which 25HC3S is a potential therapeutic agent. Zinc supplementation can be beneficial for such conditions, such as those related to the skin and gastrointestinal tract; the brain and central nervous system, the immune system, the skeleton, and the reproductive system (see, e.g., Himoto et al., Nutrients 2018, 10, 88; Grungreiff et al., Annals of Hepatology 2016 15(1) pp. 7-16; Mohammad et al., Nutr Clin Pract 20012 27(1) pp. 8-20). Magnesium deficiency is also observed in numerous pathologies, including those for which 25HC3S is a potential therapeutic agent. Magnesium supplementation can be beneficial for such conditions as celiac disease, Crohn's disease, type 2 diabetes, and conditions associated with alcohol use (see, e.g., Agus et al., J Am Soc Nephrol, 10:1616-622 (1999) and Martin et al., J Am Soc Nephrol, 20:2291-2295 (2009)).

[0442] In some embodiments, a salt of 25HC3S possesses a beneficial combination of two or more such properties, making it particularly useful, for example, as an active pharmaceutical ingredient in a pharmaceutical composition for a particular clinical application. For example, such a combination can include two or more (e.g., at least three, at least four, or at least five) properties selected from the group consisting of high solubility in a particular solvent (e.g., as discussed in the Examples), low solubility in a particular solvent (e.g., as discussed in the Examples), low hygroscopicity, high crystallinity, a high DSC endothermic transition (allowing for dry heat sterilization), high flowability, high temperature stability, and beneficial in vivo effects of the counterion of 25HC3S (particularly, such in vivo effects are beneficial in subjects suffering from a condition for which a dosage form incorporating the salt of 25HC3S is designed).

[0443] item Item 1. Salts of 25HC3S other than (i) alkali metal salts or (ii) ammonium salts.

[0444] Item 2. Salts of 25HC3S other than (i) alkali metal salts or (ii) ammonium salts, where the ammonium salt is not a choline salt.

[0445] Item 3. Crystalline salts of 25HC3S other than the crystalline sodium salt of 25HC3S.

[0446] Item 4. A crystalline salt according to Item 3 other than the crystalline choline salt of 25HC3S.

[0447] Item 5. The crystalline salt according to Item 3, which is a crystalline 25HC3S metal salt.

[0448] Item 6. The crystalline salt according to Item 5, wherein the metal is in the +1 oxidation state.

[0449] Item 7. The salt according to any one of Items 1 to 5, which is a 25HC3S metal salt, in which the metal is in either the +2 or +3 oxidation state.

[0450] Item 8. The crystalline metal salt according to Item 7, wherein the metal is an alkaline earth metal.

[0451] Item 9. The crystalline metal salt according to Item 6, wherein the metal is selected from potassium, lithium, and rubidium.

[0452] Item 10. A substantially pure salt of 25HC3S according to any one of Items 1 to 9.

[0453] Item 11. Crystalline potassium 25HC3S.

[0454] Item 12. Crystalline potassium 25HC3S according to Item 11, having an X-ray powder diffraction pattern including a peak at about 2.2° 2θ.

[0455] Item 13. Crystalline potassium 25HC3S according to Item 11 or 12, having an X-ray powder diffraction pattern including a peak at about 2.3° 2θ.

[0456] Item 14. Crystalline potassium 25HC3S according to any one of items 11 to 13, having an x-ray powder diffraction pattern including a peak at about 8.8° 2θ.

[0457] Item 15. Crystalline potassium 25HC3S according to any one of items 11 to 14, having an x-ray powder diffraction pattern including a peak at about 9.3° 2θ.

[0458] Item 16. Crystalline potassium 25HC3S according to any one of items 11 to 15, having an x-ray powder diffraction pattern including a peak at about 15.3° 2θ.

[0459] Item 17. Crystalline potassium 25HC3S according to any one of items 11 to 16, having an x-ray powder diffraction pattern including a peak at about 4.6° 2θ.

[0460] Item 18. Crystalline potassium 25HC3S according to any one of items 11 to 17, having an x-ray powder diffraction pattern including a peak at about 14.7° 2θ.

[0461] Item 19. Crystalline potassium 25HC3S according to any one of items 11 to 18, having an x-ray powder diffraction pattern including a peak at about 14.9° 2θ.

[0462] Item 20. Crystalline potassium 25HC3S according to any one of items 11 to 19, having an x-ray powder diffraction pattern including a peak at about 16.1° 2θ.

