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

Abstract

The present application provides a variety of salts of 25HC3S, including crystalline salts of 25HC3S. Provided herein are pharmaceutical formulations of the salts of the invention, and methods of treating or preventing diseases therewith, the diseases such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, and the like. Nervous system disorders, EB virus related growth, and disorders associated with fat accumulation and inflammation. Methods of making the salts of the present application are also provided.

Description

[0001] introduction

[0002] The cholesterol metabolite 5-cholestene-3β-25-diol-3-sulfate ("25HC3S") has been previously shown to reduce lipid biosynthesis and increase cholesterol secretion and degradation and is useful for treating and preventing one or more of the following diseases: non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic disorders, autoimmune disorders, inflammatory disorders, neurological disorders, Epstein-Barr virus-associated growth, and disorders associated with fat accumulation and inflammation.

[0003] Cholesterol is used by the body to make and repair cell membranes, as well as to synthesize steroid hormones and vitamin D. It is converted into bile acids in the liver. Cholesterol comes from both endogenous and exogenous sources. The average American consumes about 450 mg of cholesterol per day, and produces an additional 500 mg to 1000 mg of cholesterol in the liver and other tissues. Another source is the 500 mg to 1000 mg of bile cholesterol secreted into the intestine each day, of which about 50% is reabsorbed (enterohepatic circulation).

[0004] High serum lipid levels (hypercholesterolemia and hypertriglyceridemia) are associated with the accumulation of cholesterol in arterial walls and can lead to NAFLD and atherosclerosis. The characteristic plaques of atherosclerosis inhibit blood flow and promote thrombosis, which may ultimately cause death or severe disability due to heart attack and / or stroke. A variety of therapeutic agents have been developed to treat hyperlipidemia and are widely prescribed by doctors. Unfortunately, only about 35% of patients respond to existing therapies.

[0005] Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the United States. The condition is associated with obesity, adult-onset type 2 diabetes, a sedentary lifestyle, and a high-fat diet. The early stage of NAFLD, fatty liver, is reversible with appropriate treatment. However, if left uncontrolled, it can progress to inflammation of the liver cells (non-alcoholic steatohepatitis, or NASH), which is much more difficult to treat. If left untreated, NASH leads to irreversible scarring of liver tissue (fatty necrosis) and can potentially lead to cirrhosis, liver failure, and liver cancer.

[0006] 25HC3S has been disclosed as a pharmaceutically acceptable salt, such as a sodium salt (e.g., U.S. Pat. No. 10,144,759 and Ogawa et al., Steroids, 74, 81-87 (2009)). Crystalline solids are often more advantageous in terms of processing, storage, and stability than non-crystalline solids. However, energetics may not be favorable for rapid formation of a suitable crystalline solid, and polymorphism may make it impractical to produce a stable crystalline solid of a particular active pharmaceutical ingredient. Also provided herein are 15 salts, including crystalline salts of 25HC3S, and methods of preparing and using the salts.

[0007] Overview

[0008] In some aspects of the present application, crystalline salts of 25HC3S other than crystalline sodium 25HC3S are provided.

[0009] In other aspects of the present application, substantially pure crystalline salts of 25HC3S other than the sodium salt are provided.

[0010] In other aspects of the present application, methods for preparing crystalline salts of 25HC3S other than the sodium salt are provided.

[0011] In some aspects of the present application, metal salts of 25HC3S other than the sodium salt are provided.

[0012] In some aspects of the present application, salts of 25HC3S other than (i) alkali metal salts or (ii) ammonium salts are provided.

[0013] In another aspect of the present application, there is provided a salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt prepared by the method of the present application.

[0014] In another aspect of the present application, a crystalline salt of 25HC3S other than the sodium salt prepared by the method of the present application is provided.

[0015] In some aspects of the present application, crystalline salts of 25HC3S other than crystalline 25HC3S sodium and crystalline 25HC3S choline are provided.

[0016] In some aspects of the present application, substantially pure crystalline salts of 25HC3S other than crystalline 25HC3S sodium and crystalline 25HC3S choline are provided.

[0017] In other aspects of the present application, methods for preparing crystalline salts of 25HC3S other than the sodium salt and the choline salt are provided.

[0018] In yet another aspect of the present application, there is provided a crystalline salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt other than a choline salt.

[0019] In another aspect of the present application, a method for preparing a crystalline salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt other than a choline salt is provided.

[0020] In another aspect of the present application, there is provided a crystalline salt of 25HC3S other than the sodium salt and the choline salt prepared by the method of the present application.

[0021] In yet another aspect of the present application, a pharmaceutical composition comprising a crystalline salt of 25HC3S other than the sodium salt and a pharmaceutically acceptable excipient is provided.

[0022] In yet another aspect of the present application, there is provided a pharmaceutical composition comprising a salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt and a pharmaceutically acceptable excipient.

[0023] In another aspect of the present application, there is provided a pharmaceutical composition comprising a crystalline salt of 25HC3S other than the sodium salt and other than the choline salt and a pharmaceutically acceptable excipient.

[0024] In yet another aspect of the present application, there is provided a pharmaceutical composition comprising a salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt other than a choline salt.

[0025] In another aspect of the present application, there is provided a method for treating or preventing one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, nervous system conditions, Epstein-Barr virus-associated growth, and conditions associated with fat accumulation and inflammation, comprising administering to a patient in need thereof an effective amount of a compound of a crystalline salt of 25HC3S other than the sodium salt.

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

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

[0028] In another aspect of the present application, a method for treating or preventing one or more of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, nervous system conditions, Epstein-Barr virus-associated growth, and conditions associated with fat accumulation and inflammation is provided, comprising administering to a patient in need thereof an effective amount of a compound of a salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt other than a choline salt. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 is an X-ray powder diffraction pattern of crystalline potassium 25HC3S.

[0031] FIG1a is a magnified X-ray powder diffraction pattern of crystalline potassium 25HC3S.

[0032] FIG2 is a peak-picked X-ray powder diffraction pattern of crystalline potassium 25HC3S.

[0033] FIG2a is an enlarged peak-selected X-ray powder diffraction pattern of crystalline potassium 25HC3S.

[0034] FIG3 is an X-ray powder diffraction pattern of crystalline calcium 25HC3S.

[0035] FIG3a is a magnified X-ray powder diffraction pattern of crystalline calcium 25HC3S.

[0036] FIG4 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S calcium.

[0037] FIG4a is an enlarged peak-selected X-ray powder diffraction pattern of crystalline 25HC3S calcium.

[0038] FIG5 is an X-ray powder diffraction pattern of crystalline 25HC3S magnesium.

[0039] FIG5a is a magnified X-ray powder diffraction pattern of crystalline 25HC3S magnesium.

[0040] FIG6 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S magnesium.

[0041] FIG6a is an enlarged peak-selected X-ray powder diffraction pattern of crystalline 25HC3S magnesium.

[0042] FIG7 is an X-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium.

[0043] FIG7a is a magnified X-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium.

[0044] FIG8 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium.

[0045] FIG8a is an enlarged peak-selected X-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium.

[0046] FIG9 is an X-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylpyrrolidinium.

[0047] FIG10 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylpyrrolidinium.

[0048] FIG11 is an X-ray powder diffraction pattern of crystalline 25HC3S meglumine.

[0049] FIG11a is a magnified X-ray powder diffraction pattern of crystalline 25HC3S meglumine.

[0050] FIG12 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S meglumine.

[0051] FIG12a is an enlarged peak-selected X-ray powder diffraction pattern of crystalline 25HC3S meglumine.

[0052] FIG13 is an X-ray powder diffraction pattern of crystalline 25HC3S tris(hydroxymethyl)methylammonium.

[0053] FIG13a is a magnified X-ray powder diffraction pattern of crystalline 25HC3S tris(hydroxymethyl)methylammonium.

[0054] FIG14 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S tris(hydroxymethyl)methylammonium.

[0055] FIG14a is an enlarged peak-selected X-ray powder diffraction pattern of crystalline 25HC3S tris(hydroxymethyl)methylammonium.

[0056] FIG15 is an X-ray powder diffraction pattern of crystalline 25HC3S diethanolamine.

[0057] FIG16 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S diethanolamine.

[0058] FIG17 is an X-ray powder diffraction pattern of crystalline 25HC3S diethylammonium.

[0059] FIG18 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S diethylammonium.

[0060] FIG19 is an X-ray powder diffraction pattern of crystalline 25HC3S tert-butylammonium.

[0061] FIG20 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S tert-butylammonium.

[0062] FIG21 is an X-ray powder diffraction pattern of crystalline 25HC3S benzathine.

[0063] FIG22 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S benzathine.

[0064] FIG23 is an X-ray powder diffraction pattern of crystalline 25HC3S lysine.

[0065] FIG23a is a magnified X-ray powder diffraction pattern of crystalline 25HC3S lysine.

[0066] FIG24 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S lysine.

[0067] Figure 24a is an enlarged peak-selected X-ray powder diffraction pattern of crystalline 25HC3S lysine.

[0068] FIG25 is a TGA thermogram and a DSC thermogram of crystalline potassium 25HC3S.

[0069] FIG26 is a DVS isotherm of crystalline potassium 25HC3S.

[0070] FIG27 is an overlay of the x-ray powder diffraction patterns of crystalline potassium 25HC3S before and after DVS.

[0071] Figure 28 is 25HC3S potassium 1 H-NMR spectrum.

[0072] FIG29 is a TGA thermogram and a DSC thermogram of crystalline 25HC3S calcium.

[0073] FIG30 is a DVS isotherm of crystalline calcium 25HC3S.

[0074] FIG31 is an overlay of the x-ray powder diffraction patterns of crystalline calcium 25HC3S before and after DVS.

[0075] Figure 32 is 25HC3S calcium 1 H-NMR spectrum.

[0076] FIG33 is a TGA thermogram and a DSC thermogram of crystalline 25HC3S magnesium.

[0077] FIG34 is a DVS isotherm of crystalline 25HC3S magnesium.

[0078] FIG35 is an overlay of the x-ray powder diffraction patterns of crystalline 25HC3S magnesium before and after DVS.

[0079] Figure 36 is 25HC3S magnesium 1 H-NMR spectrum.

[0080] FIG37 is a TGA thermogram and a DSC thermogram of crystalline 25HC3S hydroxyethylammonium.

[0081] Figure 38 is a DVS isotherm of crystalline 25HC3S hydroxyethylammonium.

[0082] FIG39 is an overlay of the x-ray powder diffraction patterns of crystalline 25HC3S hydroxyethylammonium before and after DVS.

[0083] Figure 40 is a diagram of 25HC3S hydroxyethylammonium 1 H-NMR spectrum.

[0084] FIG41 is a TGA thermogram and a DSC thermogram of crystalline 25HC3S hydroxyethylpyrrolidinium.

[0085] Figure 42 is a DVS isotherm of crystalline 25HC3S hydroxyethylpyrrolidinium.

[0086] FIG43 is an overlay of the x-ray powder diffraction patterns of crystalline 25HC3S hydroxyethylpyrrolidinium before and after DVS.

[0087] Figure 44 is 25HC3S hydroxyethyl pyrrolidinium 1 H-NMR spectrum.

[0088] Figure 45 is the indexing results of crystalline 25HC3S hydroxyethylpyrrolidinium.

[0089] FIG46 is a TGA thermogram and DSC thermogram of crystalline 25HC3S meglumine.

[0090] Figure 47 is a DVS isotherm of crystalline 25HC3S meglumine.

[0091] FIG48 is an overlay of x-ray powder diffraction patterns of 25HC3S meglumine before and after DVS crystallization.

[0092] Figure 49 is a diagram of 25HC3S meglumine 1H-NMR spectrum.

[0093] FIG50 is a TGA thermogram and a DSC thermogram of crystalline 25HC3S tris(hydroxymethyl)methylammonium.

[0094] Figure 51 is a DVS isotherm of crystalline 25HC3S tris(hydroxymethyl)methylammonium.

[0095] FIG52 is an overlay of the x-ray powder diffraction patterns of crystalline 25HC3S tris(hydroxymethyl)methylammonium before and after DVS.

[0096] Figure 53 is 25HC3S trimethylol ammonium 1 H-NMR spectrum.

[0097] FIG54 is a TGA thermogram and a DSC thermogram of crystalline 25HC3S diethanolamine.

[0098] Figure 55 is a DVS isotherm of crystalline 25HC3S diethanolamine.

[0099] Figure 56 is an overlay of the x-ray powder diffraction patterns of 25HC3S diethanolamine before and after DVS crystallization.

[0100] Figure 57 is a graph showing the structure of 25HC3S diethanolamine. 1 H-NMR spectrum.

[0101] Figure 58 is the indexing results for crystalline 25HC3S diethanolamine.

[0102] Figure 59 is a TGA thermogram and DSC thermogram of crystalline 25HC3S diethylammonium.

[0103] Figure 60 is a DVS isotherm of crystalline 25HC3S diethylammonium.

[0104] Figure 61 is an overlay of the x-ray powder diffraction patterns of crystalline 25HC3S diethylammonium before and after DVS.

[0105] Figure 62 is 25HC3S diethylammonium 1 H-NMR spectrum.

[0106] Figure 63 is the indexing results of crystalline 25HC3S diethylammonium.

[0107] Figure 64 is a TGA thermogram and DSC thermogram of crystalline 25HC3S-tert-butylammonium.

[0108] Figure 65 is a DVS isotherm of crystalline 25HC3S tert-butylammonium.

[0109] Figure 66 is 25HC3S tert-butylammonium 1 H-NMR spectrum.

[0110] Figure 67 is the indexing results of crystalline 25HC3S tert-butylammonium.

[0111] FIG68 is a TGA thermogram and a DSC thermogram of crystalline 25HC3S benzathine.

[0112] Figure 69 is a DVS isotherm of crystalline 25HC3S benzathine.

[0113] FIG70 is an overlay of the x-ray powder diffraction patterns of 25HC3S benzathine DVS before and after crystallization.

[0114] Figure 71 is 25HC3S benzyl 1 H-NMR spectrum.

[0115] Figure 72 shows the indexing results of crystalline 25HC3S benzathine.

[0116] Figure 73 is a TGA thermogram and DSC thermogram of crystalline 25HC3S lysine.

[0117] Figure 74 is a DVS isotherm of crystalline 25HC3S lysine.

[0118] Figure 75 is a diagram of 25HC3S lysine 1 H-NMR spectrum.

[0119] Figure 76 is an x-ray powder diffraction pattern of crystalline 25HC3S choline.

[0120] Figure 77 is the peak-selected crystalline 25HC3S choline X-ray powder diffraction pattern.

[0121] Figure 78 is an overlay of x-ray powder diffraction patterns of crystalline 25HC3S choline before and after DVS.

[0122] Figure 79 shows the indexing results of crystalline 25HC3S choline.

[0123] Figure 80 is a DSC (bottom) and TGA (top) thermogram of crystalline 25HC3S choline.

[0124] Figure 81 is a graph showing the 25HC3S choline in solution. 1 H-NMR spectrum.

[0125] Figure 82 is a DVS isotherm of crystalline 25HC3S choline.

