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

Abstract

A number of salts of 25HC3S are provided including crystalline salts of 25HC3S. Included herein are pharmaceutical formulations of salts of the disclosure as well as methods of treating or preventing disease with same 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation. Methods for preparing salts of the disclosure are also provided.

Description

SALTS INCLUDING CRYSTALLINE SALTS OF 25-HYDROXY-CHOLEST-5-EN-3-SULFATE AND METHODS FOR PREPARING SAME INTRODUCTION

[0001] It has been shown previously that cholesterol metabolite, 5-cholesten-3β-25-diol-3- sulphate (“25HC3S”), decreases lipid biosynthesis and increases cholesterol secretion and degradation, and may be useful for the treatment and prevention of one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation.

[0002] Cholesterol is used by the body for the manufacture and repair of cell membranes, and the synthesis of steroid hormones and vitamin D, and is transformed to bile acids in the liver. There are both exogenous and endogenous sources of cholesterol. The average American consumes about 450 mg of cholesterol each day and produces an additional 500 mg to 1,000 mg in the liver and other tissues. Another source is the 500 mg to 1,000 mg of biliary cholesterol that is secreted into the intestine daily, 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 result in NAFLD and atherosclerosis. The plaques that characterize atherosclerosis inhibit blood flow and promote clot formation, and can ultimately cause death or severe disability via heart attacks and / or stroke. A number of therapeutic agents for the treatment of hyperlipidemia have been developed and are widely prescribed by physicians. Unfortunately, only about 35% of patients are responsive to the currently available therapies.

[0004] Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the United States. This condition is associated with obesity, type-II adult onset diabetes, sedentary lifestyle, and diets high in fat. The earlier stage of NAFLD, fatty liver, is potentially reversible when proper treatment steps are taken. However, left unchecked, it can progress to inflammation of liver cells (non-alcoholic steatohepatitis, or NASH) which is much more difficult to treat. Without treatment,NASH can result in irreversible scarring of liver tissue (steatonecrosis), with the potential to cause cirrhosis, liver failure, and liver cancer.

[0005] 25HC3S has been disclosed as a pharmaceutically acceptable salt, such as a sodium salt, for example (e.g., U.S. Patent 10,144,759 and Ogawa et al., Steroids, 74, 81-87 (2009)). Crystalline solids tend to be more favorable for processing, storage, and stability than non-crystalline solids. However, energetics may not favor the ready formation of suitable crystalline solids and polymorphism may make creating stable crystalline solids of a particular active pharmaceutical ingredient impractical. Fifteen salts, including crystalline salts of 25HC3S, and methods for preparing and using said salts are also provided herein. SUMMARY

[0006] In some aspects of the present disclosure, crystalline salts of 25HC3S other than crystalline 25HC3S sodium are provided.

[0007] In other aspects of the disclosure, substantially pure crystalline salts of 25HC3S other than sodium are provided.

[0008] In other aspects of the disclosure, processes for preparing crystalline salts of 25HC3S other than sodium are provided.

[0009] In some aspects of the disclosure, metal salts of 25HC3S, other than sodium, are provided.

[0010] In some aspects of the disclosure, salts of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt are provided.

[0011] In further aspects of the disclosure, salts of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt prepared by the processes of the disclosure are provided.

[0012] In additional aspects of the disclosure, crystalline salts of 25HC3S other than sodium prepared by the processes of the disclosure are provided.

[0013] In some aspects of the present disclosure, crystalline salts of 25HC3S other than crystalline 25HC3S sodium and crystalline 25HC3S choline are provided.

[0014] In some aspects of the present disclosure, substantially pure crystalline salts of 25HC3S other than crystalline 25HC3S sodium and crystalline 25HC3S choline are provided.

[0015] In additional aspects of the present disclosure, processes for preparing crystalline salts of 25HC3S other than sodium and choline are provided.

[0016] In still additional aspects of the disclosure, salts of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt other than choline, are provided.

[0017] In further aspects of the disclosure, processes for preparing crystalline salts of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt other than choline, are provided.

[0018] In additional aspects of the disclosure, crystalline salts of 25HC3S other than sodium and choline prepared by the processes of the disclosure are provided.

[0019] In still additional aspects of the disclosure, pharmaceutical compositions comprising crystalline salts of 25HC3S other than sodium and a pharmaceutically acceptable excipient are provided.

[0020] In still additional aspects of the disclosure, pharmaceutical compositions comprising salts of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt and a pharmaceutically acceptable excipient are provided.

[0021] In further aspects of the disclosure, pharmaceutical compositions comprising crystalline salts of 25HC3S other than sodium and other than choline and a pharmaceutically acceptable excipient are provided.

[0022] In still additional aspects of the disclosure, pharmaceutical compositions comprising salts of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt other than choline, are provided.

[0023] In further aspects of the disclosure, methods of treating or preventing one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation, comprising administering to a patient in need thereof an effective amount of a compound of crystalline salts of 25HC3S other than sodium are provided.

[0024] In further aspects of the disclosure, methods of treating or preventing one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation andinflammation, comprising administering to a patient in need thereof an effective amount of a compound of salts of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt are provided.

[0025] In further aspects of the disclosure, methods of treating or preventing one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation, comprising administering to a patient in need thereof an effective amount of a compound of crystalline salts of 25HC3S other than sodium or choline are provided.

[0026] In further aspects of the disclosure, methods of treating or preventing one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation, comprising administering to a patient in need thereof an effective amount of a compound of salts of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt other than choline are provided. BRIEF DESCRIPTION OF THE FIGURES

[0027] Figure 1 is an x-ray powder diffraction pattern of crystalline 25HC3S potassium.

[0028] Figure 1a is an expanded x-ray powder diffraction pattern of crystalline 25HC3S potassium.

[0029] Figure 2 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S potassium.

[0030] Figure 2a is an expanded peak-picked x-ray powder diffraction pattern of crystalline 25HC3S potassium.

[0031] Figure 3 is an x-ray powder diffraction pattern of crystalline 25HC3S calcium.

[0032] Figure 3a is an expanded x-ray powder diffraction pattern of crystalline 25HC3S calcium.

[0033] Figure 4 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S calcium.

[0034] Figure 4a is an expanded peak-picked x-ray powder diffraction pattern of crystalline 25HC3S calcium.

[0035] Figure 5 is an x-ray powder diffraction pattern of crystalline 25HC3S magnesium.

[0036] Figure 5a is an expanded x-ray powder diffraction pattern of crystalline 25HC3S magnesium.

[0037] Figure 6 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S magnesium.

[0038] Figure 6a is an expanded peak-picked x-ray powder diffraction pattern of crystalline 25HC3S magnesium.

[0039] Figure 7 is an x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium.

[0040] Figure 7a is an expanded x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium.

[0041] Figure 8 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium.

[0042] Figure 8a is an expanded peak-picked x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium.

[0043] Figure 9 is an x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylpyrrolidinium.

[0044] Figure 10 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylpyrrolidinium.

[0045] Figure 11 is an x-ray powder diffraction pattern of crystalline 25HC3S meglumine.

[0046] Figure 11a is an expanded x-ray powder diffraction pattern of crystalline 25HC3S meglumine.

[0047] Figure 12 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S meglumine.

[0048] Figure 12a is an expanded peak-picked x-ray powder diffraction pattern of crystalline 25HC3S meglumine.

[0049] Figure 13 is an x-ray powder diffraction pattern of crystalline 25HC3S tromethammonium.

[0050] Figure 13a is an expanded x-ray powder diffraction pattern of crystalline 25HC3S tromethammonium.

[0051] Figure 14 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S tromethammonium.

[0052] Figure 14a is an expanded peak-picked x-ray powder diffraction pattern of crystalline 25HC3S tromethammonium.

[0053] Figure 15 is an x-ray powder diffraction pattern of crystalline 25HC3S diethanolamine.

[0054] Figure 16 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S diethanolamine.

[0055] Figure 17 is an x-ray powder diffraction pattern of crystalline 25HC3S diethylammonium.

[0056] Figure 18 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S diethylammonium.

[0057] Figure 19 is an x-ray powder diffraction pattern of crystalline 25HC3S t- butylammonium.

[0058] Figure 20 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S t- butylammonium.

[0059] Figure 21 is an x-ray powder diffraction pattern of crystalline 25HC3S benzathine.

[0060] Figure 22 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S benzathine.

[0061] Figure 23 is an x-ray powder diffraction pattern of crystalline 25HC3S lysine.

[0062] Figure 23a is an expanded x-ray powder diffraction pattern of crystalline 25HC3S lysine.

[0063] Figure 24 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S lysine.

[0064] Figure 24a is an expanded peak-picked x-ray powder diffraction pattern of crystalline 25HC3S lysine.

[0065] Figure 25 is a TGA thermogram and DSC thermogram of crystalline 25HC3S potassium.

[0066] Figure 26 is a DVS isotherm of crystalline 25HC3S potassium.

[0067] Figure 27 is an overlay of x-ray powder diffraction patterns before and after DVS of crystalline 25HC3S potassium.

[0068] Figure 28 is a1H-NMR spectrum of 25HC3S potassium.

[0069] Figure 29 is a TGA thermogram and DSC thermogram of crystalline 25HC3S calcium.

[0070] Figure 30 is a DVS isotherm of crystalline 25HC3S calcium.

[0071] Figure 31 is an overlay of x-ray powder diffraction patterns before and after DVS of crystalline 25HC3S calcium.

[0072] Figure 32 is a1H-NMR spectrum of 25HC3S calcium.

[0073] Figure 33 is a TGA thermogram and DSC thermogram of crystalline 25HC3S magnesium.

[0074] Figure 34 is a DVS isotherm of crystalline 25HC3S magnesium.

[0075] Figure 35 is an overlay of x-ray powder diffraction patterns before and after DVS of crystalline 25HC3S magnesium.

[0076] Figure 36 is a1H-NMR spectrum of 25HC3S magnesium.

[0077] Figure 37 is a TGA thermogram and DSC thermogram of crystalline 25HC3S hydroxyethylammonium.

[0078] Figure 38 is a DVS isotherm of crystalline 25HC3S hydroxyethylammonium.

[0079] Figure 39 is an overlay of x-ray powder diffraction patterns before and after DVS of crystalline 25HC3S hydroxyethylammonium.

[0080] Figure 40 is a1H-NMR spectrum of 25HC3S hydroxyethylammonium.

[0081] Figure 41 is a TGA thermogram and DSC thermogram of crystalline 25HC3S hydroxyethylpyrrolidinium.

[0082] Figure 42 is a DVS isotherm of crystalline 25HC3S hydroxyethylpyrrolidinium.

[0083] Figure 43 is an overlay of x-ray powder diffraction patterns before and after DVS of crystalline 25HC3S hydroxyethylpyrrolidinium.

[0084] Figure 44 is a1H-NMR spectrum of 25HC3S hydroxyethylpyrrolidinium.

[0085] Figure 45 is indexing results for crystalline 25HC3S hydroxyethylpyrrolidinium.

[0086] Figure 46 is a TGA thermogram and DSC thermogram of crystalline 25HC3S meglumine.

[0087] Figure 47 is a DVS isotherm of crystalline 25HC3S meglumine.

[0088] Figure 48 is an overlay of x-ray powder diffraction patterns before and after DVS of crystalline 25HC3S meglumine.

[0089] Figure 49 is a1H-NMR spectrum of 25HC3S meglumine.

[0090] Figure 50 is a TGA thermogram and DSC thermogram of crystalline 25HC3S tromethammonium.

[0091] Figure 51 is a DVS isotherm of crystalline 25HC3S tromethammonium.

[0092] Figure 52 is an overlay of x-ray powder diffraction patterns before and after DVS of crystalline 25HC3S tromethammonium.

[0093] Figure 53 is a1H-NMR spectrum of 25HC3S tromethammonium.

[0094] Figure 54 is a TGA thermogram and DSC thermogram of crystalline 25HC3S diethanolamine.

[0095] Figure 55 is a DVS isotherm of crystalline 25HC3S diethanolamine.

[0096] Figure 56 is an overlay of x-ray powder diffraction patterns before and after DVS of crystalline 25HC3S diethanolamine.

[0097] Figure 57 is a1H-NMR spectrum of 25HC3S diethanolamine.

[0098] Figure 58 is indexing results for crystalline 25HC3S diethanolamine.

[0099] Figure 59 is a TGA thermogram and DSC thermogram of crystalline 25HC3S diethylammonium.

[0100] Figure 60 is a DVS isotherm of crystalline 25HC3S diethylammonium.

[0101] Figure 61 is an overlay of x-ray powder diffraction patterns before and after DVS of crystalline 25HC3S diethylammonium.

[0102] Figure 62 is a1H-NMR spectrum of 25HC3S diethylammonium.

[0103] Figure 63 is indexing results for crystalline 25HC3S diethylammonium.

[0104] Figure 64 is a TGA thermogram and DSC thermogram of crystalline 25HC3S t- butylammonium.

[0105] Figure 65 is a DVS isotherm of crystalline 25HC3S t-butylammonium.

[0106] Figure 66 is a1H-NMR spectrum of 25HC3S t-butylammonium.

[0107] Figure 67 is indexing results for crystalline 25HC3S t-butylammonium.

[0108] Figure 68 is a TGA thermogram and DSC thermogram of crystalline 25HC3S benzathine.

[0109] Figure 69 is a DVS isotherm of crystalline 25HC3S benzathine.

[0110] Figure 70 is an overlay of x-ray powder diffraction patterns before and after DVS of crystalline 25HC3S benzathine.

[0111] Figure 71 is a1H-NMR spectrum of 25HC3S benzathine.

[0112] Figure 72 is indexing results for crystalline 25HC3S benzathine.

[0113] Figure 73 is a TGA thermogram and DSC thermogram of crystalline 25HC3S lysine.

[0114] Figure 74 is a DVS isotherm of crystalline 25HC3S lysine.

[0115] Figure 75 is a1H-NMR spectrum of 25HC3S lysine.

[0116] Figure 76 is an x-ray powder diffraction pattern of crystalline 25HC3S choline.

[0117] Figure 77 is a peak picked crystalline 25HC3S choline x-ray powder diffraction pattern.

[0118] Figure 78 is an x-ray powder diffraction pattern overlay of crystalline 25HC3S choline before and after DVS.

[0119] Figure 79 is indexing results for crystalline 25HC3S choline.

[0120] Figure 80 is a DSC (bottom) and TGA (top) thermograms of crystalline 25HC3S choline.

[0121] Figure 81 is a1H-NMR spectrum of 25HC3S choline in solution.

[0122] Figure 82 is a DVS isotherm for crystalline 25HC3S choline.

[0123] Figure 83 is an x-ray powder diffraction pattern of crystalline 25HC3S zinc.

[0124] Figure 84 is an x-ray powder diffraction pattern of crystalline 25HC3S zinc.

[0125] Figure 85 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S zinc.

[0126] Figure 86 is a peak-picked x-ray powder diffraction pattern of crystalline 25HC3S zinc.

[0127] Figure 87 is an x-ray powder diffraction pattern overlay of crystalline 25HC3S zinc before and after DVS.

[0128] Figure 88 is a DSC thermogram and TGA thermogram for crystalline 25HC3S zinc.

[0129] Figure 89 is a1H-NMR spectrum of 25HC3S zinc.

[0130] Figure 90 is a DVS experiment of crystalline 25HC3S zinc. DETAILED DESCRIPTION

[0131] The compound 25-hydroxy-3β-cholesten-5-en-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, the compound of Formula I:

[0132] Described herein are fifteen salts of 25HC3S. The salts were prepared as set forth in Examples 27 to 41. All of the salts therein can be prepared as crystalline solids. The salts of thedisclosure include inorganic and organic salts. The inorganic salts include metal salts and the organic salts include amine salts.

[0133] Metal salts of the disclosure include monocation alkali metal salts as well as dication salts including 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, alkali metal salts of the disclosure include 25HC3S potassium, including crystalline 25HC3S potassium. 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) 25HC3S calcium, including crystalline 25HC3S calcium, and (b) 25HC3S magnesium, including crystalline 25HC3S magnesium. The disclosure further includes other metal salts of 25HC3S, including crystalline salts thereof. Other metals within the disclosures include transition metals, such as dictation transition metals, including crystalline transition metal salts of 25HC3S such as crystalline dictation transition metals salts. An exemplary dictation transition metal is zinc.

[0134] Organic salts of 25HC3S of the disclosure including crystalline organic salts of 25HC3S are disclosed herein. Examples of such organic salts include amine salts, including crystalline amine salts of 25HC3S. The amines may be aliphatic, cyclic, aromatic, or a combination thereof. The amines may be primary, secondary, tertiary, or quaternary amines. The amines may further be substituted or unsubstituted. A common substitution is with one or more alcohol groups. The alcohol may be a primary alcohol, secondary alcohol, or tertiary alcohol.

[0135] The amine functionality of the amine group may be linked to an aliphatic chain of carbons containing from between 1 and 6 carbons for example. This linker may be substituted or unsubstituted. Substitutions include alkyl, alcohol, acid, aryl, and amine groups. Also included within the salts of the disclosure are amino acid salts of the disclosure. Such amino acid salts may be naturally occurring amino acids or non-naturally occurring amino acids. An exemplified amino acid salt of the disclosure is the lysine salt of 25HC3S. In many cases herein, the amine salts are amine alcohol salts of 25HC3S including choline, hydroxyethylammonium, hydroxyethylpyrrolidinium, meglumine, tromethammonium, and diethanolamine. The amine alcohol salts of the disclosure may contain a single alcohol, or contain multiple alcohol functionalities such as diethanolamine (2 hydroxy groups), tromethammonium (3 hydroxy groups), or meglumine (5 hydroxy groups). The amine alcohols may be primary, secondary, tertiary or quaternary amine alcohol salts. Such amine alcohol salts may include cyclic amines and the amine groups may be linked to the alcohol through alkyl linkers from between 1 and 6 carbons for example.

[0136] In many cases herein, the amine salts are amine alkyl salts of 25HC3S including diethylammonium, t-butylammonium, and benzathine. The alkyl amine salts of the disclosure include primary, secondary, and tertiary amines and the alkyl chains may contain one or more carbon atoms. In many embodiments, there are two carbon atoms in the linkers. The alkyl amine salts may also be diamines. In some cases, the alkyl amine salts also have aromatic groups such as phenyl or benzyl groups.

[0137] In many embodiments, the salts of the disclosure are crystalline salts of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt. In other embodiments, the salts of the disclosure are crystalline salts of 25HC3S other than the sodium salt or the choline salt.

[0138] Substantially pure salts and crystalline salts of 25HC3S of the disclosure are further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels of other forms of25HC3S. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of the salts of 25HC3S of the disclosure.

[0139] Processes of preparing salts of 25HC3S of the disclosure are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt of 25HC3S as described in Example 41. The triethylammonium salt may then be used to create other salts of 25HC3S of the disclosure.

[0140] The present disclosure also relates to pharmaceutical compositions containing salts of 25HC3S of the disclosure. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and salts and crystalline salts of 25HC3S. Such pharmaceutical compositions may be administered orally or configured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0141] In many embodiments, the pharmaceutical compositions of the disclosure contain salts of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt provided, however, that in some embodiments, the choline salt is included within such pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises a crystalline salt of 25HC3S other than sodium and other than choline and a pharmaceutically acceptable excipient.

[0142] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts salts and crystalline salts of 25HC3S, and / or pharmaceutical compositions comprising such salts and crystalline salts of 25HC3S of the present disclosure. As used throughout the present disclosure, examples of “diabetes conditions” that may be treated include one or more of insulin resistance, insulin insufficiency, diabetes, and prediabetes. As used throughout the present 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 the present disclosure, examples of “inflammatory conditions” that may be treated include one or more of dental pulp inflammation, 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 disorder (COPD), pneumonia, chronic inflammatory lung disease, bronchitis, asthma, chronic kidney disease (CKD), nephritis, sepsis, ankylosing spondylitis, diverticulitis, and fibromyalgia. As used throughout the present disclosure, examples of “neurological conditions” that may be treated include one or more of depression, neurodegenerative disease, multiple sclerosis, Parkinson’s disease, spinocerebellar degeneration, Friedreich ataxia, ataxia-telangiectasia, progressive supranuclear palsy, Huntington’s disease, striatonigral degeneration, olivopontocerebellar atrophy, Shy-Drager syndrome, schizophrenia, schizoaffective disorder, manic-depression (bipolar) disorder, disturbed or abnormal circadian entrainment, childhood Alice in Wonderland syndrome, childhood acute cerebellar ataxia, and Alzheimer’s disease.

[0143] In many embodiments, the present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, anautoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts salts and crystalline salts of 25HC3S, 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 provided, however, that in some embodiments, the choline salt is included. In other embodiments, the 25HC3S choline used in such methods is a crystalline salt of 25HC3S other than sodium and other than choline.

[0144] Crystalline salts of 25HC3S are readily analyzed by x-ray powder diffraction. An x-ray powder diffraction pattern is an x-y 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-spacings which is related to the diffraction angle via the Bragg’s law whereby 2dsinθ = nλ where d is the d-spacing and λ is the wavelength of the incident x-ray wave. The pattern contains peaks which may be used to characterize crystalline salts of 25HC3S. Unless otherwise specified, peaks are referred to by their position on the x-axis and not their y-axis intensity. It can also occur that due to sample orientation, a peak that is present in one sample on one instrument, may not be present in another sample taken on a different instrument due to the orientation of the sample with respect to the instrument.

[0145] The data from x-ray powder diffraction may be used in multiple ways to characterize crystalline forms. For example, the entire x-ray powder diffraction pattern output from a diffractometer may be used to characterize crystalline salts of 25HC3S. A smaller subset of such data, however, may also be, and typically is, suitable for characterizing crystalline salts 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 the present application, all reported peak values are in °2θ with Cu-Kα radiation as set forth in Example 24 and Example 25. Indeed, often even a single x-ray powder diffraction peak may be used to characterize such a crystalline form. When a crystalline salt of 25HC3S herein is characterized by “one or more peaks” of an x-ray powder diffraction pattern and such peaks are listed, what is generally meant is that any combination of the peaks listed may be used to characterize a crystalline salt of 25HC3S. Further, the fact that other peaks are present in the x-ray powder diffraction pattern, generally does not negate or otherwise limit that characterization.

[0146] In addition to the variability in peak intensity, there may also be variability in the position of peaks on the x-axis. This variability can, however, typically be accounted for when reporting the positions of peaks for purposes of characterization. Such variability in the position of peaks along the x-axis may derive from several sources (e.g., sample preparation, particle size, moisture content,solvent content, instrument parameters, data analysis software, and sample orientation). For example, samples of the same crystalline material prepared under different conditions may yield slightly different diffractograms, and different x-ray instruments may operate using different parameters, and these may lead to slightly different diffraction patterns from the same crystalline solid.

[0147] Due to such sources of variability, it is common to recite x-ray diffraction peaks using the word “about” prior to the peak value in °2θ. For purposes of data reported herein, that value is generally ±0.2°2θ. This generally means that on a well-maintained instrument one would expect the variability in peak measurement to be ±0.2°2θ. Unless specified otherwise, x-ray powder diffraction peaks cited herein are generally reported with this variability of ±0.2°2θ and are generally intended to be reported with such a variability whenever disclosed herein whether the word “about” is present or not, however, variability may, in some instances, be as high as ±0.2°2θ or even higher depending on instrumentation conditions. In many of the crystalline salts of the disclosure, x-ray powder diffraction some peaks, and often intense ones at low angle such as below 5°2θ, are close together such as within 0.2°2θ, which is the same as the variability to which the peaks are reported. Due to such variability, it is possible in some measurements, the two peaks will coincide making it difficult to discern the two peaks. However, when characterizing a crystalline salt of 25HC3S of the disclosure and such characterization only specifies a single peak, then provided that single peak is within the specified variability of±0.2°2θ, it may be used to so characterize or assist in the characterization of that crystalline salt of 25HC3S, as the case may be. Furthermore, in additional embodiments of the invention, the variability in a quoted peak value or grouping of quoted peak values in °2θ is ±0.1°2θ, or even ±0.05°2θ, rather than ±0.2°2θ.

[0148] In addition to x-ray powder diffraction, several the salts of 25HC3S were observed by polarized light microscopy. Several x-ray powder diffraction patterns were also indexed. “Indexing,” as used herein, generally refers to the process of determining the size and shape of the crystallographic unit cell given the peak positions in a diffraction pattern. The term gets its name from the assignment of Miller index labels to individual peaks. For example, if all of the peaks in a pattern are indexed by a single unit cell, this can be strong evidence that the sample contains a single crystalline phase. Given the indexing solution, the unit cell volume may be calculated directly and can be useful to determine their solvation states. Indexing may also be a description of a crystalline form and provides a concise summary of all available peak positions for that phase at a particular thermodynamic state point.

[0149] Differential scanning calorimetry (DSC) is a thermoanalytical technique where the difference in the amount of heat required to increase the temperature of a sample and reference ismeasured as a function of temperature. Using this technique, phase transitions of a sample can be measured such as melting point. Decomposition can also be observed by DSC as can dehydration events. In the context of DSC measurements, there is also variability and the term “about” means ±1°C, and such variability is to be understood whether a DSC measurement is prefaced by “about” or not unless specified otherwise. Exemplary methods for collecting DSC data are set forth in Example 21. Thermogravimetric analysis, sometimes referred to as, thermal gravimetric analysis (TGA) is a thermal method of thermal analysis where the mass of a sample is measured as a function of time as the sample temperature changes. For example, when samples disclosed herein are heated and a loss in mass is measured prior to decomposition, this is likely indicative of the loss of water from the sample and may indicate that the sample being heated is a hydrate. Exemplary methods for collecting TGA are set forth in Example 23.