[0463] Item 21. Crystalline potassium 25HC3S according to any one of Items 11 to 20, having an x-ray powder diffraction pattern comprising two peaks at about 2.2° 2θ to about 2.3° 2θ that are (a) non-overlapping, (b) partially overlapping, or (c) overlapping, e.g., so as to appear as a single peak.

[0464] Item 22. Crystalline potassium 25HC3S according to Item 11, having an x-ray powder diffraction pattern substantially the same as that shown in Figure 2 or Figure 2A.

[0465] Item 23. Crystalline calcium 25HC3S.

[0466] Item 24. Crystalline calcium 25HC3S according to Item 23, having an X-ray powder diffraction pattern including a peak at about 2.2° 2θ.

[0467] Item 25. Crystalline calcium 25HC3S according to Item 23 or 24, having an X-ray powder diffraction pattern including a peak at about 4.5° 2θ.

[0468] Item 26. Crystalline 25HC3S calcium according to any one of Items 23 to 25, having an x-ray powder diffraction pattern including a peak at about 9.0° 2θ.

[0469] Item 27. Crystalline 25HC3S calcium according to any one of Items 23 to 26, having an x-ray powder diffraction pattern including a peak at about 10.0° 2θ.

[0470] Item 28. Crystalline 25HC3S calcium according to any one of Items 23 to 27, having an x-ray powder diffraction pattern including a peak at about 15.0° 2θ.

[0471] Item 29. Crystalline 25HC3S calcium according to any one of Items 23 to 28, having an x-ray powder diffraction pattern including a peak at about 15.1° 2θ.

[0472] Item 30. Crystalline 25HC3S calcium according to any one of Items 23 to 29, having an x-ray powder diffraction pattern including a peak at about 15.7° 2θ.

[0473] Item 31. Crystalline 25HC3S calcium according to any one of Items 23 to 30, having an x-ray powder diffraction pattern including a peak at about 15.4° 2θ.

[0474] Item 32. Crystalline 25HC3S calcium according to any one of Items 23 to 31, having an x-ray powder diffraction pattern including a peak at about 16.5° 2θ.

[0475] Item 33. Crystalline 25HC3S calcium according to any one of Items 23 to 32, having an x-ray powder diffraction pattern including a peak at about 18.0° 2θ.

[0476] Item 34. Crystalline 25HC3S calcium according to any one of Items 23 to 33, having an x-ray powder diffraction pattern including a peak at about 18.1° 2θ.

[0477] Item 35. Crystalline 25HC3S calcium according to any one of Items 23 to 34, having an x-ray powder diffraction pattern including a peak at about 18.4° 2θ.

[0478] Item 36. Crystalline 25HC3S calcium according to any one of Items 23 to 35, having an x-ray powder diffraction pattern including a peak at about 19.2° 2θ.

[0479] Item 37. Crystalline 25HC3S calcium according to any one of Items 23 to 36, having an x-ray powder diffraction pattern comprising two peaks at about 15.0°2θ to about 15.1°2θ that are (a) non-overlapping, (b) partially overlapping, or (c) overlapping, e.g., so as to appear as a single peak.

[0480] Item 38. Crystalline 25HC3S calcium according to Item 23, having an x-ray powder diffraction pattern substantially the same as Figure 4 or Figure 4A.

[0481] Item 39. Crystalline 25HC3S zinc.

[0482] Item 40. Crystalline zinc 25HC3S according to Item 39, having an x-ray powder diffraction pattern including a peak at about 2.1° 2θ.

[0483] Item 41. Crystalline zinc 25HC3S according to Item 39 or 40, having an x-ray powder diffraction pattern including a peak at about 2.3° 2θ.

[0484] Item 42. Crystalline zinc 25HC3S according to any one of Items 39 to 41, having an x-ray powder diffraction pattern including a peak at about 6.0° 2θ.

[0485] Item 43. Crystalline zinc 25HC3S according to any one of items 39 to 42, having an x-ray powder diffraction pattern including a peak at about 8.6° 2θ.

[0486] Item 44. Crystalline zinc 25HC3S according to any one of Items 39 to 43, having an x-ray powder diffraction pattern including a peak at about 8.9° 2θ.

[0487] Item 45. Crystalline zinc 25HC3S according to any one of Items 39 to 44, having an x-ray powder diffraction pattern including a peak at about 9.3° 2θ.