[0126] FIG83 is an X-ray powder diffraction pattern of crystalline 25HC3S zinc.

[0127] FIG84 is an X-ray powder diffraction pattern of crystalline 25HC3S zinc.

[0128] Figure 85 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S zinc.

[0129] Figure 86 is a peak-selected X-ray powder diffraction pattern of crystalline 25HC3S zinc.

[0130] FIG87 is an overlay of X-ray powder diffraction patterns of crystalline 25HC3S zinc before and after DVS.

[0131] FIG88 is a DSC thermogram and TGA thermogram of crystalline 25HC3S zinc.

[0132] Figure 89 is 25HC3S zinc 1 H-NMR spectrum.

[0133] FIG90 is a DVS experiment of crystalline 25HC3S zinc.

[0134] Details

[0135] The compound 25-hydroxy-3β-cholest-5-ene-3-sulfate (25HC3S) refers to [(3S,10R,13R,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3-yl] sulfate, that is, a compound of formula I:

[0136]

[0137] Fifteen salts of 25HC3S are described herein. The preparation methods of these salts are described in Examples 27 to 41. All salts can be prepared as crystalline solids. The salts herein include inorganic salts and organic salts. Inorganic salts include metal salts, and organic salts include amine salts.

[0138] The metal salts of the present application include monocationic alkali metal salts and dicationic salts, including alkaline earth metal salts. Disclosed herein are metal salts of 25HC3S other than sodium salts, including crystalline salts. These metal salts include alkali metals other than sodium salts. In fact, the alkali metal salts of the present application include potassium 25HC3S, including crystalline potassium 25HC3S. Other metal salts of 25HC3S include alkaline earth metal salts of 25HC3S, including crystalline alkaline earth metal salts of 25HC3S. Alkaline earth metal salts include (a) calcium 25HC3S, including crystalline calcium 25HC3S; and (b) magnesium 25HC3S, including crystalline magnesium 25HC3S. The present application also includes other metal salts of 25HC3S, including its crystalline salts. Other metals in the present application include transition metals, such as divalent transition metals, including crystalline transition metal salts of 25HC3S, such as crystalline divalent transition metal salts. An exemplary divalent transition metal is zinc.

[0139] Disclosed herein are organic salts of 25HC3S of the present application, including crystalline organic salts of 25HC3S. Examples of such organic salts include amine salts of 25HC3S, including crystalline amine salts. The amine can be aliphatic, cyclic, aromatic, or a combination thereof. The amine can be primary, secondary, tertiary, or quaternary. The amine can also be substituted or unsubstituted. Common substituents are one or more alcohol groups. The alcohol can be primary, secondary, or tertiary.

[0140] The amine functional group of the amine group can be connected to an aliphatic carbon chain containing, for example, 1 to 6 carbon atoms. The linking group can be substituted or unsubstituted. Substituents include alkyl, alcohol, acid, aryl, and amine groups. Salts of the present application also include amino acid salts of the present application. Such amino acid salts can be naturally occurring amino acids or non-naturally occurring amino acids. An exemplary amino acid salt of the present application is the lysine salt of 25HC3S. In many cases herein, the amine salt is an amine alcoholate of 25HC3S, including choline, hydroxyethylammonium, hydroxyethylpyrrolidinium, meglumine, trihydroxymethylmethylammonium, and diethanolamine. The amine alcoholates of the present application can contain a single alcohol or multiple alcohol functional groups, such as diethanolamine (2 hydroxyl groups), trihydroxymethylmethylammonium (3 hydroxyl groups), or meglumine (5 hydroxyl groups). The amine alcohol can be a primary amine, a secondary amine, a tertiary amine, or a quaternary amine alcoholate. Such amine alcoholates can include cyclic amines, and the amine group can be linked to the alcohol via an alkyl linker containing, for example, 1 to 6 carbon atoms.

[0141] In many instances herein, the amine salt is an amine alkyl salt of 25HC3S, including diethylamine, tert-butylamine, and benzathine. The alkylamine salts herein include primary, secondary, and tertiary amines, and the alkyl chain may contain one or more carbon atoms. In many embodiments, the linking group has two carbon atoms. The alkylamine salt may also be a diamine. In some instances, the alkylamine salt also contains an aromatic group, such as a phenyl or benzyl group.

[0142] In many embodiments, the salt of the present application is a crystalline salt of 25HC3S other than (i) an alkali metal salt or (ii) an ammonium salt. In other embodiments, the salt of the present application is a crystalline salt of 25HC3S other than a sodium salt or a choline salt.

[0143] The present invention further discloses substantially pure salts and crystalline salts of 25HC3S. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (except for possible trace amounts) of other forms of 25HC3S in the present invention. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less, relative to the total amount (by weight) of the salts of 25HC3S of the present invention.

[0144] Methods for preparing the salts of 25HC3S described herein are further described. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt of 25HC3S, such as described in Example 41. This triethylammonium salt can then be used to prepare other salts of 25HC3S described herein.

[0145] The present application also relates to pharmaceutical compositions comprising the salts of 25HC3S of the present application. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and salts and crystalline salts of 25HC3S. Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate-release, sustained-release, slow-release, and timed-release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0146] In many embodiments, the pharmaceutical compositions of the present application contain salts of 25HC3S other than (i) alkali metal salts or (ii) ammonium salts, provided that in some embodiments, choline salts are included in such pharmaceutical compositions. In some embodiments, the pharmaceutical compositions contain crystalline salts of 25HC3S other than sodium salts and other than choline salts and a pharmaceutically acceptable excipient.

[0147] The present application also includes methods and uses of using an effective amount of the salts and crystalline salts of 25HC3S of the present application and / or pharmaceutical compositions comprising such salts and crystalline salts of 25HC3S for treating and / or preventing diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-related growth, and conditions associated with fat accumulation and inflammation. In the present application, examples of "diabetic conditions" that can be treated include one or more of insulin resistance, insulin deficiency, diabetes, and prediabetes. In the present application, examples of "autoimmune conditions" that can be treated include one or more of hepatitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis. As used in this application, examples of "inflammatory conditions" that can be treated include pulpitis, periodontal disease, skin inflammation, psoriasis, ulcerative colitis, osteoarthritis, inflammatory bowel disease (IBD), Crohn's disease, irritable bowel syndrome (IBS), Alzheimer's disease, Parkinson's disease, pancreatitis (acute and / or chronic), hepatitis (viral and / or non-viral), atherosclerosis, myocarditis, idiopathic lung disease (IPD), chronic obstructive pulmonary disease (COPD), pneumonia, chronic inflammatory lung disease, bronchitis, asthma, chronic kidney disease (CKD), nephritis, sepsis, ankylosing spondylitis, diverticulitis, and fibromyalgia. As used herein, examples of "neurological disorders" that can be treated include one or more of depression, neurodegenerative diseases, multiple sclerosis, Parkinson's disease, spinocerebellar degeneration, Friedreich's ataxia, ataxia telangiectasia, progressive supranuclear palsy, Huntington's disease, striatonigral degeneration, olivopontocerebellar atrophy, Shy-Derrick syndrome, schizophrenia, schizoaffective disorder, manic-depressive (bipolar) disorder, circadian rhythm disturbances or abnormalities, childhood Alice in Wonderland syndrome, childhood acute cerebellar ataxia, and Alzheimer's disease.

[0148] In many embodiments, the present application also includes methods and uses of using an effective amount of the salts and crystalline salts of 25HC3S of the present application and / or pharmaceutical compositions comprising such salts and crystalline salts of 25HC3S to treat and / or prevent diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, nervous system conditions, Epstein-Barr virus-associated growth, and conditions associated with fat accumulation and inflammation, wherein 25HC3S is not (i) an alkali metal salt or (ii) an ammonium salt, but with the proviso that in some embodiments, it comprises a choline salt. In other embodiments, the 25HC3S choline used in such methods is a crystalline salt of 25HC3S other than the sodium salt and the choline salt.

[0149] The crystalline salt of 25HC3S can be readily analyzed by X-ray powder diffraction. An X-ray powder diffraction pattern is an xy plot, with °2θ (diffraction angle) on the x-axis and intensity on the y-axis. The x-axis can also be expressed as a d-spacing, which is related to the diffraction angle via Bragg's law: 2dsinθ = nλ, where d is the d-spacing and λ is the wavelength of the incident X-ray wave. This plot contains peaks that can be used to characterize the crystalline salt of 25HC3S. Unless otherwise specified, peaks refer to their position on the x-axis, not their y-axis intensity. This can also occur due to sample orientation: a peak present in one sample collected on one instrument may not be present in another sample collected on a different instrument, depending on the sample's orientation relative to the instrument.

[0150] X-ray powder diffraction data can be used to characterize a crystalline form in a variety of ways. For example, the entire X-ray powder diffraction pattern output by a diffractometer can be used to characterize a crystalline salt of 25HC3S. However, smaller subsets of such data are also generally applicable to characterizing a crystalline salt of 25HC3S. For example, a collection of one or more peaks in the pattern can be used to characterize a crystalline salt of 25HC3S. In this application, all reported peaks are expressed in degrees 2θ using Cu-Kα radiation, as shown in Examples 24 and 25. In practice, even a single X-ray powder diffraction peak can often be used to characterize such a crystalline form. Herein, when a crystalline salt of 25HC3S is characterized by "one or more peaks" in an X-ray powder diffraction pattern and such peaks are listed, it generally means that any combination of the listed peaks can be used to characterize the crystalline salt of 25HC3S. Furthermore, the presence of additional peaks in the X-ray powder diffraction pattern does not generally negate or otherwise limit such characterization.

[0151] In addition to variations in peak intensity, there may also be variations in the position of the peaks along the x-axis. However, such variations can usually be accounted for when reporting peak positions for characterization purposes. Variations in the position of peaks along the x-axis can arise from a variety of factors (e.g., sample preparation, particle size, moisture content, solvent content, instrument parameters, data analysis software, and sample orientation). For example, the same sample of crystalline material prepared under different conditions may produce slightly different diffraction patterns, and different x-ray instruments can be operated with different parameters, which can result in slightly different diffraction patterns for the same crystalline solid.

[0152] Due to the presence of such difference factors, when describing x-ray diffraction peaks, the word "about" is usually used before the peak (in ° 2θ). For the data reported herein, this value is usually ± 0.2 ° 2θ. This usually means that on a well-maintained instrument, the difference in peak measurement is expected to be ± 0.2 ° 2θ. Unless otherwise stated, the x-ray powder diffraction peaks cited herein are usually reported with a difference of ± 0.2 ° 2θ, and whether or not the word "about" is present, this document is intended to report with such differences. However, in some cases, the difference may be as high as ± 0.2 ° 2θ or even higher, depending on the instrument conditions. In many crystalline salts of the present application, x-ray powder diffraction shows that some peaks (usually strong peaks at low angles (e.g., less than 5 ° 2θ)) are close to each other, for example, within 0.2 ° 2θ, which is the same as the variability of the reported peaks. Due to this variability, in some measurements, two peaks may overlap, making it difficult to distinguish between the two peaks. However, when characterizing the crystalline salt of 25HC3S of the present application and the characterization specifies only a single peak, then as long as the single peak is within the specified variability of ±0.2° 2θ, it can be used to characterize or assist in the characterization of the crystalline salt of 25HC3S (as the case may be). Moreover, in other embodiments of the present invention, the variability of the peak or peak group (in ° 2θ) quoted is ±0.1° 2θ, or even ±0.05° 2θ, rather than ±0.2° 2θ.

[0153] In addition to X-ray powder diffraction patterns, several salts of 25HC3S were observed using polarizing microscopy. Furthermore, several X-ray powder diffraction patterns were indexed. As used herein, "indexing" generally refers to the process of determining the size and shape of a crystal unit cell based on the positions of the peaks in a diffraction pattern. The term derives its name from the assignment of Miller index labels to individual peaks. For example, if all peaks in a pattern are indexed by a single unit cell, this can be strong evidence that the sample contains a single crystalline phase. Given an indexed solution, the unit cell volume can be directly calculated, which can be used to determine their solvation state. An index can also be a description of the crystalline form, briefly summarizing all available peak positions for that phase at a specific thermodynamic point.

[0154] Differential Scanning Calorimetry (DSC) is a thermal analysis technique in which the difference in the amount of heat required to raise the temperature of a sample and a reference is measured as a function of temperature. Phase changes in a sample, such as melting point, can be measured using this technique. Decomposition and dehydration events can also be observed with DSC. Variability also exists in the case of DSC measurements, and the term "about" means ±1°C, and unless otherwise stated, this variability should be understood regardless of whether the DSC measurement result is preceded by "about". An exemplary method for collecting DSC data is listed in Example 21. Thermogravimetric analysis, sometimes also called thermogravimetric analysis (TGA), is a thermal method of thermal analysis in which the change in mass of a sample over time is measured as the temperature of the sample is changed. For example, when a sample disclosed herein is heated and a loss of mass is measured before decomposition, this may indicate a loss of water from the sample and may indicate that the heated sample is a hydrate. An exemplary method for collecting TGA is shown in Example 23.

[0155] Dynamic vapor sorption (DVS) measures the absorption of vapor (usually water) by a sample when it is exposed to varying relative humidity. When relative humidity decreases, the loss of moisture can also be measured. Hygroscopic materials tend to absorb moisture more easily than non-hygroscopic materials. In some cases, hydrates absorb moisture to form higher order hydrates (e.g., a monohydrate converts to a dihydrate when exposed to increased humidity). In some salts of the present application, moisture is retained when exposed in a DVS experiment, and little or no weight loss is observed after heating from room temperature. In such experiments, the hygroscopicity of the salt under study may indicate that a hydrate has been formed from an anhydrous substance. In other salts of the present application, weight loss was observed upon heating, indicating that the salt was in the form of a hydrate when such thermal experiments began. An exemplary method for collecting DVS data is listed in Example 26.

[0156] Inductively coupled plasma optical emission spectrometry (ICP-OES) is an analytical technique used to detect chemical elements. It can be used to determine the relative stoichiometry of compounds and was used in this article to determine the metal salts of 25HC3S.

[0157] Regarding the inorganic salt of 25HC3S, an exemplary inorganic salt of the present application is a metal salt of 25HC3S and potassium. Crystalline potassium 25HC3S can be prepared as described in Example 27. The X-ray powder diffraction pattern of crystalline potassium 25HC3S is shown in Figure 1, with an enlarged view thereof in Figure 1A. A selected peak X-ray powder diffraction pattern is shown in Figure 2, with an enlarged view thereof in Figure 2A. Table 1 shows selected peaks from Figure 2.

[0158] Peaks of Crystallized Potassium 25HC3S in Table 1-Figure 2

[0159]

[0160]

[0161]

[0162] Crystalline potassium 25HC3S can be characterized by a variety of analytical techniques, including X-ray powder diffraction. The X-ray powder diffraction pattern of crystalline potassium 25HC3S, or a portion thereof, can be used to identify crystalline potassium 25HC3S. Crystalline potassium 25HC3S contains multiple X-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline potassium 25HC3S.