[0150] Dynamic vapor sorption (DVS) measures the uptake of a vapor (typically water) by a sample when exposed to a changing relative humidity. The loss of water can also be measured as relative humidity is decreased. Hygroscopic materials tend to absorb water more readily than non- hygroscopic. In some cases, hydrates absorb water to create higher-order hydrates (e.g., a monohydrate converting to a dihydrate when exposed to increasingly humid conditions). In some salts of the disclosure, moisture is retained upon exposure in a DVS experiment and little or no weight loss is observed upon heating from room temperature. In such experiments, the hygroscopic nature of the salt under study may indicate the formation of a hydrate from an anhydrate. In other salts of the disclosure, weight loss is observed upon heating which suggests that the form of the salt at the beginning of such a thermal experiment was a hydrate. Exemplary methods for collecting DVS data are set forth in Example 26.

[0151] Inductively coupled plasma optical emission spectrometry (ICP-OES), is an analytical technique used for the detection of chemical elements. It can be used to determine the relative stoichiometry of a compound and was used herein to identify metal salts of 25HC3S.

[0152] Turning to inorganic salts of 25HC3S, an exemplary inorganic salt of the disclosure is the metal salt of 25HC3S with potassium. Crystalline 25HC3S potassium, may be prepared as set forth in Example 27. An x-ray powder diffraction pattern of crystalline 25HC3S potassium can be found in Figure 1 with an expanded pattern at Figure 1A. A peak-picked x-ray powder diffraction pattern can be seen in Figure 2, with an expanded pattern at Figure 2A. Table 1 shows picked peaks from Figure 2.Table 1 - Peaks of Crystalline 25HC3S Potassium of Figure 2 %)

[0153] Crystalline 25HC3S potassium may be characterized by various analytical techniques, including by x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S potassium or portions thereof, may be used to identify crystalline 25HC3S potassium. Crystalline 25HC3S potassium contains various x-ray powder diffraction peaks which alone or together may help identify the presence of crystalline 25HC3S potassium.

[0154] In some cases, crystalline 25HC3S potassium may be characterized by an x-ray powder diffraction pattern having one or more of the peaks in Figure 2. The two most intense peaks in Figure 2 are the peaks at about 2.2˚2θ and about 2.3˚2θ. In Figure 2, these peaks appear very close together due to their respective intensities, but the XRPD instrumentation is able to distinguish them. However, it is possible that due to the variability associated with these XRPD peaks that when analyzed they may appear “underneath” each other as a single peak. Because such a single apparent peak is possible, in many cases herein, the crystalline 25HC3S potassium is characterized, at least in part, by having an x- ray powder diffraction pattern comprising two peaks from about 2.2°2θ to about 2.3°2θ that are: (a) non-overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak.

[0155] ICP-OES results are consistent with a 1:1 stoichiometry of potassium ion to 25HC3S ion. For example, a peak at about 2.2°2θ may be used to characterize crystalline 25HC3S potassium. In these and other cases, crystalline 25HC3S potassium 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.

[0156] In some cases, crystalline 25HC3S potassium may be characterized by an x-ray powder diffraction pattern having a peak at about 2.3°2θ. In these and other cases, crystalline 25HC3S potassium may be further characterized by one or more peaks at about 2.2°2θ, about 8.8°2θ, about 9.3θ°2θ, and about 15.3°2θ.

[0157] In some cases, crystalline 25HC3S potassium may be characterized by an x-ray powder diffraction pattern having a peak at about 8.8°2θ. In these and other cases, crystalline 25HC3S potassium 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.

[0158] In some cases, crystalline 25HC3S potassium may be characterized by an x-ray powder diffraction pattern having a peak at about 9.3°2θ. In these and other cases, crystalline 25HC3Spotassium 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.

[0159] In some cases, crystalline 25HC3S potassium may be characterized by an x-ray powder diffraction pattern having a peak at about 15.3°2θ. In these and other cases, crystalline 25HC3S potassium 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.

[0160] In some cases, crystalline 25HC3S potassium 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 instance, the crystalline 25HC3S potassium may be further characterized by a peak at about 4.6°2θ. Alternatively, or additionally, the crystalline 25HC3S potassium may be further characterized by a peak at about 14.7°2θ. Alternatively, or additionally, the crystalline 25HC3S potassium may be further characterized by a peak at about 14.9°2θ. Alternatively, or additionally, the crystalline 25HC3S potassium may be further characterized by a peak at about 16.1°2θ.

[0161] In some cases, crystalline 25HC3S potassium may be characterized by an x-ray powder diffraction pattern substantially the same as that set forth in Figure 2 or Figure 2A.

[0162] Crystalline 25HC3S potassium has limited hygroscopicity per DVS, gaining only 2% water as a relative humidity approaches 95% RH (Figure 26), and exhibits weight loss of about 0.9% to about 100°C and a further 1.7% between 100°C to 180°C by TGA (Figure 25). These data suggest crystalline 25HC3S potassium is an anhydrate. Figure 27 is an overlay showing the XRPD diffractogram before and after DVS of crystalline 25HC3S potassium. There is not an appreciable change in the pattern. Figure 28 is the1H-NMR spectrum of a solution of 25HC3S potassium which is consistent with the chemical structure. The DSC thermogram also in Figure 25 exhibits small endotherms at near 100°C and then again at 139°C. Additional endotherms at 198°C and 244°C are observed with decomposition believed to be at 198°C.

[0163] Substantially pure crystalline 25HC3S potassium is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S potassium. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S potassium present.

[0164] Processes of preparing 25HC3S potassium are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, atriethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S potassium as set forth in Example 27.

[0165] The present disclosure also relates to pharmaceutical compositions containing 25HC3S potassium, including crystalline 25HC3S potassium, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S potassium, including crystalline 25HC3S potassium. Such pharmaceutical compositions may be administered orally or configured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0166] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S potassium, including crystalline 25HC3S potassium and / or pharmaceutical compositions comprising crystalline 25HC3S potassium of the present disclosure.

[0167] Another exemplary salt of the disclosure is the metal salt of 25HC3S with calcium. Crystalline 25HC3S calcium may be prepared as set forth in Example 28. An x-ray powder diffraction pattern of crystalline 25HC3S calcium can be found in Figure 3 with an expanded pattern at Figure 3A. A peak-picked x-ray powder diffraction pattern can be seen in Figure 4, with an expanded pattern at Figure 4A. Table 2 shows picked peaks from Figure 4. The calcium salt of 25HC3S contains 2 ions of 25HC3S for every ion of calcium, as confirmed by ICP-OES. It thus can also be referred to as a hemicalcium salt. Table 2 – Peaks of Crystalline 25HC3S Calcium of Figure 4 ˚2θ d (Å) I t it (%)1495 ± 020 5919 ± 0079 28 8 5 0 8 0 1 8

[0168] Crystalline 25HC3S calcium may be characterized by various analytical techniques, including by x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S calcium or portions thereof, may be used to identify crystalline 25HC3S calcium. Crystalline 25HC3S calcium contains various x-ray powder diffraction peaks which alone or together may help identify the presence of crystalline 25HC3S calcium.

[0169] Two of the most intense peaks in Figure 4 are the peaks at about 15.0˚2θ and about 15.1˚2θ. In Figure 4, these peaks appear very close together due to their respective intensities, but the XRPD instrumentation is able to distinguish them. However, it is possible that due to the variability associated with these XRPD peaks that when analyzed they may appear “underneath” each other as a single peak. Because such a single apparent peak is possible, in many cases herein, the crystalline25HC3S calcium is characterized, at least in part, by having an x-ray powder diffraction pattern comprising two peaks from about 15.0°2θ to about 15.1°2θ that are: (a) non-overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak. In some cases, crystalline 25HC3S calcium may be characterized by an x-ray powder diffraction pattern having one or more of the peaks in Figure 4. For example, a peak at about 2.2°2θ may be used to characterize crystalline 25HC3S calcium. In these and other cases, crystalline 25HC3S calcium 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.

[0170] In some cases, 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, crystalline 25HC3S calcium may be further characterized by one or more peaks at about, 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.

[0171] In some cases, 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 cases, crystalline 25HC3S calcium may be further characterized by one or more peaks at about, 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.

[0172] In some cases, 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 cases, crystalline 25HC3S calcium may be further characterized by one or more peaks at about, 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.

[0173] In some cases, 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 cases, crystalline 25HC3S calcium may be further characterized by one or more peaks at about, 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.

[0174] In some cases, 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 cases, crystalline 25HC3S calcium may be further characterized by one or more peaks at about, 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.

[0175] In some cases, 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 cases, crystalline 25HC3S calcium may be further characterized by one or more peaks at about, 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.

[0176] In some cases, 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 instance, 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 additionally, the crystalline 25HC3S calcium may be further characterized by a peak at about 19.2°2θ.

[0177] In some cases, crystalline 25HC3S calcium may be characterized by an x-ray powder diffraction pattern substantially the same as that in Figure 4 or Figure 4A.

[0178] Crystalline 25HC3S calcium may occur as a hydrate. A sample of crystalline 25HC3S calcium lost about 0.9% weight between room temperature and about 93°C with a further 3.1% weight loss between about 112°C and about 130°C as shown in the TGA thermogram of Figure 29. The TGA data were calculated based on the 25HC3S salt having one ion of 25HC3S and 0.5 ion of calcium. The weight loss is presumed to be due to water at these temperatures. Thus, 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) thus suggesting a monohydrate. According to the DVS data in Figure 30, a higher water uptake may occur when exposed to a higher relative humidity which may result in a dihydrate as seen by the plateau in the DVS isotherm. Thus, the data indicate crystalline 25HC3S calcium may exist in several hydrated states including a monohydrate or a dihydrate, when referenced to a single ion of 25HC3S and a half ion of calcium dication per salt molecule. In Example 28, for instance, the crystalline 25HC3S hemicalcium salt prepared contained two equivalents of water.

[0179] A DSC thermogram of crystalline 25HC3S calcium indicated a small endothermic peak at about 70.3°C and a larger one at about 130°C, with decomposition associated with the higher value (Figure 29). Figure 31 is an overlay showing the XRPD diffractogram before and after DVS of crystalline 25HC3S calcium. There is not an appreciable change in the pattern. Figure 32 is the1H- NMR spectrum of a solution of 25HC3S calcium which is consistent with the chemical structure.

[0180] Substantially pure crystalline 25HC3S calcium is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S calcium. Examples of tracelevels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S calcium present.

[0181] Processes of preparing 25HC3S calcium are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S calcium as set forth in Example 28.

[0182] The present disclosure also relates to pharmaceutical compositions containing 25HC3S calcium, including crystalline 25HC3S calcium, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S calcium, including crystalline 25HC3S calcium. Such pharmaceutical compositions may be administered orally or configured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0183] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S calcium, including crystalline 25HC3S calcium and / or pharmaceutical compositions comprising crystalline 25HC3S calcium of the present disclosure.

[0184] Another exemplary inorganic salt of the disclosure is the metal salt of 25HC3S with zinc. Crystalline 25HC3S zinc may be prepared as set forth in Example 29. An x-ray powder diffraction pattern of crystalline 25HC3S zinc can be found in Figure 83 with an expanded pattern at Figure 84. A peak-picked x-ray powder diffraction pattern can be seen in Figure 85, with an expanded pattern at Figure 86. Table 3 shows picked peaks from Figure 85. The zinc salt of 25HC3S contains 2 ions of 25HC3S for every ion of zinc, as confirmed by ICP-OES, confirming the presence of zinc at 5.72% which comports with calculations of 5.98% zinc at 0.5 mol / mol. It thus may also, like with calcium, be referred to as a hemizinc salt. The1H-NMR spectrum of 25HC3S zinc is consistent with chemical structure as seen in Figure 89.

[0185] The two most intense peaks in Figure 85 are the peaks at about 2.1˚2θ and about 2.3˚2θ. In Figure 85, these peaks appear very close together due to their respective intensities, but the XRPD instrumentation is able to distinguish them. However, it is possible that due to the variability associated with these XRPD peaks that when analyzed they may appear “underneath” each other as a single peak. Because such a single peak is possible, in many cases herein, the crystalline 25HC3S zinc is characterized, at least in part, by having an x-ray powder diffraction pattern comprising two peaks from about 2.1˚2θ to about 2.3˚2θ that are: (a) non-overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak. An expanded diffraction pattern can be found in Figure 86 to help show other peaks in the pattern.

[0186] In some cases, crystalline 25HC3S zinc may be characterized by an x-ray powder diffraction pattern having a peak at about 2.1°2θ. In these and other cases, crystalline 25HC3S zinc may 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.

[0187] In some cases, crystalline 25HC3S zinc may be characterized by an x-ray powder diffraction pattern having a peak at about 2.3°2θ. In these and other cases, crystalline 25HC3S zincmay 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.

[0188] In some case, crystalline 25HC3S zinc 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, crystalline 25HC3S zinc 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.

[0189] In some cases, crystalline 25HC3S zinc 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 a peak at about 18.3°2θ. In these and other cases, crystalline 25HC3S zinc 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, and about 18.8°2θ.

[0190] In some cases, crystalline 25HC3S zinc may be characterized by an x-ray powder diffraction pattern having a peak at about 6.0°2θ. In these and other cases, crystalline 25HC3S zinc 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.

[0191] In some cases, crystalline 25HC3S zinc may be characterized by an x-ray powder diffraction pattern having a peak at about 8.6°2θ. In these and other cases, crystalline 25HC3S zinc may 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.

[0192] In some cases, crystalline 25HC3S zinc may be characterized by an x-ray powder diffraction pattern having a peak at about 8.9°2θ. In these and other cases, crystalline 25HC3S zinc 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.

[0193] In some cases, crystalline 25HC3S zinc may be characterized by an x-ray powder diffraction pattern having a peak at about 9.3°2θ. In these and other cases, crystalline 25HC3S zinc 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 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0194] In some cases, crystalline 25HC3S zinc may be characterized by an x-ray powder diffraction pattern having a peak at about 15.1°2θ. In these and other cases, crystalline 25HC3S zinc 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.

[0195] In some cases, crystalline 25HC3S zinc may be characterized by an x-ray powder diffraction pattern having a peak at about 18.3°2θ. In these and other cases, crystalline 25HC3S zincmay 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.

[0196] In some cases, crystalline 25HC3S zinc may be characterized by an x-ray powder diffraction pattern having a peak at about 18.8°2θ. In these and other cases, crystalline 25HC3S zinc 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 18.3°2θ.

[0197] In some cases, crystalline 25HC3S zinc 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 a peak at about 6.0°2θ. In these and other cases, crystalline 25HC3S zinc 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.

[0198] In some cases, crystalline 25HC3S zinc 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, a peak about 6.0°2θ, and a peak at about 8.6°2θ. In these and other cases, crystalline 25HC3S zinc 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.

[0199] In some cases, crystalline 25HC3S zinc 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, a peak about 6.0°2θ, a peak about 8.6°2θ, and a peak at about 9.3°2θ. In these and other cases, crystalline 25HC3S zinc 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.

[0200] In some cases, crystalline 25HC3S zinc 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 a peak at about 6.0°2θ, a peak at about 8.6°2θ, a peak at about 9.3°2θ, and a peak at about 15.1°2θ. In these and other cases, crystalline 25HC3S zinc may be further characterized by one or more peaks at 8.9°2θ, about 18.3°2θ, and about 18.8°2θ.

[0201] In some cases, crystalline 25HC3S zinc 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, a peak at about 6.0°2θ, a peak at about 8.6°2θ, a peak at about 9.3°2θ, about 15.1°2θ, and a peak at about 18.3°2θ. In these and other cases, crystalline 25HC3S zinc may be further characterized by one or more peaks at about 8.9°2θ, and about 18.8°2θ.

[0202] In some cases, crystalline 25HC3S zinc 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, a peak about6.0°2θ, a peak at about 8.6°2θ, a peak at about 9.3°2θ, a peak at about 15.1°2θ, a peak at about 18.3°2θ, and a peak at about 18.8°2θ. In these and other cases, crystalline 25HC3S zinc may be further characterized by a peak at about 8.9°2θ.

[0203] In some cases, crystalline 25HC3S zinc may 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.

[0204] In some cases, crystalline 25HC3S zinc 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 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.

[0205] In some cases, crystalline 25HC3S zinc may 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.

[0206] In some cases, crystalline 25HC3S zinc may 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.

[0207] In some cases, crystalline 25HC3S zinc may 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.

[0208] In some cases, crystalline 25HC3S zinc may 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.

[0209] In some cases, crystalline 25HC3S zinc 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.

[0210] In some cases, crystalline 25HC3S zinc 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.

[0211] In some cases, crystalline 25HC3S zinc may be characterized by an x-ray powder diffraction pattern having a peak at about 18.8°2θ.

[0212] In some cases, crystalline 25HC3S zinc may be characterized by an x-ray powder diffraction pattern substantially the same as that found in Figure 85 or Figure 86.

[0213] Crystalline 25HC3S zinc, may exhibit a differential scanning calorimetry (DSC) thermogram having one or more small endothermic peaks at about 68°C, about 86°C, and about 102°Cas seen in Figure 88. It is believed that the thermal decomposition of crystalline 25HC3S zinc occurs at about 119°C, which can be seen as a large endothermic peak in Figure 88.

[0214] Crystalline 25HC3S zinc may be hydrated. In such cases, the water of hydration may be part of the crystal structure such that crystalline 25HC3S is a hydrate. A sample of crystalline 25HC3S zinc lost about 5.1% of its weight between room temperature and about 130°C as shown in the TGA thermogram of Figure 88. The weight loss is presumed to be due to water at these temperatures. For the zinc salt, a water content of 5.1% is associated with approximately 1.5 moles of water per mole of 25HC3S. The TGA data were calculated based on the zinc 25HC3S salt having one ion of 25HC3S and one-half ion of zinc.

[0215] The DVS data indicate that water uptake may also stabilize at about 2.8% (change in mass) between 25% and 45% relative humidity, suggesting a hydrate of 0.75 moles of water per mole of 25HC3S or a monohydrate, and also at about 4.5 to 5% (change in mass) above 45% relative humidity, suggesting a sesquihydrate or dihydrate; when referenced to a single ion of 25HC3S and a half ion of zinc dication per salt molecule. Accordingly, in many cases, the level of hydration may include between 0.75 and 2 water molecules (including 1 and 1.5) on average per ion of 25HC3S in crystalline 25HC3S zinc. A weight loss of 11.5% in the TGA was further observed from about 131°C to about 190°C, which may be due to decomposition. The XRPD pattern of crystalline zinc before and after DVS is shown in Figure 87. Figure 89 is the1H-NMR spectrum which is consistent with the chemical structure.

[0216] Substantially pure crystalline 25HC3S zinc is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S zinc. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S zinc present.

[0217] Processes of preparing 25HC3S zinc are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S zinc as set forth in Example 29.

[0218] The preparation of the triethylammonium salt of 25HC3S may be accomplished, for example, by passing a mixture of triethylammonium chloride and triethylamine through a column and treating with a solvent such as an alcohol until neutral pH. Separately, crystalline 25HC3S sodium may bedissolved in a solvent such as an alcohol. The solution may then be passed through the same column previously exposed to triethylamine and combined with the triethylammonium solution. Isolating resulting solids such as under vacuum or by drying may then provide crystalline 25HC3S triethylammonium salt which may be homogenized, for example, with a mortar and pestle. A suitable alcohol for this process includes methanol.

[0219] 25HC3S zinc, including crystalline 25HC3S zinc, may be prepared by starting with 25HC3S sodium, converting to a second s of 25HC3S such as the triethylammonium salt, and then converting that second salt of 25HC3S to 25HC3S zinc, including crystalline 25HC3S zinc. The preparation of crystalline 25HC3S zinc may be accomplished by preparing a suspension of a triethylammonium salt of 25HC3S in a suitable solvent such as acetonitrile and treating with a zinc source such as zinc chloride in water to form 25HC3S zinc including crystalline 25HC3S zinc. The 25HC3S zinc may be purified such as 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 increase the yield. Further, processing may include stirring and / or further treatment with a suitable solvent as acetonitrile. Additional processing such as drying under vacuum or otherwise may also be performed to assist in the preparation of crystalline 25HC3S zinc. The disclosure further includes crystalline 25HC3S zinc made by the processes described herein.

[0220] The present disclosure also relates to pharmaceutical compositions containing 25HC3S zinc, including crystalline 25HC3S zinc, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S zinc, including crystalline 25HC3S zinc. Such pharmaceutical compositions may be administered orally or configured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0221] As discussed elsewhere herein, the 25HC3S zinc of the disclosure advantageously provides supplementary zinc to patients suffering from the conditions targeted by the 25HC3S. As discussed elsewhere herein, zinc deficiency can contribute to these conditions and it may be advantageous to provide zinc alongside the 25HC3S in the course of therapy. Hence, the 25HC3S zinc surprisingly and beneficially combines advantageous salt / crystalline form properties along with intrinsic ability beneficially to provide zinc supplementation in course of conducting methods of treatment.

[0222] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S zinc, including crystalline 25HC3S zinc and / or pharmaceutical compositions comprising crystalline 25HC3S zinc of the present disclosure.

[0223] Another exemplary inorganic salt of the disclosure is the metal salt of 25HC3S with magnesium. Crystalline 25HC3S magnesium, may be prepared as set forth in Example 30. An x-ray powder diffraction pattern of crystalline 25HC3S magnesium can be found in Figure 5 with an expanded pattern at Figure 5A. A peak-picked x-ray powder diffraction pattern can be seen in Figure 6, with an expanded pattern at Figure 6A. An expanded peak Table 4 shows picked peaks from Figure 6. The magnesium salt contains 2 ions of 25HC3S for every magnesium ion as confirmed by ICP-EOS. It thus can be referred to as a hemimagnesium salt. Table 4 – Peaks of Crystalline 25HC3S Magnesium of Figure 6 Å

[0224] Crystalline 25HC3S magnesium may be characterized by various analytical techniques, including by x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S magnesium or portions thereof, may be used to identify crystalline 25HC3S magnesium. Crystalline 25HC3S magnesium contains various x-ray powder diffraction peaks which alone or together may help identify the presence of crystalline 25HC3S magnesium.

[0225] In some cases, crystalline 25HC3S magnesium may be characterized by an x-ray powder diffraction pattern having one or more of the peaks in Figure 6. For example, a peak at about 2.2°2θ may 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.

[0226] In some cases, crystalline 25HC3S magnesium may be characterized by an x-ray powder diffraction pattern having a peak at about 6.6°2θ. In these and other cases, crystalline 25HC3S calcium 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.

[0227] In some cases, crystalline 25HC3S magnesium may be characterized by an x-ray powder diffraction pattern having a peak at about 8.9°2θ. In these and other cases, crystalline 25HC3S calcium 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.

[0228] In some cases, crystalline 25HC3S magnesium may be characterized by an x-ray powder diffraction pattern having a peak at about 15.1°2θ. In these and other cases, crystalline 25HC3S calcium 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.

[0229] 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 instance, the crystalline 25HC3S magnesium may be further characterized by a peak at about 15.6°2θ. Alternatively, or additionally, the crystalline 25HC3S magnesium may be further characterized by a peak at about 16.4°2θ. Alternatively, or additionally, the crystalline 25HC3S magnesium may be further characterized by a peak at about 17.6°2θ. Alternatively, or additionally, the crystalline 25HC3S magnesium may be further characterized by a peak at about 17.8°2θ.

[0230] 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.

[0231] Crystalline 25HC3S magnesium may occur as a hydrate. A sample of crystalline 25HC3S magnesium lost about 1.5% weight up to 127°C as shown in the TGA thermogram of Figure 33. The weight loss is presumed to be due to water at these temperatures. Thus, for the magnesium salt, a 1.5% water loss is associated with approximately 0.5 moles of water per mole of 25HC3S. The plateau-like regions in the DVS of Figure 34 of crystalline 25HC3S magnesium at around 5% to 7% mass change suggest that higher hydrates of 1.5 to 2 moles of water per mole of 25HC3S are also available, such as a sesquihydrate or dihydrate. Thus, the data indicate crystalline 25HC3S magnesium may exist as a hydrate such as a hemihydrate, sesquihydrate, or dihydrate, as referenced to a single ion of 25HC3S and a half ion of magnesium dication per salt molecule. The x-ray powder diffractograms before and after DVS are shown in Figure 35. The 1H-NMR spectrum in Figure 36 is consistent with the chemical structure of 25HC3S magnesium. The DSC thermogram exhibits an endothermic peak at about 139ºC, which is believed to be due to decomposition.

[0232] Substantially pure crystalline 25HC3S magnesium is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S magnesium. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S magnesium present.

[0233] Processes of preparing 25HC3S magnesium are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S magnesium as set forth in Example 30.

[0234] The present disclosure also relates to pharmaceutical compositions containing 25HC3S magnesium, including crystalline 25HC3S magnesium, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S magnesium, including crystalline 25HC3S magnesium. Such pharmaceutical compositions may be administered orally or configured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0235] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S magnesium, including crystalline 25HC3S magnesium and / or pharmaceutical compositions comprising crystalline 25HC3S magnesium of the present disclosure.

[0236] Another exemplary organic salt of the 25HC3S of the disclosure is the organic amine hydroxyethylammonium salt of 25HC3S. A preparation of crystalline hydroxyethylammonium salt of 25HC3S is found in Example 31. An x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium can be found in Figure 7 with an expanded pattern at Figure 7A. A peak- picked x-ray powder diffraction pattern can be seen in Figure 8, with an expanded pattern at Figure 8A. Table 5 shows picked peaks from Figure 8. Table 5 – Peaks of Crystalline 25HC3S Hydroxyethylammonium of Figure 8 ˚2θ d (Å) I i (%)

[0237] Crystalline 25HC3S hydroxyethylammonium may be characterized by various analytical techniques, including by x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium or portions thereof, may be used to identify crystalline 25HC3S hydroxyethylammonium. Crystalline 25HC3S hydroxyethylammonium contains various x- ray powder diffraction peaks which alone or together may help identify the presence of crystalline 25HC3S hydroxyethylammonium.