[0488] Item 46. Crystalline zinc 25HC3S according to any one of Items 39 to 45, having an x-ray powder diffraction pattern including a peak at about 15.1° 2θ.

[0489] Item 47. Crystalline zinc 25HC3S according to any one of items 39 to 46, having an x-ray powder diffraction pattern including a peak at about 18.3° 2θ.

[0490] Item 48. Crystalline zinc 25HC3S according to any one of items 39 to 47, having an x-ray powder diffraction pattern including a peak at about 18.8° 2θ.

[0491] Item 49. Crystalline zinc 25HC3S according to any one of items 39 to 48, having an x-ray powder diffraction pattern including two peaks at about 2.1°2θ to about 2.3°2θ that are (a) non-overlapping, (b) partially overlapping, or (c) overlapping, e.g., so as to appear as a single peak.

[0492] Item 50. Crystalline zinc 25HC3S according to Item 40, having an x-ray powder diffraction pattern further including a peak at about 6.0° 2θ.

[0493] Item 51. Crystalline zinc 25HC3S according to Item 40, having an x-ray powder diffraction pattern further including a peak at about 8.6° 2θ.

[0494] Item 52. Crystalline zinc 25HC3S according to Item 40, having an x-ray powder diffraction pattern further including a peak at about 8.9° 2θ.

[0495] Item 53. Crystalline zinc 25HC3S according to Item 40, having an x-ray powder diffraction pattern further including a peak at about 9.3° 2θ.

[0496] Item 54. Crystalline zinc 25HC3S according to Item 40, having an x-ray powder diffraction pattern further including a peak at about 15.1° 2θ.

[0497] Item 55. Crystalline zinc 25HC3S according to Item 40, having an x-ray powder diffraction pattern further including a peak at about 18.3° 2θ.

[0498] Item 56. Crystalline zinc 25HC3S according to Item 40, having an x-ray powder diffraction pattern further including a peak at about 18.8° 2θ.

[0499] Item 57. Crystalline zinc 25HC3S according to Item 50, having an x-ray powder diffraction pattern further including a peak at about 8.6° 2θ.

[0500] Item 58. Crystalline zinc 25HC3S according to Item 50, having an x-ray powder diffraction pattern further including a peak at about 8.9° 2θ.

[0501] Item 59. Crystalline zinc 25HC3S according to Item 50, having an x-ray powder diffraction pattern further including a peak at about 9.3° 2θ.

[0502] Item 60. Crystalline zinc 25HC3S according to Item 50, having an x-ray powder diffraction pattern further including a peak at about 15.1° 2θ.

[0503] Item 61. Crystalline zinc 25HC3S according to Item 50, having an x-ray powder diffraction pattern further including a peak at about 18.3° 2θ.

[0504] Item 62. Crystalline zinc 25HC3S according to Item 50, having an x-ray powder diffraction pattern further including a peak at about 18.8° 2θ.

[0505] Item 63. Crystalline zinc 25HC3S according to Item 57, having an x-ray powder diffraction pattern further including a peak at about 8.9° 2θ.

[0506] Item 64. Crystalline zinc 25HC3S according to Item 57, having an x-ray powder diffraction pattern further including a peak at about 9.3° 2θ.

[0507] Item 65. Crystalline zinc 25HC3S according to Item 57, having an x-ray powder diffraction pattern further including a peak at about 15.1° 2θ.

[0508] Item 66. Crystalline zinc 25HC3S according to Item 57, having an x-ray powder diffraction pattern further including a peak at about 18.3° 2θ.

[0509] Item 67. Crystalline zinc 25HC3S according to Item 57, having an x-ray powder diffraction pattern further including a peak at about 18.8° 2θ.

[0510] Item 68. Crystalline zinc 25HC3S according to Item 63, having an x-ray powder diffraction pattern further including a peak at about 9.3° 2θ.

[0511] Item 69. Crystalline zinc 25HC3S according to Item 63, having an x-ray powder diffraction pattern further including a peak at about 15.1° 2θ.

[0512] Item 70. Crystalline zinc 25HC3S according to Item 63, having an x-ray powder diffraction pattern further including a peak at about 18.3° 2θ.

[0513] Item 71. Crystalline zinc 25HC3S according to Item 63, having an x-ray powder diffraction pattern further including a peak at about 18.8° 2θ.

[0514] Item 72. Crystalline zinc 25HC3S according to Item 68, having an x-ray powder diffraction pattern further including a peak at about 15.1° 2θ.