[0163] In some cases, crystalline potassium 25HC3S can be characterized by an X-ray powder diffraction pattern having one or more peaks as shown in Figure 2. The two most intense peaks in Figure 2 are at approximately 2.2° 2θ and approximately 2.3° 2θ. In Figure 2, these two peaks appear very close due to their respective intensities, but the XRPD instrument is able to distinguish them. However, due to the variability associated with these XRPD peaks, they may appear as single peaks located "below" each other when analyzed. Because such a single apparent peak is possible, in many cases herein, crystalline potassium 25HC3S is characterized, at least in part, by an X-ray powder diffraction pattern comprising two peaks located between approximately 2.2° 2θ and approximately 2.3° 2θ that: (a) do not overlap; (b) partially overlap; or (c) superimpose, thereby appearing as a single peak.

[0164] ICP-OES results are consistent with a 1:1 stoichiometric ratio of potassium ions to 25HC3S ions. For example, a peak at approximately 2.2° 2θ can be used to characterize crystalline potassium 25HC3S. In these and other cases, crystalline potassium 25HC3S can also be characterized by one or more peaks at approximately 2.3° 2θ, approximately 8.8° 2θ, approximately 9.3° 2θ, and approximately 15.3° 2θ.

[0165] In some cases, crystalline potassium 25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 2.3° 2θ. In these and other cases, crystalline potassium 25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 8.8° 2θ, about 9.3° 2θ, and about 15.3° 2θ.

[0166] In some cases, crystalline potassium 25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 8.8° 2θ. In these and other cases, crystalline potassium 25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 2.3° 2θ, about 9.3° 2θ, and about 15.3° 2θ.

[0167] In some cases, crystalline potassium 25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 9.3° 2θ. In these and other cases, crystalline potassium 25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 2.3° 2θ, about 8.8° 2θ, and about 15.3° 2θ.

[0168] In some cases, crystalline potassium 25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 15.3° 2θ. In these and other cases, crystalline potassium 25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 2.3° 2θ, about 8.8° 2θ, and about 9.3° 2θ.

[0169] In some cases, crystalline potassium 25HC3S can also be characterized by one or more peaks at about 4.6° 2θ, 14.7° 2θ, 14.9° 2θ, and about 16.1° 2θ. For example, crystalline potassium 25HC3S can also be characterized by a peak at about 4.6° 2θ. Alternatively or additionally, crystalline potassium 25HC3S can also be characterized by a peak at about 14.7° 2θ. Alternatively or additionally, crystalline potassium 25HC3S can also be characterized by a peak at about 14.9° 2θ. Alternatively or additionally, crystalline potassium 25HC3S can also be characterized by a peak at about 16.1° 2θ.

[0170] In some cases, the crystalline potassium 25HC3S can be characterized by an x-ray powder diffraction pattern substantially the same as shown in FIG. 2 or FIG. 2A .

[0171] Crystalline potassium 25HC3S has limited hygroscopicity as determined by DVS, absorbing only 2% water when the relative humidity approaches 95% RH (Figure 26), and exhibits a weight loss of approximately 0.9% at approximately 100°C and an additional 1.7% weight loss between 100°C and 180°C according to TGA (Figure 25). These data indicate that crystalline potassium 25HC3S is anhydrous. Figure 27 is an overlay of the XRPD diffraction patterns of crystalline potassium 25HC3S before and after DVS. There is no significant change in the pattern. Figure 28 is a graph of the XRPD diffraction patterns of potassium 25HC3S solution. 1 The H-NMR spectrum is consistent with its chemical structure. The DSC thermogram in Figure 25 also shows a small endotherm near 100°C, which then reappears at 139°C. Additional endotherms are observed at 198°C and 244°C, suggesting that decomposition occurs at 198°C.

[0172] Further disclosed herein is substantially pure crystalline potassium 25HC3S. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of potassium 25HC3S in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of potassium 25HC3S present (by weight).

[0173] Methods for preparing potassium 25HC3S are further described herein. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt, for example, as described in Example 41. This triethylammonium salt can then be used to prepare potassium 25HC3S, as described in Example 27.

[0174] The present application also relates to pharmaceutical compositions comprising potassium 25HC3S (including crystalline potassium 25HC3S) as disclosed herein. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and potassium 25HC3S (including crystalline potassium 25HC3S). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

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

[0176] Another exemplary salt of the present application is a metal salt of 25HC3S and calcium. Crystalline calcium 25HC3S can be prepared according to the method described in Example 28. The X-ray powder diffraction pattern of crystalline calcium 25HC3S is shown in Figure 3, with Figure 3A being an enlarged view. A selected peak X-ray powder diffraction pattern is shown in Figure 4, with Figure 4A being an enlarged view. Table 2 shows selected peaks from Figure 4. ICP-OES confirmed that the calcium salt of 25HC3S contains two 25HC3S ions for each calcium ion. Therefore, it can also be referred to as a hemi-calcium salt.

[0177] Table 2 - Peaks of Crystallized 25HC3S Calcium in Figure 4

[0178]

[0179]

[0180] Crystalline calcium 25HC3S can be characterized by a variety of analytical techniques, including x-ray powder diffraction. An x-ray powder diffraction pattern of crystalline calcium 25HC3S, or a portion thereof, can be used to identify crystalline calcium 25HC3S. Crystalline calcium 25HC3S comprises multiple x-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline calcium 25HC3S.

[0181] The two most intense peaks in Figure 4 are at approximately 15.0° 2θ and approximately 15.1° 2θ. In Figure 4, these two peaks appear very close due to their respective intensities, but the XRPD instrument is able to distinguish them. However, due to the variability associated with these XRPD peaks, they may appear as single peaks located "below" each other when analyzed. Because such a single apparent peak is possible, in many cases herein, crystalline calcium 25HC3S is characterized, at least in part, by an X-ray powder diffraction pattern comprising two peaks located at approximately 15.0° 2θ to approximately 15.1° 2θ that: (a) do not overlap; (b) partially overlap; or (c) superimpose, thereby appearing as a single peak. In some cases, crystalline calcium 25HC3S can be characterized by an X-ray powder diffraction pattern having one or more of the peaks in Figure 4. For example, a peak at approximately 2.2° 2θ can be used to characterize crystalline calcium 25HC3S. In these and other cases, crystalline calcium 25HC3S may also be 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.

[0182] In some cases, the crystalline calcium 25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 4.5° 2θ. In these and other cases, the crystalline calcium 25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 9.0° 2θ, about 10.0° 2θ, about 15.0° 2θ, about 15.1° 2θ, and about 15.7° 2θ.

[0183] In some cases, the crystalline calcium 25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 9.0° 2θ. In these and other cases, the crystalline calcium 25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 4.5° 2θ, about 10.0° 2θ, about 15.0° 2θ, about 15.1° 2θ, and about 15.7° 2θ.

[0184] In some cases, the crystalline calcium 25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 10.0° 2θ. In these and other cases, the crystalline calcium 25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 4.5° 2θ, about 9.0° 2θ, about 15.0° 2θ, about 15.1° 2θ, and about 15.7° 2θ.

[0185] In some cases, the crystalline calcium 25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 15.0° 2θ. In these and other cases, the crystalline calcium 25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 4.5° 2θ, about 9.0° 2θ, about 10.0° 2θ, about 15.1° 2θ, and about 15.7° 2θ.

[0186] In some cases, the crystalline calcium 25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 15.1° 2θ. In these and other cases, the crystalline calcium 25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 4.5° 2θ, about 9.0° 2θ, about 10.0° 2θ, about 15.0° 2θ, and about 15.7° 2θ.

[0187] In some cases, the crystalline calcium 25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 15.7° 2θ. In these and other cases, the crystalline calcium 25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 4.5° 2θ, about 9.0° 2θ, about 10.0° 2θ, about 15.0° 2θ, and about 15.1° 2θ.

[0188] In some cases, crystalline calcium 25HC3S can also be characterized by one or more peaks at about 15.4° 2θ, 16.5° 2θ, 18.0° 2θ, 18.1° 2θ, 18.4° 2θ, and about 19.2° 2θ. For example, crystalline calcium 25HC3S can also be characterized by a peak at about 15.4° 2θ. Alternatively or additionally, crystalline calcium 25HC3S can also be characterized by a peak at about 16.5° 2θ. Alternatively or additionally, crystalline calcium 25HC3S can also be characterized by a peak at about 18.0° 2θ. Alternatively or additionally, crystalline calcium 25HC3S can also be characterized by a peak at about 18.1° 2θ. Alternatively or additionally, crystalline calcium 25HC3S can also be characterized by a peak at about 18.4° 2θ. Alternatively or additionally, crystalline calcium 25HC3S can also be characterized by a peak at about 19.2° 2θ.

[0189] In some cases, the crystalline calcium 25HC3S can be characterized by an x-ray powder diffraction pattern substantially the same as that of FIG. 4 or FIG. 4A .

[0190] Crystalline calcium 25HC3S can exist as a hydrate. As shown in the TGA thermogram in Figure 29, a sample of crystalline calcium 25HC3S loses approximately 0.9% weight between room temperature and approximately 93°C, and loses a further 3.1% weight between approximately 112°C and approximately 130°C. The TGA data are calculated based on a 25HC3S salt containing one 25HC3S ion and 0.5 calcium ions. The weight loss is presumably due to water at these temperatures. Therefore, for the calcium salt, a 3-4% water loss is associated with approximately 1 mole of water per mole of 25HC3S ion (i.e., 0.5 moles of divalent calcium ions), indicating a monohydrate. Based on the DVS data in Figure 30, higher water uptake may occur upon exposure to higher relative humidity, potentially resulting in a dihydrate, as indicated by the plateau in the DVS isotherm. Thus, the data indicate that crystalline 25HC3S calcium may exist in a variety of hydration states, including monohydrate or dihydrate, when reference is made to one 25HC3S ion and half a divalent calcium ion per salt molecule. For example, in Example 28, the crystalline 25HC3S hemi-calcium salt prepared contained two equivalents of water.

[0191] The DSC thermogram of crystalline 25HC3S calcium shows a small endothermic peak at about 70.3°C and a larger endothermic peak at about 130°C, with decomposition associated with the higher values ​​(Figure 29). Figure 31 is an overlay of the XRPD diffraction patterns of crystalline 25HC3S calcium before and after DVS. There is no significant change in the pattern. Figure 32 is a superposition of the XRPD diffraction patterns of 25HC3S calcium solution. 1 The H-NMR spectrum is consistent with the chemical structure.

[0192] Further disclosed herein is substantially pure crystalline calcium 25HC3S. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of calcium 25HC3S in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of calcium 25HC3S present (by weight).

[0193] Methods for preparing calcium 25HC3S are further described herein. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt, for example, as described in Example 41. This triethylammonium salt can then be used to prepare calcium 25HC3S, as described in Example 28.

[0194] The present application also relates to pharmaceutical compositions comprising 25HC3S calcium (including crystalline 25HC3S calcium) as described herein. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and 25HC3S calcium (including crystalline 25HC3S calcium). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0195] The present application also includes methods and uses of using an effective amount of 25HC3S calcium (including crystalline 25HC3S calcium) of the present application and / or a pharmaceutical composition comprising crystalline 25HC3S calcium to treat and / or prevent diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0196] Another exemplary inorganic salt of the present application is a metal salt of 25HC3S and zinc. Crystalline 25HC3S zinc can be prepared according to the method described in Example 29. The x-ray powder diffraction pattern of crystalline 25HC3S zinc is shown in Figure 83, and its enlarged view is shown in Figure 84. The x-ray powder diffraction pattern of the selected peaks is shown in Figure 85, and its enlarged view is shown in Figure 86. Table 3 shows the peaks selected from Figure 85. It was confirmed by ICP-OES that for every 1 zinc ion in the zinc salt of 25HC3S, there were 2 25HC3S ions, thereby confirming the presence of 5.72% zinc, which is consistent with the calculated zinc content of 5.98% at a concentration of 0.5 mol / mol. Therefore, it can also be called a semi-zinc salt like calcium. 25HC3S zinc 1 The H-NMR spectrum was consistent with the chemical structure shown in FIG89 .

[0197] Table 3 - Peaks of Crystallized 25HC3S Zinc in Figure 85

[0198]

[0199]

[0200] The two strongest peaks in Figure 85 are at about 2.1 ° 2θ and about 2.3 ° 2θ. In Figure 85, these two peaks appear very close due to their respective intensities, but the XRPD instrument is able to distinguish them. However, due to the variability associated with these XRPD peaks, they may appear as single peaks located "below" each other when analyzed. Because such single peaks are possible, in many cases herein, crystalline 25HC3S zinc is characterized at least in part by an X-ray powder diffraction pattern containing two peaks located at approximately 2.1 ° 2θ to about 2.3 ° The two peaks are: (a) non-overlapping; (b) partially overlapping; or (c) superimposed, thereby appearing as a single peak. An enlarged diffraction pattern can be seen in Figure 86 to help illustrate the other peaks in the figure.

[0201] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having a peak at about 2.1° 2θ. In these and other cases, the crystalline 25HC3S zinc can also be 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.

[0202] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having a peak at about 2.3° 2θ. In these and other cases, the crystalline 25HC3S zinc can also be 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.

[0203] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1° 2θ and about 2.3° 2θ. In these and other cases, the crystalline 25HC3S zinc can be characterized by one or more peaks at about 6.0° 2θ, about 8.6° 2θ, about 8.9° 2θ, about 9.3° 2θ, about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0204] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1° 2θ and about 2.3° 2θ and a peak at about 18.3° 2θ. In these and other cases, the crystalline 25HC3S zinc can be characterized by one or more peaks at about 6.0° 2θ, about 8.6° 2θ, about 8.9° 2θ, about 9.3° 2θ, about 15.1° 2θ, and about 18.8° 2θ.

[0205] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having a peak at about 6.0° 2θ. In these and other cases, the crystalline 25HC3S zinc can also be 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.

[0206] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having a peak at about 8.6° 2θ. In these and other cases, the crystalline 25HC3S zinc can also be 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.

[0207] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having a peak at about 8.9° 2θ. In these and other cases, the crystalline 25HC3S zinc can also be 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.

[0208] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having a peak at about 9.3° 2θ. In these and other cases, the crystalline 25HC3S zinc can also be 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.

[0209] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having a peak at about 15.1° 2θ. In these and other cases, the crystalline 25HC3S zinc can also be 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.

[0210] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having a peak at about 18.3° 2θ. In these and other cases, the crystalline 25HC3S zinc can also be 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.

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

[0212] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1° 2θ and about 2.3° 2θ, and a peak at about 6.0° 2θ. In these and other cases, the crystalline 25HC3S zinc can also be 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.

[0213] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1° 2θ and about 2.3° 2θ, a peak at about 6.0° 2θ, and a peak at about 8.6° 2θ. In these and other cases, the crystalline 25HC3S zinc can also be 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.