[0238] In some cases, crystalline 25HC3S hydroxyethylammonium may be characterized by an x-ray powder diffraction pattern having one or more of the peaks in Figure 8. For example, a peak at about 2.1°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°2θ.

[0239] Crystalline 25HC3S hydroxyethylammonium is hygroscopic as determined by DVS in Figure 38. It shows a weight loss of about 2.7% from ambient to 134°C by TGA in Figure 37. A1H- NMR spectrum in Figure 40 shows isopropyl alcohol in the solution, and which is consistent with the preparation of the salt as per Example 31, where the salt was prepared with isopropyl alcohol. Thus, it is believed, without being bound by theory, that the crystalline 25HC3S hydroxyethylammonium salt may exist as an isopropyl alcohol solvate. An x-ray analysis after DVS in Figure 39 also showed the appearance of extra x-ray powder diffraction peaks which may indicate additional physical transformation. A DSC of crystalline 25HC3S hydroxyethylammonium can be found in Figure 37 showing multiple endotherms.

[0240] Substantially pure crystalline 25HC3S hydroxyethylammonium is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels of, other forms of 25HC3S hydroxyethylammonium. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S hydroxyethylammonium present.

[0241] Processes of preparing 25HC3S hydroxyethylammonium are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S hydroxyethylammonium as set forth in Example 31.

[0242] The present disclosure also relates to pharmaceutical compositions containing 25HC3S hydroxyethylammonium, including crystalline 25HC3S hydroxyethylammonium, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S hydroxyethylammonium, including crystalline 25HC3S hydroxyethylammonium. Such pharmaceutical compositions may be administered orally or configured to be delivered as any effective conventional dosage forms, including, for example, immediate,sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0243] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S hydroxyethylammonium, including crystalline 25HC3S hydroxyethylammonium and / or pharmaceutical compositions comprising crystalline 25HC3S hydroxyethylammonium of the present disclosure.

[0244] An exemplary organic salt of the 25HC3S of the disclosure is the organic amine tromethammonium salt of 25HC3S. A preparation of crystalline tromethammonium salt of 25HC3S is found in Example 32. An x-ray powder diffraction pattern of crystalline 25HC3S tromethammonium can be found in Figure 13 with an expanded pattern at Figure 13A. A peak-picked x-ray powder diffraction pattern can be seen in Figure 14, with an expanded pattern at Figure 14A. Table 6 shows picked peaks from Figure 14. Table 6 – Peaks of Crystalline 25HC3S Tromethammonium of Figure 14 Å

[0245] Crystalline 25HC3S tromethammonium may be characterized by various analytical techniques, including by x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S tromethammonium or portions thereof, may be used to identify crystalline 25HC3S tromethammonium. Crystalline 25HC3S tromethammonium contains various x-ray powder diffraction peaks which alone or together may help identify the presence of crystalline 25HC3S tromethammonium.

[0246] In some cases, crystalline 25HC3S tromethammonium may be characterized by an x- ray powder diffraction pattern having one or more of the peaks in Figure 14 or Figure 14A. For example, a peak at about 1.9°2θ may be used to characterize crystalline 25HC3S tromethammonium. In these and other cases, crystalline 25HC3S tromethammonium may be further characterized by a peak at about 2.1°2θ or at about 3.8°2θ.

[0247] The two most intense peaks in Figure 14 are the peaks at about 1.9˚2θ and about 2.1˚2θ. In Figure 14, these peaks appear very close together due to their respective intensities, but the XRPD instrumentation is able to distinguish them. However, it is possible that due to the variability associatedwith these XRPD peaks that when analyzed they may appear “underneath” each other as a single peak. Because such a single apparent peak is possible, in many cases herein, the crystalline 25HC3S tromethammonium is characterized, at least in part, by having an x-ray powder diffraction pattern comprising two peaks from about 1.9°2θ to about 2.1°2θ that are: (a) non-overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak. In some cases, crystalline 25HC3S tromethammonium may be characterized by an x-ray powder diffraction pattern having a peak at about 2.1°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 3.8°2θ.

[0248] In some cases, crystalline 25HC3S tromethammonium 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.

[0249] In some cases, crystalline 25HC3S tromethammonium may be further characterized by one or more peaks at about 4.2°2θ and about 15.4°2θ. For instance, the crystalline 25HC3S tromethammonium may be further characterized by a peak at about 4.2°2θ. Alternatively, or additionally, the crystalline 25HC3S tromethammonium may be further characterized by a peak at about 15.4°2θ.

[0250] In some cases, crystalline 25HC3S tromethammonium may be characterized by an x- ray powder diffraction pattern substantially the same as that of Figure 14 or Figure 14A.

[0251] Crystalline 25HC3S tromethammonium is hygroscopic as seen by DVS in Figure 51. Indeed, it takes up over 20% by weight water when exposed to a relative humidity of around 95%. By TGA in Figure 50, only about a 0.3% weight loss is observed up to about 80°C suggesting an anhydrate under ambient conditions. Further, the x-ray powder diffraction patterns before and after DVS are presented in Figure 52 and show some potential differences which may be due to its hygroscopicity. A DSC of crystalline 25HC3S tromethammonium can be found in Figure 50 showing multiple small endotherms at about 54°C, about 114°C, and about 158°C followed by decomposition at about 184°C. The1H-NMR structure of Figure 53 is consistent with chemical structure.

[0252] Substantially pure crystalline 25HC3S tromethammonium is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S tromethammonium. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S tromethammonium present.

[0253] Processes of preparing 25HC3S tromethammonium are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S tromethammonium as set forth in Example 32.

[0254] The present disclosure also relates to pharmaceutical compositions containing 25HC3S tromethammonium, including crystalline 25HC3S tromethammonium, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S tromethammonium, including crystalline 25HC3S tromethammonium. Such pharmaceutical compositions may be administered orally or configured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0255] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S tromethammonium, including crystalline 25HC3S tromethammonium and / or pharmaceutical compositions comprising crystalline 25HC3S tromethammonium of the present disclosure.

[0256] An exemplary organic salt of the 25HC3S of the disclosure is the organic amino acid lysine salt of 25HC3S. A preparation of crystalline l-lysine salt of 25HC3S is found in Example 33. An x-ray powder diffraction pattern of crystalline 25HC3S lysine can be found in Figure 23 with an expanded pattern at Figure 23A. A peak-picked x-ray powder diffraction pattern can be seen in Figure 24, with an expanded pattern at Figure 24A. Table 7 shows picked peaks from Figure 24. Table 7 - Peaks of Crystalline 25HC3S Lysine of Figure 24

[0257] Crystalline 25HC3S lysine may be characterized by various analytical techniques, including by x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S lysine or portions thereof, may be used to identify crystalline 25HC3S lysine. Crystalline 25HC3S lysine contains various x-ray powder diffraction peaks which alone or together may help identify the presence of crystalline 25HC3S lysine. In some cases, crystalline 25HC3S lysine may be characterized by an x-ray powder diffraction pattern having one or more of the peaks in Figure 24. For example, a peak at about 1.5°2θ may be used to characterize crystalline 25HC3S lysine. In these and other cases, crystalline 25HC3S lysine may 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.

[0258] In some cases, 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, 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.

[0259] In some cases, 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, 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.

[0260] In some cases, 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, 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.

[0261] In some cases, 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, crystalline 25HC3S lysinemay 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.

[0262] 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 instance, the crystalline 25HC3S lysine may be further characterized by a peak at about 3.2°2θ. Alternatively, or additionally, the crystalline 25HC3S lysine may be further characterized by a peak at about 10.0°2θ. Alternatively, or additionally, the crystalline 25HC3S lysine may be further characterized by a peak at about 12.2°2θ. Alternatively, or additionally, the crystalline 25HC3S lysine may be further characterized by a peak at about 15.2°2θ.

[0263] In some cases, crystalline 25HC3S lysine may be characterized by an x-ray powder diffraction pattern substantially the same as that of Figure 24.

[0264] Crystalline 25HC3S lysine is hygroscopic above 75% relative humidity by DVS in Figure 74. The TGA, as seen in Figure 73, shows weight loss of about 1.5% weight loss to 100ºC and further weight loss of about 8.8% between 100°C and 235ºC, some of which may be due to water loss and some due to decomposition. Thus, it is possible that crystalline 25HC3S lysine exists as a hydrate. A DSC of crystalline 25HC3S lysine can be found in Figure 73 showing a weak endotherm at about 96°C. It is believed that the endotherm at about 186°C is indicative of decomposition. The1H-NMR spectrum of Figure 75 is consistent with chemical structure.

[0265] Substantially pure crystalline 25HC3S lysine is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S lysine. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S lysine present.

[0266] Processes of preparing 25HC3S lysine are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S lysine as set forth in Example 33.

[0267] The present disclosure also relates to pharmaceutical compositions containing 25HC3S lysine, including crystalline 25HC3S lysine, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S lysine, including crystalline 25HC3S lysine. Such pharmaceutical compositions may be administered orally orconfigured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0268] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S lysine, including crystalline 25HC3S lysine and / or pharmaceutical compositions comprising crystalline 25HC3S lysine of the present disclosure.

[0269] An exemplary organic salt of the 25HC3S of the disclosure is the organic amine meglumine salt of 25HC3S. A preparation of crystalline meglumine salt of 25HC3S is found in Example 34. An x-ray powder diffraction pattern of crystalline 25HC3S meglumine can be found in Figure 11 with an expanded pattern at Figure 11A. A peak-picked x-ray powder diffraction pattern can be seen in Figure 12, with an expanded pattern at Figure 12A. Table 8 shows picked peaks from Figure 12. Table 8 – Peaks of Crystalline 25HC3S Meglumine of Figure 12 ˚2θ d (Å) I i (%)

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

[0271] In some cases, crystalline 25HC3S meglumine may be characterized by an x-ray powder diffraction pattern having one or more of the peaks in Figure 12. For example, a peak at about 1.7°2θ may be used to characterize crystalline 25HC3S meglumine. In these and other cases, 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.

[0272] In some cases, 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 cases, 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.

[0273] In some cases, 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 cases, 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.

[0274] In some cases, 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 cases, 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.

[0275] In some cases, 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 cases, 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.

[0276] 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 instance, the crystalline 25HC3S meglumine may be further characterized by a peak at about 8.6°2θ. Alternatively, or additionally, the crystalline 25HC3S meglumine may be further characterized by a peak at about 14.5°2θ. Alternatively, or additionally, the crystalline 25HC3S meglumine may be further characterized by a peak at about 15.1°2θ. Alternatively, or additionally, the crystalline 25HC3S meglumine may be further characterized by a peak at about 17.5°2θ. Alternatively, or additionally, the crystalline 25HC3S meglumine may be further characterized by a peak at about 18.2°2θ.

[0277] In some cases, crystalline 25HC3S meglumine can be characterized by an x-ray powder diffraction pattern substantially the same as that of Figure 12 or Figure 12A.

[0278] Crystalline 25HC3S meglumine picks up moisture in a DVS experiment as seen in Figure 47 showing a water uptake of about 3% and has a stable plateau at about that level of water uptake above 35% relative humidity. This level of water uptake is consistent with the formation of a monohydrate. The TGA, starting with ambient, shows a loss of about 3.2% loss of moisture which is also consistent with a monohydrate. An x-ray powder diffraction pattern before and after DVS is attached as Figure 48. A DSC of crystalline 25HC3S meglumine can be found in Figure 46 showing multiple weak endotherms from between 50°C and 90°C. The solution state1H-NMR spectrum of the salt shows some additional peaks between 4.3 and 5.5 ppm and is otherwise consistent with chemical structure.

[0279] Substantially pure crystalline 25HC3S meglumine is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S meglumine. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S meglumine present.

[0280] Processes of preparing 25HC3S meglumine are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S meglumine as set forth in Example 34.

[0281] The present disclosure also relates to pharmaceutical compositions containing 25HC3S meglumine, including crystalline 25HC3S meglumine, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3Smeglumine, including crystalline 25HC3S meglumine. Such pharmaceutical compositions may be administered orally or configured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0282] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S meglumine, including crystalline 25HC3S meglumine and / or pharmaceutical compositions comprising crystalline 25HC3S meglumine of the present disclosure.

[0283] An exemplary organic salt of the 25HC3S of the disclosure is the organic cyclic amine hydroxyethylpyrrolidinium salt of 25HC3S. A preparation of crystalline hydroxyethylpyrrolidinium salt of 25HC3S is found in Example 35. An x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylpyrrolidinium can be found in Figure 9. A peak-picked x-ray powder diffraction pattern can be seen in Figure 10. Table 9 shows picked peaks from Figure 10. Table 9 – Peaks of Crystalline 25HC3S Hydroxyethylpyrrolidinium of Figure 10 Å

[0284] Crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by various analytical techniques, including by x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylpyrrolidinium or portions thereof, may be used to identify crystalline 25HC3S hydroxyethylpyrrolidinium. Crystalline 25HC3S hydroxyethylpyrrolidinium contains various x-ray powder diffraction peaks which alone or together may help identify the presence of crystalline 25HC3S hydroxyethylpyrrolidinium.

[0285] In some cases, crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction pattern having one or more of the peaks in Figure 10. For example, a peak at about 3.8°2θ may be used to characterize crystalline 25HC3S hydroxyethylpyrrolidinium. In these and other cases, crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by oneor 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.

[0286] Two of the most intense peaks in Figure 10 are the peaks at about 7.5˚2θ and about 7.6˚2θ. In Figure 10, these peaks appear very close together due to their respective intensities, but the XRPD instrumentation is able to distinguish them. However, it is possible that due to the variability associated with these XRPD peaks that when analyzed they may appear “underneath” each other as a single peak. Because such a single apparent peak is possible, in many cases herein, the crystalline 25HC3S hydroxyethylpyrrolidinium is characterized, at least in part, by having an x-ray powder diffraction pattern comprising two peaks from about 7.5°2θ to about 7.6°2θ that are: (a) non- overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak.

[0287] Two of the most intense peaks in Figure 10 are the peaks at about 8.2˚2θ and about 8.6˚2θ. In Figure 10, these peaks appear very close together due to their respective intensities, but the XRPD instrumentation is able to distinguish them. However, it is possible that due to the variability associated with these XRPD peaks that when analyzed they may appear “underneath” each other as a single peak. Because such a single apparent peak is possible, in many cases herein, the crystalline 25HC3S hydroxyethylpyrrolidinium is characterized, at least in part, by having an x-ray powder diffraction pattern comprising two peaks from about 8.2°2θ to about 8.6°2θ that are: (a) non- overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak.

[0288] In some cases, 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 cases, 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.

[0289] In some cases, crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction pattern having a peak at about 7.6°2θ. In these and other cases, crystalline 25HC3S hydroxyethylpyrrolidinium 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.

[0290] In some cases, crystalline 25HC3S hydroxyethylpyrrolidinium 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 hydroxyethylpyrrolidinium may be further characterized by one or more peaks at about 3.8°2θ, about 7.5°2θ, about 7.6°2θ, about 8.6°2θ, about 12.4°2θ, about 13.3°2θ, and about 15.0°2θ.

[0291] In some cases, crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction pattern having a peak at about 8.6°2θ. In these and other cases, crystalline25HC3S 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 12.4°2θ, about 13.3°2θ, and about 15.0°2θ.

[0292] In some cases, crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction pattern having a peak at 12.4°2θ. In these and other cases, 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.

[0293] In some cases, crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction pattern having a peak at 13.3°2θ. In these and other cases, 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.

[0294] In some cases, crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction pattern having a peak at 15.0°2θ. In these and other cases, 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 cases, crystalline 25HC3S hydroxyethylpyrrolidinium may be characterized by an x-ray powder diffraction substantially the same as that of Figure 10.

[0295] 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 instance, the crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by a peak at about 10.5°2θ. Alternatively, or additionally, the crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by a peak at about 15.3°2θ. Alternatively, or additionally, the crystalline 25HC3S hydroxyethylpyrrolidinium may be further characterized by a peak at about 15.6°2θ. Alternatively, or additionally, the 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.

[0296] Crystalline 25HC3S hydroxyethylpyrrolidinium picks up and retains moisture as seen in a DVS experiment. In Figure 42, a stable plateau in the DVS shows a water content of 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 into a hydrate at such relative humidities including at levels which would be consistent with a monohydrate. Weightloss upon heating up to 181°C is limited to about 0.1% as seen by TGA in Figure 41 suggesting the salt is an anhydrate at ambient and thus converts to a hydrate upon exposure to suitable relative humidity by DVS. After the DVS experiment in Figure 42, the XRPD pattern seen in Figure 43 is different than before which is also consistent with a form change such as from an anhydrate to a hydrate. By solution1H-NMR in Figure 44, there is an extra proton at 5.3 ppm which is unaccounted for in the structure, but the spectrum is otherwise consistent with chemical structure. Thus, it is believed, without being bound by theory, that crystalline 25HC3S hydroxyethylpyrrolidinium may exist both as an anhydrate and as a hydrate, such as a monohydrate at a suitable relative humidity. A DSC of crystalline 25HC3S hydroxyethylpyrrolidinium can be found in Figure 41 showing a weak endotherm at about 40°C and another endotherm at about 140°C followed by what is presumably decomposition at about 181°C.

[0297] The x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylpyrrolidinium was successfully indexed, indicating the pattern represents a single crystalline phase with the results set forth in Figure 45. The indexing result reveals crystalline 25HC3S hydroxyethylpyrrolidinium to have a triclinic cell with a cell volume consistent with an anhydrous form and the salt stoichiometries provided. The formula volume was determined to be 3416.8 Å3 / cell (±5%) and can accommodate 1 mol / mol hydroxyethylpyrrolidinium. Several cell parameters are set forth in Table 10 below.

[0298] Table 10 – Indexing Summary for Crystalline 25HC3S Hydroxyethylpyrrolidinium

[0299] Substantially pure crystalline 25HC3S hydroxyethylpyrrolidinium is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S hydroxyethylpyrrolidinium. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S hydroxyethylpyrrolidinium present.

[0300] Processes of preparing 25HC3S hydroxyethylpyrrolidinium are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S hydroxyethylpyrrolidinium as set forth in Example 35.

[0301] The present disclosure also relates to pharmaceutical compositions containing 25HC3S hydroxyethylpyrrolidinium, including crystalline 25HC3S hydroxyethylpyrrolidinium, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S hydroxyethylpyrrolidinium, including crystalline 25HC3S hydroxyethylpyrrolidinium Such pharmaceutical compositions may be administered orally orconfigured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0302] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S hydroxyethylpyrrolidinium, including crystalline 25HC3S hydroxyethylpyrrolidinium and / or pharmaceutical compositions comprising crystalline 25HC3S hydroxyethylpyrrolidinium of the present disclosure.

[0303] An exemplary organic salt of the 25HC3S of the disclosure is the organic aliphatic amine diethylammonium salt of 25HC3S. A preparation of crystalline diethylammonium salt of 25HC3S is found in Example 36. An x-ray powder diffraction pattern of crystalline 25HC3S diethylammonium can be found in Figure 17. A peak-picked x-ray powder diffraction pattern can be seen in Figure 18. Table 11 shows picked peaks from Figure 18. Table 11 – Peaks of Crystalline 25HC3S Diethylammonium of Figure 18

[0304] Crystalline 25HC3S diethylammonium may be characterized by various analytical techniques, including by x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S diethylammonium or portions thereof, may be used to identify crystalline 25HC3S diethylammonium. Crystalline 25HC3S diethylammonium contains various x-ray powder diffraction peaks which alone or together may help identify the presence of crystalline 25HC3S diethylammonium.

[0305] In some cases, crystalline 25HC3S diethylammonium may be characterized by an x-ray powder diffraction pattern having one or more of the 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.

[0306] In some cases, 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 cases, 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.

[0307] In some cases, 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 cases, 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.

[0308] In some cases, 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 cases, 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.

[0309] In some cases, 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 cases, 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.

[0310] In some cases, 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, 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.

[0311] In some cases, crystalline 25HC3S diethylammonium may be characterized by an x-ray powder diffraction pattern having a peak at 15.4°2θ. In these and other cases, 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.

[0312] In some cases, crystalline 25HC3S diethylammonium may be characterized by an x-ray powder diffraction pattern having a peak at 17.2°2θ. In these and other cases, 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.

[0313] In some cases, crystalline 25HC3S diethylammonium can be characterized by an x-ray powder diffraction pattern substantially the same as that of Figure 18.

[0314] Crystalline 25HC3S diethylammonium picks up moisture in a DVS experiment as seen in Figure 60 showing it to be hygroscopic above 85% relative humidity. As seen by TGA in Figure59, weight loss upon heating up to 204°C is limited to about 0.1% suggesting an anhydrate structure at ambient. A DSC of crystalline 25HC3S diethylammonium can be found in Figure 59 showing an endotherm at about 134°C and one at about 215°C. The x-ray powder diffraction pattern before and after DVS is shown in Figure 61 and the1H-NMR spectrum of Figure 62 is consistent with chemical structure.

[0315] The x-ray powder diffraction pattern of crystalline 25HC3S diethylammonium was successfully indexed, indicating the pattern represents a single crystalline phase with the results set forth in Figure 63. The indexing result reveals crystalline 25HC3S diethylammonium to have an orthorhombic cell with a cell volume of 3293.3 Å3(±5%), consistent with an anhydrous form and the salt stoichiometry provided. Several cell parameters are set forth in Table 12 below. Table 12 – Indexing Summary for Crystalline 25HC3S Diethylammonium

[0316] Substantially pure crystalline 25HC3S diethylammonium is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S diethylammonium.Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S diethylammonium present.

[0317] Processes of preparing 25HC3S diethylammonium are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S diethylammonium as set forth in Example 36.

[0318] The present disclosure also relates to pharmaceutical compositions containing 25HC3S diethylammonium, including crystalline 25HC3S diethylammonium, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S diethylammonium, including crystalline 25HC3S diethylammonium. Such pharmaceutical compositions may be administered orally or configured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0319] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S diethylammonium, including crystalline 25HC3S diethylammonium and / or pharmaceutical compositions comprising crystalline 25HC3S diethylammonium of the present disclosure.

[0320] An exemplary organic salt of the 25HC3S of the disclosure is the organic amine diethanolamine salt of 25HC3S. A preparation of crystalline diethanolamine salt of 25HC3S is found in Example 37. An x-ray powder diffraction pattern of crystalline 25HC3S diethanolamine can be found in Figure 15. A peak-picked x-ray powder diffraction pattern can be seen in Figure 16. Table 13 shows picked peaks from Figure 16. Table 13 – Peaks of Crystalline 25HC3S Diethanolamine of Figure 16

[0321] Crystalline 25HC3S diethanolamine may be characterized by various analytical techniques, including by x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S diethanolamine or portions thereof, may be used to identify crystalline 25HC3Sdiethanolamine. Crystalline 25HC3S diethanolamine contains various x-ray powder diffraction peaks which alone or together may help identify the presence of crystalline 25HC3S diethanolamine.

[0322] In some cases, crystalline 25HC3S diethanolamine may be characterized by an x-ray powder diffraction pattern having one or more of the peaks in Figure 16. For example, a peak at about 3.8°2θ may 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.

[0323] Two of the most intense peaks in Figure 16 are the peaks at about 7.7˚2θ and about 8.1˚2θ. In Figure 16, these peaks appear very close together due to their respective intensities, but the XRPD instrumentation is able to distinguish them. However, it is possible that due to the variability associated with these XRPD peaks that when analyzed they may appear “underneath” each other as a single peak. Because such a single apparent peak is possible, in many cases herein, the crystalline 25HC3S diethanolamine is characterized, at least in part, by having an x-ray powder diffraction pattern comprising two peaks from about 7.7°2θ to about 8.1°2θ that are: (a) non-overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak.

[0324] In some cases, 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, 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.

[0325] In some cases, 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, 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.

[0326] In some cases, 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 cases, 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.

[0327] In some cases, 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 cases, 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.

[0328] In some cases, 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, 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.

[0329] In some cases, crystalline 25HC3S diethanolamine may be characterized by an x-ray powder diffraction pattern substantially the same as that of Figure 16.

[0330] Crystalline 25HC3S diethanolamine picks up moisture in a DVS experiment in Figure 55 shows hygroscopicity above about 75% relative humidity. Negligible weight loss was observed upon heating up to 175°C by TGA as seen in Figure 54 Suggesting an anhydrate structure at ambient. By solution1H-NMR in Figure 57, there is an extra proton at 5.3 ppm which is unaccounted for in the structure, but otherwise is consistent with chemical structure. A DSC of crystalline 25HC3S diethanolamine can be found in Figure 54 shows a single isotherm at about 181°C. The x-ray powder diffraction patterns both before and after DVS are shown in Figure 56.

[0331] The x-ray powder diffraction pattern of crystalline 25HC3S diethanolamine was successfully indexed, indicating the pattern represents a single crystalline phase with the results set forth in Figure 58. The indexing result reveals crystalline 25HC3S diethanolamine to have a monoclinic cell with a cell volume of 3294.1 Å3(±5%), consistent with an anhydrous form and the salt stoichiometries provided. Several cell parameters are set forth in Table 14 below.Table 14 – Indexing Summary for Crystalline 25HC3S Diethanolamine

[0332] Substantially pure crystalline 25HC3S diethanolamine is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S diethanolamine. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S diethanolamine present.

[0333] Processes of preparing 25HC3S diethanolamine are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S diethanolamine as set forth in Example 37.