[0515] Item 73. Crystalline zinc 25HC3S according to Item 68, having an x-ray powder diffraction pattern further including a peak at about 18.3° 2θ.

[0516] Item 74. Crystalline zinc 25HC3S according to Item 68, having an x-ray powder diffraction pattern further including a peak at about 18.8° 2θ.

[0517] Item 75. Crystalline zinc 25HC3S according to Item 72, having an x-ray powder diffraction pattern further including a peak at about 18.3° 2θ.

[0518] Item 76. Crystalline zinc 25HC3S according to Item 72, having an x-ray powder diffraction pattern further including a peak at about 18.8° 2θ.

[0519] Item 77. The crystalline zinc 25HC3S according to Item 39, having an X-ray powder diffraction pattern including one or more peaks selected from about 2.1°2θ, about 2.3°2θ, about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0520] Item 78. Crystalline zinc 25HC3S according to Item 39, having an X-ray powder diffraction pattern including one or more peaks selected from about 2.3°2θ, about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0521] Item 79. The crystalline zinc 25HC3S according to Item 39, having an X-ray powder diffraction pattern including one or more peaks selected from about 6.0°2θ, about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0522] Item 80. Crystalline zinc 25HC3S according to Item 39, having an X-ray powder diffraction pattern including one or more peaks selected from about 8.6°2θ, about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0523] Item 81. The crystalline zinc 25HC3S according to Item 39, having an x-ray powder diffraction pattern including one or more peaks selected from about 8.9°2θ, about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0524] Item 82. Crystalline zinc 25HC3S according to Item 39, having an x-ray powder diffraction pattern including one or more peaks selected from about 9.3° 2θ, about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0525] Item 83. Crystalline zinc 25HC3S according to Item 39, having an x-ray powder diffraction pattern including one or more peaks selected from about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0526] Item 84. Crystalline zinc 25HC3S according to Item 39, having an x-ray powder diffraction pattern including one or more peaks selected from about 18.3° 2θ and about 18.8° 2θ.

[0527] Item 85. Crystalline zinc 25HC3S according to Item 39, having an x-ray powder diffraction pattern including a peak at about 18.8° 2θ.

[0528] Item 86. Crystalline zinc 25HC3S according to Item 39, having an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern shown in Figure 85 or Figure 86.

[0529] Item 87. Crystalline 25HC3S magnesium.

[0530] Item 88. Crystalline magnesium 25HC3S according to Item 87, having an x-ray powder diffraction pattern including a peak at about 2.2° 2θ.

[0531] Item 89. Crystalline magnesium 25HC3S according to Item 87 or 88, having an x-ray powder diffraction pattern including a peak at about 4.4° 2θ.

[0532] Item 90. Crystalline 25HC3S magnesium according to any one of Items 87 to 89, having an x-ray powder diffraction pattern including a peak at about 6.6° 2θ.

[0533] Item 91. Crystalline 25HC3S magnesium according to any one of Items 87 to 90, having an x-ray powder diffraction pattern including a peak at about 8.9° 2θ.

[0534] Item 92. Crystalline 25HC3S magnesium according to any one of Items 87 to 91, having an x-ray powder diffraction pattern including a peak at about 15.1° 2θ.

[0535] Item 93. Crystalline magnesium 25HC3S according to any one of items 87 to 92, having an x-ray powder diffraction pattern including a peak at about 15.6° 2θ.

[0536] Item 94. Crystalline 25HC3S magnesium according to any one of Items 87 to 93, having an x-ray powder diffraction pattern including a peak at about 16.4° 2θ.

[0537] Item 95. Crystalline 25HC3S magnesium according to any one of Items 87 to 94, having an x-ray powder diffraction pattern including a peak at about 17.6° 2θ.

[0538] Item 96. Crystalline 25HC3S magnesium according to any one of Items 87 to 95, having an x-ray powder diffraction pattern including a peak at about 17.8° 2θ.

[0539] Item 97. Crystalline 25HC3S magnesium according to Item 87, having an x-ray powder diffraction pattern substantially the same as Figure 6 or Figure 6A.

[0540] Item 98. Organic salts of 25HC3S other than ammonium salts of 25HC3S.

[0541] Item 99. Crystalline organic salt of 25HC3S.

[0542] Item 100. The organic salt according to Item 98 or 99, which is an amine salt of 25HC3S.