[0214] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1° 2θ and about 2.3° 2θ, a peak at about 6.0° 2θ, a peak at about 8.6° 2θ, and a peak at about 9.3° 2θ. In these and other cases, the crystalline 25HC3S zinc can also be characterized by one or more peaks at about 8.9° 2θ, about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0215] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1° 2θ and about 2.3° 2θ, as well as 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, the crystalline 25HC3S zinc can also be characterized by one or more peaks at 8.9° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0216] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1° 2θ and about 2.3° 2θ, a peak at about 6.0° 2θ, a peak at about 8.6° 2θ, a peak at about 9.3° 2θ, a peak at about 15.1° 2θ, and a peak at about 18.3° 2θ. In these and other cases, the crystalline 25HC3S zinc can also be characterized by one or more peaks at about 8.9° 2θ and about 18.8° 2θ.

[0217] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1° 2θ and about 2.3° 2θ, a peak at about 6.0° 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, the crystalline 25HC3S zinc can also be characterized by a peak at about 8.9° 2θ.

[0218] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 2.1° 2θ, about 2.3° 2θ, about 6.0° 2θ, about 8.6° 2θ, about 8.9° 2θ, about 9.3° 2θ, about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0219] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or two peaks between about 2.1° 2θ and about 2.3° 2θ and 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.

[0220] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 6.0° 2θ, about 8.6° 2θ, about 8.9° 2θ, about 9.3° 2θ, about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0221] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 8.6° 2θ, about 8.9° 2θ, about 9.3° 2θ, about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0222] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 8.9° 2θ, about 9.3° 2θ, about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0223] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 9.3° 2θ, about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0224] In some cases, the crystalline 25HC3S zinc can 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.

[0225] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 18.3° 2Θ and about 18.8° 2Θ.

[0226] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern having a peak at about 18.8° 2θ.

[0227] In some cases, the crystalline 25HC3S zinc can be characterized by an x-ray powder diffraction pattern substantially the same as FIG. 85 or FIG. 86 .

[0228] Crystalline 25HC3S zinc can exhibit a differential scanning calorimetry (DSC) thermogram having one or more small endothermic peaks at about 68°C, about 86°C, and about 102°C, as shown in Figure 88. It is believed that thermal decomposition of crystalline 25HC3S zinc occurs at about 119°C, which can be observed as a large endothermic peak in Figure 88.

[0229] Crystalline zinc-25HC3S may be hydrated. In this case, the water of hydration may be part of the crystal structure, thus the crystalline 25HC3S is hydrated. As shown in the TGA thermogram in Figure 88, a sample of crystalline zinc-25HC3S loses approximately 5.1% of its weight between room temperature and approximately 130°C. It is speculated that the weight loss is due to water at these temperatures. For the zinc salt, a 5.1% water content corresponds to approximately 1.5 moles of water per mole of 25HC3S. The TGA data are calculated based on a zinc-25HC3S salt containing one 25HC3S ion and half a zinc ion.

[0230] The DVS data indicate that the water uptake may also stabilize at approximately 2.8% (mass change) between 25% and 45% relative humidity, indicating 0.75 moles of water per mole of 25HC3S or monohydrate; above 45% relative humidity, the water uptake may also stabilize at approximately 4.5% to 5% (mass change), which, when referenced to one 25HC3S ion and half a zinc divalent ion per salt molecule, indicates a sesquihydrate or dihydrate. Therefore, in many cases, the hydration level of each 25HC3S zinc ion in the crystallized 25HC3S may include an average of 0.75 to 2 water molecules (including 1 and 1.5). A further weight loss of 11.5% was observed in the TGA at about 131°C to about 190°C, which may be due to decomposition. Figure 87 shows the XRPD patterns of the crystalline zinc before and after DVS, and Figure 89 shows the XRPD patterns of the crystalline zinc before and after DVS. 1The H-NMR spectrum is consistent with the chemical structure.

[0231] Further disclosed herein is substantially pure crystalline zinc-25HC3S. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of zinc-25HC3S in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of zinc-25HC3S present (by weight).

[0232] Methods for preparing zinc 25HC3S are further described herein. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt, for example, as described in Example 41. This triethylammonium salt can then be used to prepare zinc 25HC3S, as described in Example 29.

[0233] The preparation of the 25HC3S triethylammonium salt can be accomplished, for example, by passing a mixture of triethylammonium chloride and triethylamine through a column and treating with a solvent (e.g., an alcohol) to a neutral pH. Alternatively, crystalline 25HC3S sodium can be dissolved in a solvent (e.g., an alcohol). This solution is then passed through the same column that was previously exposed to triethylamine and combined with the triethylammonium solution. The resulting solid is isolated (e.g., under vacuum or by drying) to yield the crystalline 25HC3S triethylammonium salt, which can be homogenized using, for example, a mortar and pestle. Suitable alcohols for this method include methanol.

[0234] The preparation of zinc 25HC3S (including crystalline zinc 25HC3S) is carried out as follows: starting with sodium 25HC3S, converting it to a second salt of 25HC3S (e.g., a triethylammonium salt), and then converting the second salt of 25HC3S to zinc 25HC3S (including crystalline zinc 25HC3S). Crystalline zinc 25HC3S can be prepared by preparing a suspension of the triethylammonium salt of 25HC3S in a suitable solvent (e.g., acetonitrile), followed by treatment with a zinc source (e.g., aqueous zinc chloride) to form zinc 25HC3S (including crystalline zinc 25HC3S). Zinc 25HC3S can be purified by washing with a suitable solvent (e.g., acetonitrile). Further treatment with a zinc source (e.g., aqueous zinc chloride) can improve the yield. Furthermore, the treatment process can include stirring and / or further treatment with a suitable solvent (e.g., acetonitrile). Other treatments, such as vacuum drying, can also be performed to assist in the preparation of crystalline zinc 25HC3S. The present application also includes crystalline zinc 25HC3S prepared by the methods described herein.

[0235] The present application also relates to pharmaceutical compositions comprising 25HC3S zinc (including crystalline 25HC3S zinc) as disclosed herein. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and 25HC3S zinc (including crystalline 25HC3S zinc). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0236] As described elsewhere herein, the 25HC3S zinc of the present application advantageously provides zinc supplementation to patients suffering from conditions targeted by 25HC3S. As described elsewhere herein, zinc deficiency can contribute to these conditions, and therefore providing zinc concurrently with 25HC3S during treatment can be advantageous. Thus, 25HC3S zinc surprisingly and advantageously combines favorable salt / crystal form properties with an inherent ability to advantageously provide zinc supplementation during treatment methods.

[0237] The present application also includes methods and uses of using an effective amount of 25HC3S zinc (including crystalline 25HC3S zinc) of the present application and / or a pharmaceutical composition comprising crystalline 25HC3S zinc to treat and / or prevent diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0238] Another exemplary inorganic salt of the present application is a metal salt of 25HC3S and magnesium. Crystalline 25HC3S magnesium can be prepared according to the method described in Example 30. The X-ray powder diffraction pattern of crystalline 25HC3S magnesium is shown in Figure 5, with Figure 5A being an enlarged view. A selected peak X-ray powder diffraction pattern is shown in Figure 6, with Figure 6A being an enlarged view. The enlarged peaks in Table 4 show selected peaks from Figure 6. ICP-EOS confirmed that each magnesium ion in this magnesium salt corresponds to two 25HC3S ions. Therefore, this magnesium salt can be referred to as a hemi-magnesium salt.

[0239] Table 4 - Peaks of Crystallized 25HC3S Magnesium in Figure 6

[0240]

[0241] Crystalline magnesium-25HC3S can be characterized by a variety of analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline magnesium-25HC3S, or a portion thereof, can be used to identify crystalline magnesium-25HC3S. Crystalline magnesium-25HC3S comprises multiple x-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline magnesium-25HC3S.

[0242] In some cases, crystalline magnesium-25HC3S can be characterized by an x-ray powder diffraction pattern having one or more of the peaks in FIG6 . For example, a peak at about 2.2° 2θ can be used to characterize crystalline magnesium-25HC3S. In these and other cases, crystalline magnesium-25HC3S can also be 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 magnesium-25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 4.4° 2θ. In these and other cases, crystalline calcium-25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 6.6° 2θ, about 8.9° 2θ, and about 15.1° 2θ.

[0243] In some cases, the crystalline magnesium-25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 6.6° 2θ. In these and other cases, the crystalline calcium-25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 4.4° 2θ, about 8.9° 2θ, and about 15.1° 2θ.

[0244] In some cases, the crystalline magnesium-25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 8.9° 2θ. In these and other cases, the crystalline calcium-25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 4.4° 2θ, about 6.6° 2θ, and about 15.1° 2θ.

[0245] In some cases, the crystalline magnesium-25HC3S can be characterized by an x-ray powder diffraction pattern having a peak at about 15.1° 2θ. In these and other cases, the crystalline calcium-25HC3S can also be characterized by one or more peaks at about 2.2° 2θ, about 4.4° 2θ, about 6.6° 2θ, and about 8.9° 2θ.

[0246] In some cases, crystalline 25HC3S magnesium can also be characterized by one or more peaks at about 15.6° 2θ, 16.4° 2θ, 17.6° 2θ, and about 17.8° 2θ. For example, crystalline 25HC3S magnesium can also be characterized by a peak at about 15.6° 2θ. Alternatively or additionally, crystalline 25HC3S magnesium can also be characterized by a peak at about 16.4° 2θ. Alternatively or additionally, crystalline 25HC3S magnesium can also be characterized by a peak at about 17.6° 2θ. Alternatively or additionally, crystalline 25HC3S magnesium can also be characterized by a peak at about 17.8° 2θ.

[0247] Crystalline 25HC3S magnesium can be characterized by an x-ray powder diffraction pattern substantially the same as that of FIG. 6 or FIG. 6A .

[0248] Crystalline 25HC3S magnesium may exist in the form of a hydrate. As shown in the TGA thermogram of Figure 33, a sample of crystalline 25HC3S magnesium loses about 1.5% of its weight at temperatures up to 127°C. It is speculated that the weight loss is due to water at these temperatures. Therefore, for the magnesium salt, a 1.5% water loss is associated with about 0.5 moles of water per mole of 25HC3S. The plateau region at about 5% to 7% mass change in the DVS of crystalline 25HC3S magnesium in Figure 34 indicates that higher hydrates containing 1.5 to 2 moles of water per mole of 25HC3S, such as sesquihydrate or dihydrate, are also present. Therefore, the data indicate that crystalline 25HC3S magnesium may exist in the form of a hydrate, such as a hemihydrate, sesquihydrate, or dihydrate, as referenced by one 25HC3S ion and half a magnesium divalent ion per salt molecule. Figure 35 shows the x-ray powder diffraction patterns before and after DVS. The DVS in Figure 36 1 The H-NMR spectrum was consistent with the chemical structure of 25HC3S magnesium. The DSC thermogram showed an endothermic peak at approximately 139°C, which was believed to be caused by decomposition.

[0249] Further disclosed herein is substantially pure crystalline magnesium-25HC3S. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of magnesium-25HC3S in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of magnesium-25HC3S present (by weight).

[0250] Methods for preparing magnesium-25HC3S are further described herein. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt, such as described in Example 41. This triethylammonium salt can then be used to prepare magnesium-25HC3S, as described in Example 30.

[0251] The present application also relates to pharmaceutical compositions comprising magnesium 25HC3S (including crystalline magnesium 25HC3S) as disclosed herein. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and magnesium 25HC3S (including crystalline magnesium 25HC3S). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0252] The present application also includes methods and uses of using an effective amount of 25HC3S magnesium (including crystalline 25HC3S magnesium) of the present application and / or a pharmaceutical composition comprising crystalline 25HC3S magnesium to treat and / or prevent diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0253] Another exemplary organic salt of 25HC3S of the present application is an organic amine hydroxyethylammonium salt of 25HC3S. A method for preparing the crystalline hydroxyethylammonium salt of 25HC3S is provided in Example 31. An X-ray powder diffraction pattern of crystalline hydroxyethylammonium 25HC3S is provided in FIG7 , with FIG7A being an enlarged view.

[0254] The X-ray powder diffraction pattern of the selected peaks is given in Figure 8, wherein Figure 8A is an enlarged view. Table 5 shows the peaks selected from Figure 8.

[0255] Table 5-Figure 8 Peaks of crystallized 25HC3S hydroxyethylammonium

[0256]

[0257] Crystalline 25HC3S hydroxyethylammonium can be characterized by a variety of analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylammonium, or a portion thereof, can be used to identify crystalline 25HC3S hydroxyethylammonium. Crystalline 25HC3S hydroxyethylammonium comprises multiple x-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline 25HC3S hydroxyethylammonium.

[0258] In some cases, crystalline 25HC3S hydroxyethylammonium can 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 can be used to characterize crystalline 25HC3S hydroxyethylammonium. In these and other cases, crystalline 25HC3S hydroxyethylammonium can also be characterized by a peak at about 8.6° 2θ.

[0259] In Figure 38, the crystalline 25HC3S hydroxyethylammonium was determined to be hygroscopic by DVS. In Figure 37, the weight loss from ambient temperature to 134°C was shown to be approximately 2.7%. 1 The H-NMR spectrum shows the presence of isopropanol in the solution, which is consistent with the preparation of the salt according to Example 31, in which the salt was prepared using isopropanol. Therefore, without being bound by theory, it is believed that the crystalline 25HC3S hydroxyethylammonium salt may exist as an isopropanol solvate. The X-ray analysis after DVS in Figure 39 also shows the appearance of additional X-ray powder diffraction peaks, which may indicate the occurrence of additional physical transformations. The DSC of the crystalline 25HC3S hydroxyethylammonium in Figure 37 shows multiple endothermic peaks.

[0260] Further disclosed herein is substantially pure crystalline 25HC3S hydroxyethylammonium. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of 25HC3S hydroxyethylammonium in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of 25HC3S hydroxyethylammonium present (based on weight).

[0261] Methods for preparing 25HC3S hydroxyethylammonium are further described herein. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt, such as described in Example 41. This triethylammonium salt can then be used to prepare 25HC3S hydroxyethylammonium, as described in Example 31.

[0262] The present application also relates to pharmaceutical compositions comprising 25HC3S hydroxyethylammonium (including crystalline 25HC3S hydroxyethylammonium) as disclosed herein. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and 25HC3S hydroxyethylammonium (including crystalline 25HC3S hydroxyethylammonium). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0263] The present application also includes methods and uses of using an effective amount of 25HC3S hydroxyethylammonium (including crystalline 25HC3S hydroxyethylammonium) of the present application and / or a pharmaceutical composition comprising crystalline 25HC3S hydroxyethylammonium to treat and / or prevent diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0264] An exemplary organic salt of 25HC3S in the present application is the organic amine tris(hydroxymethyl)methylammonium salt of 25HC3S. A method for preparing the crystalline tris(hydroxymethyl)methylammonium salt of 25HC3S is provided in Example 32. An X-ray powder diffraction pattern of crystalline tris(hydroxymethyl)methylammonium 25HC3S is provided in FIG13 , with FIG13A being an enlarged view. A selected peak X-ray powder diffraction pattern is provided in FIG14 , with FIG14A being an enlarged view. Table 6 shows selected peaks from FIG14 .