[0334] The present disclosure also relates to pharmaceutical compositions containing 25HC3S diethanolamine, including crystalline 25HC3S diethanolamine, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S diethanolamine, including crystalline 25HC3S diethanolamine. Such pharmaceutical compositions may be administered orally or configured to be delivered as any effective conventional dosage forms including for example immediate sustained-release slow and timed-release oralpreparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0335] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S diethanolamine, including crystalline 25HC3S diethanolamine and / or pharmaceutical compositions comprising crystalline 25HC3S diethanolamine of the present disclosure.

[0336] An exemplary organic salt of the 25HC3S of the disclosure is the organic amine t- butylammonium salt of 25HC3S. A preparation of crystalline t-butylammonium salt of 25HC3S is found in Example 38. An x-ray powder diffraction pattern of crystalline 25HC3S t-butylammonium can be found in Figure 19. A peak-picked x-ray powder diffraction pattern can be seen in Figure 20. Table 15 shows picked peaks from Figure 20. Table 15 – Peaks of Crystalline 25HC3S t-Butylammonium of Figure 20 ˚2θ d space (Å) Intensity (%)

[0337] Crystalline 25HC3S t-butylammonium may be characterized by various analytical techniques, including by x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S t-butylammonium or portions thereof, may be used to identify crystalline 25HC3S t- butylammonium. Crystalline 25HC3S t-butylammonium contains various x-ray powder diffraction peaks which alone or together may help identify the presence of crystalline 25HC3S t-butylammonium.

[0338] In some cases, crystalline 25HC3S t-butylammonium may be characterized by an x-ray powder diffraction pattern having one or more of the 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.

[0339] In some cases, 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 cases, 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.

[0340] In some cases, 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 cases, 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.

[0341] In some cases, 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 cases, 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.

[0342] In some cases, 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 cases, 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.

[0343] In some cases, 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 cases, 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.

[0344] In some cases, 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 cases, 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.

[0345] 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 instance, the crystalline 25HC3S t-butylammonium may be further characterized by a peak at about 10.3°2θ. Alternatively, or additionally, the crystalline 25HC3S t-butylammonium may be further characterized by a peak at about 10.5°2θ. Alternatively, or additionally, the crystalline 25HC3S t-butylammonium may be further characterized by a peak at about 13.8°2θ. Alternatively, or additionally, the 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.

[0346] In some cases, crystalline 25HC3S t-butylammonium may be characterized by an x-ray powder diffraction pattern substantially the same as that of Figure 20.

[0347] Crystalline 25HC3S t-butylammonium has a low hygroscopicity, from 5% to 95% relative humidity as seen by DVS in Figure 65. A weight loss of 0.2% was measured by heating up to 200°C by TGA in Figure 64 suggesting an anhydrate structure at ambient. A DSC of crystalline25HC3S t-butylammonium can be found in Figure 64 shows isotherms near 205°C and 218°C. The1H-NMR spectrum of Figure 66 is consistent with chemical structure.

[0348] The x-ray powder diffraction pattern of crystalline 25HC3S t-butylammonium was successfully indexed, indicating the pattern represents a single crystalline phase with the results set forth in Figure 67. The indexing result reveals crystalline 25HC3S t-butylammonium to have a monoclinic cell with a cell volume of 3333.3 Å3(±5%), consistent with an anhydrous form and the salt stoichiometries provided. Several cell parameters are set forth in Table 16 below. Table 16 – Indexing Summary for Crystalline 25HC3S t-Butylammonium

[0349] Substantially pure crystalline 25HC3S t-butylammonium is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S t-butylammonium. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S t-butylammonium present.

[0350] Processes of preparing 25HC3S t-butylammonium are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, atriethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S t-butylammonium as set forth in Example 38.

[0351] The present disclosure also relates to pharmaceutical compositions containing 25HC3S t-butylammonium, including crystalline 25HC3S t-butylammonium, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S t-butylammonium, including crystalline 25HC3S t-butylammonium. Such pharmaceutical compositions may be administered orally or configured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0352] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S t-butylammonium, including crystalline 25HC3S t-butylammonium and / or pharmaceutical compositions comprising crystalline 25HC3S t-butylammonium of the present disclosure.

[0353] An exemplary organic salt of the 25HC3S of the disclosure is the organic amine benzathine salt of 25HC3S. A preparation of crystalline benzathine salt of 25HC3S is found in Example 39. An x-ray powder diffraction pattern of crystalline 25HC3S benzathine can be found in Figure 21. A peak-picked x-ray powder diffraction pattern can be seen in Figure 22. Table 17 shows picked peaks from Figure 22. The benzathine salt of 25HC3S contains 2 ions of 25HC3S for every ion of benzathine, as confirmed by ICP-OES. Table 17 – Peaks of Crystalline 25HC3S Benzathine of Figure 22 ˚2θ d (Å) I t it (%)

[0354] Crystalline 25HC3S benzathine may be characterized by various analytical techniques, including by x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S benzathine or portions thereof, may be used to identify crystalline 25HC3S benzathine. Crystalline 25HC3S benzathine contains various x-ray powder diffraction peaks which alone or together may help identify the presence of crystalline 25HC3S benzathine.

[0355] In some cases, crystalline 25HC3S benzathine may be characterized by an x-ray powder diffraction pattern having one or more of the peaks in Figure 22. For example, a peak at about 4.1°2θ may 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.

[0356] 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.

[0357] 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.

[0358] 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.

[0359] 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.

[0360] 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 instance, the crystalline 25HC3S benzathine may be further characterized by a peak at about 7.3°2θ. Alternatively, or additionally, the crystalline 25HC3S benzathine may be further characterized by a peak at about 15.1°2θ. Alternatively, or additionally, the crystalline 25HC3S benzathine may be further characterized by a peak at about 16.4°2θ. Alternatively, or additionally, the crystalline 25HC3S benzathine may be further characterized by a peak at about 17.8°2θ. Alternatively, or additionally, the crystalline 25HC3S benzathine may be further characterized by a peak at about 20.4°2θ.

[0361] In some cases, crystalline 25HC3S benzathine has an x-ray powder diffraction pattern substantially the same as that of Figure 22.

[0362] Crystalline 25HC3S benzathine has a low hygroscopicity, from 5% to 95% relative humidity. A weight loss of 0.4% was measured by heating up to 200°C as seen by TGA as see in Figure 68 suggesting that crystalline 25HC3S benzathine is an anhydrate at ambient. A DSC of crystalline 25HC3S benzathine can be found in Figure 68 shows isotherms near 216°C and 223°C. The DVS isotherm of Figure 69 indicates low hygroscopicity. The x-ray powder diffraction patterns before and after DVS are shown in Figure 70. The1H-NMR spectrum of Figure 71 is consistent with chemical structure.

[0363] The x-ray powder diffraction pattern of crystalline 25HC3S benzathine was successfully indexed, indicating the pattern represents a single crystalline phase with the results set forth in Figure 72. The indexing result reveals crystalline 25HC3S benzathine to have a monoclinic cell with a cellvolume of 3394.0 Å3(±5%), consistent with an anhydrous form and the salt stoichiometries provided. Several cell parameters are set forth in Table 18 below. Table 18 – Indexing Summary for Crystalline 25HC3S Benzathine

[0364] Substantially pure crystalline 25HC3S benzathine is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S benzathine. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S benzathine present.

[0365] Processes of preparing 25HC3S benzathine are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S benzathine as set forth in Example 39.

[0366] The present disclosure also relates to pharmaceutical compositions containing 25HC3S benzathine, including crystalline 25HC3S benzathine, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S benzathine including crystalline 25HC3S benzathine Such pharmaceutical compositions may beadministered orally or configured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0367] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S benzathine, including crystalline 25HC3S benzathine and / or pharmaceutical compositions comprising crystalline 25HC3S benzathine of the present disclosure.

[0368] An exemplary organic salt of the 25HC3S of the disclosure is the organic amine choline salt of 25HC3S. A preparation of crystalline choline salt of 25HC3S is found in Example 40. An x- ray powder diffraction pattern of crystalline 25HC3S choline can be found in Figure 76. A peak-picked x-ray powder diffraction pattern can be seen in Figure 77. Table 19 shows picked peaks from Figure 77. Table 19 – Peaks of Crystalline 25HC3S Choline of Figure 77 ˚2θ d (Å) I i (%)

[0369] Crystalline 25HC3S choline may be characterized by various analytical techniques, including by x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S choline or portions thereof, may be used to identify crystalline 25HC3S choline. Crystalline 25HC3S choline contains various x-ray powder diffraction peaks which alone or together may help identify the presence of crystalline 25HC3S choline.

[0370] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 3.9°2θ. In these and other cases, crystalline 25HC3S choline may 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.

[0371] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 7.8°2θ. In these and other cases, crystalline 25HC3S choline may 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.

[0372] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 9.5°2θ. In these and other cases, crystalline 25HC3S choline may 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.

[0373] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 10.1°2θ. In these and other cases, crystalline 25HC3S choline may 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.

[0374] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 11.0°2θ. In these and other cases, crystalline 25HC3S choline may 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.

[0375] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 12.2°2θ. In these and other cases, crystalline 25HC3S choline may 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.

[0376] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 13.7°2θ. In these and other cases, crystalline 25HC3S choline may 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.

[0377] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 14.7°2θ. In these and other cases, crystalline 25HC3S cholinemay 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.

[0378] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 15.1°2θ. In these and other cases, crystalline 25HC3S choline may 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.

[0379] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 15.8°2θ. In these and other cases, crystalline 25HC3S choline may 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.

[0380] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 16.3°2θ. In these and other cases, crystalline 25HC3S choline may 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.

[0381] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 19.1°2θ. In these and other cases, crystalline 25HC3S choline may 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.

[0382] In some cases, crystalline 25HC3S choline may 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 cases, crystalline 25HC3S choline may 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.

[0383] In some cases, crystalline 25HC3S choline may 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 cases, crystalline 25HC3S choline may be further characterized by an x-ray powder diffraction patternhaving 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.

[0384] In some cases, crystalline 25HC3S choline may 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 cases, crystalline 25HC3S choline may 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.

[0385] In some cases, crystalline 25HC3S choline may 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 cases, crystalline 25HC3S choline may 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.

[0386] In some cases, crystalline 25HC3S choline may 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 cases, crystalline 25HC3S choline may 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.

[0387] In some cases, crystalline 25HC3S choline may 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 cases, crystalline 25HC3S choline may 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.

[0388] In some cases, crystalline 25HC3S choline may 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 cases, crystalline 25HC3S choline may 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.

[0389] In some cases, crystalline 25HC3S choline may 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, crystalline 25HC3S choline may 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.

[0390] In some cases, crystalline 25HC3S choline may 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, crystalline 25HC3S choline may 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.

[0391] In some cases, crystalline 25HC3S choline may 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 cases, crystalline 25HC3S choline may be further characterized by an x- ray powder diffraction pattern having a peak at about 19.1°2θ.

[0392] In some cases, crystalline 25HC3S choline may 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.

[0393] In some cases, crystalline 25HC3S choline may 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.

[0394] In some cases, crystalline 25HC3S choline may 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.

[0395] In some cases, crystalline 25HC3S choline may 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.

[0396] In some cases, crystalline 25HC3S choline may 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.

[0397] In some cases, crystalline 25HC3S choline may 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.

[0398] In some cases, crystalline 25HC3S choline may 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.

[0399] In some cases, crystalline 25HC3S choline may 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.

[0400] In some cases, crystalline 25HC3S choline may 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.

[0401] In some cases, crystalline 25HC3S choline may 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.

[0402] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having one or more peaks at 16.3°2θ and about 19.1°2θ.

[0403] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having a peak at about 19.1°2θ.

[0404] In some cases, crystalline 25HC3S choline may be characterized by an x-ray powder diffraction pattern having substantially the same pattern as that found in Figure 77.

[0405] The x-ray powder diffraction pattern of crystalline 25HC3S choline was successfully indexed, indicating the pattern represents a single crystalline phase with the results set forth in Figure 79. The indexing result reveals crystalline 25HC3S choline to have an orthorhombic cell with a cell volume of 3371.5 Å3(±5%), consistent with an anhydrous form. Several cell parameters are set forth in Table 20 belowTable 20 – Indexing Summary for Crystalline 25HC3S Choline

[0406] A DSC thermogram of crystalline 25HC3S choline in Figure 80 indicated endothermic peaks at about 198°C and about 220°C. The TGA thermogram of Figure 80 indicates that there is negligible weight loss up to 198°C.

[0407] Without being bound by theory, it is believed that crystalline 25HC3S choline is an anhydrate, meaning that there is no water of crystallization in the unit cell. This does not preclude the possibility of other water being present in a solid comprising crystalline 25HC3S choline. In addition, crystalline 25HC3S choline is not appreciably hygroscopic up to about 95% relative humidity, increasing in weight by only about 0.5% up to this relative humidity as evidenced by a dynamic vapor sorption experiment according to Example 26 and whose results are shown in Figure 82. Further, the x-ray powder diffraction pattern of crystalline 25HC3S choline does not appreciably change after DVS as shown in Figure 78. Only a 0.5% weight gain was observed on going from 5% to 95% relative humidity and a 0.5% weight loss was observed on the return to 5% relative humidity indicating no hysteresis. Such low hygroscopicity indicates good stability under such stresses, which, as further discussed elsewhere herein, may make it suitably stable for pharmaceutical processing. Indeed, the present disclosure thus further includes stable crystalline 25HC3S choline. Such stability includes, for example sufficiently stable crystalline 25HC3S choline to be formulated for patient delivery The1H-NMR spectrum is, other than a peak at 5.3ppm, consistent with structure as seen in Figure 81. There is no evidence of residual solvent. An overlap of a methylene from chlorine at about 3.8 ppm appears to be 1 mol / mol choline.

[0408] The choline salt has the additional advantage over the sodium salt and some other salts of 25HC3S in that the choline counterion has additional beneficial properties. For example, choline is an essential nutrient and lack of choline has been indicated as a cause of fat and cholesterol build up in the liver. Further, 25HC3S choline forms crystals of better quality and diffraction than those of the prior art. Lastly, crystalline 25HC3S choline is less hygroscopic, and thus more physically stable than, for example, crystalline 25HC3S sodium. Crystalline 25HC3S sodium stabilizes as a hydrate when exposed to humid conditions. In particular, monohydrates, dihydrates, and variable hydrates of crystalline 25HC3S sodium have been prepared. Form I, a hydrate, has been found to be hygroscopic and may form a liquid crystal at high water activities (e.g., above 0.73). Another hydrate, Form II, is stable at relative humidities between about 21% and about 30%. By comparison, under conditions going up to about 95% relative humidity, only about 0.5% water by weight is absorbed indicating crystalline 25HC3S choline is stable as an anhydrate.

[0409] Substantially pure crystalline 25HC3S choline is further disclosed. “Substantially pure,” as described herein, generally refers to a form herein that is present without any appreciable amounts, other than potentially trace levels, of other forms of 25HC3S choline. Examples of trace levels include not more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less in total relative to the total amount (based on weight) of 25HC3S choline present.

[0410] Processes of preparing 25HC3S choline are further described herein. In some cases, one may first prepare a sodium salt of 25HC3S. Examples of such preparation are set forth herein. The sodium salt of 25HC3S, which may be crystalline, may be converted into, for example, a triethylammonium salt as described in Example 41. The triethylammonium salt may then be used to create 25HC3S choline as set forth in Example 40.

[0411] The present disclosure also relates to pharmaceutical compositions containing 25HC3S choline, including crystalline 25HC3S choline, as disclosed herein. Such pharmaceutical compositions are comprised of one or more pharmaceutically acceptable excipients and 25HC3S choline, including crystalline 25HC3S choline. Such pharmaceutical compositions may be administered orally or configured to be delivered as any effective conventional dosage forms, including, for example, immediate, sustained-release, slow and timed-release oral preparations, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0412] As discussed elsewhere herein, and demonstrated in the Examples, the 25HC3S choline of the disclosure has surprisingly low hygroscopicity, including in comparison to other salt forms of 25HC3S. The 25HC3S choline can therefore advantageously manufactured and may be utilized in the preparation of pharmaceutical formulations, and particularly in the preparation of dosage forms for oral administration (e.g., solid dosage forms, such as tablets, capsules (each of which includes immediate release, sustained release or timed release formulations), pills, powders, or granules.

[0413] Still further, the 25HC3S choline of the disclosure also advantageously provides supplementary choline to patients suffering from the conditions targeted by the 25HC3S. As discussed elsewhere herein, choline deficiency can contribute to these conditions and it may be advantageous to provide choline alongside the 25HC3S in the course of therapy. Hence, the 25HC3S choline surprisingly and beneficially combines advantageous salt form properties, contributing for instance to the preparation of oral dosage forms particularly well suited for treating certain conditions, along with intrinsic ability beneficially to provide choline supplementation in course of conducting methods of treatment using the said oral dosage forms.

[0414] The present disclosure further includes methods and uses for treating and / or preventing diseases (e.g., in humans) such as one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation with effective amounts 25HC3S choline, including crystalline 25HC3S choline and / or pharmaceutical compositions comprising crystalline 25HC3S choline of the present disclosure.

[0415] An exemplary organic salt of the 25HC3S of the disclosure is the organic amine triethylammonium salt of 25HC3S. A preparation of triethylammonium salt of 25HC3S is found in Example 41.

[0416] The preparation of the triethylammonium salt of 25HC3S may be accomplished, for example, by passing a mixture of triethylammonium chloride and triethylamine through a column and treating with a solvent such as an alcohol until neutral pH. Separately, crystalline 25HC3S sodium may be dissolved in a solvent such as an alcohol. The solution may then be passed through the same column previously exposed to triethylamine and combined with the triethylammonium solution. Isolating resulting solids such as under vacuum or by drying may then provide crystalline 25HC3Striethylammonium salt which may be homogenized, for example, with a mortar and pestle. A suitable alcohol for this process includes methanol.

[0417] 25HC3S choline, including crystalline 25HC3S choline, may be prepared by starting with 25HC3S sodium, converting to a second salt of 25HC3S such as the triethylammonium salt, and then converting that second salt of 25HC3S to 25HC3S choline, including crystalline 25HC3S choline. The preparation of crystalline 25HC3S choline may be accomplished by preparing a suspension of a triethylammonium salt of 25HC3S in a suitable solvent such as acetonitrile and treating with a choline source such as aqueous choline hydroxide to form 25HC3S choline including crystalline 25HC3S choline. The 25HC3S choline may be purified such as by rinsing with a suitable solvent. Additional processing such as drying under vacuum or otherwise may also be performed. The disclosure further includes crystalline 25HC3S choline made by the processes described herein.

[0418] Methods for preparing 25-hydroxy-cholesten-5-en-3-sulfate (25HC3S)

[0419] Methods for preparing 25-hydroxy-cholesten-5-en-3-sulfate, such as 25-hydroxy-3β- cholesten-5-en-3-sulfate (25HC3S) are described herein. Although many of the teachings herein involve a sulfate in the 3β position, the teachings of the present disclosure are also generally applicable to a sulfate in the 3α position. The components used in each step of the subject methods for preparing 25-hydroxy-3β-cholesten-5-en-3-sulfate described herein may be a purified composition or a crude composition as desired. The term “purified” is used in its conventional sense to refer to a composition where at least some isolation or purification process has been conducted, such as for example, filtration or aqueous workup of a reaction mixture. In certain instances, purification includes at least one of liquid chromatography, recrystallization, distillation (e.g., azeotropic distillation) and other type of compound purification. For example, compounds as 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. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. Mobile phases may be chosen from polar solvents and non-polar solvents. In some cases, the mobile phase includes a polar solvent. In some cases, the polar solvent is chosen from chloroform, dichloromethane, tetrahydrofuran, dichloroethane, acetone, dioxane, ethyl acetate, dimethylsulfoxide, aniline, diethylamine, nitromethane, acetonitrile, pyridine, isopropanol, ethanol, methanol, ethylene glycol, acetic acid and water. In some cases, the mobile phase includes a non-polar solvent. In some cases, the non-polar solvent is chosen from diethyl ether, toluene, benzene, pentane, hexanes, cyclohexane, petroleum ether and carbon tetrachloride. See, e.g., Introduction to ModernLiquid Chromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed. E. Stahl, Springer-Verlag, New York, 1969.

[0420] In some cases, a reaction mixture is used in a subsequent step in the methods described herein as a crude mixture where no purification or other workup of the reaction mixture has been conducted. In certain instances, the crude mixture includes the compound of interest in sufficient purity such as where the reaction mixture includes the compound of interest in a purity of 70% or greater, such as 75% or greater, such as 80% or greater, such as 85% or greater, such as 90% or greater, such as 95% or greater, such as 97% or greater, such as 99% or greater, such as 99.5% or greater, such as 99.9% or greater, such as 99.99% or greater and including 99.999% or greater, relative to the crude reaction mixture (apart from solvent when present), as determined by chromatography (e.g., HPLC or SFC), nuclear magnetic resonance spectroscopy (e.g.,1H NMR or13C NMR) or a combination thereof. In some cases, the compound of interest is present in the reaction mixture in an amount that is 30% by weight or greater relative to the crude reaction mixture (apart from solvent when present), such as 40% by weight or greater, such as 50% by weight or greater, such as 60% by weight or greater, such as 70% by weight or greater, such as 75% by weight or greater, such as by 80% by weight or greater, such as 85% by weight or greater, such as 90% by weight or greater, such as 95% by weight or greater, such as 97% by weight or greater, such as 99% by weight or greater, such as 99.5% by weight or greater, such as 99.9% by weight or greater, such as 99.99% by weight or greater and including 99.999% by weight or greater relative to the crude reaction mixture, and may range from 5% by weight to 99.999% by weight, such as 30% by weight to 99.99% by weight, 40% by weight to 99.9% by weight, 50% by weight to 99% by weight, 70% by weight to 95% by weight, 75% by weight to 90% by weight, 80% by weight to 99% by weight, or 80% by weight to 95% by weight. In some cases, the compound of interest is present at 30 mol% or greater in the crude reaction mixture (apart from solvent when present), such as 40 mol% or greater, such as 50 mol% or greater, such as 60 mol% or greater, such as 70 mol% or greater, such as 75 mol% or greater, such as by 80 mol% or greater, such as 85 mol% or greater, such as 90 mol% or greater, such as 95 mol% or greater, such as 97 mol% or greater, such as 99 mol% or greater, such as 99.5 mol% or greater, such as 99.9 mol% or greater, such as 99.99 mol% or greater and including 99.999 mol% or greater relative to the crude reaction mixture, and may range from 30 mol% to 99.999 mol%, such as 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%.

[0421] Methods for preparing a metal salt of 25-hydroxy-3β-cholesten-5-en-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]phenanthren-3-yl] sulfate metal salt) according to the present disclosure include contacting 25-hydroxy-(3β)-cholest-5-en-3-ol with a sulfating agent to produce a 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt; and contacting the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt with at least one metal salt to produce the 5-cholesten-3β,25-diol 3-sulfate metal salt can be achieved such as in (Scheme Ia).

[0422] The 25-hydroxy-(3β)-cholest-5-en-3-ol may be sulfated by contacting with a sulfating agent (Scheme IA1). In some cases, the sulfating reagent is chosen from sulfur trioxide complexes, sulfuric acid compounds, sulfonic acid compounds, and sulfonate compounds. In some cases, the sulfating reagent is chosen from sulfur trioxide dimethyl formamide, sulfur trioxide triethylamine, and sulfur trioxide trimethylamine. In some cases, the sulfating reagent includes from sulfuric acid and acetic anhydride and pyridine. In some cases, the sulfating reagent includes sulfur trioxide triethylamine and pyridine. In some cases, the sulfating reagent is chosen from 1) chlorosulfonic acidand pyridine and 2) chlorosulfonic acid and 2,6-lutidine. In some cases, the sulfating reagent is ethyl chlorosulfonate.

[0423] The 25-hydroxy-(3β)-cholest-5-en-3-ol may be sulfated at a temperature that ranges from -10 °C to 50 °C, such as from -5 °C to 45 °C, such as from -4 °C to 40 °C, such as from -3 °C to 35 °C, such as from -2 °C to 30 °C, such as from -1 °C to 25 °C, and including from 0 °C to 20 °C. The reaction may be carried out for a duration that ranges from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, such as from 0.7 hours to 18 hours, such as from 0.8 hours to 17 hours, such as from 0.9 hours to 16 hours and including from 1 hour to 15 hours. The amount of sulfating agent used relative to the 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, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and including 10 equivalents or more, and may range from 0.001 equivalents to 10 equivalents, such as 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 8 equivalents, 0.9 equivalents to 5 equivalents, 1.3 equivalents to 10 equivalents, 1.3 equivalents to 8 equivalents, 1.3 equivalents to 5 equivalents, 1.5 equivalents to 10 equivalents, 1.5 equivalents to 8 equivalents, 1.5 equivalents to 5 equivalents, 2 equivalents to 10 equivalents, 2 equivalents to 8 equivalents, 2 equivalents to 5 equivalents, or 1 equivalent to 2 equivalents, 1 equivalents to 1.5 equivalents, or 1.1 to 1.2 equivalents, relative to the 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0424] In some cases, methods include sulfating the 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent where the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product exhibits low solubility. In some cases, the 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in at least one solvent where the 25- hydroxy-(3β)-cholest-5-en-3-sulfate product exhibits a solubility of 100 mmol / L or less, such as 90 mmol / L or less, such as 80 mmol / L or less, such as 70 mmol / L or less, such as 60 mmol / L or less, suchas 50 mmol / L or less, such as 40 mmol / L or less, such as 30 mmol / L or less, such as 20 mmol / L or less, such as 10 mmol / L or less, and including sulfating the 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent where the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product exhibits a solubility of 5 mmol / L or less. In some cases, the 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in at least one solvent where 25-hydroxy-(3β)-cholest-5-en-3-sulfate product precipitates after formation. In some cases, the at least one solvent is chosen from chloroform, methylene chloride, acetone, acetonitrile, toluene, tetrahydrofuran, and methyltetrahydrofuran.