[0543] Item 101. The crystalline salt of 25HC3S described in Item 100.

[0544] Item 102. The salt of 25HC3S according to Item 100 or 101, wherein the amine is aliphatic, cyclic, aromatic, or a combination thereof.

[0545] Item 103. The salt of 25HC3S according to any one of items 100 to 102, wherein the amine comprises a primary, secondary, or tertiary amino group, or a combination thereof.

[0546] Item 104. The salt according to any one of Items 100 to 103, wherein the amine is substituted.

[0547] Item 105. The salt according to item 104, wherein the substitution is one or more of an alcohol, alkyl, aryl or even amine group.

[0548] Item 106. The salt according to Item 105, wherein the substitution is one or more alcohol groups.

[0549] Item 107. The salt according to Item 106, wherein the one or more alcohol groups are selected from primary, secondary, and tertiary alcohol groups.

[0550] Item 108. The salt according to any one of items 100 to 107, wherein the amine group contains 1, 2, 3, or 4 carbon atoms covalently linked to the nitrogen of the amine group.

[0551] Item 109. The salt according to Item 108, wherein the linked carbon atom comprises a cyclic alkyl.

[0552] Item 110. The organic salt according to any one of Items 100 to 109, wherein the amine is an amino acid.

[0553] Item 111. The salt according to Item 110, wherein the amino acid is a natural amino acid, for example, a proteinogenic amino acid.

[0554] Item 112. The salt according to Item 110, wherein the amino acid is an unnatural amino acid.

[0555] Item 113. A salt of 25HC3S according to Item 105, wherein the substitution is an aryl group.

[0556] Item 114. A salt of 25HC3S according to Item 113, wherein the aryl group comprises phenyl or benzyl.

[0557] Item 115. A salt of 25HC3S according to any one of Items 100 to 114, which contains a cyclic amine.

[0558] Item 116. The salt according to any one of Items 100 to 114, wherein the oxidation state of the amine is +1.

[0559] Item 117. The salt according to any one of Items 100 to 114, wherein the oxidation state of the amine is +2.

[0560] Item 118. The salt according to any one of items 100 to 114, wherein the amine contains one or more alcohol groups.

[0561] Item 119. The salt according to Item 118, wherein the amine contains two alcohol groups.

[0562] Item 120. The salt according to Item 118, wherein the amine contains three alcohol groups.

[0563] Item 121. The salt according to Item 118, wherein the amine contains four or more alcohol groups.

[0564] Item 122. The salt according to Item 118, wherein the amine contains five alcohol groups.

[0565] Item 123. The salt according to any one of items 118 to 122, wherein the alcohol group is a primary alcohol.

[0566] Item 124. The salt according to any one of items 100 to 123, wherein the amine contains two amine groups.

[0567] Item 125. The salt according to Item 124, wherein at least one amine group is a secondary amine group.

[0568] Item 126. The salt according to Item 125, wherein the two amine groups are secondary amine groups.

[0569] Item 127. The salt according to any one of items 124 to 216, further comprising at least one aryl group.

[0570] Item 128. The salt according to any one of items 124 to 216, further comprising at least two aryl groups.

[0571] Item 129. The salt according to Item 108 or 109, wherein at least one amine group is bonded to one, two, or three independently substituted or unsubstituted alkyl groups.

[0572] Item 130. The salt according to Item 129, wherein the alkyl group contains 1, 2, 3, 4, 5, or 6 carbon atoms.

[0573] Item 131. The salt according to Item 130, wherein at least one alkyl group is substituted with an aryl group.

[0574] Item 132. The salt according to Item 131, wherein the aryl group is phenyl or benzyl.

[0575] Item 133. 25HC3S Hydroxyethylammonium.

[0576] Item 134. Crystalline 25HC3S Hydroxyethylammonium.

[0577] Item 135. Crystalline 25HC3S hydroxyethylammonium according to Item 134, having an X-ray powder diffraction pattern including a peak at about 2.1° 2θ.

[0578] Item 136. Crystalline 25HC3S hydroxyethylammonium according to Item 135, having an x-ray powder diffraction pattern further including a peak at about 8.6° 2θ.

[0579] Item 137. Crystalline 25HC3S hydroxyethylammonium according to Item 134, having an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of Figure 8 or Figure 8A.

[0580] Item 138. 25HC3S Tromethammonium.

[0581] Item 139. Crystalline 25HC3S tromethammonium.