[0265] Table 6 - Peaks of Crystallized 25HC3S Tris(hydroxymethyl)methylammonium in Figure 14

[0266]

[0267]

[0268]

[0269] Crystalline 25HC3S tris(hydroxymethyl)methylammonium can be characterized by a variety of analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S tris(hydroxymethyl)methylammonium, or a portion thereof, can be used to identify crystalline 25HC3S tris(hydroxymethyl)methylammonium. Crystalline 25HC3S tris(hydroxymethyl)methylammonium comprises multiple x-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline 25HC3S tris(hydroxymethyl)methylammonium.

[0270] In some cases, crystalline 25HC3S tris(hydroxymethyl)methanium can 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 can be used to characterize crystalline 25HC3S tris(hydroxymethyl)methanium. In these and other cases, crystalline 25HC3S tris(hydroxymethyl)methanium can also be characterized by a peak at about 2.1° 2θ or at about 3.8° 2θ.

[0271] The two most intense peaks in Figure 14 are at approximately 1.9° 2θ and approximately 2.1° 2θ. In Figure 14, these two peaks appear very close due to their respective intensities, but the XRPD instrument is able to distinguish them. However, due to the variability associated with these XRPD peaks, they may appear as single peaks located "below" each other when analyzed. Because such a single apparent peak is possible, in many cases herein, crystalline 25HC3S tris(hydroxymethyl)methylammonium is characterized at least in part by an X-ray powder diffraction pattern comprising two peaks located between approximately 1.9° 2θ and approximately 2.1° 2θ that: (a) do not overlap; (b) partially overlap; or (c) overlap, thereby appearing as a single peak. In some cases, crystalline 25HC3S tris(hydroxymethyl)methylammonium can be characterized by an X-ray powder diffraction pattern having a peak at approximately 2.1° 2θ. In these and other cases, crystalline calcium 25HC3S may also be characterized by one or more peaks at about 1.9° 2Θ and about 3.8° 2Θ.

[0272] In some cases, crystalline 25HC3S tris(hydroxymethyl)methylammonium can 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 can also be characterized by one or more peaks at about 1.9° 2θ and about 2.1° 2θ.

[0273] In some cases, crystalline 25HC3S tris(hydroxymethyl)methanium can also be characterized by one or more peaks at about 4.2° 2θ and about 15.4° 2θ. For example, crystalline 25HC3S tris(hydroxymethyl)methanium can also be characterized by a peak at about 4.2° 2θ. Alternatively or additionally, crystalline 25HC3S tris(hydroxymethyl)methanium can also be characterized by a peak at about 15.4° 2θ.

[0274] In some cases, crystalline 25HC3S tris(hydroxymethyl)methylammonium can be characterized by an x-ray powder diffraction pattern substantially the same as that of FIG. 14 or FIG. 14A .

[0275] As shown in FIG51 , crystalline 25HC3S tris(hydroxymethyl)methylammonium was observed to be hygroscopic by DVS. In fact, it absorbed over 20% by weight of water when exposed to a relative humidity of about 95%. As can be seen by TGA in FIG50 , only about 0.3% weight loss was observed up to about 80°C, indicating an anhydrous form under ambient conditions. Additionally, FIG52 shows the x-ray powder diffraction patterns before and after DVS, and illustrates some potential differences that may be due to its hygroscopic nature. The DSC of crystalline 25HC3S tris(hydroxymethyl)methylammonium can be found in FIG50 , showing multiple small endothermic peaks at about 54°C, about 114°C, and about 158°C, followed by decomposition at about 184°C. FIG53 1 The H-NMR structure was consistent with the chemical structure.

[0276] Further disclosed herein is substantially pure crystalline 25HC3S tris(hydroxymethyl)methylammonium. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of 25HC3S tris(hydroxymethyl)methylammonium in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of 25HC3S tris(hydroxymethyl)methylammonium present (based on weight).

[0277] Methods for preparing 25HC3S tris(hydroxymethyl)methylammonium are further described herein. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt, such as described in Example 41. This triethylammonium salt can then be used to prepare 25HC3S tris(hydroxymethyl)methylammonium, as described in Example 32.

[0278] The present application also relates to pharmaceutical compositions comprising 25HC3S trihydroxymethylamine (including crystalline 25HC3S trihydroxymethylamine) disclosed herein. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and 25HC3S trihydroxymethylamine (including crystalline 25HC3S trihydroxymethylamine). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0279] The present application also includes methods and uses for treating and / or preventing diseases (e.g., in humans) using an effective amount of 25HC3S trihydroxymethylmethanaminium (including crystalline 25HC3S trihydroxymethylmethanaminium) of the present application and / or a pharmaceutical composition comprising crystalline 25HC3S trihydroxymethylmethanaminium, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0280] An exemplary organic salt of 25HC3S herein is the organic amino acid lysine salt of 25HC3S. A method for preparing the crystalline l-lysine salt of 25HC3S is provided in Example 33. An X-ray powder diffraction pattern of crystalline 25HC3S lysine is provided in FIG23 , with FIG23A being an enlarged view. A selected peak X-ray powder diffraction pattern is provided in FIG24 , with FIG24A being an enlarged view. Table 7 shows selected peaks from FIG24 .

[0281] Table 7 - Peaks of crystallized 25HC3S lysine in Figure 24

[0282]

[0283] Crystalline 25HC3S-lysine can be characterized by a variety of analytical techniques, including x-ray powder diffraction. An x-ray powder diffraction pattern of crystalline 25HC3S-lysine, or a portion thereof, can be used to identify crystalline 25HC3S-lysine. Crystalline 25HC3S-lysine comprises multiple x-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline 25HC3S-lysine. In some cases, crystalline 25HC3S-lysine can be characterized by an x-ray powder diffraction pattern having one or more of the peaks in Figure 24. For example, a peak at approximately 1.5° 2θ can be used to characterize crystalline 25HC3S-lysine. In these and other cases, crystalline 25HC3S-lysine can also be characterized by one or more peaks at approximately 7.0° 2θ, approximately 10.7° 2θ, approximately 11.8° 2θ, and approximately 16.8° 2θ.

[0284] In some cases, the crystalline 25HC3S lysine can be characterized by an x-ray powder diffraction pattern having a peak at about 7.0° 2θ. In these and other cases, the crystalline 25HC3S lysine can also be characterized by one or more peaks at about 1.5° 2θ, about 10.7° 2θ, about 11.8° 2θ, and about 16.8° 2θ.

[0285] In some cases, the crystalline 25HC3S lysine can be characterized by an x-ray powder diffraction pattern having a peak at about 10.7° 2θ. In these and other cases, the crystalline 25HC3S lysine can also be characterized by one or more peaks at about 1.5° 2θ, about 7.0° 2θ, about 11.8° 2θ, and about 16.8° 2θ.

[0286] In some cases, the crystalline 25HC3S lysine can be characterized by an x-ray powder diffraction pattern having a peak at about 11.8° 2θ. In these and other cases, the crystalline 25HC3S lysine can also be characterized by one or more peaks at about 1.5° 2θ, about 7.0° 2θ, about 10.7° 2θ, and about 16.8° 2θ.

[0287] In some cases, the crystalline 25HC3S lysine can be characterized by an x-ray powder diffraction pattern having a peak at about 16.8° 2θ. In these and other cases, the crystalline 25HC3S lysine can also be characterized by one or more peaks at about 1.5° 2θ, about 7.0° 2θ, about 10.7° 2θ, and about 11.8° 2θ.

[0288] In some cases, crystalline 25HC3S Lysine can also be characterized by one or more peaks at about 3.2° 2θ, about 10.0° 2θ, about 12.2° 2θ, and about 15.2° 2θ. For example, crystalline 25HC3S Lysine can also be characterized by a peak at about 3.2° 2θ. Alternatively or additionally, crystalline 25HC3S Lysine can also be characterized by a peak at about 10.0° 2θ. Alternatively or additionally, crystalline 25HC3S Lysine can also be characterized by a peak at about 12.2° 2θ. Alternatively or additionally, crystalline 25HC3S Lysine can also be characterized by a peak at about 15.2° 2θ.

[0289] In some cases, crystalline 25HC3S lysine can be characterized by an x-ray powder diffraction pattern substantially the same as that in Figure 24.

[0290] In FIG74 , the DVS method determined that crystalline 25HC3S-lysine is hygroscopic at relative humidity above 75%. As shown in FIG73 , TGA shows a weight loss of about 1.5% at 100°C and a further weight loss of about 8.8% between 100°C and 235°C, some of which may be due to water loss and some to decomposition. Therefore, crystalline 25HC3S-lysine may exist in the form of a hydrate. The DSC of crystalline 25HC3S-lysine can be observed in FIG73 , showing a weak endothermic peak at about 96°C. It is believed that the endothermic peak at about 186°C indicates that decomposition has occurred. FIG75 1 The H-NMR spectrum was consistent with the chemical structure.

[0291] Further disclosed herein is substantially pure crystalline 25HC3S-lysine. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of 25HC3S-lysine present in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of 25HC3S-lysine present (based on weight).

[0292] Methods for preparing 25HC3S-lysine are further described herein. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt, for example, as described in Example 41. This triethylammonium salt can then be used to prepare 25HC3S-lysine, as described in Example 33.

[0293] The present application also relates to pharmaceutical compositions comprising 25HC3S lysine (including crystalline 25HC3S lysine) disclosed herein. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and 25HC3S lysine (including crystalline 25HC3S lysine). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0294] The present application also includes methods and uses of using an effective amount of 25HC3S lysine (including crystalline 25HC3S lysine) of the present application and / or a pharmaceutical composition comprising crystalline 25HC3S lysine to treat and / or prevent diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, nervous system conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0295] An exemplary organic salt of 25HC3S herein is the organic amine meglumine salt of 25HC3S. A method for preparing the crystalline meglumine salt of 25HC3S is provided in Example 34. The X-ray powder diffraction pattern of crystalline 25HC3S meglumine is shown in Figure 11, with Figure 11A being an enlarged view. A selected peak X-ray powder diffraction pattern is shown in Figure 12, with Figure 12A being an enlarged view. Table 8 shows selected peaks from Figure 12.

[0296] Table 8 - Peaks of Crystallized 25HC3S Meglumine in Figure 12

[0297]

[0298] Crystalline 25HC3S meglumine can be characterized by a variety of analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S meglumine, or a portion thereof, can be used to identify crystalline 25HC3S meglumine. Crystalline 25HC3S meglumine comprises multiple x-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline 25HC3S meglumine.

[0299] In some cases, crystalline 25HC3S meglumine can 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 can be used to characterize crystalline 25HC3S meglumine. In these and other cases, crystalline 25HC3S meglumine can also be characterized by one or more peaks at about 3.5° 2θ, about 5.2° 2θ, about 14.9° 2θ, and about 24.2° 2θ.

[0300] In some cases, crystalline 25HC3S meglumine can 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 can also be characterized by one or more peaks at about 1.7° 2θ, about 5.2° 2θ, about 14.9° 2θ, and about 24.2° 2θ.

[0301] In some cases, crystalline 25HC3S meglumine can 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 can also be characterized by one or more peaks at about 1.7° 2θ, about 3.5° 2θ, about 14.9° 2θ, and about 24.2° 2θ.

[0302] In some cases, crystalline 25HC3S meglumine can 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 can also be characterized by one or more peaks at about 1.7° 2θ, about 3.5° 2θ, about 5.2° 2θ, and about 24.2° 2θ.

[0303] In some cases, crystalline 25HC3S meglumine can 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 can also be characterized by one or more peaks at about 1.7° 2θ, about 3.5° 2θ, about 5.2° 2θ, and about 14.9° 2θ.

[0304] In some cases, crystalline 25HC3S meglumine can also be characterized by one or more peaks at about 8.6° 2θ, about 14.5° 2θ, about 15.1° 2θ, about 17.5° 2θ, and about 18.2° 2θ. For example, crystalline 25HC3S meglumine can also be characterized by a peak at about 8.6° 2θ. Alternatively or additionally, crystalline 25HC3S meglumine can also be characterized by a peak at about 14.5° 2θ. Alternatively or additionally, crystalline 25HC3S meglumine can also be characterized by a peak at about 15.1° 2θ. Alternatively or additionally, crystalline 25HC3S meglumine can also be characterized by a peak at about 17.5° 2θ. Alternatively or additionally, crystalline 25HC3S meglumine can also be characterized by a peak at about 18.2° 2θ.

[0305] In some cases, crystalline 25HC3S meglumine can be characterized by an x-ray powder diffraction pattern substantially the same as that in Figure 12 or Figure 12A.

[0306] As shown in Figure 47, crystalline 25HC3S meglumine absorbs water during the DVS experiment at a level of approximately 3%, with a plateau of water uptake at approximately this level above 35% relative humidity. This water uptake is consistent with the formation of a monohydrate. TGA from ambient temperature shows a water loss of approximately 3.2%, which is also consistent with the formation of a monohydrate. Figure 48 shows X-ray powder diffraction patterns before and after DVS. Figure 46 shows the DSC of crystalline 25HC3S meglumine, which shows multiple weak endothermic peaks between 50°C and 90°C. The solution state of this salt is 1 The H-NMR spectrum showed some additional peaks between 4.3 and 5.5 ppm, which were otherwise consistent with the chemical structure.

[0307] Further disclosed herein is substantially pure crystalline 25HC3S meglumine. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of 25HC3S meglumine in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of 25HC3S meglumine present (based on weight).

[0308] Methods for preparing 25HC3S meglumine are further described herein. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt, for example, as described in Example 41. This triethylammonium salt can then be used to prepare 25HC3S meglumine, as described in Example 34.

[0309] The present application also relates to pharmaceutical compositions comprising 25HC3S meglumine (including crystalline 25HC3S meglumine) disclosed herein. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and 25HC3S meglumine (including crystalline 25HC3S meglumine). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0310] The present application also includes methods and uses of using an effective amount of 25HC3S meglumine (including crystalline 25HC3S meglumine) of the present application and / or a pharmaceutical composition comprising crystalline 25HC3S meglumine to treat and / or prevent diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0311] An exemplary organic salt of 25HC3S herein is the organic cyclic amine hydroxyethylpyrrolidinium salt of 25HC3S. A method for preparing the crystalline hydroxyethylpyrrolidinium salt of 25HC3S is provided in Example 35. FIG9 shows an X-ray powder diffraction pattern of crystalline 25HC3S hydroxyethylpyrrolidinium. FIG10 shows a selected peak X-ray powder diffraction pattern. Table 9 shows selected peaks from FIG10.