[0425] In some cases, methods include sulfating the 25-hydroxy-(3β)-cholest-5-en-3-ol in a manner sufficient to reduce or eliminate bis-sulfation of the 25-hydroxy-(3β)-cholest-5-en-3-ol. In some instances, the 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 of the reaction product formed by contacting the 25-hydroxy-(3β)-cholest-5-en-3-ol with the sulfating agent, such as 9% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 5% by weight or less, such as 4% by weight or less, such as 3% by weight or less, such as 2% by weight or less, such as 1% by weight or less, such as 0.5% by weight or less, such as 0.1% by weight or less, such as 0.01% by weight or less, such as 0.001% by weight or less, and including where the 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated and the bis-sulfate product is formed in an amount that is 0.0001% by weight or less, and may range from 10% by weight to 0.001% by weight, such as 10% by weight to 0.1% by weight, 10% by weight to 1% by weight , 10% by weight to 2% by weight, 8% by weight to 0.001% by weight, 8% by weight to 0.1% by weight, 8% by weight to 1% by weight, 8% by weight to 2% by weight, 6% by weight to 0.001% by weight, 6% by weight to 0.1% by weight, 6% by weight to 1% by weight, 6% by weight to 2% by weight, 4% by weight to 0.001% by weight, 4% by weight to 0.1% by weight, 4% by weight to 1% by weight, 4% by weight to 2% by weight, 3% by weight to 0.001% by weight, 3% by weight to 0.1% by weight, 3% by weight to 1% by weight, 2% by weight to 0.001% by weight, 2% by weight to 0.1% by weight, or 2% by weight to 1% by weight.

[0426] In some cases, the ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate to the 5-cholesten-3β-25-diol-disulfate formed is 10:1 or more, such as 25:1 or more, such as 50:1 or more, such as 100:1 or more, such as such as 250:1 or more, such as 500:1 or more, such as 1000:1 or more, such as 2500:1 or more, such as 5000:1 or more, such as 10,000:1 or more, such as 25,000:1 or more, such as 50,000:1 or more, such as 100,000:1 or more, such as 106:1 or more, such as 107:1 or more, such as 108:1 or more, and including where the ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate to the 5-cholesten-3β-25-diol-disulfate formed is 109:1 or more, and may range from a ratio by weight of 10:1 to a ratio by weight of 109:1, such as a ratio of weight of 10:1 to a ratio of weight of 106:1, a ratio of weight of 10:1 to a ratio of weight of 103:1, a ratio of weight of 10:1 to a ratio of weight of 100:1, a ratio of weight of 100:1 to a ratio of weight of 109:1, a ratio of weight of 100:1 to a ratio of weight of 106:1, a ratio of weight of 100:1 to a ratio of weight of 103:1, a ratio of weight of 250:1 to a ratio of weight of 109:1, a ratio of weight of 250:1 to a ratio of weight of 106:1, a ratio of weight of 250:1 to a ratio of weight of 103:1, a ratio of weight of 500:1 to a ratio of weight of 109:1, a ratio of weight of 500:1 to a ratio of weight of 106:1, a ratio of weight of 500:1 to a ratio of weight of 103:1, a ratio of weight of 103:1 to a ratio of weight of 109:1, a ratio of weight of 103:1 to a ratio of weight of 106:1, or a ratio of weight of 250:1 to a ratio of weight of 103:1.

[0427] In some cases, the 5-cholesten-3β-25-diol-disulfate formed when sulfating 25-hydroxy- (3β)-cholest-5-en-3-ol remains solubilized in the at least one solvent. In some cases, the 5-cholesten- 3β-25-diol-disulfate has high solubility in the at least one solvent. In some instances, the 5-cholesten- 3β-25-diol-disulfate exhibits a solubility of 500 mmol / L or more in the at least one solvent, such as 600 mmol / L or more, such as 700 mmol / L or more, such as 800 mmol / L or more, such as 900 mmol / L, or more and including a solubility of 1 mol / L or more in the at least one solvent.

[0428] In certain cases, methods further include separating the 25-hydroxy-(3β)-cholest-5-en- 3-sulfate product from the bis-sulfate product (i.e., 5-cholesten-3β-25-diol-disulfate). In some cases, the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product is separated from the bis-sulfate product by vacuum filtration. In some cases, the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product is separated from the bis-sulfate product by recrystallization of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product. In some cases, the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product is separated from the bis-sulfate product by chromatography (e.g., silica column).

[0429] In some cases, the 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in a reaction mixture having a pH that ranges from 5.0 to 8.0, such as a pH from 5.1 to 7.9, such as a pH from 5.2 to 7.8, such as a pH from 5.3 to 7.7, such as a pH from 5.4 to 7.6, such as a pH from 5.5 to 7.5, such as a pH from 5.6 to 7.4, such as a pH from 5.7 to 7.3, such as a pH from 5.8 to 7.2, such as a pH from 5.9 to 7.1, and including sulfating the 25-hydroxy-(3β)-cholest-5-en-3-ol in a reaction mixture having a pH of from 6.0 to 7.0.

[0430] In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in the presence of a 25- hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt. In certain cases, the 25-hydroxy-(3β)- cholest-5-en-3-sulfate organic cationic salt is present as particles (e.g., seed crystals of 25-hydroxy- (3β)-cholest-5-en-3-sulfate organic cationic salt produced in a previous reaction or purified reaction batch). In some cases, sulfating 25-hydroxy-(3β)-cholest-5-en-3-ol in the presence of 25-hydroxy- (3β)-cholest-5-en-3-sulfate organic cationic salt (e.g., as particles) is sufficient to reduce the solubility of 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt produced by reaction of the sulfating agent with 25-hydroxy-(3β)-cholest-5-en-3-ol as compared to the solubility when the 25-hydroxy-(3β)- cholest-5-en-3-sulfate organic cationic salt is not present. In certain cases, the solubility of 25-hydroxy- (3β)-cholest-5-en-3-sulfate organic cationic salt produced in the reaction mixture is reduced as compared to the solubility when the added 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt is not present by 5% or more, such as by 10% or more, such as by 25% or more, such as by 50% or more, such as by 75% or more, such as by 90% or more and including by reducing the solubility of the produced 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt by 99% or more. The size of the particles of 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt added to the reaction mixture may vary and may have a dimension (e.g., length, width or diameter) of 0.01 mm or more, such as 0.025 mm or more, such as 0.05 mm or more, such as 0.075 mm or more, such as 0.1 mm or more, such as 0.25 mm or more, such as 0.5 mm or more, such as 0.75 mm or more, such as 1 mm or more, such as 2 mm or more, such as 3 mm or more, such as 4 mm or more and including 5 mm or more. In some cases, the particles of 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt are added to the reaction mixture immediately after contacting the sulfating agent with the 25-hydroxy- (3β)-cholest-5-en-3-ol. In some cases, the particles of 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt are added to the reaction mixture 1 minute or more after contacting the sulfating agent with the 25-hydroxy-(3β)-cholest-5-en-3-ol, such as 5 minutes or more, such as 10 minutes or more, such as 15 minutes or more, such as 20 minutes or more, such as 30 minutes or more, such as 40 minutes or more, such as 50 minutes or more and including adding the particles of 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt to the reaction mixture 60 minutes or more after contacting the sulfating agent with the 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0431] In certain cases, the sulfating agent is characterized prior to contacting with the 25- hydroxy-(3β)-cholest-5-en-3-ol. In some cases, characterizing the sulfating agent includes determining the extent of degradation of the sulfating agent prior to contacting with the 25-hydroxy-(3β)-cholest- 5-en-3-ol. In certain cases, determining the extent of degradation of the sulfating reagent includes determining the amount of impurity in the sulfating reagent prior to contacting with the 25-hydroxy- (3β)-cholest-5-en-3-ol.

[0432] In some instances, the degradation of the sulfating agent is determined by proton nuclear magnetic resonance spectroscopy (1H-NMR). Proton NMR spectroscopy of the sulfating agent may be conducted in at least one deuterated solvent. In certain cases, the at least one deuterated solvent is deuterated acetone ((CD3)2CO). In certain cases, the at least one deuterated solvent is not deuterated benzene (C6D6). In certain cases, the at least one deuterated solvent is not deuterated acetonitrile (CD3CN). In certain cases, the at least one deuterated solvent is not deuterated chloroform (CD3Cl).

[0433] In some instances, methods for determining the extent of degradation include integrating one or more peaks in the1H-NMR spectrum at a chemical shift of from 9.2 ppm to 9.3 ppm and calculating the impurity level of the sulfating agent based on the integrated peaks. In certain instances, methods for determining the extent of degradation include integrating one or more peaks in the1H-NMR spectrum at a chemical shift of about 9.25 ppm and calculating the impurity level of the sulfating agent based on the integrated peaks. In some cases, the sulfating agent is contacted with the 25-hydroxy-(3β)-cholest-5-en-3-ol when the impurity level of the sulfating agent is below a predetermined threshold, such as where the impurity level is 25% or less as determined by integrating one or more peaks in the proton NMR spectrum at a chemical shift of from 9.2 ppm to 9.3 ppm, such as 24% or less, such as 23% or less, such as 22% or less, such as 21% or less, such as 20% or less, such as 19% or less, such as 18% or less, such as 17% or less, such as 16% or less, such as 15% or less, such as 14% or less such as 13% or less, such as 12% or less, such as 11% or less, such as 10% or less, such as 9% or less, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 4% or less, such as 3% or less such as 2% or less and including where the impurity level is 1% or less as determined by integrating one or more peaks in the proton NMR spectrum at a chemical shift of from 9.2 ppm to 9.3 ppm. In some cases, the sulfating agent is not contacted with the 25-hydroxy- (3β)-cholest-5-en-3-ol when the impurity level is above a predetermined threshold, such as where the impurity level is 25% or more as determined by integrating one or more peaks in the proton NMRspectrum at a chemical shift of from 9.2 ppm to 9.3 ppm, such as 26% or more, such as 27% or more, such as 28% or more, such as 29% or more, such as 30% or more, such as 31% or more, such as 32% or more, such as 33% or more, such as 34% or more and including where the impurity level is 35% or more as determined by integrating one or more peaks in the proton NMR spectrum at a chemical shift of from 9.2 ppm to 9.3 ppm.

[0434] In certain cases, the generated 25-hydroxy-(3β)-cholest-5-en-3-sulfate product includes one or more byproducts. In some cases, the byproduct is 5-cholesten-3β-25-diol-disulfate. In some cases, 5-cholesten-3β-25-diol-disulfate byproduct is present in the composition produced by sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol in an amount relative to the 25-hydroxy-(3β)-cholest-5-en-3- sulfate of 10% by weight or less, such as 9% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 5% by weight or less, such as 4% by weight or less, such as 3% by weight or less, such as 2% by weight or less, such as 1% by weight or less, such as 0.5% by weight or less, such as 0.1% by weight or less, such as 0.01% by weight or less, such as 0.001% by weight or less, and including where 5-cholesten-3β-25-diol-disulfate byproduct is present in the composition produced by sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol in an amount of 0.001% by weight or less, and may range from 0.1% by weight to 50% by weight, such as 0.5% by weight to 20% by weight or 1% by weight to 12% by weight. In some cases, the ratio by weight of the 25- hydroxy-(3β)-cholest-5-en-3-sulfate to the 5-cholesten-3β-25-diol-disulfate byproduct formed is 10:1 or more, such as 25:1 or more, such as 50:1 or more, such as 100:1 or more, such as such as 250:1 or more, such as 500:1 or more, such as 1000:1 or more, such as 2500:1 or more, such as 5000:1 or more, such as 10,000:1 or more, such as 25,000:1 or more, such as 50,000:1 or more, such as 100,000:1 or more, such as 106:1 or more, such as 107:1 or more, such as 108:1 or more, and including where the ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate to the 5-cholesten-3β-25-diol-disulfate formed is 109:1 or more. In some cases, the ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3- sulfate and the 5-cholesten-3β-25-diol-disulfate formed ranges from 10:1 to 109:1, such as from 100:1 to 108:1, such as from 1000:1 to 107:1, and including from 10000:1 to 106:1.

[0435] Aspects of the present disclosure also include compositions having 25-hydroxy-(3β)- cholest-5-en-3-sulfate and 5-cholesten-3β-25-diol-disulfate that is present in the composition in an amount relative to the 25-hydroxy-(3β)-cholest-5-en-3-sulfate of 10% by weight or less, such as 9% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 5% by weight or less, such as 4% by weight or less, such as 3% by weight or less, such as 2% by weight or less, such as 1% by weight or less, such as 0.5% by weight or less, such as 0.1%by weight or less, such as 0.01% by weight or less, such as 0.001% by weight or less, and including 0.001% by weight or less, and may range from 10% by weight to 0.001% by weight, such as 10% by weight to 0.1% by weight, 10% by weight to 1% by weight, 10% by weight to 2% by weight, 8% by weight to 0.001% by weight, 8% by weight to 0.1% by weight, 8% by weight to 1% by weight, 8% by weight to 2% by weight, 6% by weight to 0.001% by weight, 6% by weight to 0.1% by weight, 6% by weight to 1% by weight, 6% by weight to 2% by weight, 4% by weight to 0.001% by weight, 4% by weight to 0.1% by weight, 4% by weight to 1% by weight, 4% by weight to 2% by weight, 3% by weight to 0.001% by weight, 3% by weight to 0.1% by weight, 3% by weight to 1% by weight, 2% by weight to 0.001% by weight, 2% by weight to 0.1% by weight, or 2% by weight to 1% by weight.

[0436] In some cases, compositions include a ratio by weight of the 25-hydroxy-(3β)-cholest- 5-en-3-sulfate and the 5-cholesten-3β-25-diol-disulfate of 10:1 or more, such as 25:1 or more, such as 50:1 or more, such as 100:1 or more, such as such as 250:1 or more, such as 500:1 or more, such as 1000:1 or more, such as 2500:1 or more, such as 5000:1 or more, such as 10,000:1 or more, such as 25,000:1 or more, such as 50,000:1 or more, such as 100,000:1 or more, such as 106:1 or more, such as 107:1 or more, such as 108:1 or more, and including where the ratio by weight of the 25-hydroxy-(3β)- cholest-5-en-3-sulfate to the 5-cholesten-3β-25-diol-disulfate in the composition is 109:1 or more. In some cases, compositions include a ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate and the 5-cholesten-3β-25-diol-disulfate that ranges from 10:1 to 109:1, such as from 100:1 to 108:1, such as from 1000:1 to 107:1, and including from 10000:1 to 106:1.

[0437] In some cases, the byproduct is sulfated desmosterol (Structure IB).

[0438] 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]phenanthren-3-yl] sulfate) is present in the composition produced by sulfation of 25- hydroxy-(3β)-cholest-5-en-3-ol in an amount relative to the 25-hydroxy-(3β)-cholest-5-en-3-sulfate of10% by weight or less, such as 9% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 5% by weight or less, such as 4% by weight or less, such as 3% by weight or less, such as 2% by weight or less, such as 1% by weight or less, such as 0.5% by weight or less, such as 0.1% by weight or less, such as 0.01% by weight or less, such as 0.001% by weight or less, and including where sulfated desmosterol is present in the composition produced by sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol in an amount relative to the 25-hydroxy- (3β)-cholest-5-en-3-sulfate of 0.001% by weight or less, and may range from 0.1% by weight to 10% by weight, such as 0.2% by weight to 5% by weight or 0.3% by weight to 3% by weight. In some cases, the ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate to the sulfated desmosterol formed is 10:1 or more, such as 25:1 or more, such as 50:1 or more, such as 100:1 or more, such as such as 250:1 or more, such as 500:1 or more, such as 1000:1 or more, such as 2500:1 or more, such as 5000:1 or more, such as 10,000:1 or more, such as 25,000:1 or more, such as 50,000:1 or more, such as 100,000:1 or more, such as 106:1 or more, such as 107:1 or more, such as 108:1 or more, and including where the ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate to the sulfated desmosterol formed is 109:1 or more. In some cases, the ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3- sulfate and the sulfated desmosterol formed ranges from 10:1 to 109:1, such as from 100:1 to 108:1, such as from 1000:1 to 107:1 and including from 10000:1 to 106:1.

[0439] Aspects of the present disclosure also include compositions having 25-hydroxy-(3β)- cholest-5-en-3-sulfate and sulfated desmosterol that is present in the composition in an amount relative to the 25-hydroxy-(3β)-cholest-5-en-3-sulfate of 10% by weight or less, such as 9% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 5% by weight or less, such as 4% by weight or less, such as 3% by weight or less, such as 2% by weight or less, such as 1% by weight or less, such as 0.5% by weight or less, such as 0.1% by weight or less, such as 0.01% by weight or less, such as 0.001% by weight or less, and including 0.001% w / w or less relative to the 25-hydroxy-(3β)-cholest-5-en-3-sulfate, and may range from 10% by weight to 0.001% by weight, such as 10% by weight to 0.1% by weight, 10% by weight to 1% by weight, 10% by weight to 2% by weight, 8% by weight to 0.001% by weight, 8% by weight to 0.1% by weight, 8% by weight to 1% by weight, 8% by weight to 2% by weight, 6% by weight to 0.001% by weight, 6% by weight to 0.1% by weight, 6% by weight to 1% by weight, 6% by weight to 2% by weight, 4% by weight to 0.001% by weight, 4% by weight to 0.1% by weight, 4% by weight to 1% by weight, 4% by weight to 2% by weight, 3% by weight to 0.001% by weight, 3% by weight to 0.1% by weight, 3% by weight to1% by weight, 2% by weight to 0.001% by weight, 2% by weight to 0.1% by weight, or 2% by weight to 1% by weight.

[0440] In some cases, compositions include a ratio by weight of the 25-hydroxy-(3β)-cholest- 5-en-3-sulfate and the sulfated desmosterol of 10:1 or more, such as 25:1 or more, such as 50:1 or more, such as 100:1 or more, such as such as 250:1 or more, such as 500:1 or more, such as 1000:1 or more, such as 2500:1 or more, such as 5000:1 or more, such as 10,000:1 or more, such as 25,000:1 or more, such as 50,000:1 or more, such as 100,000:1 or more, such as 106:1 or more, such as 107:1 or more, such as 108:1 or more, and including where the ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3- sulfate to the sulfated desmosterol in the composition is 109:1 or more. In some cases, compositions include a ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate and the sulfated desmosterol that ranges from 10:1 to 109:1, such as from 100:1 to 108:1, such as from 1000:1 to 107:1 and including from 10000:1 to 106:1.

[0441] In some cases, the byproduct of sulfating the 25-hydroxy-(3β)-cholest-5-en-3-ol that is present in the 25-hydroxy-(3β)-cholest-5-en-3-sulfate composition is a thermal degradation product. In some cases, the byproduct is identified by relative retention time when the components of the 25- hydroxy-(3β)-cholest-5-en-3-sulfate composition are separated by liquid chromatography (e.g., HPLC). In certain cases, the byproduct is sulfated desmosterol, a compound having a retention time of about 18.3 minutes when the components of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate composition are separated by HPLC operating at about 45 ºC with a C8 stationary phase and separates the components of the composition with a first mobile phase comprising a buffer (e.g., an aqueous buffer of sodium phosphate) and a second mobile phase comprising one or more organic solvents (see e.g., Tables 13 and 14 below). In some cases, the first mobile phase is an aqueous buffer. In certain cases, the first mobile phase includes sodium phosphate. In some cases, the second mobile phase is chosen 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 / minute. In some cases, the flow rate of the second mobile phase is about 1.0 mL / minute or more. In some cases, 25-hydroxy-(3β)-cholest-5-en-3-sulfate has a retention time of about 7.7 minutes under the same HPLC conditions. In some cases, the byproduct is a compound having a retention time of about 37.7 minutes when the components of the 25-hydroxy- (3β)-cholest-5-en-3-sulfate composition are separated by HPLC operating at about 45 ºC with a C8 stationary phase and separates the components of the composition with a first mobile phase comprising a buffer (e.g., an aqueous buffer of sodium phosphate) and a second mobile phase comprising one or more organic solvents (see e.g., Tables 13 and 14 below). While not wishing to be bound by theory, itis 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 certain cases, the first mobile phase includes sodium phosphate. In some cases, the second mobile phase is chosen 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 / minute. In some cases, the flow rate of the second mobile phase is about 1.0 mL / minute or more. In some cases, and 25-hydroxy-(3β)-cholest-5-en-3-sulfate has a retention time of about 7.7 minutes under the same HPLC conditions such 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).

[0442] Aspects of the present disclosure also include compositions having 25-hydroxy-(3β)- cholest-5-en-3-sulfate and one or more byproducts of sulfating the 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, the one or more byproducts are present in the composition in an amount relative to the 25-hydroxy-(3β)-cholest-5-en-3-sulfate of 10% by weight or less, such as 9% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 5% by weight or less, such as 4% by weight or less, such as 3% by weight or less, such as 2% by weight or less, such as 1% by weight or less, such as 0.5% by weight or less, such as 0.1% by weight or less, such as 0.01% by weight or less, such as 0.001% by weight or less, and including 0.001% by weight or less, and may range from 0.1% by weight to 5% by weight, such as 0.2% by weight to 10% by weight or 0.3% by weight to 15% by weight. In some cases, compositions include 25-hydroxy-(3β)-cholest-5- en-3-sulfate and the one or more byproducts in an amount relative to the 25-hydroxy-(3β)-cholest-5- en-3-sulfate that ranges from 0.0001% by weight to 10% by weight, such as from 0.005% by weight to 9.5% by weight, such as from 0.001% to 9.0% by weight, such as from 0.05% by weight to 8.5% by weight, such as from 0.1% by weight to 8.0% by weight, such as from 0.5% by weight to 7.5% by weight, such as from 1% by weight to 7% by weight, such as from 1.5% by weight to 6.5% by weight, and including from 2% by weight to 6% by weight.

[0443] In some cases, the ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate to the one or more byproducts formed is 10:1 or more, such as 25:1 or more, such as 50:1 or more, such as 100:1 or more, such as such as 250:1 or more, such as 500:1 or more, such as 1000:1 or more, such as 2500:1 or more, such as 5000:1 or more, such as 10,000:1 or more, such as 25,000:1 or more, such as 50,000:1 or more, such as 100,000:1 or more, such as 106:1 or more, such as 107:1 or more, such as 108:1 or more, and including where the ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate to the one or more byproducts formed is 109:1 or more. In some cases, the ratio by weight of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate and the one or more byproducts formed ranges from 10:1 to 109:1, such as from 100:1 to 108:1, such as from 1000:1 to 107:1, and including from 10000:1 to 106:1.

[0444] In some cases, the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt is a 25- hydroxy-(3β)-cholest-5-en-3-sulfate pyridinium salt (Scheme IA2).

[0445] In certain cases, the sulfating agent is contacted with an anhydride prior to contacting with the 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, the anhydride is chosen from acetic anhydride, trifluoroacetic anhydride and triflic anhydride. The amount of anhydride relative to the 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, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and including 10 equivalents or more, and may range from 0.001 equivalents to 10 equivalents, such as 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 8 equivalents, 0.9 equivalents to 5 equivalents, 1.3 equivalents to 10 equivalents, 1.3 equivalents to 8 equivalents, 1.3 equivalents to 5equivalents, 1.5 equivalents to 10 equivalents, 1.5 equivalents to 8 equivalents, 1.5 equivalents to 5 equivalents, 2 equivalents to 10 equivalents, 2 equivalents to 8 equivalents, 2 equivalents to 5 equivalents, 0.1 equivalent to 1.5 equivalents, 0.5 equivalents to 1.1 equivalents, or 0.1 equivalent to 1 equivalent relative to the 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0446] In some cases, methods include quenching (i.e., deactivating) unreacted sulfating agent after producing the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic 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 the 25-hydroxy- (3β)-cholest-5-en-3-ol may vary and may be 1 equivalent or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, such as 6 equivalents or more, such as 7 equivalents or more, such as 8 equivalents or more, such as 9 equivalents or more, such as 10 equivalents or more, such as 15 equivalents or more, such as 20 equivalents or more and including 25 equivalents or more.

[0447] In certain cases, quenching the reactivity of unreacted sulfating agent includes adding water to the reaction mixture followed by the addition of 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 certain cases, the at least one base is pyridine. The pyridine may be added to the reaction mixture 1 minute or more after adding the water, such as 5 minutes or more, such as 10 minutes or more, such as 15 minutes or more, such as 30 minutes or more, such as 45 minutes or more, such as 60 minutes or more, such as 90 minutes or more, such as 120 minutes or more, such as 150 minutes or more, such as 180 minutes or more, such as 210 minutes or more and including 240 minutes or more after adding the water to the reaction mixture. In certain cases, pyridine is added to the reaction mixture 60 minutes after adding the water. The amount of pyridine added to the reaction mixture relative to the amount of sulfating agent may vary and may be 0.001 equivalents or more, such as 0.005 equivalents or more, such as 0.01 equivalents or more, such as 0.05 equivalents or more, such as 0.1 equivalents or more, such as 0.5 equivalents or more, such as 1 equivalent or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, such as 6 equivalents or more and including 10 equivalents or more.