[0582] Item 140. The crystalline 25HC3S tromethammonium salt according to Item 139, having an X-ray powder diffraction pattern including a peak at about 1.9° 2θ.

[0583] Item 141. The crystalline 25HC3S tromethammonium salt according to Item 139 or 140, having an X-ray powder diffraction pattern including a peak at about 2.1° 2θ.

[0584] Item 142. The crystalline 25HC3S tromethammonium salt according to any one of Items 139 to 141, having an X-ray powder diffraction pattern including a peak at about 3.8° 2θ.

[0585] Item 143. The crystalline 25HC3S tromethammonium salt according to any one of Items 139 to 142, having an X-ray powder diffraction pattern including peaks at about 4.2° 2θ and / or about 15.4° 2θ.

[0586] Item 144. The crystalline 25HC3S tromethammonium salt according to any one of Items 139 to 143, having an X-ray powder diffraction pattern including two peaks at about 1.9°2θ to about 2.1°2θ that are (a) non-overlapping, (b) partially overlapping, or (c) overlapping, e.g., so as to appear as a single peak.

[0587] Item 145. The crystalline 25HC3S tromethammonium salt according to Item 139, having an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of Figure 14 or Figure 14A.

[0588] Item 146. 25HC3S Lysine.

[0589] Item 147. Crystalline 25HC3S Lysine.

[0590] Item 148. The crystalline 25HC3S lysine salt according to Item 147, having an x-ray powder diffraction pattern including a peak at about 1.5° 2θ.

[0591] Item 149. The crystalline 25HC3S lysine salt according to Item 147 or 148, having an x-ray powder diffraction pattern including a peak at about 7.0° 2θ.

[0592] Item 150. The crystalline 25HC3S lysine salt of any one of items 147 to 149, having an x-ray powder diffraction pattern including a peak at about 10.7° 2θ.

[0593] Item 151. The crystalline 25HC3S lysine salt of any one of items 147 to 150, having an x-ray powder diffraction pattern including a peak at about 11.8° 2θ.

[0594] Item 152. The crystalline 25HC3S lysine salt of any one of items 147 to 151, having an x-ray powder diffraction pattern including a peak at about 16.8° 2θ.

[0595] Item 153. The crystalline 25HC3S lysine salt of any one of items 147 to 152, having an x-ray powder diffraction pattern including a peak at about 3.2° 2θ.

[0596] Item 154. The crystalline 25HC3S lysine salt of any one of items 147 to 153, having an x-ray powder diffraction pattern including a peak at about 10.0° 2θ.

[0597] Item 155. The crystalline 25HC3S lysine salt of any one of items 147 to 154, having an x-ray powder diffraction pattern including a peak at about 12.2° 2θ.

[0598] Item 156. The crystalline 25HC3S lysine salt of any one of items 147 to 155, having an x-ray powder diffraction pattern including a peak at about 15.2° 2θ.

[0599] Item 157. The crystalline 25HC3S lysine salt according to Item 147, having an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of Figure 24 or Figure 24A.

[0600] Item 158. 25HC3S Meglumine.

[0601] Item 159. Crystalline 25HC3S Meglumine.

[0602] Item 160. The crystalline 25HC3S meglumine salt according to Item 159, having an x-ray powder diffraction pattern including a peak at about 1.7° 2θ.

[0603] Item 161. The crystalline 25HC3S meglumine salt according to Item 159 or 160, having an x-ray powder diffraction pattern including a peak at about 3.5° 2θ.

[0604] Item 162. The crystalline 25HC3S meglumine salt according to any one of items 159 to 161, having an x-ray powder diffraction pattern including a peak at about 5.2° 2θ.

[0605] Item 163. The crystalline 25HC3S meglumine salt of any one of items 159 to 162, having an x-ray powder diffraction pattern including a peak at about 14.9° 2θ.

[0606] Item 164. The crystalline 25HC3S meglumine salt according to any one of items 159 to 163, having an x-ray powder diffraction pattern including a peak at about 24.2° 2θ.

[0607] Item 165. The crystalline 25HC3S meglumine salt of any one of items 159 to 164, having an x-ray powder diffraction pattern including a peak at about 8.6° 2θ.

[0608] Item 166. The crystalline 25HC3S meglumine salt of any one of items 159 to 165, having an x-ray powder diffraction pattern including a peak at about 14.5° 2θ.