[0312] Table 9 - Peaks of Crystallized 25HC3S Hydroxyethylpyrrolidinium in Figure 10

[0313]

[0314] 17.42±0.20 5.087±0.058 47

[0315] 18.18±0.20 4.876±0.053 12

[0316] 18.71±0.20 4.739±0.050 8

[0317] 18.98±0.20 4.672±0.049 23

[0318] 19.23±0.20 4.612±0.048 30

[0319] 20.01±0.20 4.434±0.044 16

[0320] 20.22±0.20 4.389±0.043 9

[0321] 20.62±0.20 4.304±0.041 12

[0322] 20.91±0.20 4.245±0.040 45

[0323] 21.42±0.20 4.145±0.038 12

[0324] 21.73±0.20 4.087±0.037 18

[0325] 22.36±0.20 3.972±0.035 8

[0326] 22.93±0.20 3.875±0.033 17

[0327] 23.14±0.20 3.841±0.033 15

[0328] 23.72±0.20 3.748±0.031 14

[0329] 24.11±0.20 3.688±0.030 9

[0330] 24.28±0.20 3.663±0.030 9

[0331] 25.00±0.20 3.559±0.028 14

[0332] 25.73±0.20 3.460±0.026 8

[0333] 26.78±0.20 3.326±0.024 12

[0334] 27.39±0.20 3.253±0.023 6

[0335] 27.74±0.20 3.213±0.023 9

[0336] 28.48±0.20 3.132±0.022 6

[0337] 28.87±0.20 3.090±0.021 7

[0338] Crystalline 25HC3S-hydroxyethylpyrrolidinium can be characterized by a variety of analytical techniques, including X-ray powder diffraction. The X-ray powder diffraction pattern of crystalline 25HC3S-hydroxyethylpyrrolidinium, or a portion thereof, can be used to identify crystalline 25HC3S-hydroxyethylpyrrolidinium. Crystalline 25HC3S-hydroxyethylpyrrolidinium comprises multiple X-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline 25HC3S-hydroxyethylpyrrolidinium.

[0339] In some cases, crystalline 25HC3S-hydroxyethylpyrrolidinium can be characterized by an x-ray powder diffraction pattern having one or more of the peaks in Figure 10. For example, a peak at approximately 3.8° 2θ can be used to characterize crystalline 25HC3S-hydroxyethylpyrrolidinium. In these and other cases, crystalline 25HC3S-hydroxyethylpyrrolidinium can also be characterized by one or more peaks at approximately 7.5° 2θ, approximately 7.6° 2θ, approximately 8.2° 2θ, approximately 8.6° 2θ, approximately 12.4° 2θ, approximately 13.3° 2θ, and approximately 15.0° 2θ.

[0340] The two most intense peaks in Figure 10 are at approximately 7.5° 2θ and approximately 7.6° 2θ. In Figure 10, these two peaks appear very close due to their respective intensities, but the XRPD instrument is able to distinguish them. However, due to the variability associated with these XRPD peaks, they may appear to be single peaks located "below" each other when analyzed. Because such a single apparent peak is possible, in many cases herein, crystalline 25HC3S-hydroxyethylpyrrolidinium is characterized, at least in part, by an X-ray powder diffraction pattern comprising two peaks located between approximately 7.5° 2θ and approximately 7.6° 2θ that: (a) do not overlap; (b) partially overlap; or (c) superimpose, thereby appearing as a single peak.

[0341] The two strongest peaks in Figure 10 are approximately 8.2 ° 2θ and about 8.6 ° 2θ. In Figure 10, these two peaks appear very close due to their respective intensities, but the XRPD instrument is able to distinguish them. However, due to the variability associated with these XRPD peaks, they may appear to be single peaks located "below" each other when analyzed. Because such a single apparent peak is possible, in many cases herein, crystalline 25HC3S-hydroxyethylpyrrolidinium is characterized at least in part by an X-ray powder diffraction pattern that contains two peaks located at about 8.2° 2θ to about 8.6° 2θ that: (a) do not overlap; (b) partially overlap; or (c) superimpose, thereby appearing as a single peak.

[0342] In some cases, the crystalline 25HC3S-hydroxyethylpyrrolidinium can be characterized by an x-ray powder diffraction pattern having a peak at about 7.5° 2θ. In these and other cases, the crystalline 25HC3S-hydroxyethylpyrrolidinium can also be 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.

[0343] In some cases, the crystalline 25HC3S-hydroxyethylpyrrolidinium can be characterized by an x-ray powder diffraction pattern having a peak at about 7.6° 2θ. In these and other cases, the crystalline 25HC3S-hydroxyethylpyrrolidinium can also be 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.

[0344] In some cases, the crystalline 25HC3S-hydroxyethylpyrrolidinium can be characterized by an x-ray powder diffraction pattern having a peak at about 8.2° 2θ. In these and other cases, the crystalline 25HC3S-hydroxyethylpyrrolidinium can also be 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.

[0345] In some cases, the crystalline 25HC3S hydroxyethyl pyrrolidinium can be characterized by an x-ray powder diffraction pattern having a peak at about 8.6° 2θ. In these and other cases, the crystalline 25HC3S hydroxyethyl pyrrolidinium can also be 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.

[0346] In some cases, the crystalline 25HC3S hydroxyethyl pyrrolidinium can be characterized by an x-ray powder diffraction pattern having a peak at 12.4° 2θ. In these and other cases, the crystalline 25HC3S hydroxyethyl pyrrolidinium can also be 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.

[0347] In some cases, the crystalline 25HC3S hydroxyethyl pyrrolidinium can be characterized by an x-ray powder diffraction pattern having a peak at 13.3° 2θ. In these and other cases, the crystalline 25HC3S hydroxyethyl pyrrolidinium can also be 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.

[0348] In some cases, the crystalline 25HC3S-hydroxyethylpyrrolidinium can be characterized by an x-ray powder diffraction pattern having a peak at 15.0° 2θ. In these and other cases, the crystalline 25HC3S-hydroxyethylpyrrolidinium can also be 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, the crystalline 25HC3S-hydroxyethylpyrrolidinium can be characterized by an x-ray powder diffraction pattern substantially the same as that of FIG. 10 .

[0349] In some cases, crystalline 25HC3S-hydroxyethylpyrrolidinium can also be characterized by one or more peaks at about 10.5° 2θ, about 15.3° 2θ, about 15.6° 2θ, about 16.3° 2θ, about 16.7° 2θ, and about 20.9° 2θ. For example, crystalline 25HC3S-hydroxyethylpyrrolidinium can also be characterized by a peak at about 10.5° 2θ. Alternatively or additionally, crystalline 25HC3S-hydroxyethylpyrrolidinium can also be characterized by a peak at about 15.3° 2θ. Alternatively or additionally, crystalline 25HC3S-hydroxyethylpyrrolidinium can also be characterized by a peak at about 15.6° 2θ. Alternatively or additionally, crystalline 25HC3S-hydroxyethylpyrrolidinium can also be characterized by a peak at about 16.3° 2θ. Alternatively or additionally, crystalline 25HC3S hydroxyethylpyrrolidinium may also be characterized by a peak at about 16.7° 2θ. Alternatively or additionally, crystalline 25HC3S hydroxyethylpyrrolidinium may also be characterized by a peak at about 20.9° 2θ.

[0350] Crystalline 25HC3S hydroxyethyl pyrrolidinium absorbs and retains water during the DVS experiment. In Figure 42, the stable plateau in the DVS shows a water content of about 2.5% to about 6% over a relative humidity range of about 25% to 75%. These data suggest that crystalline 25HC3S hydroxyethyl pyrrolidinium may convert to hydrates at this relative humidity, including levels consistent with a monohydrate. The TGA in Figure 41 shows that the weight loss upon heating to as high as 181°C is limited to about 0.1%, indicating that the salt is anhydrous at room temperature and therefore converts to a hydrate upon exposure to the appropriate relative humidity in the DVS. After the DVS experiment in Figure 42 is completed, the XRPD pattern in Figure 43 is different from that before the experiment, which is also consistent with a form change (e.g., from anhydrous to hydrate). The solution in Figure 44 1 H-NMR spectra revealed an additional proton at 5.3 ppm that was not explained in the structure, but the rest of the spectrum was consistent with the chemical structure. Therefore, without being bound by theory, it is believed that crystalline 25HC3S-hydroxyethylpyrrolidinium may exist as both an anhydrate and a hydrate (e.g., a monohydrate) at appropriate relative humidity. The DSC of crystalline 25HC3S-hydroxyethylpyrrolidinium can be seen in Figure 41 , showing a weak endothermic peak at approximately 40°C and another endothermic peak at approximately 140°C, followed by a presumed decomposition at approximately 181°C.

[0351] The X-ray powder diffraction pattern of crystalline 25HC3S-hydroxyethylpyrrolidinium was successfully indexed, indicating that the pattern represents a single crystalline phase, and the results are shown in Figure 45. The indexing results show that crystalline 25HC3S-hydroxyethylpyrrolidinium has a triclinic unit cell, and its unit cell volume is consistent with the anhydrous form and the stoichiometry of the provided salt. Its molecular formula volume was determined to be / unit cell (±5%), which can accommodate 1 mol / mol of hydroxyethylpyrrolidinium. Table 10 below lists some unit cell parameters.

[0352] Table 10 - Indexing Summary of Crystalline 25HC3S Hydroxyethylpyrrolidinium

[0353]

[0354] Further disclosed herein is substantially pure crystalline 25HC3S-hydroxyethylpyrrolidinium. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of 25HC3S-hydroxyethylpyrrolidinium present. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of 25HC3S-hydroxyethylpyrrolidinium present (based on weight).

[0355] Further described herein are methods for preparing 25HC3S-hydroxyethylpyrrolidinium. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to a triethylammonium salt, such as described in Example 41. This triethylammonium salt can then be used to prepare 25HC3S-hydroxyethylpyrrolidinium, as described in Example 35.

[0356] The present application also relates to pharmaceutical compositions comprising the 25HC3S-hydroxyethylpyrrolidinium disclosed herein (including crystalline 25HC3S-hydroxyethylpyrrolidinium). Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and 25HC3S-hydroxyethylpyrrolidinium (including crystalline 25HC3S-hydroxyethylpyrrolidinium). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0357] The present application also includes methods and uses of using an effective amount of the 25HC3S-hydroxyethylpyrrolidinium of the present application (including crystalline 25HC3S-hydroxyethylpyrrolidinium) and / or a pharmaceutical composition comprising crystalline 25HC3S-hydroxyethylpyrrolidinium to treat and / or prevent diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0358] An exemplary organic salt of 25HC3S herein is the organic aliphatic amine diethylammonium salt of 25HC3S. A method for preparing the crystalline diethylammonium salt of 25HC3S is provided in Example 36. FIG17 shows an X-ray powder diffraction pattern of crystalline diethylammonium 25HC3S. FIG18 shows a selected peak X-ray powder diffraction pattern. Table 11 shows the selected peaks in FIG18.

[0359] Table 11 - Peaks of crystallized 25HC3S diethylammonium in Figure 18

[0360]

[0361]

[0362] Crystalline 25HC3S-diethylammonium can be characterized by a variety of analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S-diethylammonium, or a portion thereof, can be used to identify crystalline 25HC3S-diethylammonium. Crystalline 25HC3S-diethylammonium comprises multiple x-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline 25HC3S-diethylammonium.

[0363] In some cases, crystalline 25HC3S diethylammonium can 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 can be used to characterize crystalline 25HC3S diethylammonium. In these and other cases, crystalline 25HC3S diethylammonium can also be 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.

[0364] In some cases, crystalline 25HC3S diethylammonium can 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 can also be 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.

[0365] In some cases, crystalline 25HC3S diethylammonium can 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 can also be 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.

[0366] In some cases, crystalline 25HC3S diethylammonium can 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 can also be 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.

[0367] In some cases, crystalline 25HC3S diethylammonium can 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 can also be 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.

[0368] In some cases, crystalline 25HC3S diethylammonium can be characterized by an x-ray powder diffraction pattern having a peak at 12.3° 2θ. In these and other cases, crystalline 25HC3S diethylammonium can also be 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, about 15.4° 2θ, and about 17.2° 2θ.

[0369] In some cases, crystalline 25HC3S diethylammonium can be characterized by an x-ray powder diffraction pattern having a peak at 15.4° 2θ. In these and other cases, crystalline 25HC3S diethylammonium can also be 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.

[0370] In some cases, the crystalline 25HC3S diethylammonium can be characterized by an x-ray powder diffraction pattern having a peak at 17.2° 2θ. In these and other cases, the crystalline 25HC3S diethylammonium can also be 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.

[0371] In some cases, crystalline 25HC3S diethylammonium can be characterized by an x-ray powder diffraction pattern substantially the same as that of FIG. 18 .

[0372] As shown in Figure 60, crystalline 25HC3S diethylammonium absorbs water during the DVS experiment, indicating that it is hygroscopic at relative humidity exceeding 85%. As shown in the TGA in Figure 59, after heating to 204°C, the weight loss is limited to about 0.1%, indicating that it is anhydrous under ambient conditions. The DSC of crystalline 25HC3S diethylammonium is shown in Figure 59, showing an endothermic peak at about 134°C and an endothermic peak at about 215°C. Figure 61 shows the X-ray powder diffraction patterns before and after DVS, and Figure 62 shows the X-ray powder diffraction patterns before and after DVS. 1 The H-NMR spectrum was consistent with the chemical structure.

[0373] The X-ray powder diffraction pattern of crystalline 25HC3S diethylammonium has been successfully indexed, indicating that the pattern represents a single crystalline phase, as shown in Figure 63. The indexing results show that crystalline 25HC3S diethylammonium has an orthorhombic unit cell with a unit cell volume of (±5%), consistent with the stoichiometry of the anhydrous form and the supplied salt. Table 12 below lists some unit cell parameters.

[0374] Table 12 - Indexing Summary of Crystalline 25HC3S Diethylammonium

[0375]

[0376] Further disclosed herein is substantially pure crystalline 25HC3S diethylammonium. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of 25HC3S diethylammonium in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of 25HC3S diethylammonium present (based on weight).

[0377] Methods for preparing 25HC3S diethylammonium are further described herein. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt, such as described in Example 41. This triethylammonium salt can then be used to prepare 25HC3S diethylammonium, as described in Example 36.

[0378] The present application also relates to pharmaceutical compositions comprising 25HC3S diethylammonium (including crystalline 25HC3S diethylammonium) disclosed herein. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and 25HC3S diethylammonium (including crystalline 25HC3S diethylammonium). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0379] The present application also includes methods and uses of using an effective amount of 25HC3S diethylammonium (including crystalline 25HC3S diethylammonium) of the present application and / or a pharmaceutical composition comprising crystalline 25HC3S diethylammonium to treat and / or prevent diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, nervous system conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0380] An exemplary organic salt of 25HC3S herein is the organic amine diethanolamine salt of 25HC3S. A method for preparing the crystalline diethanolamine salt of 25HC3S is provided in Example 37. FIG15 shows an X-ray powder diffraction pattern of crystalline 25HC3S diethanolamine. FIG16 shows a selected peak X-ray powder diffraction pattern. Table 13 shows the selected peaks in FIG16.

[0381] Table 13 - Peaks of Crystallized 25HC3S Diethanolamine in Figure 16

[0382]

[0383]

[0384] Crystalline 25HC3S-diethanolamine can be characterized by a variety of analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S-diethanolamine, or a portion thereof, can be used to identify crystalline 25HC3S-diethanolamine. Crystalline 25HC3S-diethanolamine contains multiple x-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline 25HC3S-diethanolamine.

[0385] In some cases, crystalline 25HC3S-diethanolamine can 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 can be used to characterize crystalline 25HC3S-diethanolamine. In these and other cases, crystalline 25HC3S-diethanolamine can also be 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.