[0448] In some cases, the unreacted sulfating agent in the reaction mixture is quenched under slow agitation. In certain cases, quenching the unreacted sulfating agent under slow agitation includes stirring the reaction mixture in a manner sufficient to maintain agglomerates of the unreacted sulfating agent in the reaction mixture. In some cases, slow agitation of the reaction mixture is sufficient suchthat agglomerates of unreacted sulfating agent reduce in size during quenching by 10% or less, such as by 9% or less, such as by 8% or less, such as by 7% or less, such as by 6% or less, such as by 5% or less, such as by 4% or less, such as by 3% or less, such as by 2% or less, such as by 1% or less and including where the reaction mixture is slowly agitated such that agglomerates of unreacted sulfating agent reduce in size during quenching by 0.1% or less. In certain cases, slow agitation of the reaction mixture is sufficient such that agglomerates of unreacted sulfating agent remain at the bottom of the reaction flask during quenching. In certain cases, slow agitation of the reaction mixture is sufficient such that little to no agglomerates of unreacted sulfating agent is present in the stirring vortex of the agitated reaction mixture.

[0449] In some cases, methods include purifying the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt prior to contacting the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt with the at least one metal salt. In some cases, the purified 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt has a purity of 97% or greater, such as a purity of 98% or greater, such as a purity of 99% or greater, such as purity of 99.5% or greater, such as purity of 99.7% or greater, such as a purity of 99.9% or greater and including a purity of 99.99% or greater. In certain cases, the purified 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt has one or more by-products of sulfation (e.g., by-products from sulfating the 25-hydroxy-(3β)-cholest-5-en-3-ol) where the one or more by- products is present in an amount of 5% w / w or less relative to the 25-hydroxy-(3β)-cholest-5-en-3- sulfate organic cationic salt, such as 4% w / w or less, such as 3% w / w or less, such as 2% w / w or less, such as 1% w / w or less, such as in an amount of 0.9% w / w or less, such as 0.8% w / w or less, such as 0.7% w / w or less, such as 0.6% w / w or less, such as 0.5% w / w or less, such as 0.4% w / w or less, such as 0.3% w / w or less, such as 0.2% w / w or less, such as 0.1% w / w or less, such as 0.05% w / w or less, such as 0.01% w / w or less and including being present in an amount of 0.001% w / w or less relative to the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic 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-en- 3-sulfate organic cationic salt composition in an amount of 1% w / w or less relative to the 25-hydroxy- (3β)-cholest-5-en-3-sulfate organic cationic salt, such as in an amount of 0.9% w / w or less, such as 0.8% w / w or less, such as 0.7% w / w or less, such as 0.6% w / w or less, such as 0.5% w / w or less, such as 0.4% w / w or less, such as 0.3% w / w or less, such as 0.2% w / w or less, such as 0.1% w / w or less, such as 0.05% w / w or less, such as 0.01% w / w or less and including being present in an amount of 0.001% w / w or less relative to the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt.

[0450] In some cases, the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt is purified by liquid chromatography. In some cases, purifying the 25-hydroxy-(3β)-cholest-5-en-3- sulfate organic cationic salt includes liquid chromatography using a silica gel stationary phase (e.g., a silica gel plug column, ≥5 mass equivalents). In some cases, the 25-hydroxy-(3β)-cholest-5-en-3- sulfate organic cationic salt is purified using the silica gel stationary phase and a mobile phase that includes pyridine. In certain cases, the mobile phase includes methylene chloride, methanol, and pyridine. In certain cases, the mobile phase includes a mixture of methylene chloride-methanol (85:15) and pyridine (1%).

[0451] 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 certain cases, the combined fractions are concentrated by distillation. In certain cases, the combined fractions are concentrated under vacuum. In certain cases, the combined fractions are concentrated by distillation under vacuum.

[0452] In some cases, the combined fractions are contacted with one or more particles of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt (e.g., particles from a previously purified sample of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt). In some cases, contacting the particles of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt with the combined fractions is sufficient to precipitate 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt in the combined fractions. In some cases, contacting particles of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt with the combined fractions includes adding the particles during distillation of the combined fractions. In some cases, the particles of 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt are added to the combined fractions before distilling the combined fractions. In some cases, the particles of 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt are added to the combined fractions while distilling the combined fractions, such as 1 minute or more after beginning the distillation, such as 5 minutes or more, such as 10 minutes or more, such as 15 minutes or more, such as 20 minutes or more, such as 30 minutes or more, such as 40 minutes or more, such as 50 minutes or more and including adding the particles of 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt to the combined fractions 60 minutes or more after beginning the distillation of the combined fractions. In certain cases, the combined fractions are distilled under constant pressure, such as where the pressure changes by 10% or less, such as by 9% or less, such as by 8% or less, such as by 7% or less, such as by 6% or less, such as by 5% or less, such as by 4% or less, such as by 3% or less, such as by 2% or less, such as by 1% or less and including by 0.1% or less. In some cases, the pressure during distillation changes by 10 inHg or less, such as by 9 inHg or less, such as by 8 inHg or less,such as by 7 inHg or less, such as by 6 inHg or less, such as by 5 inHg or less, such as by 4 inHg or less, such as by 3 inHg or less, such as by 2 inHg or less, such as by 1 inHg or less, such as by 0.5 inHg or less, such as by 0.1 inHg or less, such as by 0.05 inHg or less and including by 0.01 inHg or less. In some cases, the combined fractions are distilled under a reduced pressure wherein the pressure is maintained between 15 inHg to 30 inHg, such as from 17.5 inHg to 27.5 inHg, such as from 20 inHg to 25 inHg, such as from 21 inHg and 24 inHg and including maintained at a pressure of from 22 inHg to 23 inHg.

[0453] In some cases, the combined fractions are concentrated under vacuum and the concentrated combined fractions are contacted with a composition containing particles of the 25- hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt. In certain cases, the concentrated combined fractions are contacted with a composition containing particles of the 25-hydroxy-(3β)-cholest-5-en-3- sulfate organic cationic salt and at least one solvent. In certain cases, the at least one solvent is chosen from tetrahydrofurans, such as 2-methyltetrahydrofuran. The concentrated combined fractions may be contacted with the composition containing the particles of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt over a duration of 0.001 minutes or more, such as over 0.005 minutes or more, such as over 0.01 minutes or more, such as over 0.05 minutes or more, such as over 0.1 minutes or more, such as over 0.5 minutes or more, such as over 1 minute or more, such as over 2 minutes or more, such as over 3 minutes or more, such as over 4 minutes or more, such as over 5 minutes or more, such as over 10 minutes or more, such as over 15 minutes or more, such as over 30 minutes or more, such as over 45 minutes or more and including over 60 minutes or more. In certain cases, the combined fractions are added dropwise to a composition containing 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt in 2-methyltetrahydrofuran.

[0454] In some cases, the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt is contacted with a metal salt to produce the 25-hydroxy-(3β)-cholest-5-en-3-sulfate metal salt (Scheme IB1).

[0455] In some cases, methods to produce the 25-hydroxy-(3β)-cholest-5-en-3-sulfate metal salt includes contacting the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt with at least one sodium salt. In some cases, the at least one sodium salt is chosen from sodium acetate, sodium iodide, sodium chloride, sodium hydroxide and sodium methoxide. The 25-hydroxy-(3β)-cholest-5- en-3-sulfate organic cationic salt may be contacted with the metal salt at a temperature that ranges from -10 °C to 75 °C, such as from -5 °C to 70 °C, such as from -4 °C to 65 °C, such as from -3 °C to 60 °C, such as from -2 °C to 55 °C, such as from -1 °C to 50 °C, such as from 0 °C to 45 °C, such as from 5 °C to 40 °C, and including from 10 °C to 35 °C.

[0456] The reaction may be carried out for a duration that ranges from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, such as from 0.7 hours to 18 hours, such as from 0.8 hours to 17 hours, such as from 0.9 hours to 16 hours, and including from 1 hours to 15 hours. The amount of metal salt used relative to the 25-hydroxy-(3β)-cholest-5-en-3- sulfate organic cationic salt may vary and may be 0.0001 equivalents or more, such as 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and including 10equivalents or more, and may range from 0.001 equivalents to 10 equivalents, such as 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 equivalents to 10 equivalents, 1 equivalents to 8 equivalents, 1 equivalents to 6 equivalents, 1 equivalents to 4 equivalents, 1 equivalents 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, or 2 equivalents to 3 equivalents, 1 equivalent to 100 equivalents, 1 equivalent to 5 equivalents, 1 equivalent to 2 equivalents.

[0457] In some cases, methods include contacting the 25-hydroxy-(3β)-cholest-5-en-3-sulfate pyridinium salt with sodium iodide to produce a 25-hydroxy-(3β)-cholest-5-en-3-sulfate sodium salt (Scheme IB2).

[0458] In some cases, methods for preparing 25-hydroxy-3β-cholesten-5-en-3-sulfate include contacting 25-hydroxy-(3β)-cholest-5-en-3-ol with a sulfur trioxide-pyridine complex to produce a 25- hydroxy-(3β)-cholest-5-en-3-sulfate pyridinium salt; and contacting the 25-hydroxy-(3β)-cholest-5-en- 3-sulfate pyridinium salt with a sodium salt to produce the 5-cholesten-3β,25-diol 3-sulfate sodium salt (Scheme Ib). Scheme Ib

[0459] In some cases, methods for preparing 25-hydroxy-3β-cholesten-5-en-3-sulfate include contacting (3β)-cholest-5-en-3-ol with a sulfating agent to produce a first (3β)-cholest-5-en-3-sulfate organic cationic salt; contacting the first (3β)-cholest-5-en-3-sulfate organic cationic salt with an organic base to produce a second (3β)-cholest-5-en-3-sulfate organic cationic salt; oxidizing the second (3β)-cholest-5-en-3-sulfate organic cationic salt in the presence of at least one surfactant to produce a 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt; generating a 25-hydroxy-(3β)- cholest-5-en-3-sulfate organic cationic salt from the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt by deoxygenation; and contacting the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt with at least one metal salt to produce the 5-cholesten-3β,25-diol 3-sulfate metal salt (Scheme IIa). Scheme IIa

[0460] In some cases, cholesterol is sulfated with a sulfating agent (Scheme IIA1). In some cases, the sulfating agent is chosen from sulfur trioxide complexes, sulfuric acid 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 chosen from sulfur trioxide dimethylformamide, sulfur trioxide triethylamine, and sulfur trioxide trimethylamine. In some cases, the sulfating agent is sulfuric acid and acetic anhydride and pyridine. In some cases, the sulfating agent is chosen from chlorosulfonic acid and pyridine. In some cases, the sulfating agent is chosen from chlorosulfonic acid and 2,6-lutidine. In some cases, the sulfating agent is chosen from ethyl chlorosulfonate.

[0461] Cholesterol may be sulfated at a temperature that ranges from 0 °C to 100 °C, such as from 5 °C to 95 °C, such as from 10 °C to 90 °C, such as from 15 °C to 85 °C, such as from 20 °C to 80 °C, such as from 25 °C to 75 °C, and including from 30 °C to 70 °C. The reaction may be carried out for a duration that ranges from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, and including from 0.7 hours to 18 hours. The amount of sulfating agent used relative to cholesterol may vary and may be 0.0001 equivalents or more, such as 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and including 10 equivalents or more, and may range from 0.001 equivalents to 10 equivalents, such as 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 equivalents to 10 equivalents, 1 equivalents to 8 equivalents, 1 equivalents to 6 equivalents, 1 equivalents to 4 equivalents, 1 equivalents 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 30 equivalents, 1 equivalent to 5 equivalents, or 1 equivalent to 2 equivalents.

[0462] In some cases, the first (3β)-cholest-5-en-3-sulfate organic cationic salt is a (3β)- cholest-5-en-3-sulfate pyridinium salt (Scheme IIA2).

[0463] In some cases, the first (3β)-cholest-5-en-3-sulfate organic cationic salt (Structure IIA) is contacted with an organic base to produce a second (3β)-cholest-5-en-3-sulfate organic cationic salt (Structure IIB) (Scheme IIB1).

[0464] In some cases, the organic base contacted with the first (3β)-cholest-5-en-3-sulfate organic cationic salt is chosen from a hydroxide base. In some cases, the hydroxide base is chosen from tetraethylammonium hydroxide, tetra-butylammonium hydroxide, tetrapropylammonium hydroxide and tetramethylammonium hydroxide. In some cases, the second (3β)-cholest-5-en-3- sulfate organic cationic salt is chosen from a tetraethylammonium cationic salt, a tetra-butylammonium cationic salt, a tetrapropylammonium cationic salt and a tetramethylammonium cationic salt. In some cases, the organic base is contacted with the first (3β)-cholest-5-en-3-sulfate organic cationic salt at a temperature that ranges from -10 °C to 75 °C, such as from -5 °C to 70 °C, such as from -4 °C to 65 °C, such as from -3 °C to 60 °C, such as from -2 °C to 55 °C, such as from -1 °C to 50 °C and including from 0 °C to 15 °C. The reaction may be carried out for a duration that ranges from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, such as from 0.7 hours to 18 hours, such as from 0.8 hours to 17 hours, such as from 0.9 hours to 16 hours, and including from 1 hour to 15 hours. The amount of the organic base used relative to the first (3β)-cholest-5-en-3- sulfate organic cationic salt may vary and may be 0.0001 equivalents or more, such as 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more, such as 2 equivalents or more, such as 3equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and including 10 equivalents or more, and may range from 0.001 equivalents to 10 equivalents, such as 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 equivalents to 10 equivalents, 1 equivalents to 8 equivalents, 1 equivalents to 6 equivalents, 1 equivalents to 4 equivalents, 1 equivalents 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 10 equivalents, 1 equivalent to 5 equivalents, or 1 equivalent to 2 equivalents.

[0465] In certain cases, methods include contacting the first (3β)-cholest-5-en-3-sulfate organic cationic salt with tetra-butylammonium hydroxide to generate a (3β)-cholest-5-en-3-sulfate tetra- butylammonium cationic salt (Structure IIB1) (Scheme IIB2).

[0466] In some cases, the second (3β)-cholest-5-en-3-sulfate organic cationic salt is oxidized to produce a 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt (Structure IIC) (Scheme IIC1).

[0467] In some cases, oxidizing the second (3β)-cholest-5-en-3-sulfate organic cationic salt includes contacting the second (3β)-cholest-5-en-3-sulfate organic cationic salt with a composition having an oxidizing agent and at least one surfactant.

[0468] In some cases, the at least one surfactant is chosen from non-ionic surfactants, anionic surfactants, cationic surfactants and zwitterionic surfactants. Non-ionic surfactants may be chosen 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 non-ionic surfactants. Anionic surfactants may be chosen from surfactants having an anionic functional head group, such as a sulfonate, phosphate, sulfate or carboxylate head group-containing surfactant. For example, anionic surfactants may be chosen from alkyl sulfates such as ammonium lauryl sulfate, dioctyl sodium sulfosuccinate, perfluorooctanesulfonate, perfluorononanoate, perfluorooctanoate, a linear alkylbenzene sulfonate, an alkyl-aryl ether phosphate, sodium lauryl ether sulfate, lignosulfonate or sodium stearate, among other anionic surfactants. Cationic surfactants may be chosen from surfactants having a cationic functional head group, such as a pyridinium or a quaternary ammonium head group. For example, cationic surfactants may be chosen from cetyltrimethylammonium hydrogen sulfate, tetra-butylammonium hydrogen sulfate, cetyltrimethylammonium bromide, tetrat-butylammonium bromide, tetrat- butylammonium iodide, tetrabutylphosphonium bromide, tetraoctylammonium bromide, tetraoctylammonium iodide, benzyltriethylammonium chloride, benzyltriethylammonium bromide, benzylcetyldimethylammonium chloride or benzylcetyldimethylammonium bromide. Zwitterionic surfactants include both cationic and anionic centers, such as a sultaine (e.g., 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate) or a betaine (e.g., cocamidopropyl betaine).In certain cases, the at least one surfactant is an Extran laboratory soap, La Parisienne soap or DL-α- tocopherol methoxypolyethylene glycol succinate (e.g., TPGS-750-M-2).

[0469] The amount of surfactant used relative to the second (3β)-cholest-5-en-3-sulfate organic cationic salt may vary, where in some instances, 0.0001 equivalents or more of the surfactant is used, such as 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and including 10 equivalents or more of the surfactant, and may range from 0.001 equivalents to 10 equivalents, such as 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 equivalents to 10 equivalents, 1 equivalents to 8 equivalents, 1 equivalents to 6 equivalents, 1 equivalents to 4 equivalents, 1 equivalents 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 equivalent to 5 equivalents, 0.15 equivalents to 1 equivalent, or 0.2 equivalents to 0.3 equivalents.

[0470] In some cases, oxidizing the second (3β)-cholest-5-en-3-sulfate organic cationic salt includes contacting the second (3β)-cholest-5-en-3-sulfate organic cationic salt with an oxidizing agent and at least one ketone in the presence of at least one surfactant.

[0471] In some instances, the at least one ketone is chosen from tetrahydrothiopyran-4-one 1,1- dioxide and halogenated ketones. In some cases, the halogenated ketones are chosen from 1,1,1- trifluoro-2-butanone, 4,4-difluorocyclohexanone, 2-2-2-4’-tetrafluoroacetophenone, and 1,1,1- trifluoroacetone. In certain cases, the at least one ketone is 1,1,1-trifluoro-2-butanone. The amount of ketone used relative to the oxidizing agent in the subject reaction may vary, and may be 1 equivalent or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, such as 6 equivalents or more, such as 7 equivalents or more, such as 8 equivalents or more, such as 9 equivalents or more, such as 10 equivalents or more, such as 15equivalents or more, such as 20 equivalents or more, such as 25 equivalents or more, such as 30 equivalents or more, such as 35 equivalents or more, and including 50 equivalents or more of the ketone, and may range from 1 equivalent to 50 equivalents, such as 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 equivalent to 50 equivalents, 2 equivalent to 35 equivalents, 2 equivalent to 25 equivalents, 2 equivalent to 15 equivalents, 2 equivalent to 10 equivalents, 2 equivalent to 8 equivalents, 2 equivalent to 5 equivalents, 4 equivalent to 50 equivalents, 4 equivalent to 35 equivalents, 4 equivalent to 25 equivalents, 4 equivalent to 15 equivalents, 4 equivalent to 10 equivalents, 4 equivalent to 8 equivalents, 1 equivalent to 50 equivalents, 2 equivalent to 25 equivalents, or 5 equivalents to 10 equivalents.

[0472] In certain cases, the ketone is further purified before use. For example, the ketone may be purified by distillation prior to use. In some instances, the reactivity of the ketone is tested (e.g., tested for impurities by1H-NMR) in order to determine whether purification may be required.

[0473] In certain cases, oxidizing the second (3β)-cholest-5-en-3-sulfate organic cationic salt includes contacting the second (3β)-cholest-5-en-3-sulfate organic cationic 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, ranging from 0.0000001% w / v or more of the reaction mixture, such as 0.000001% w / v or more, such 0.00001% w / v or more, such as 0.0001% w / v or more, such as 0.001% w / v, such as 0.01% w / v or more, such as 0.1% w / v, such as 0.05% w / v or more, such as 0.1% w / v or more, such as 0.5% w / v or more, such as 1% w / v or more, such as 5% w / v or more, such as 10% w / v or more, such as 15% w / v or more, and including 25% w / v or more of the reaction mixture, and may range from 0.0000001% w / v to 25% w / v, such as 0.0000001% w / v to 15% w / v, 0.0000001% w / v to 10% w / v, 0.0000001% w / v to 5% w / v, 0.0000001% w / v to 1% w / v, 0.001% w / v to 25% w / v, 0.001% w / v to 15% w / v, 0.001% w / v to 10% w / v, 0.001% w / v to 5% w / v, 0.001% w / v to 1% w / v, 0.1% w / v to 25% w / v, 0.1% w / v to 15% w / v, 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.

[0474] The second (3β)-cholest-5-en-3-sulfate organic cationic salt may be oxidized at a temperature that ranges from -25 °C to 50 °C, such as from -20 °C to 45 °C, such as from -15 °C to 40 °C, such as from -10 °C to 35 °C, such as from -5 °C to 30 °C, such as from -1 °C to 25 °C, and including from 0 °C to 15 °C. In certain cases, the second (3β)-cholest-5-en-3-sulfate organic cationic salt is oxidized at a temperature of from 0 °C to 5 °C. Where the reaction mixture includes an amountof water, the reaction may be conducted at a temperature that is from -10 °C to 50 °C, such as from -5 °C to 45 °C, such as from 0 °C to 40 °C, such as from 0 °C to 35 °C, such as from 0 °C to 30 °C, such as from 0 °C to 25 °C, such as from 0 °C to 20 °C, such as from 0 °C to 15 °C, and including from 0 °C to 10 °C.

[0475] The second (3β)-cholest-5-en-3-sulfate organic cationic salt may be oxidized at a pH that ranges from 5 to 7.5, such as a pH of from 5.5 to 7.0 and including a pH of from 5.5 to 6.5. In some cases, where the reaction mixture contains water (e.g., in a biphasic solvent system), the pH ranges from 5.0 to 6.0, such as a pH of from 5.0 to 5.9, such as a pH of from 5.0 to 5.8, such as a pH of from 5.0 to 5.7, such as a pH from 5.0 to 5.6, and including a pH of from 5.0 to 5.5.

[0476] The reaction may be carried out for a duration that ranges from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, such as from 0.7 hours to 18 hours, such as from 0.8 hours to 17 hours, such as from 0.9 hours to 16 hours, and including from 1 hours to 15 hours.

[0477] In some instances, the second (3β)-cholest-5-en-3-sulfate organic cationic 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, methods include contacting the potassium peroxymonosulfate with at least one ketone in the presence of at least one surfactant to form a separate oxidative reactive mixture and adding the oxidative reactive mixture to the second (3β)-cholest-5-en- 3-sulfate organic cationic salt. In these cases, the potassium peroxymonosulfate may be contacted with the at least one ketone in the presence of the at least one surfactant for a duration of 0.1 minute or more before contacting the oxidative reactive mixture with the second (3β)-cholest-5-en-3-sulfate organic cationic salt, such as 1 minute or more, such as 2 minutes or more, such as 3 minutes or more, such as 5 minutes or more, and including 10 minutes or more, and the time may range from 2 minutes to 180 minutes, such as 3 minutes to 120 minutes or 4 minutes to 60 minutes. In certain instances, the potassium peroxymonosulfate may be contacted with the at least one ketone in the presence of the at least one surfactant to form a separate oxidative reactive mixture and immediately contacting the oxidative reactive mixture with the second (3β)-cholest-5-en-3-sulfate organic cationic salt. The oxidative reactive mixture may be formed at a temperature that ranges from -10 °C to 50 °C, such as from -5 °C to 45 °C, such as from -4 °C to 40 °C, such as from -3 °C to 35 °C, such as from -2 °C to 30 °C, such as from -1 °C to 25 °C and including from 0 °C to 15 °C. Where the oxidative reactive mixture is not immediately contacted with the second (3β)-cholest-5-en-3-sulfate organic cationic salt,the oxidative reactive mixture may be maintained at a temperature that ranges from -10 °C to 50 °C, such as from -5 °C to 45 °C, such as from -4 °C to 40 °C, such as from -3 °C to 35 °C, such as from - 2 °C to 30 °C, such as from -1 °C to 25 °C, and including from 0 °C to 15 °C.

[0478] In some cases, methods further include adding the oxidative reactive mixture to the second (3β)-cholest-5-en-3-sulfate organic cationic salt. In some instances, methods include adding dropwise the oxidative reactive mixture to the second (3β)-cholest-5-en-3-sulfate organic cationic salt. In some instances, the oxidative reactive mixture is added to the second (3β)-cholest-5-en-3-sulfate organic cationic salt in metered amounts. The metered amounts may be added continuously or at predetermined time intervals (e.g., every 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, or some other interval). In some instances, the oxidative reactive mixture is added to the second (3β)- cholest-5-en-3-sulfate organic cationic salt by controlled addition, such as with a mechanically or computer-controlled pump, e.g., syringe pump. In some cases, methods include generating the oxidative reactive mixture and adding a composition containing the second (3β)-cholest-5-en-3-sulfate organic cationic salt to the oxidative reactive mixture. In some instances, methods include adding dropwise the second (3β)-cholest-5-en-3-sulfate organic cationic salt to the oxidative reactive mixture. In some instances, the second (3β)-cholest-5-en-3-sulfate organic cationic salt is added to the oxidative reactive mixture in metered amounts. The metered amounts may be added continuously or at predetermined time intervals (e.g., every 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, or some other interval). In some instances, the second (3β)-cholest-5-en-3-sulfate organic cationic salt is added to the oxidative reactive mixture by controlled addition, such as with a mechanically or computer-controlled pump, e.g., syringe pump.

[0479] In certain cases, oxidizing the second (3β)-cholest-5-en-3-sulfate organic cationic salt includes contacting the second (3β)-cholest-5-en-3-sulfate organic cationic salt with at least one oxidative species. In some instances, the at least one oxidative species is chosen from dioxiranes. In some instances, the dioxiranes are generated in situ in a composition having the second (3β)-cholest- 5-en-3-sulfate organic cationic salt. In some instances, the dioxiranes are generated separately (e.g., in a separate reaction container, e.g., flask) and added to the composition having the second (3β)-cholest- 5-en-3-sulfate organic cationic salt.