[0609] Item 167. The crystalline 25HC3S meglumine salt of any one of items 159 to 166, having an x-ray powder diffraction pattern including a peak at about 15.1° 2θ.

[0610] Item 168. The crystalline 25HC3S meglumine salt of any one of items 159 to 167, having an x-ray powder diffraction pattern including a peak at about 17.5° 2θ.

[0611] Item 169. The crystalline 25HC3S meglumine salt of any one of items 159 to 168, having an x-ray powder diffraction pattern including a peak at about 18.2° 2θ.

[0612] Item 170. The crystalline 25HC3S meglumine salt according to Item 159, having an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of Figure 12 or Figure 12A.

[0613] Item 171. 25HC3S Hydroxyethylpyrrolidinium.

[0614] Item 172. Crystalline 25HC3S Hydroxyethylpyrrolidinium.

[0615] Item 173. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to Item 172, having an X-ray powder diffraction pattern including a peak at about 3.8° 2θ.

[0616] Item 174. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to Item 172 or 173, having an X-ray powder diffraction pattern including a peak at about 7.5° 2θ.

[0617] Item 175. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 174, having an x-ray powder diffraction pattern including a peak at about 7.6° 2θ.

[0618] Item 176. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 175, having an x-ray powder diffraction pattern including a peak at about 8.2° 2θ.

[0619] Item 177. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 176, having an x-ray powder diffraction pattern including a peak at about 8.6° 2θ.

[0620] Item 178. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 177, having an x-ray powder diffraction pattern including a peak at about 12.4° 2θ.

[0621] Item 179. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 178, having an x-ray powder diffraction pattern including a peak at about 13.3° 2θ.

[0622] Item 180. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 179, having an x-ray powder diffraction pattern including a peak at about 15.0° 2θ.

[0623] Item 181. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 180, having an x-ray powder diffraction pattern including a peak at about 10.5° 2θ.

[0624] Item 182. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 181, having an x-ray powder diffraction pattern including a peak at about 15.3° 2θ.

[0625] Item 183. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 182, having an x-ray powder diffraction pattern including a peak at about 15.6° 2θ.

[0626] Item 184. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 183, having an x-ray powder diffraction pattern including a peak at about 16.3° 2θ.

[0627] Item 185. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 184, having an x-ray powder diffraction pattern including a peak at about 16.7° 2θ.

[0628] Item 186. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 185, having an x-ray powder diffraction pattern including a peak at about 20.9° 2θ.

[0629] Item 187. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of Items 172 to 186, having an X-ray powder diffraction pattern comprising two peaks at about 7.5°2θ to about 7.6°2θ that are (a) non-overlapping, (b) partially overlapping, or (c) overlapping, e.g., so as to appear as a single peak.

[0630] Item 188. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 187, having an X-ray powder diffraction pattern including two peaks at about 8.2°2θ to about 8.6°2θ that are (a) non-overlapping, (b) partially overlapping, or (c) overlapping, e.g., so as to appear as a single peak.

[0631] Item 189. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to Item 172, having an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of Figure 10.

[0632] Item 190. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to any one of items 172 to 189, wherein the unit cell of the crystalline salt is triclinic.

[0633] Item 191. The formula volume of the unit cell is approximately 3417 Å 3 Item 190. The crystalline 25HC3S hydroxyethylpyrrolidinium salt according to item 190, wherein the 25HC3S hydroxyethylpyrrolidinium salt is a lattice.

[0634] Item 192. 25HC3S Diethylammonium.

[0635] Item 193. Crystalline 25HC3S diethylammonium.

[0636] Item 194. Crystalline 25HC3S diethylammonium according to Item 193, having an x-ray powder diffraction pattern including a peak at about 3.8° 2θ.

[0637] Item 195. Crystalline 25HC3S diethylammonium according to Item 193 or 194, having an X-ray powder diffraction pattern including a peak at about 7.9° 2θ.

[0638] Item 196. The crystalline 25HC3S diethylammonium salt according to any one of Items 193 to 195, having an x-ray powder diffraction pattern including a peak at about 8.6° 2θ.

[0639] Item 197. Crystalline 25HC3S diethylammonium according to any one of items 193 to 196, having an x-ray powder diffraction pattern including a peak at about 9.6° 2θ.

[0640] Item 198. Crystalline 25HC3S diethylammonium according to any one of items 193 to 197, having an x-ray powder diffraction pattern including a peak at about 10.9° 2θ.