[0386] The two most intense peaks in Figure 16 are at about 7.7° 2θ and about 8.1° 2θ. In Figure 16, these two peaks appear very close due to their respective intensities, but the XRPD instrument is able to distinguish them. However, due to the variability associated with these XRPD peaks, they may appear to be single peaks located "below" each other when analyzed. Because such a single apparent peak is possible, in many cases herein, crystalline 25HC3S-diethanolamine is characterized at least in part by an X-ray powder diffraction pattern that contains two peaks located at about 7.7° 2θ to about 8.1° 2θ that: (a) do not overlap; (b) partially overlap; or (c) superimpose, thereby appearing as a single peak.

[0387] In some cases, the crystalline 25HC3S diethanolamine can be characterized by an x-ray powder diffraction pattern having a peak at about 7.7° 2θ. In these and other cases, the crystalline 25HC3S diethanolamine can also be 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.

[0388] In some cases, the crystalline 25HC3S diethanolamine can be characterized by an x-ray powder diffraction pattern having a peak at about 8.1° 2θ. In these and other cases, the crystalline 25HC3S diethanolamine can also be 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.

[0389] In some cases, the crystalline 25HC3S diethanolamine can be characterized by an x-ray powder diffraction pattern having a peak at about 8.2° 2θ. In these and other cases, the crystalline 25HC3S diethanolamine can also be 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.

[0390] In some cases, the crystalline 25HC3S diethanolamine can be characterized by an x-ray powder diffraction pattern having a peak at about 8.8° 2θ. In these and other cases, the crystalline 25HC3S diethanolamine can also be 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.

[0391] In some cases, the crystalline 25HC3S diethanolamine can be characterized by an x-ray powder diffraction pattern having a peak at 14.6° 2θ. In these and other cases, the crystalline 25HC3S diethanolamine can also be 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.

[0392] In some cases, crystalline 25HC3S diethanolamine can be characterized by an x-ray powder diffraction pattern substantially the same as that of FIG. 16 .

[0393] The DVS experiment in Figure 55 shows that crystalline 25HC3S diethanolamine absorbs water above about 75% relative humidity and exhibits hygroscopicity. As shown in Figure 54, TGA observed that there was almost no weight loss when heated to 175°C, indicating that it is anhydrous at room temperature. The solution in Figure 57 1 H-NMR shows an additional proton at 5.3 ppm that is not accounted for in the structure, but is otherwise consistent with the chemical structure. Figure 54 shows the DSC of crystalline 25HC3S diethanolamine, which exhibits a single isotherm at approximately 181°C. Figure 56 shows the X-ray powder diffraction patterns before and after DVS.

[0394] The X-ray powder diffraction pattern of crystalline 25HC3S diethanolamine has been successfully indexed, indicating that the pattern represents a single crystalline phase, as shown in Figure 58. The indexing results show that crystalline 25HC3S diethanolamine has a monoclinic unit cell with a unit cell volume of The stoichiometry is consistent with the anhydrous form and the supplied salt. Table 14 below lists some unit cell parameters.

[0395] Table 14 - Indexing Summary of Crystalline 25HC3S Diethanolamine

[0396]

[0397] Further disclosed herein is substantially pure crystalline 25HC3S-diethanolamine. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of 25HC3S-diethanolamine present in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of 25HC3S-diethanolamine present (based on weight).

[0398] Methods for preparing 25HC3S-diethanolamine are further described herein. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt, such as described in Example 41. This triethylammonium salt can then be used to prepare 25HC3S-diethanolamine, as described in Example 37.

[0399] The present application also relates to pharmaceutical compositions comprising 25HC3S diethanolamine (including crystalline 25HC3S diethanolamine) disclosed herein. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and 25HC3S diethanolamine (including crystalline 25HC3S diethanolamine). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0400] The present application also includes methods and uses of using an effective amount of 25HC3S diethanolamine (including crystalline 25HC3S diethanolamine) of the present application and / or a pharmaceutical composition comprising crystalline 25HC3S diethanolamine to treat and / or prevent diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0401] An exemplary organic salt of 25HC3S herein is the organic amine tert-butylammonium salt of 25HC3S. A method for preparing the crystalline tert-butylammonium salt of 25HC3S is provided in Example 38. FIG19 shows an X-ray powder diffraction pattern of crystalline tert-butylammonium 25HC3S. FIG20 shows a selected peak X-ray powder diffraction pattern. Table 15 shows the selected peaks in FIG20.

[0402] Table 15 - Peaks of Crystallized 25HC3S Tert-Butyl Ammonium in Figure 20

[0403]

[0404] 21.15±0.20 4.197±0.039 22

[0405] 21.57±0.20 4.116±0.038 11

[0406] 22.68±0.20 3.917±0.034 5

[0407] 22.96±0.20 3.871±0.033 6

[0408] 23.33±0.20 3.810±0.032 4

[0409] 23.82±0.20 3.733±0.031 11

[0410] 24.36±0.20 3.651±0.030 5

[0411] 24.76±0.20 3.593±0.029 4

[0412] 25.28±0.20 3.520±0.027 6

[0413] 25.99±0.20 3.426±0.026 4

[0414] 26.68±0.20 3.338±0.025 3

[0415] 26.96±0.20 3.304±0.024 3

[0416] 27.43±0.20 3.249±0.023 3

[0417] 28.17±0.20 3.166±0.022 3

[0418] 28.94±0.20 3.082±0.021 3

[0419] Crystalline 25HC3S-tert-butylammonium can be characterized by a variety of analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S-tert-butylammonium, or a portion thereof, can be used to identify crystalline 25HC3S-tert-butylammonium. Crystalline 25HC3S-tert-butylammonium comprises multiple x-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline 25HC3S-tert-butylammonium.

[0420] In some cases, crystalline 25HC3S-tert-butylammonium can 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 can be used to characterize crystalline 25HC3S-tert-butylammonium. In these and other cases, crystalline 25HC3S-tert-butylammonium can also be 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.

[0421] In some cases, the crystalline 25HC3S-tert-butylammonium can be characterized by an x-ray powder diffraction pattern having a peak at about 8.0° 2θ. In these and other cases, the crystalline 25HC3S-tert-butylammonium can also be 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.

[0422] In some cases, the crystalline 25HC3S-tert-butylammonium can be characterized by an x-ray powder diffraction pattern having a peak at about 11.5° 2θ. In these and other cases, the crystalline 25HC3S-tert-butylammonium can also be 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.

[0423] In some cases, the crystalline 25HC3S-tert-butylammonium can be characterized by an x-ray powder diffraction pattern having a peak at about 12.2° 2θ. In these and other cases, the crystalline 25HC3S-tert-butylammonium can also be 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.

[0424] In some cases, the crystalline 25HC3S-tert-butylammonium can be characterized by an x-ray powder diffraction pattern having a peak at about 14.4° 2θ. In these and other cases, the crystalline 25HC3S-tert-butylammonium can also be 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.

[0425] In some cases, the crystalline 25HC3S-tert-butylammonium can be characterized by an x-ray powder diffraction pattern having a peak at 15.4° 2θ. In these and other cases, the crystalline 25HC3S-tert-butylammonium can also be 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.

[0426] In some cases, the crystalline 25HC3S-tert-butylammonium can be characterized by an x-ray powder diffraction pattern having a peak at 16.3° 2θ. In these and other cases, the crystalline 25HC3S-tert-butylammonium can also be 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.

[0427] In some cases, crystalline 25HC3S-tert-butylammonium can also be characterized by one or more peaks at about 10.3° 2θ, about 10.5° 2θ, about 13.8° 2θ, about 16.8° 2θ, about 17.0° 2θ, and about 17.4° 2θ. For example, crystalline 25HC3S-tert-butylammonium can also be characterized by a peak at about 10.3° 2θ. Alternatively or additionally, crystalline 25HC3S-tert-butylammonium can also be characterized by a peak at about 10.5° 2θ. Alternatively or additionally, crystalline 25HC3S-tert-butylammonium can also be characterized by a peak at about 13.8° 2θ. Alternatively or additionally, crystalline 25HC3S-tert-butylammonium can also be characterized by a peak at about 16.8° 2θ. Alternatively or additionally, crystalline 25HC3S-tert-butylammonium can also be characterized by a peak at about 17.0° 2θ. Alternatively or additionally, crystalline 25HC3S-tert-butylammonium may also be characterized by a peak at approximately 17.4° 2θ.

[0428] In some cases, crystalline 25HC3S-tert-butylammonium can be characterized by an x-ray powder diffraction pattern substantially the same as that of FIG. 20 .

[0429] Crystalline 25HC3S tert-butylammonium is less hygroscopic, as shown in Figure 65, where relative humidity ranges from 5% to 95% were observed by DVS. In Figure 64, a weight loss of 0.2% was observed by TGA after heating to 200°C, indicating an anhydrous structure under ambient conditions. The DSC trace of crystallized 25HC3S tert-butylammonium is shown in Figure 64, which shows isotherms near 205°C and 218°C. 1 The H-NMR spectrum was consistent with the chemical structure.

[0430] The X-ray powder diffraction pattern of crystalline 25HC3S tert-butylammonium has been successfully indexed, indicating that the pattern represents a single crystalline phase, as shown in Figure 67. The indexing results show that crystalline 25HC3S tert-butylammonium has a monoclinic unit cell with a unit cell volume of The stoichiometry is consistent with the anhydrous form and the supplied salt. Table 16 below lists some unit cell parameters.

[0431] Table 16 - Indexing Summary of Crystalline 25HC3S Tert-Butylammonium

[0432]

[0433] Further disclosed herein is substantially pure crystalline 25HC3S-tert-butylammonium. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of 25HC3S-tert-butylammonium in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of 25HC3S-tert-butylammonium present (by weight).

[0434] Methods for preparing 25HC3S-tert-butylammonium are further described herein. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt, such as described in Example 41. This triethylammonium salt can then be used to prepare 25HC3S-tert-butylammonium, as described in Example 38.

[0435] The present application also relates to pharmaceutical compositions comprising 25HC3S-tert-butylammonium (including crystalline 25HC3S-tert-butylammonium) disclosed herein. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and 25HC3S-tert-butylammonium (including crystalline 25HC3S-tert-butylammonium). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0436] The present application also includes methods and uses for treating and / or preventing diseases (e.g., in humans) using an effective amount of 25HC3S tert-butylammonium (including crystalline 25HC3S tert-butylammonium) and / or a pharmaceutical composition comprising crystalline 25HC3S tert-butylammonium of the present application, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0437] An exemplary 25HC3S organic salt of the present application is a 25HC3S organic amine benzathine salt. A method for preparing a crystalline benzathine salt of 25HC3S is provided in Example 39. Figure 21 shows an X-ray powder diffraction pattern of crystalline 25HC3S benzathine. Figure 22 shows a selected peak X-ray powder diffraction pattern. Table 17 shows selected peaks from Figure 22. ICP-OES confirmed that the benzathine salt of 25HC3S contained two 25HC3S ions for each benzathine ion.

[0438] Table 17 - Peaks of Crystallized 25HC3S Benzathine in Figure 22

[0439]

[0440] 15.06±0.20 5.878±0.078 16

[0441] 15.24±0.20 5.809±0.076 18

[0442] 16.18±0.20 5.474±0.067 100

[0443] 16.41±0.20 5.397±0.065 52

[0444] 17.04±0.20 5.199±0.061 11

[0445] 17.81±0.20 4.976±0.055 28

[0446] 18.32±0.20 4.839±0.052 13

[0447] 18.77±0.20 4.724±0.050 4

[0448] 19.18±0.20 4.624±0.048 5

[0449] 19.68±0.20 4.507±0.045 12

[0450] 20.35±0.20 4.360±0.042 30

[0451] 21.34±0.20 4.160±0.039 7

[0452] 21.73±0.20 4.087±0.037 11

[0453] 22.70±0.20 3.914±0.034 6

[0454] 23.36±0.20 3.805±0.032 7

[0455] 23.66±0.20 3.758±0.031 6

[0456] 24.44±0.20 3.639±0.029 8

[0457] 25.20±0.20 3.531±0.028 4

[0458] 26.71±0.20 3.335±0.025 7

[0459] 27.39±0.20 3.254±0.023 4

[0460] 28.96±0.20 3.080±0.021 3

[0461] 29.76±0.20 3.000±0.020 10

[0462] 30.39±0.20 2.939±0.019 11

[0463] 31.17±0.20 2.867±0.018 6

[0464] Crystalline 25HC3S-benzylthion can be characterized by a variety of analytical techniques, including x-ray powder diffraction. The x-ray powder diffraction pattern of crystalline 25HC3S-benzylthion, or a portion thereof, can be used to identify crystalline 25HC3S-benzylthion. Crystalline 25HC3S-benzylthion contains multiple x-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline 25HC3S-benzylthion.

[0465] In some cases, crystalline 25HC3S benzathine can 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 can be used to characterize crystalline 25HC3S benzathine. In these and other cases, crystalline 25HC3S benzathine can also be characterized by one or more peaks at about 6.9° 2θ, about 8.2° 2θ, about 12.3° 2θ, and about 16.2° 2θ.

[0466] In some cases, the crystalline 25HC3S benzathine can be characterized by an x-ray powder diffraction pattern having a peak at about 6.9° 2θ. In these and other cases, the crystalline 25HC3S benzathine can also be characterized by one or more peaks at about 4.1° 2θ, about 8.2° 2θ, about 12.3° 2θ, and about 16.2° 2θ.

[0467] In some cases, the crystalline 25HC3S benzathine can be characterized by an x-ray powder diffraction pattern having a peak at about 8.2° 2θ. In these and other cases, the crystalline 25HC3S benzathine can also be characterized by one or more peaks at about 4.1° 2θ, about 6.9° 2θ, about 12.3° 2θ, and about 16.2° 2θ.

[0468] In some cases, the crystalline 25HC3S benzathine can be characterized by an x-ray powder diffraction pattern having a peak at about 12.3° 2θ. In these and other cases, the crystalline 25HC3S benzathine can also be characterized by one or more peaks at about 4.1° 2θ, about 6.9° 2θ, and about 16.2° 2θ.

[0469] In some cases, the crystalline 25HC3S benzathine can be characterized by an x-ray powder diffraction pattern having a peak at about 16.2° 2θ. In these and other cases, the crystalline 25HC3S benzathine can also be characterized by one or more peaks at about 4.1° 2θ, about 6.9° 2θ, and about 12.3° 2θ.

[0470] In some cases, crystalline 25HC3S benzathine can also be characterized by one or more peaks at about 7.3° 2θ, about 15.1° 2θ, about 16.4° 2θ, about 17.8° 2θ, and about 20.4° 2θ. For example, crystalline 25HC3S benzathine can also be characterized by a peak at about 7.3° 2θ. Alternatively or additionally, crystalline 25HC3S benzathine can also be characterized by a peak at about 15.1° 2θ. Alternatively or additionally, crystalline 25HC3S benzathine can also be characterized by a peak at about 16.4° 2θ. Alternatively or additionally, crystalline 25HC3S benzathine can also be characterized by a peak at about 17.8° 2θ. Alternatively or additionally, crystalline 25HC3S benzathine can also be characterized by a peak at about 20.4° 2θ.