[0480] In certain cases, the second (3β)-cholest-5-en-3-sulfate organic cationic salt is oxidized in the presence of at least one base. In certain cases, the at least one base is chosen from weak bases. In some cases, the at least one base is chosen from potassium hydrogen carbonate, sodium hydrogen carbonate, potassium phenoxide, sodium citrate buffer, sodium phosphate buffer, potassium formateand potassium acetate. In certain cases, the at least one base is potassium hydrogen carbonate. In some cases, the at least one base may be added to the reaction mixture over time, such as in metered amounts where the base is added at predetermined time intervals (e.g., every 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, or some other interval). In some cases, the at least one base may be a composition having water where the base present in the composition may be 0.0000001% w / v or more of the composition, such as 0.000001% w / v or more, such as 0.00001% w / v or more, such as 0.0001% w / v or more, such as 0.001% w / v or more, such as 0.01% w / v or more, such as 0.05% w / v or more, such as 0.1% w / v or more, such as 0.5% w / v or more, such as 1% w / v or more, such as 5% w / v or more, such as 10% w / v or more, such as 15% w / v or more, and including 25% w / v or more of the composition, and may range from 0.0000001% w / v to 25% w / v, such as 0.0000001% w / v to 15% w / v, 0.0000001% w / v to 10% w / v, 0.0000001% w / v to 5% w / v, 0.0000001% w / v to 1% w / v, 0.001% w / v to 25% w / v, 0.001% w / v to 15% w / v, 0.001% w / v to 10% w / v, 0.001% w / v to 5% w / v, 0.001% w / v to 1% w / v, 0.1% w / v to 25% w / v, 0.1% w / v to 15% w / v, 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 20% w / v, 0.2% w / v to 15% w / v, or 0.3% w / v to 10% w / v. In certain cases, the at least one base may be an aqueous potassium hydrogen carbonate composition.

[0481] In certain cases, the second (3β)-cholest-5-en-3-sulfate organic cationic salt is oxidized by contacting with oxone in the presence of cetyltrimethylammonium hydrogen sulfate (CTAHS) followed by adding trifluorobutanone and potassium hydrogen sulfate to form 25-hydroxy-(3β)- cholest-(5,6-epoxy)-3-sulfate organic cationic salt (Scheme IIC2).

[0482] In certain instances, methods include forming an oxidative species in situ with the second (3β)-cholest-5-en-3-sulfate organic cationic salt, such as by contacting potassium peroxymonosulfate and trifluorobutanone in the presence of cetyltrimethylammonium hydrogen sulfate (CTAHS) in a reaction mixture with the second (3β)-cholest-5-en-3-sulfate organic cationic salt. In certain cases, forming an oxidative species in situ with the second (3β)-cholest-5-en-3-sulfate organic cationic salt includes forming a dioxirane in situ with the second (3β)-cholest-5-en-3-sulfate organic cationic salt.

[0483] In certain cases, methods include forming a dioxirane in a separate reaction and adding the dioxirane to the second (3β)-cholest-5-en-3-sulfate organic cationic salt. In these cases, the potassium peroxymonosulfate may be contacted with the trifluorobutanone in the presence of cetyltrimethylammonium hydrogen sulfate (CTAHS) for a duration of 0.1 minute or more before contacting the reactive composition with the second (3β)-cholest-5-en-3-sulfate organic cationic salt, such as 1 minute or more, such as 2 minutes or more, such as 3 minutes or more, such as 5 minutes or more, and including 10 minutes or more), and the time may range from 0.01 minutes to 120 minutes, such as 0.1 minutes to 90 minutes or 0.5 minutes to 60 minutes. In certain instances, the potassium peroxymonosulfate may be contacted with trifluorobutanone in the presence of cetyltrimethylammonium hydrogen sulfate (CTAHS) to form the oxidative reactive composition, which is immediately contacted with the second (3β)-cholest-5-en-3-sulfate organic cationic salt.

[0484] The 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt is deoxygenated to produce a 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt (Structure IID) (Scheme IID1).

[0485] In some cases, generating 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt from the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt includes deoxygenation by contacting the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt with zinc. In certain instances, the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic 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 chosen from iodine and metal halides. In some cases, the metal halide is chosen from sodium iodide and lithium iodide. In some cases, the at least one acid is chosen from weak acids. In some cases, the at least one acid is chosen from acetic acid, hydrochloric acid, citric acid, para-toluene sulfonic acid, formic acid and methane sulfonic acid.

[0486] The amount of reagent used to deoxygenate the 25-hydroxy-(3β)-cholest-(5,6-epoxy)- 3-sulfate organic cationic salt may vary, where in some instances, 0.0001 equivalents or more of reagent relative to the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt is used, such as 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and including 10 equivalents or more, and may range from 0.001 equivalents to 10 equivalents, such as 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 equivalents to 10 equivalents, 1 equivalents to 8 equivalents, 1 equivalents to 6 equivalents, 1 equivalents to 4 equivalents, 1 equivalents 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.

[0487] The 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt may be deoxygenated at a temperature that ranges from 10 °C to 75 °C such as from 5 °C to 70 °C such asfrom -4 °C to 65 °C, such as from -3 °C to 60 °C, such as from -2 °C to 55 °C, such as from -1 °C to 50 °C and including from 0 °C to 25 °C. The reaction may be carried out for a duration that ranges from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, such as from 0.7 hours to 18 hours, such as from 0.8 hours to 17 hours, such as from 0.9 hours to 16 hours, and including from 1 hours to 15 hours.

[0488] In certain instances, methods include contacting the 25-hydroxy-(3β)-cholest-(5,6- epoxy)-3-sulfate organic cationic salt with zinc in the presence of iodine and acetic acid to generate the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt (Scheme IID2). S h IID2

[0489] In some cases, the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt (Structure IID) is contacted with a metal salt to produce the 25-hydroxy-(3β)-cholest-5-en-3-sulfate metal salt (Structure IIE) (Scheme IIE1).

[0490] In some cases, methods to produce the 25-hydroxy-(3β)-cholest-5-en-3-sulfate metal salt include contacting the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt with at least one sodium salt. In some cases, the at least one sodium salt is chosen from sodium acetate, sodium iodide, sodium chloride, sodium hydroxide and sodium methoxide. The 25-hydroxy-(3β)-cholest-5- en-3-sulfate organic cationic salt may be contacted with the metal salt at a temperature that ranges from -10 °C to 75 °C, such as from -5 °C to 70 °C, such as from -4 °C to 65 °C, such as from -3 °C to 60 °C, such as from -2 °C to 55 °C, such as from -1 °C to 50 °C, such as from 0 °C to 45 °C, such as from 5 °C to 40 °C, and including from 10 °C to 35 °C.

[0491] The reaction may be carried out for a duration that ranges from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, such as from 0.7 hours to 18 hours, such as from 0.8 hours to 17 hours, such as from 0.9 hours to 16 hours, and including from 1 hours to 15 hours. The amount of metal salt used relative to the 25-hydroxy-(3β)-cholest-5-en-3- sulfate organic cationic salt may vary and may be 0.0001 equivalents or more, such as 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and including 10 equivalents or more, and may range from 0.001 equivalents to 10 equivalents, such as 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 equivalents to 10 equivalents, 1 equivalents to 8 equivalents, 1 equivalents to 6 equivalents, 1 equivalents to 4 equivalents, 1 equivalents 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 1 equivalent to 7 equivalents.

[0492] In some cases, methods include contacting the 25-hydroxy-(3β)-cholest-5-en-3-sulfate pyridinium salt with sodium iodide to produce a 25-hydroxy-(3β)-cholest-5-en-3-sulfate sodium salt (Scheme IIE2).

[0493] In some embodiments, salts of 25HC3S, including crystalline salts of 25HC3S, have relatively high solubility, which is useful for making concentrated solutions, e.g., 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 25HC3S diethylammonium, which has relatively high solubility in water and saline; crystalline 25HC3S hydroxyethylpyrrolidinium, which has relatively high solubility in dextrose, and crystalline 25HC3S zinc, which has relatively high solubility in ethanol.

[0494] In some embodiments, salts of 25HC3S have relatively high solubility, which is useful for making concentrated solutions, e.g., for topical administration. For example, crystalline 25HC3S zinc has relatively high solubility in ethanol, which is a well-known topical penetration enhancer and may be used in transdermal delivery systems.

[0495] In some embodiments, salts of 25HC3S have relatively high solubility, which is useful for making concentrated solutions, e.g., for oral administration. For example, crystalline 25HC3S zinc has relatively high solubility in ethanol.

[0496] In some embodiments, salts of 25HC3S have relatively low solubility, which may be useful, e.g., 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 at least because of their low solubility in saline: crystalline 25HC3S potassium, crystalline 25HC3Scalcium, 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 at least because of their low solubility in 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 because of its low solubility in Fasted State Simulated Intestinal Fluid (FaSSIF) and in Fed State Simulated Intestinal Fluid (FeSSIF).

[0497] In some embodiments, salts of 25HC3S may be orally bioavailable. For example, salts of 25HC3S that have high solubility 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 have high solubility 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 have high solubility 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.

[0498] In some embodiments, the salt of 25HC3S has high solubility in a solvent, which may be used during synthesis. For instance, as shown in the Examples, the 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 may be used for synthesis, and for spray drying drug-polymer dispersions for amorphous preparation of the drug substance to enhance oral bioavailability. Crystalline 25HC3S hydroxyethylpyrrolidinium has relatively high solubility in acetonitrile (ACN).

[0499] In some embodiments, salts of 25HC3S are non-hygroscopic, which facilitates handling of the drug substance at ambient conditions and avoids the need for special precautions, such as the need to handle in low humidity conditions, or handle in a dry environment, or keep in a tightly closed container. The manufacturing step of weighing these drug substance salts at ambient conditions is non-problematic, since there is no concern of the weighing changes on the balance due to moisture uptake. Also, the containers of these salts can be opened and closed multiple times at ambient conditions without the concerns of the powder changing composition due to water absorptions. The non- hygroscopic nature of these salts also allows for the preparation of wet granulations for oral tablet and capsule products, and minimizes the possibility for a polymorph or other solid-form conversion such as hydrate formation. For instance, as shown in the Examples, crystalline 25HC3S t-butylammonium, crystalline 25HC3S benzathine, and crystalline 25HC3S choline salts of 25HC3S gain less than 0.5% water at 95% relative humidity. In addition, as shown in the DVS isotherms, when the crystalline 25HC3S t-butylammonium, crystalline 25HC3S benzathine, and crystalline 25HC3S choline salts of 25HC3S gain small amount of water as the relative humidity is increased to 95%, they reversibly lose all that water as the relative humidity is reduced to 5%.

[0500] In some embodiments, the salt of 25HC3S is highly crystalline, which can be advantageous from a processing perspective, for example. Crystalline 25HC3S hydroxyethylpyrrolidinium, crystalline 25HC3S diethylammonium, crystalline 25HC3S diethanolamine, crystalline 25HC3S t- butylammonium, crystalline 25HC3S benzathine, and crystalline 25HC3S choline salts are highly crystalline. The XRPD patterns were successfully indexed by single unit cells and provide a robust description of the crystalline forms through tentative crystallographic unit cell parameters. The formula unit volumes from the indexing results are all consistent with anhydrous forms and the expected salt stoichiometry.

[0501] In some embodiments, the salt of 25HC3S has a relatively high DSC (differential scanning calorimetry) endothermic transition (indicative of thermal degradation or solid-state transformation). While not wishing to be bound by theory, this property may allow for dry heat sterilization (e.g., 160° for 2 hours) of the drug substance, to facilitate preparation of sterilized dosage forms. For instance, as shown in the Examples, the first significant endothermic transition 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 may be sterilized by dry heat processing.

[0502] In some embodiments, the powder of salts of 25HC3S have relatively good flowability, which may be useful during manufacturing. For instance, as shown in the Examples, crystalline 25HC3S hydroxyethylammonium and crystalline 25HC3S lysine salts have relatively good flowability.

[0503] In some embodiments, the salts of 25HC3S have good temperature stability. As shown in the Examples, the following salts of 25HC3S have good, stressed stability at 80°C: crystalline 25HC3Sdiethylammonium, crystalline 25HC3S t-butylammonium, crystalline 25HC3S choline, crystalline 25HC3S diethanolamine, crystalline 25HC3S tromethammonium, and crystalline 25HC3S lysine.

[0504] In some embodiments, the counterion of 25HC3S may have beneficial effects in vivo. For instance, the choline salt may be beneficial because choline deficiency has also been implicated in such conditions related to fat accumulation and inflammation, with choline supplementation being suggested as potentially desirable in 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). The zinc salt of 25HC3S may be beneficial because zinc is an essential trace element required for cell growth, development and differentiation. Zinc deficiency is observed in many pathological conditions, including those for which 25HC3S is a potential therapeutic agent. Zinc supplementation may be beneficial in such conditions, such as those related to the skin and gastrointestinal tract; brain and central nervous system, immune, skeletal, and reproductive system (see, e.g., Himoto et al. Nutrients 2018, 10, 88; Grüngreiff et al. Annals of Hepatology 201615(1) 7-16; Mohammad et al. Nutr Clin Pract 2001227(1) 8-20). Magnesium deficiency is also observed in many pathological conditions, including those for which 25HC3S is a potential therapeutic agent. Magnesium supplementation may be beneficial in such conditions, such as celiac disease, Chrohn’s disease, type 2 diabetes, and conditions related to alcohol use (see, e.g., Agus et al., J Am Soc Nephrol, 10:1616-1622 (1999) and Martin et al., J Am Soc Nephrol, 20:2291-2295 (2009)).

[0505] In some embodiments, the salt of 25HC3S is one that has a beneficial combination of two or more such properties, which for instance renders it particularly useful for application as the active pharmaceutical ingredient in a pharmaceutical composition for a particular clinical application. Such a combination, for instance, may include two or more (e.g., at least 3, at least 4, or at least 5) properties selected from the group consisting of high solubility in a particular solvent (e.g. those studied in the Examples), low solubility in a particular solvent (e.g. those studied in the Examples), low hygroscopicity, high crystallinity, high DSC endothermic transition (allowing for dry heat sterilization), high flowability, high temperature stability, and beneficial in vivo effects of the counterion of the 25HC3S (particularly where those in vivo effects are beneficial in subjects suffering from the condition(s) for which a dosage form incorporating the salt of 25HC3S has been designed). CLAUSES

[0506] Clause 1. A salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt.

[0507] Clause 2. A salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt wherein the ammonium salt is not a choline salt.

[0508] Clause 3. A crystalline salt of 25HC3S other than a crystalline sodium salt of 25HC3S.

[0509] Clause 4. The crystalline salt of clause 3, other than a crystalline choline salt of 25HC3S.

[0510] Clause 5. The crystalline salt of clause 3, which is a crystalline 25HC3S metal salt.

[0511] Clause 6. The crystalline salt of clause 5, wherein the metal is in the +1 oxidation state.

[0512] Clause 7. The salt of any one of clauses 1 to 5, which is a 25HC3S metal salt wherein the metal is in either the +2 oxidation state or +3 oxidation state.

[0513] Clause 8. The crystalline metal salt of clause 7, wherein the metal is an alkaline earth metal.

[0514] Clause 9. The crystalline metal salt of clause 6, wherein the metal is selected from potassium, lithium, and rubidium.

[0515] Clause 10. A substantially pure salt of 25HC3S of any one of clauses 1 to 9.

[0516] Clause 11. Crystalline 25HC3S potassium.

[0517] Clause 12. The crystalline 25HC3S potassium of clause 11, having an x-ray powder diffraction pattern comprising a peak at about 2.2°2θ.

[0518] Clause 13. The crystalline 25HC3S potassium of clause 11 or 12, having an x-ray powder diffraction pattern comprising a peak at about 2.3°2θ.

[0519] Clause 14. The crystalline 25HC3S potassium of any one of clauses 11 to 13, having an x-ray powder diffraction pattern comprising a peak at about 8.8°2θ.

[0520] Clause 15. The crystalline 25HC3S potassium of any one of clauses 11 to 14, having an x-ray powder diffraction pattern comprising a peak at about 9.3°2θ.

[0521] Clause 16. The crystalline 25HC3S potassium of any one of clauses 11 to 15, having an x-ray powder diffraction pattern comprising a peak at about 15.3°2θ.

[0522] Clause 17. The crystalline 25HC3S potassium of any one of clauses 11 to 16, having an x-ray powder diffraction pattern comprising a peak at about 4.6°2θ.

[0523] Clause 18. The crystalline 25HC3S potassium of any one of clauses 11 to 17, having an x-ray powder diffraction pattern comprising a peak at about 14.7°2θ.

[0524] Clause 19. The crystalline 25HC3S potassium of any one of clauses 11 to 18, having an x-ray powder diffraction pattern comprising a peak at about 14.9°2θ.

[0525] Clause 20. The crystalline 25HC3S potassium of any one of clauses 11 to 19, having an x-ray powder diffraction pattern comprising a peak at about 16.1°2θ.

[0526] Clause 21. The crystalline 25HC3S potassium of any one of clauses 11 to 20, having an x-ray powder diffraction pattern comprising two peaks from about 2.2°2θ to about 2.3°2θ that are: (a) non-overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak.

[0527] Clause 22. The crystalline 25HC3S potassium of clause 11 having substantially the same x-ray powder diffraction pattern of Figure 2 or Figure 2A.

[0528] Clause 23. Crystalline 25HC3S calcium.

[0529] Clause 24. The crystalline 25HC3S calcium of clause 23, having an x-ray powder diffraction pattern comprising a peak at about 2.2°2θ.

[0530] Clause 25. The crystalline 25HC3S calcium of clause 23 or 24, having an x-ray powder diffraction pattern comprising a peak at about 4.5°2θ.

[0531] Clause 26. The crystalline 25HC3S calcium of any one of clauses 23 to 25, having an x-ray powder diffraction pattern comprising a peak at about 9.0°2θ.

[0532] Clause 27. The crystalline 25HC3S calcium of any one of clauses 23 to 26, having an x-ray powder diffraction pattern comprising a peak at about 10.0°2θ.

[0533] Clause 28. The crystalline 25HC3S calcium of any one of clauses 23 to 27, having an x-ray powder diffraction pattern comprising a peak at about 15.0°2θ.

[0534] Clause 29. The crystalline 25HC3S calcium of any one of clauses 23 to 28, having an x-ray powder diffraction pattern comprising a peak at about 15.1°2θ.

[0535] Clause 30. The crystalline 25HC3S calcium of any one of clauses 23 to 29, having an x-ray powder diffraction pattern comprising a peak at about 15.7°2θ.

[0536] Clause 31. The crystalline 25HC3S calcium of any one of clauses 23 to 30, having an x-ray powder diffraction pattern comprising a peak at about 15.4°2θ.

[0537] Clause 32. The crystalline 25HC3S calcium of any one of clauses 23 to 31, having an x-ray powder diffraction pattern comprising a peak at about 16.5°2θ.

[0538] Clause 33. The crystalline 25HC3S calcium of any one of clauses 23 to 32, having an x-ray powder diffraction pattern comprising a peak at about 18.0°2θ.

[0539] Clause 34. The crystalline 25HC3S calcium of any one of clauses 23 to 33, having an x-ray powder diffraction pattern comprising a peak at about 18.1°2θ.

[0540] Clause 35. The crystalline 25HC3S calcium of any one of clauses 23 to 34, having an x-ray powder diffraction pattern comprising a peak at about 18.4°2θ.

[0541] Clause 36. The crystalline 25HC3S calcium of any one of clauses 23 to 35, having an x-ray powder diffraction pattern comprising a peak at about 19.2°2θ.

[0542] Clause 37. The crystalline 25HC3S calcium of any one of clauses 23 to 36, having an x-ray powder diffraction pattern comprising two peaks from about 15.0°2θ to about 15.1°2θ that are: (a) non-overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak.

[0543] Clause 38. The crystalline 25HC3S calcium of clause 23 having substantially the same x-ray powder diffraction pattern of Figure 4 or Figure 4A.

[0544] Clause 39. Crystalline 25HC3S zinc.

[0545] Clause 40. The crystalline 25HC3S zinc of clause 39, having an x-ray powder diffraction pattern comprising a peak at about 2.1°2θ.

[0546] Clause 41. The crystalline 25HC3S zinc of clause 39 or 40, having an x-ray powder diffraction pattern comprising a peak at about 2.3°2θ.

[0547] Clause 42. The crystalline 25HC3S zinc of any one of clauses 39 to 41, having an x- ray powder diffraction pattern comprising a peak at about 6.0°2θ.

[0548] Clause 43. The crystalline 25HC3S zinc of any one of clauses 39 to 42, having an x- ray powder diffraction pattern comprising a peak at about 8.6°2θ.

[0549] Clause 44. The crystalline 25HC3S zinc of any one of clauses 39 to 43, having an x- ray powder diffraction pattern comprising a peak at about 8.9°2θ.

[0550] Clause 45. The crystalline 25HC3S zinc of any one of clauses 39 to 44, having an x- ray powder diffraction pattern comprising a peak at about 9.3°2θ.

[0551] Clause 46. The crystalline 25HC3S zinc of any one of clauses 39 to 45, having an x- ray powder diffraction pattern comprising a peak at about 15.1°2θ.

[0552] Clause 47. The crystalline 25HC3S zinc of any one of clauses 39 to 46, having an x- ray powder diffraction pattern comprising a peak at about 18.3°2θ.

[0553] Clause 48. The crystalline 25HC3S zinc of any one of clauses 39 to 47, having an x- ray powder diffraction pattern comprising a peak at about 18.8°2θ.

[0554] Clause 49. The crystalline 25HC3S zinc of any one of clauses 39 to 48, having an x- ray powder diffraction pattern comprising two peaks from about 2.1°2θ to about 2.3°2θ that are: (a) non-overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak.

[0555] Clause 50. The crystalline 25HC3S zinc of clause 40, having an x-ray powder diffraction pattern further comprising a peak at about 6.0°2θ.

[0556] Clause 51. The crystalline 25HC3S zinc of clause 40, having an x-ray powder diffraction pattern further comprising a peak at about 8.6°2θ.

[0557] Clause 52. The crystalline 25HC3S zinc of clause 40, having an x-ray powder diffraction pattern further comprising a peak at about 8.9°2θ.

[0558] Clause 53. The crystalline 25HC3S zinc of clause 40, having an x-ray powder diffraction pattern further comprising a peak at about 9.3°2θ.

[0559] Clause 54. The crystalline 25HC3S zinc of clause 40, having an x-ray powder diffraction pattern further comprising a peak at about 15.1°2θ.

[0560] Clause 55. The crystalline 25HC3S zinc of clause 40, having an x-ray powder diffraction pattern further comprising a peak at about 18.3°2θ.

[0561] Clause 56. The crystalline 25HC3S zinc of clause 40, having an x-ray powder diffraction pattern further comprising a peak at about 18.8°2θ.

[0562] Clause 57. The crystalline 25HC3S zinc of clause 50, having an x-ray powder diffraction pattern further comprising a peak at about 8.6°2θ.

[0563] Clause 58. The crystalline 25HC3S zinc of clause 50, having an x-ray powder diffraction pattern further comprising a peak at about 8.9°2θ.

[0564] Clause 59. The crystalline 25HC3S zinc of clause 50, having an x-ray powder diffraction pattern further comprising a peak at about 9.3°2θ.

[0565] Clause 60. The crystalline 25HC3S zinc of clause 50, having an x-ray powder diffraction pattern further comprising a peak at about 15.1°2θ.

[0566] Clause 61. The crystalline 25HC3S zinc of clause 50, having an x-ray powder diffraction pattern further comprising a peak at about 18.3°2θ.

[0567] Clause 62. The crystalline 25HC3S zinc of clause 50, having an x-ray powder diffraction pattern further comprising a peak at about 18.8°2θ.

[0568] Clause 63. The crystalline 25HC3S zinc of clause 57, having an x-ray powder diffraction pattern further comprising a peak at about 8.9°2θ.

[0569] Clause 64. The crystalline 25HC3S zinc of clause 57, having an x-ray powder diffraction pattern further comprising a peak at about 9.3°2θ.

[0570] Clause 65. The crystalline 25HC3S zinc of clause 57, having an x-ray powder diffraction pattern further comprising a peak at about 15.1°2θ.

[0571] Clause 66. The crystalline 25HC3S zinc of clause 57, having an x-ray powder diffraction pattern further comprising a peak at about 18.3°2θ.

[0572] Clause 67. The crystalline 25HC3S zinc of clause 57, having an x-ray powder diffraction pattern further comprising a peak at about 18.8°2θ.

[0573] Clause 68. The crystalline 25HC3S zinc of clause 63, having an x-ray powder diffraction pattern further comprising a peak at about 9.3°2θ.

[0574] Clause 69. The crystalline 25HC3S zinc of clause 63, having an x-ray powder diffraction pattern further comprising a peak at about 15.1°2θ.

[0575] Clause 70. The crystalline 25HC3S zinc of clause 63, having an x-ray powder diffraction pattern further comprising a peak at about 18.3°2θ.

[0576] Clause 71. The crystalline 25HC3S zinc of clause 63, having an x-ray powder diffraction pattern further comprising a peak at about 18.8°2θ.

[0577] Clause 72. The crystalline 25HC3S zinc of clause 68, having an x-ray powder diffraction pattern further comprising a peak at about 15.1°2θ.

[0578] Clause 73. The crystalline 25HC3S zinc of clause 68, having an x-ray powder diffraction pattern further comprising a peak at about 18.3°2θ.

[0579] Clause 74. The crystalline 25HC3S zinc of clause 68, having an x-ray powder diffraction pattern further comprising a peak at about 18.8°2θ.

[0580] Clause 75. The crystalline 25HC3S zinc of clause 72, having an x-ray powder diffraction pattern further comprising a peak at about 18.3°2θ.

[0581] Clause 76. The crystalline 25HC3S zinc of clause 72, having an x-ray powder diffraction pattern further comprising a peak at about 18.8°2θ.

[0582] Clause 77. The crystalline 25HC3S zinc of clause 39, having an x-ray powder diffraction pattern comprising 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.

[0583] Clause 78. The crystalline 25HC3S zinc of clause 39, having an x-ray powder diffraction pattern comprising 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.

[0584] Clause 79. The crystalline 25HC3S zinc of clause 39, having an x-ray powder diffraction pattern comprising 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.

[0585] Clause 80. The crystalline 25HC3S zinc of clause 39, having an x-ray powder diffraction pattern comprising 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.