[0641] Item 199. The crystalline 25HC3S diethylammonium salt according to any one of items 193 to 198, having an x-ray powder diffraction pattern including a peak at about 12.3° 2θ.

[0642] Item 200. Crystalline 25HC3S diethylammonium according to any one of items 193 to 199, having an x-ray powder diffraction pattern including a peak at about 15.4° 2θ.

[0643] Item 201. Crystalline 25HC3S diethylammonium according to any one of items 193 to 200, having an x-ray powder diffraction pattern including a peak at about 17.2° 2θ.

[0644] Item 202. Crystalline 25HC3S diethylammonium according to Item 193, having an x-ray powder diffraction pattern substantially the same as the x-ray powder diffraction pattern of Figure 18.

[0645] Item 203. Crystalline 25HC3S diethylammonium according to any one of Items 193 to 202, wherein the unit cell of the crystalline salt is orthorhombic.

[0646] Item 204. The formula volume of the unit cell is approximately 3293 Å 3 Item 203. The crystalline 25HC3S diethylammonium salt according to item 203, which is a lattice.

[0647] Item 205. 25HC3S Diethanolamine.

[0648] Item 206. Crystalline 25HC3S diethanolamine.

[0649] Item 207. Crystalline 25HC3S diethanolamine according to Item 206, having an x-ray powder diffraction pattern including a peak at about 3.8° 2θ.

[0650] Item 208. Crystalline 25HC3S diethanolamine according to Item 206 or 207, having an x-ray powder diffraction pattern including a peak at about 7.7° 2θ.

[0651] Item 209. Crystalline 25HC3S diethanolamine according to any one of items 206 to 208, having an x-ray powder diffraction pattern including a peak at about 8.1° 2θ.

[0652] Item 210. Crystalline 25HC3S diethanolamine according to any one of items 206 to 209, having an x-ray powder diffraction pattern including a peak at about 8.8° 2θ.

[0653] Item 211. Crystalline 25HC3S diethanolamine according to any one of items 206 to 210, having an x-ray powder diffraction pattern including a peak at about 14.6° 2θ.

[0654] Item 212. Crystalline 25HC3S diethanolamine according to any one of items 206 to 211, having an x-ray powder diffraction pattern including a peak at about 15.2° 2θ.

[0655] Item 213. Crystalline 25HC3S diethanolamine according to any one of items 206 t...

Claims

1. (i) A salt of 25HC3S other than an alkali metal salt or (ii) an ammonium salt.

2. (i) an alkali metal salt or (ii) a salt of 25HC3S other than the ammonium salt, wherein the ammonium salt is not the choline salt.

3. Crystalline salts of 25HC3S other than the crystalline sodium salt of 25HC3S.

4. 4. A crystalline salt according to claim 3 other than the crystalline choline salt of 25HC3S.

5. 4. The crystalline salt of claim 3, which is a crystalline 25HC3S metal salt.

6. 6. The crystalline salt of claim 5, wherein the metal is in the +1 oxidation state.

7. 6. The salt of claim 1, which is a 25HC3S metal salt, wherein the metal is in the +2 oxidation state.

8. 8. The crystalline metal salt of claim 7, wherein the metal is an alkaline earth metal.

9. 7. The crystalline metal salt of claim 6, wherein the metal is selected from potassium, lithium, and rubidium.

10. 10. A substantially pure salt of 25HC3S according to any one of claims 1 to 9.

11. Crystalline potassium 25HC3S.

12. Crystalline 25HC3S zinc.

13. Organic salts of 25HC3S other than the ammonium salt of 25HC3S.

14. Crystalline organic salt of 25HC3S.

15. 15. A pharmaceutical composition comprising a 25HC3S compound according to any one of claims 1 to 14 and at least one pharmaceutically acceptable excipient.

16. A method for treating or preventing one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation, comprising administering to a patient in need thereof an effective amount of a 25HC3S compound described in any one of claims 1 to 14.

17. 25HC3S compound according to any one of claims 1 to 14 for use as a medicine.

18. 25HC3S compound according to any one of claims 1 to 14 for use in a method for treating or preventing one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation and conditions associated with fat accumulation and inflammation.

19. Use of a 25HC3S compound described in any one of claims 1 to 14 in the manufacture of a medicament for use in a method for treating or preventing one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated proliferation, and conditions associated with fat accumulation and inflammation.

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