[0586] Clause 81. The crystalline 25HC3S zinc of clause 39, having an x-ray powder diffraction pattern comprising 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.

[0587] Clause 82. The crystalline 25HC3S zinc of clause 39, having an x-ray powder diffraction pattern comprising one or more peaks selected from about 9.3°2θ, about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0588] Clause 83. The crystalline 25HC3S zinc of clause 39, having an x-ray powder diffraction pattern comprising one or more peaks selected from about 15.1°2θ, about 18.3°2θ, and about 18.8°2θ.

[0589] Clause 84. The crystalline 25HC3S zinc of clause 39, having an x-ray powder diffraction pattern comprising one or more peaks selected from about 18.3°2θ and about 18.8°2θ.

[0590] Clause 85. The crystalline 25HC3S zinc of clause 39, having an x-ray powder diffraction pattern comprising a peak at about 18.8°2θ.

[0591] Clause 86. The crystalline 25HC3S zinc of clause 39, having an x-ray powder diffraction pattern substantially the same as that found in Figure 85 or Figure 86.

[0592] Clause 87. Crystalline 25HC3S magnesium.

[0593] Clause 88. The crystalline 25HC3S magnesium of clause 87, having an x-ray powder diffraction pattern comprising a peak at about 2.2°2θ.

[0594] Clause 89. The crystalline 25HC3S magnesium of clause 87 or 88, having an x-ray powder diffraction pattern comprising a peak at about 4.4°2θ.

[0595] Clause 90. The crystalline 25HC3S magnesium of any one of clauses 87 to 89, having an x-ray powder diffraction pattern comprising a peak at about 6.6°2θ.

[0596] Clause 91. The crystalline 25HC3S magnesium of any one of clauses 87 to 90, having an x-ray powder diffraction pattern comprising a peak at about 8.9°2θ.

[0597] Clause 92. The crystalline 25HC3S magnesium of any one of clauses 87 to 91, having an x-ray powder diffraction pattern comprising a peak at about 15.1°2θ.

[0598] Clause 93. The crystalline 25HC3S magnesium of any one of clauses 87 to 92, having an x-ray powder diffraction pattern comprising a peak at about 15.6°2θ.

[0599] Clause 94. The crystalline 25HC3S magnesium of any one of clauses 87 to 93, having an x-ray powder diffraction pattern comprising a peak at about 16.4°2θ.

[0600] Clause 95. The crystalline 25HC3S magnesium of any one of clauses 87 to 94, having an x-ray powder diffraction pattern comprising a peak at about 17.6°2θ.

[0601] Clause 96. The crystalline 25HC3S magnesium of any one of clauses 87 to 95, having an x-ray powder diffraction pattern comprising a peak at about 17.8°2θ.

[0602] Clause 97. The crystalline 25HC3S magnesium of clause 87 having substantially the same x-ray powder diffraction pattern of Figure 6 or Figure 6A.

[0603] Clause 98. An organic salt of 25HC3S other than an ammonium salt of 25HC3S.

[0604] Clause 99. A crystalline organic salt of 25HC3S.

[0605] Clause 100. The organic salt of clause 98 or 99 which is an amine salt of 25HC3S.

[0606] Clause 101. The crystalline salt of 25HC3S of clause 100.

[0607] Clause 102. The salt of 25HC3S of clause 100 or 101 wherein the amine is aliphatic, cyclic, aromatic, or a combination thereof.

[0608] Clause 103. The salt of 25HC3S of any one of clauses 100 to 102 wherein the amine comprises a primary, secondary, or tertiary amine group, or a combination thereof.

[0609] Clause 104. The salt of any one of clauses 100 to 103 wherein the amine is substituted.

[0610] Clause 105. The salt of clause 104, wherein the substitution is one or more alcohol, alkyl, aryl, or further amine groups.

[0611] Clause 106. The salt of clause 105, wherein the substitution is one or more alcohol groups.

[0612] Clause 107. The salt of clause 106, wherein the one or more alcohol groups are selected from primary, secondary, and tertiary alcohol groups.

[0613] Clause 108. The salt of any one of clauses 100 to 107, wherein the amine group contains 1, 2, 3, or 4 carbon atoms covalently linked to the nitrogen of the amine group.

[0614] Clause 109. The salt of clause 108, wherein the linked carbon atoms include a cyclic alkyl.

[0615] Clause 110. The organic salt of any one of clauses 100 to 109 wherein the amine is an amino acid.

[0616] Clause 111. The salt of clause 110, wherein the amino acid is naturally occurring amino acid, for example a proteinogenic amino acid.

[0617] Clause 112. The salt of clause 110, wherein the amino acid is a non-naturally occurring amino acid.

[0618] Clause 113. The salt of 25HC3S of clause 105, wherein the substitution is an aryl group.

[0619] Clause 114. The salt of 25HC3S of clause 113, wherein the aryl group comprises phenyl or benzyl.

[0620] Clause 115. The salt of 25HC3S of any one of clauses 100 to 114, comprising a cyclic amine.

[0621] Clause 116. The salt of any one of clauses 100 to 114, wherein the oxidation state of the amine is +1.

[0622] Clause 117. The salt of any one of clauses 100 to 114, wherein the oxidation state of the amine is +2.

[0623] Clause 118. The salt of any one of clauses 100 to 114, wherein the amine comprises one or more alcohol groups.

[0624] Clause 119. The salt of clause 118, wherein the amine comprises two alcohol groups.

[0625] Clause 120. The salt of clause 118, wherein the amine comprises three alcohol groups.

[0626] Clause 121. The salt of clause 118, wherein the amine comprises four or more alcohol groups.

[0627] Clause 122. The salt of clause 118, wherein the amine comprises five alcohol groups.

[0628] Clause 123. The salt of any one of clauses 118 to 122 wherein the alcohol groups are primary alcohols.

[0629] Clause 124. The salt of any one of clauses 100 to 123, wherein the amine comprises two amine groups.

[0630] Clause 125. The salt of clause 124, wherein at least one amine group is a secondary amine group.

[0631] Clause 126. The salt of clause 125, wherein two amine groups are secondary amine groups.

[0632] Clause 127. The salt of any one of clauses 124 to 216 further comprising at least one aryl group.

[0633] Clause 128. The salt of any one of clauses 124 to 216, further comprising at least two aryl groups.

[0634] Clause 129. The salt of clause 108 or 109, wherein the at least one amine group is bonded to one, two, or three independently substituted or unsubstituted alkyl groups.

[0635] Clause 130. The salt of clause 129, wherein the alkyl groups contain 1, 2, 3, 4, 5, or 6 carbon atoms.

[0636] Clause 131. The salt of clause 130, wherein at least one alkyl group is substituted with an aryl group.

[0637] Clause 132. The salt of clause 131, wherein the aryl group is phenyl or benzyl.

[0638] Clause 133. 25HC3S hydroxyethylammonium.

[0639] Clause 134. Crystalline 25HC3S hydroxyethylammonium.

[0640] Clause 135. The crystalline 25HC3S hydroxyethylammonium of clause 134, having an x-ray powder diffraction pattern comprising a peak at about 2.1°2θ.

[0641] Clause 136. The crystalline 25HC3S hydroxyethylammonium of clause 135, having an x-ray powder diffraction pattern further comprising a peak at about 8.6°2θ.

[0642] Clause 137. The crystalline 25HC3S hydroxyethylammonium of clause 134 having an x-ray powder diffraction pattern substantially the same as that of Figure 8 or Figure 8A.

[0643] Clause 138. 25HC3S tromethammonium.

[0644] Clause 139. Crystalline 25HC3S tromethammonium.

[0645] Clause 140. The crystalline 25HC3S tromethammonium salt of clause 139, having an x-ray powder diffraction pattern comprising a peak at about 1.9°2θ.

[0646] Clause 141. The crystalline 25HC3S tromethammonium salt of clause 139 or 140, having an x-ray powder diffraction pattern comprising a peak at about 2.1°2θ.

[0647] Clause 142. The crystalline 25HC3S tromethammonium salt of any one of clauses 139 to 141, having an x-ray powder diffraction pattern comprising a peak at about 3.8°2θ.

[0648] Clause 143. The crystalline 25HC3S tromethammonium salt of any one of clauses 139 to 142, having an x-ray powder diffraction pattern comprising a peak at about 4.2°2θ and / or about 15.4°2θ.

[0649] Clause 144. The crystalline 25HC3S tromethammonium salt of any one of clauses 139 to 143, having an x-ray powder diffraction pattern comprising two peaks from about 1.9°2θ to about 2.1°2θ that are: (a) non-overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak.

[0650] Clause 145. The crystalline 25HC3S tromethammonium salt of clause 139 having an x-ray powder diffraction pattern substantially the same as that of Figure 14 or Figure 14A.

[0651] Clause 146. 25HC3S lysine.

[0652] Clause 147. Crystalline 25HC3S lysine.

[0653] Clause 148. The crystalline 25HC3S lysine salt of clause 147, having an x-ray powder diffraction pattern comprising a peak at about 1.5°2θ.

[0654] Clause 149. The crystalline 25HC3S lysine salt of clause 147 or 148, having an x-ray powder diffraction pattern comprising a peak at about 7.0°2θ.

[0655] Clause 150. The crystalline 25HC3S lysine salt of any one of clauses 147 to 149, having an x-ray powder diffraction pattern comprising a peak at about 10.7°2θ.

[0656] Clause 151. The crystalline 25HC3S lysine salt of any one of clauses 147 to 150, having an x-ray powder diffraction pattern comprising a peak at about 11.8°2θ.

[0657] Clause 152. The crystalline 25HC3S lysine salt of any one of clauses 147 to 151, having an x-ray powder diffraction pattern comprising a peak at about 16.8°2θ.

[0658] Clause 153. The crystalline 25HC3S lysine salt of any one of clauses 147 to 152, having an x-ray powder diffraction pattern comprising a peak at about 3.2°2θ.

[0659] Clause 154. The crystalline 25HC3S lysine salt of any one of clauses 147 to 153, having an x-ray powder diffraction pattern comprising a peak at about 10.0°2θ.

[0660] Clause 155. The crystalline 25HC3S lysine salt of any one of clauses 147 to 154, having an x-ray powder diffraction pattern comprising a peak at about 12.2°2θ.

[0661] Clause 156. The crystalline 25HC3S lysine salt of any one of clauses 147 to 155, having an x-ray powder diffraction pattern comprising a peak at about 15.2°2θ.

[0662] Clause 157. The crystalline 25HC3S lysine salt of clause 147 having an x-ray powder diffraction pattern substantially the same as that of Figure 24 or Figure 24A.

[0663] Clause 158. 25HC3S meglumine.

[0664] Clause 159. Crystalline 25HC3S meglumine.

[0665] Clause 160. The crystalline 25HC3S meglumine salt of clause 159, having an x-ray powder diffraction pattern comprising a peak at about 1.7°2θ.

[0666] Clause 161. The crystalline 25HC3S meglumine salt of clause 159 or 160, having an x-ray powder diffraction pattern comprising a peak at about 3.5°2θ.

[0667] Clause 162. The crystalline 25HC3S meglumine salt of any one of clauses 159 to 161, having an x-ray powder diffraction pattern comprising a peak at about 5.2°2θ.

[0668] Clause 163. The crystalline 25HC3S meglumine salt of any one of clauses 159 to 162, having an x-ray powder diffraction pattern comprising a peak at about 14.9°2θ.

[0669] Clause 164. The crystalline 25HC3S meglumine salt of any one of clauses 159 to 163, having an x-ray powder diffraction pattern comprising a peak at about 24.2°2θ.

[0670] Clause 165. The crystalline 25HC3S meglumine salt of any one of clauses 159 to 164, having an x-ray powder diffraction pattern comprising a peak at about 8.6°2θ.

[0671] Clause 166. The crystalline 25HC3S meglumine salt of any one of clauses 159 to 165, having an x-ray powder diffraction pattern comprising a peak at about 14.5°2θ.

[0672] Clause 167. The crystalline 25HC3S meglumine salt of any one of clauses 159 to 166, having an x-ray powder diffraction pattern comprising a peak at about 15.1°2θ.

[0673] Clause 168. The crystalline 25HC3S meglumine salt of any one of clauses 159 to 167, having an x-ray powder diffraction pattern comprising a peak at about 17.5°2θ.

[0674] Clause 169. The crystalline 25HC3S meglumine salt of any one of clauses 159 to 168, having an x-ray powder diffraction pattern comprising a peak at about 18.2°2θ.

[0675] Clause 170. The crystalline 25HC3S meglumine salt of clause 159, having an x-ray powder diffraction pattern substantially the same as that of Figure 12 or Figure 12A.

[0676] Clause 171. 25HC3S hydroxyethylpyrrolidinium.

[0677] Clause 172. Crystalline 25HC3S hydroxyethylpyrrolidinium.

[0678] Clause 173. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of clause 172, having an x-ray powder diffraction pattern comprising a peak at about 3.8°2θ.

[0679] Clause 174. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of clause 172 or 173, having an x-ray powder diffraction pattern comprising a peak at about 7.5°2θ.

[0680] Clause 175. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 174, having an x-ray powder diffraction pattern comprising a peak at about 7.6°2θ.

[0681] Clause 176. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 175, having an x-ray powder diffraction pattern comprising a peak at about 8.2°2θ.

[0682] Clause 177. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 176, having an x-ray powder diffraction pattern comprising a peak at about 8.6°2θ.

[0683] Clause 178. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 177, having an x-ray powder diffraction pattern comprising a peak at about 12.4°2θ.

[0684] Clause 179. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 178, having an x-ray powder diffraction pattern comprising a peak at about 13.3°2θ.

[0685] Clause 180. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 179, having an x-ray powder diffraction pattern comprising a peak at about 15.0°2θ.

[0686] Clause 181. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 180, having an x-ray powder diffraction pattern comprising a peak at about 10.5°2θ.

[0687] Clause 182. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 181, having an x-ray powder diffraction pattern comprising a peak at about 15.3°2θ.

[0688] Clause 183. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 182, having an x-ray powder diffraction pattern comprising a peak at about 15.6°2θ.

[0689] Clause 184. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 183, having an x-ray powder diffraction pattern comprising a peak at about 16.3°2θ.

[0690] Clause 185. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 184, having an x-ray powder diffraction pattern comprising a peak at about 16.7°2θ.

[0691] Clause 186. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 185, having an x-ray powder diffraction pattern comprising a peak at about 20.9°2θ.

[0692] Clause 187. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 186, having an x-ray powder diffraction pattern comprising two peaks from about 7.5°2θ to about 7.6°2θ that are: (a) non-overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak.

[0693] Clause 188. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 187, having an x-ray powder diffraction pattern comprising two peaks from about 8.2°2θ to about 8.6°2θ that are: (a) non-overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak.

[0694] Clause 189. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of clause 172, having an x-ray powder diffraction pattern substantially the same as that of Figure 10.

[0695] Clause 190. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of clauses 172 to 189, wherein the unit cell of the crystalline salt is triclinic.

[0696] Clause 191. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of clause 190, wherein the formula volume of the unit cell is about 3417Å3 / cell.

[0697] Clause 192. 25HC3S diethylammonium.

[0698] Clause 193. Crystalline 25HC3S diethylammonium.

[0699] Clause 194. The crystalline 25HC3S diethylammonium of clause 193, having an x-ray powder diffraction pattern comprising a peak at about 3.8°2θ.

[0700] Clause 195. The crystalline 25HC3S diethylammonium of clause 193 or 194, having an x-ray powder diffraction pattern comprising a peak at about 7.9°2θ.

[0701] Clause 196. The crystalline 25HC3S diethylammonium of any one of clauses 193 to 195, having an x-ray powder diffraction pattern comprising a peak at about 8.6°2θ.

[0702] Clause 197. The crystalline 25HC3S diethylammonium of any one of clauses 193 to 196, having an x-ray powder diffraction pattern comprising a peak at about 9.6°2θ.

[0703] Clause 198. The crystalline 25HC3S diethylammonium of any one of clauses 193 to 197, having an x-ray powder diffraction pattern comprising a peak at about 10.9°2θ.

[0704] Clause 199. The crystalline 25HC3S diethylammonium of any one of clauses 193 to 198, having an x-ray powder diffraction pattern comprising a peak at about 12.3°2θ.

[0705] Clause 200. The crystalline 25HC3S diethylammonium of any one of clauses 193 to 199, having an x-ray powder diffraction pattern comprising a peak at about 15.4°2θ.

[0706] Clause 201. The crystalline 25HC3S diethylammonium of any one of clauses 193 to 200, having an x-ray powder diffraction pattern comprising a peak at about 17.2°2θ.

[0707] Clause 202. The crystalline 25HC3S diethylammonium of clause 193, having an x-ray powder diffraction pattern substantially the same as that of Figure 18.

[0708] Clause 203. The crystalline 25HC3S diethylammonium of any one of clauses 193 to 202, wherein the unit cell of the crystalline salt is orthorhombic.

[0709] Clause 204. The crystalline 25HC3S diethylammonium salt of clause 203, wherein the formula volume of the unit cell is about 3293Å3 / cell.

[0710] Clause 205. 25HC3S diethanolamine.

[0711] Clause 206. Crystalline 25HC3S diethanolamine.

[0712] Clause 207. The crystalline 25HC3S diethanolamine of clause 206, having an x-ray powder diffraction pattern comprising a peak at about 3.8°2θ.

[0713] Clause 208. The crystalline 25HC3S diethanolamine of clause 206 or 207, having an x-ray powder diffraction pattern comprising a peak at about 7.7°2θ.

[0714] Clause 209. The crystalline 25HC3S diethanolamine of any one of clauses 206 to 208, having an x-ray powder diffraction pattern comprising a peak at about 8.1°2θ.

[0715] Clause 210. The crystalline 25HC3S diethanolamine of any one of clauses 206 to 209, having an x-ray powder diffraction pattern comprising a peak at about 8.8°2θ.

[0716] Clause 211. The crystalline 25HC3S diethanolamine of any one of clauses 206 to 210, having an x-ray powder diffraction pattern comprising a peak at about 14.6°2θ.

[0717] Clause 212. The crystalline 25HC3S diethanolamine of any one of clauses 206 to 211, having an x-ray powder diffraction pattern comprising a peak at about 15.2°2θ.

[0718] Clause 213. The crystalline 25HC3S diethanolamine of any one of clauses 206 to 212, having an x-ray powder diffraction pattern comprising two peaks from about 7.7°2θ to about 8.1°2θ that are: (a) non-overlapping; (b) partly overlapping; or (c) superimposed such as to appear as a single peak.

[0719] Clause 214. The crystalline 25HC3S diethanolamine of clause 206, having an x-ray powder diffraction pattern substantially the same as that of Figure 16.

[0720] Clause 215. The crystalline 25HC3S diethanolamine of any one of clauses 206 to 214, wherein the unit cell of the crystalline salt is monoclinic.

[0721] Clause 216. The crystalline 25HC3S diethanolamine salt of clause 215, wherein the formula volume of the unit cell is about 3294Å3 / cell.

[0722] Clause 217. 25HC3S t-butylammonium.

[0723] Clause 218. Crystalline 25HC3S t-butylammonium.

[0724] Clause 219. The crystalline 25HC3S t-butylammonium of clause 218, having an x-ray powder diffraction pattern comprising a peak at about 4.0°2θ.

[0725] Clause 220. The crystalline 25HC3S t-butylammonium of clause 218 or 219, having an x-ray powder diffraction pattern comprising a peak at about 8.0°2θ.

[0726] Clause 221. The crystalline 25HC3S t-butylammonium of any one of clauses 218 to 220, having an x-ray powder diffraction pattern comprising a peak at about 11.5°2θ.

[0727] Clause 222. The crystalline 25HC3S t-butylammonium of any one of clauses 218 to 221, having an x-ray powder diffraction pattern comprising a peak at about 12.2°2θ.

[0728] Clause 223. The crystalline 25HC3S t-butylammonium of any one of clauses 218 to 222, having an x-ray powder diffraction pattern comprising a peak at about 14.4°2θ.

[0729] Clause 224. The crystalline 25HC3S t-butylammonium of any one of clauses 218 to 223, having an x-ray powder diffraction pattern comprising a peak at about 15.4°2θ.

[0730] Clause 225. The crystalline 25HC3S t-butylammonium of any one of clauses 218 to 224, having an x-ray powder diffraction pattern comprising a peak at about 16.3°2θ.

[0731] Clause 226. The crystalline 25HC3S t-butylammonium of any one of clauses 218 to 225, having an x-ray powder diffraction pattern comprising a peak at about 10.3°2θ.

[0732] Clause 227. The crystalline 25HC3S t-butylammonium of any one of clauses 218 to 226, having an x-ray powder diffraction pattern comprising a peak at about 10.5°2θ.

[0733] Clause 228. The crystalline 25HC3S t-butylammonium of any one of clauses 218 to 227, having an x-ray powder diffraction pattern comprising a peak at about 13.8°2θ.

[0734] Clause 229. The crystalline 25HC3S t-butylammonium of any one of clauses 218 to 228, having an x-ray powder diffraction pattern comprising a peak at about 16.8°2θ.

[0735] Clause 230. The crystalline 25HC3S t-butylammonium of any one of clauses 218 to 229, having an x-ray powder diffraction pattern comprising a peak at about 17.0°2θ.

[0736] Clause 231. The crystalline 25HC3S t-butylammonium of any one of clauses 218 to 230, having an x-ray powder diffraction pattern comprising a peak at about 17.4°2θ.

[0737] Clause 232. The crystalline 25HC3S t-butylammonium of clause 218, having an x-ray powder diffraction pattern substantially the same as that of Figure 20.

[0738] Clause 233. The crystalline 25HC3S t-butylammonium of any one of clauses 218 to 232, wherein the unit cell of the crystalline salt is monoclinic.

[0739] Clause 234. The crystalline 25HC3S t-butylammonium salt of clause 233, wherein the formula volume of the unit cell is about 3333Å3 / cell.

[0740] Clause 235. 25HC3S benzathine.

[0741] Clause 236. Crystalline 25HC3S benzathine.

[0742] Clause 237. The crystalline 25HC3S benzathine of clause 236, having an x-ray powder diffraction pattern comprising a peak at about 4.1°2θ.

[0743] Clause 238. The crystalline 25HC3S benzathine of clause 236 or 237, having an x-ray powder diffraction pattern comprising a peak at about 6.9°2θ.

[0744] Clause 239. The crystalline 25HC3S benzathine of any one of clauses 236 to 238, having an x-ray powder diffraction pattern comprising a peak at about 8.2°2θ.

[0745] Clause 240. The crystalline 25HC3S benzathine of any one of clauses 236 to 239, having an x-ray powder diffraction pattern comprising a peak at about 12.3°2θ.

[0746] Clause 241. The crystalline 25HC3S benzathine of any one of clauses 236 to 240, having an x-ray powder diffraction pattern comprising a peak at about 16.2°2θ.

[0747] Clause 242. The crystalline 25HC3S benzathine of any one of clauses 236 to 241, having an x-ray powder diffraction pattern comprising a peak at about 7.3°2θ.

[0748] Clause 243. The crystalline 25HC3S benzathine of any one of clauses 236 to 242, having an x-ray powder diffraction pattern comprising a peak at about 15.1°2θ.

[0749] Clause 244. The crystalline 25HC3S benzathine of any one of clauses 236 to 243, having an x-ray powder diffraction pattern comprising a peak at about 16.4°2θ.

[0750] Clause 245. The crystalline 25HC3S benzathine of any one of clauses 236 to 244, having an x-ray powder diffraction pattern comprising a peak at about 17.8°2θ.

[0751] Clause 246. The crystalline 25HC3S benzathine of any one of clauses 236 to 245, having an x-ray powder diffraction pattern comprising a peak at about 20.4°2θ.

[0752] Clause 247. The crystalline 25HC3S benzathine of clause 236, having an x-ray powder diffraction pattern substantially the same as that of Figure 22.

[0753] Clause 248. The crystalline 25HC3S benzathine of any one of clauses 236 to 247, wherein the unit cell of the crystalline salt is monoclinic.

[0754] Clause 249. The crystalline 25HC3S benzathine of clause 248, wherein the formula volume of the unit cell is about 3394Å3 / cell.

[0755] Clause 250. 25HC3S choline.

[0756] Clause 251. Crystalline 25HC3S choline.

[0757] Clause 252. The crystalline 25HC3S choline of clause 251, having an x-ray powder diffraction pattern comprising a peak at about 3.9°2θ.

[0758] Clause 253. The crystalline 25HC3S choline of clause 251 or 252, having an x-ray powder diffraction pattern comprising a peak at about 7.8°2θ.

[0759] Clause 254. The crystalline 25HC3S choline of any one of clauses 251 to 253, having an x-ray powder diffraction pattern com...

Claims

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

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

3. A crystalline salt of 25HC3S other than a crystalline sodium salt of 25HC3S.

4. The crystalline salt of claim 3, other than a crystalline choline salt of 25HC3S.

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

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

7. The salt of any one of claims 1 to 5, which is a 25HC3S metal salt wherein the metal is in the +2 oxidation state.

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

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

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

11. Crystalline 25HC3S potassium.

12. Crystalline 25HC3S zinc.

13. An organic salt of 25HC3S other than an ammonium salt of 25HC3S.

14. A crystalline organic salt of 25HC3S.

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

16. A method of treating or preventing one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation, comprising administering to a patient in need thereof an effective amount of a 25HC3S compound of any one of claims 1-14.

17. A 25HC3S compound of any one of claims 1-14 for use as a medicament.

18. A 25HC3S compound of any one of claims 1-14 for use in a method for treating or preventing one or more of 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation.

19. Use of a 25HC3S compound of 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 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, a diabetes condition, an autoimmune condition, an inflammatory condition, a neurological condition, Epstein Barr virus-related growth, and conditions related to fat accumulation and inflammation.

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