[0471] In some cases, the crystalline 25HC3S benzathine has an x-ray powder diffraction pattern substantially the same as that in FIG. 22 .

[0472] Crystalline 25HC3S-benzyl oxaline has low hygroscopicity at relative humidity levels of 5% to 95%. As shown in Figure 68, a 0.4% weight loss was observed by TGA upon heating to 200°C, indicating that crystalline 25HC3S-benzyl oxaline is anhydrous under ambient conditions. The DSC of crystalline 25HC3S-benzyl oxaline shown in Figure 68 shows isotherms near 216°C and 223°C. The DVS isotherm in Figure 69 indicates low hygroscopicity. Figure 70 shows X-ray powder diffraction patterns before and after DVS treatment. Figure 71 shows 1 The H-NMR spectrum was consistent with the chemical structure.

[0473] The X-ray powder diffraction pattern of crystalline 25HC3S benzathine has been successfully indexed, indicating that the pattern represents a single crystalline phase, as shown in Figure 72. The indexing results show that crystalline 25HC3S benzathine has a monoclinic unit cell with a unit cell volume of (±5%), consistent with the anhydrous form and the supplied salt stoichiometry. Table 18 below lists some unit cell parameters.

[0474] Table 18 - Indexing Summary of Crystalline 25HC3S Benzathine

[0475]

[0476] Further disclosed herein is substantially pure crystalline 25HC3S-benzylthion. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (other than trace amounts) of other forms of 25HC3S-benzylthion in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less, relative to the total amount of 25HC3S-benzylthion present (by weight).

[0477] Methods for preparing 25HC3S-benzylthion are further described herein. In some cases, the sodium salt of 25HC3S may be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) may be converted to the triethylammonium salt, for example, as described in Example 41. This triethylammonium salt can then be used to prepare 25HC3S-benzylthion, as described in Example 39.

[0478] The present application also relates to pharmaceutical compositions comprising 25HC3S benzathine (including crystalline 25HC3S benzathine) disclosed herein. Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and 25HC3S benzathine (including crystalline 25HC3S benzathine). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0479] The present application also includes methods and uses for treating and / or preventing diseases (e.g., in humans) using an effective amount of 25HC3S benzathine (including crystalline 25HC3S benzathine) of the present application and / or a pharmaceutical composition comprising crystalline 25HC3S benzathine, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, neurological conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0480] An exemplary organic salt of 25HC3S of the present application is the organic amine choline salt of 25HC3S. A method for preparing the crystalline choline salt of 25HC3S is provided in Example 40. FIG76 shows an X-ray powder diffraction pattern of crystalline 25HC3S choline. FIG77 shows an X-ray powder diffraction pattern of selected peaks. Table 19 shows the selected peaks in FIG77.

[0481] Table 19 - Peaks of Crystallized 25HC3S Choline in Figure 77

[0482]

[0483]

[0484] Crystalline 25HC3S-choline can be characterized by a variety of analytical techniques, including X-ray powder diffraction. The X-ray powder diffraction pattern of crystalline 25HC3S-choline, or a portion thereof, can be used to identify crystalline 25HC3S-choline. Crystalline 25HC3S-choline contains multiple X-ray powder diffraction peaks that, individually or in combination, can help identify the presence of crystalline 25HC3S-choline.

[0485] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 3.9° 2θ. In these and other cases, the crystalline 25HC3S choline can also be 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.

[0486] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 7.8° 2θ. In these and other cases, the crystalline 25HC3S choline can also be 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.

[0487] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 9.5° 2θ. In these and other cases, the crystalline 25HC3S choline can also be 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.

[0488] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 10.1° 2θ. In these and other cases, the crystalline 25HC3S choline can also be 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.

[0489] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 11.0° 2θ. In these and other cases, the crystalline 25HC3S choline can also be 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.

[0490] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 12.2° 2θ. In these and other cases, the crystalline 25HC3S choline can also be 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.

[0491] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 13.7° 2θ. In these and other cases, the crystalline 25HC3S choline can also be 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.

[0492] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 14.7° 2θ. In these and other cases, the crystalline 25HC3S choline can also be 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.

[0493] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 15.1° 2θ. In these and other cases, the crystalline 25HC3S choline can also be 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.

[0494] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 15.8° 2θ. In these and other cases, the crystalline 25HC3S choline can also be 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.

[0495] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 16.3° 2θ. In these and other cases, the crystalline 25HC3S choline can also be 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.

[0496] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 19.1° 2θ. In these and other cases, the crystalline 25HC3S choline can also be 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.

[0497] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9° 2θ and about 7.8° 2θ. In these and other cases, the crystalline 25HC3S choline can also 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.

[0498] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9° 2θ, about 7.8° 2θ, and about 9.5° 2θ. In these and other cases, the crystalline 25HC3S choline can also 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.

[0499] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9° 2θ, about 7.8° 2θ, about 9.5° 2θ, and about 10.1° 2θ. In these and other cases, the crystalline 25HC3S choline can also 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.

[0500] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9° 2θ, about 7.8° 2θ, about 9.5° 2θ, about 10.1° 2θ, and about 11.0° 2θ. In these and other cases, the crystalline 25HC3S choline can also 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.

[0501] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9° 2θ, about 7.8° 2θ, about 9.5° 2θ, about 10.1° 2θ, about 11.0° 2θ, and about 12.2° 2θ. In these and other cases, the crystalline 25HC3S choline can also 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.

[0502] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9° 2θ, about 7.8° 2θ, about 9.5° 2θ, about 10.1° 2θ, about 11.0° 2θ, about 12.2° 2θ, and about 13.7° 2θ. In these and other cases, the crystalline 25HC3S choline can also 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.

[0503] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9° 2θ, about 7.8° 2θ, about 9.5° 2θ, about 10.1° 2θ, about 11.0° 2θ, about 12.2° 2θ, about 13.7° 2θ, and about 14.7° 2θ. In these and other cases, the crystalline 25HC3S choline can also 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.

[0504] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9° 2θ, about 7.8° 2θ, about 9.5° 2θ, about 10.1° 2θ, about 11.0° 2θ, about 12.2° 2θ, about 13.7° 2θ, about 14.7° 2θ, and about 15.1° 2θ. In these and other cases, the crystalline 25HC3S choline can also 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.

[0505] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9° 2θ, about 7.8° 2θ, about 9.5° 2θ, about 10.1° 2θ, about 11.0° 2θ, about 12.2° 2θ, about 13.7° 2θ, about 14.7° 2θ, about 15.1° 2θ, and about 15.8° 2θ. In these and other cases, the crystalline 25HC3S choline can also be characterized by an x-ray powder diffraction pattern having one or more peaks at about 16.3° 2θ and about 19.1° 2θ.

[0506] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9° 2θ, about 7.8° 2θ, about 9.5° 2θ, about 10.1° 2θ, about 11.0° 2θ, about 12.2° 2θ, about 13.7° 2θ, about 14.7° 2θ, about 15.1° 2θ, about 15.8° 2θ, and about 16.3° 2θ. In these and other cases, the crystalline 25HC3S choline can also be characterized by an x-ray powder diffraction pattern having a peak at about 19.1° 2θ.

[0507] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having peaks at about 3.9°2θ, about 7.8°2θ, about 9.5°2θ, about 10.1°2θ, about 11.0°2θ, about 12.2°2θ, about 13.7°2θ, about 14.7°2θ, about 15.1°2θ, about 15.8°2θ, about 16.3°2θ, and about 19.1°2θ.

[0508] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 7.8° 2θ, about 9.5° 2θ, about 10.1° 2θ, about 11.0° 2θ, about 12.2° 2θ, about 13.7° 2θ, about 14.7° 2θ, about 15.1° 2θ, about 15.8° 2θ, about 16.3° 2θ, and about 19.1° 2θ.

[0509] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 9.5° 2θ, about 10.1° 2θ, about 11.0° 2θ, about 12.2° 2θ, about 13.7° 2θ, about 14.7° 2θ, about 15.1° 2θ, about 15.8° 2θ, about 16.3° 2θ, and about 19.1° 2θ.

[0510] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 10.1° 2θ, about 11.0° 2θ, about 12.2° 2θ, about 13.7° 2θ, about 14.7° 2θ, about 15.1° 2θ, about 15.8° 2θ, about 16.3° 2θ, and about 19.1° 2θ.

[0511] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 11.0° 2θ, about 12.2° 2θ, about 13.7° 2θ, about 14.7° 2θ, about 15.1° 2θ, about 15.8° 2θ, about 16.3° 2θ, and about 19.1° 2θ.

[0512] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 12.2° 2θ, about 13.7° 2θ, about 14.7° 2θ, about 15.1° 2θ, about 15.8° 2θ, about 16.3° 2θ, and about 19.1° 2θ.

[0513] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 13.7° 2θ, about 14.7° 2θ, about 15.1° 2θ, about 15.8° 2θ, about 16.3° 2θ, and about 19.1° 2θ.

[0514] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 14.7° 2θ, about 15.1° 2θ, about 15.8° 2θ, about 16.3° 2θ, and about 19.1° 2θ.

[0515] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 15.1° 2θ, about 15.8° 2θ, about 16.3° 2θ, and about 19.1° 2θ.

[0516] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at about 15.8° 2θ, about 16.3° 2θ, and about 19.1° 2θ.

[0517] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having one or more peaks at 16.3° 2Θ and about 19.1° 2Θ.

[0518] In some cases, the crystalline 25HC3S choline can be characterized by an x-ray powder diffraction pattern having a peak at about 19.1° 2θ.

[0519] In some cases, crystalline 25HC3S choline can be characterized by having an x-ray powder diffraction pattern substantially the same as that shown in Figure 77.

[0520] The X-ray powder diffraction pattern of crystalline 25HC3S choline has been successfully indexed, indicating that the pattern represents a single crystalline phase, as shown in Figure 79. The indexing results show that crystalline 25HC3S choline has an orthorhombic unit cell with a unit cell volume of The following table 20 lists some unit cell parameters.

[0521] Table 20 - Indexing Summary of Crystalline 25HC3S Choline

[0522]

[0523] The DSC thermogram of crystalline 25HC3S choline in Figure 80 shows endothermic peaks at about 198° C. and about 220° C. The TGA thermogram in Figure 80 shows that the weight loss is almost negligible up to 198° C.

[0524] Without being bound by theory, crystalline 25HC3S choline is believed to be anhydrate, meaning that there is no water of crystallization in the unit cell. This does not exclude the possibility that additional water may be present in the solid comprising crystalline 25HC3S choline. Furthermore, crystalline 25HC3S choline exhibits no significant hygroscopicity up to a relative humidity of approximately 95%, with a weight increase of only approximately 0.5% up to this relative humidity, as confirmed by dynamic vapor sorption experiments in Example 26, the results of which are shown in FIG82 . Furthermore, the X-ray powder diffraction pattern of crystalline 25HC3S choline showed no significant changes after DVS, as shown in FIG78 . Upon increasing the relative humidity from 5% to 95%, only a 0.5% weight increase was observed, while upon returning the relative humidity to 5%, a 0.5% weight loss was observed, indicating no hysteresis. This low hygroscopicity indicates good stability under such stresses, which, as discussed further elsewhere herein, may contribute to its stability suitable for pharmaceutical processing. Indeed, the present application also encompasses stable crystalline 25HC3S choline. Such stability includes, for example, crystalline 25HC3S choline that is sufficiently stable to be formulated for patient delivery. 1 The H-NMR spectrum was consistent with the structure shown in Figure 81 except for the peak at 5.3 ppm. There was no evidence of residual solvent. The overlap of the chlorine and methylene groups at approximately 3.8 ppm appeared to be 1 mol / mol choline.

[0525] The choline salt offers additional advantages over the sodium salt and some other salts of 25HC3S due to the beneficial properties of the choline counterion. For example, choline is an essential nutrient, and choline deficiency has been implicated in the accumulation of liver fat and cholesterol. Furthermore, the crystals formed from 25HC3S choline exhibit superior quality and diffraction properties compared to existing technologies. Finally, crystalline 25HC3S choline exhibits low hygroscopicity, resulting in improved physical stability compared to, for example, crystalline sodium 25HC3S. Crystalline sodium 25HC3S is stable as a hydrate when exposed to moisture. Specifically, monohydrate, dihydrate, and various hydrates of crystalline sodium 25HC3S have been prepared. Form I (hydrate) has been found to be hygroscopic and may form liquid crystals at high water activities (e.g., above 0.73). Another hydrate (Form II) is stable at relative humidity levels of approximately 21% to 30%. In contrast, at relative humidity up to about 95%, only about 0.5% by weight of water was absorbed, indicating that crystalline 25HC3S choline is stable as an anhydrate.

[0526] The present invention further discloses substantially pure crystalline 25HC3S choline. As used herein, "substantially pure" generally refers to the absence of any appreciable amount (except for trace amounts that may be present) of other forms of 25HC3S choline in the form herein. Examples of trace amounts include no more than about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1% or less, relative to the total amount of 25HC3S choline present (based on weight).

[0527] Methods for preparing 25HC3S choline are further described herein. In some cases, the sodium salt of 25HC3S can be prepared first. Examples of such preparations are listed herein. The sodium salt of 25HC3S (which may be crystalline) can be converted to the triethylammonium salt, such as described in Example 41. This triethylammonium salt can then be used to prepare 25HC3S choline, as described in Example 40.

[0528] The present application also relates to pharmaceutical compositions comprising the 25HC3S choline disclosed herein (including crystalline 25HC3S choline). Such pharmaceutical compositions are composed of one or more pharmaceutically acceptable excipients and 25HC3S choline (including crystalline 25HC3S choline). Such pharmaceutical compositions can be administered orally or formulated for delivery as any effective conventional dosage form, including, for example, immediate release, sustained release, slow release, and timed release oral formulations, parenteral, topical, nasal, ocular, ocular, sublingual, rectal, vaginal, and the like.

[0529] As described elsewhere herein and demonstrated in the Examples, the 25HC3S choline of the present application has surprisingly low hygroscopicity, including compared to other salt forms of 25HC3S. Therefore, 25HC3S choline can be advantageously produced and used to prepare pharmaceutical formulations, particularly dosage forms for oral administration (e.g., solid dosage forms such as tablets, capsules (each of which includes immediate release, sustained release, slow release, and timed release formulations), pills, powders, or granules).

[0530] Furthermore, the 25HC3S choline of the present application can also provide supplemental choline to patients suffering from the conditions targeted by 25HC3S. As described elsewhere herein, choline deficiency can contribute to these conditions, and therefore it may be advantageous to provide choline concurrently with 25HC3S during treatment. Thus, 25HC3S choline surprisingly and advantageously combines the advantageous properties of a salt form, for example, facilitating the preparation of oral dosage forms particularly suitable for treating certain conditions, with the inherent ability to provide choline supplementation during treatment methods using such oral dosage forms.

[0531] The present application also includes methods and uses of using an effective amount of 25HC3S choline (including crystalline 25HC3S choline) of the present application and / or a pharmaceutical composition comprising crystalline 25HC3S choline to treat and / or prevent diseases (e.g., in humans), such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), leptin resistance, leptin deficiency, diabetic conditions, autoimmune conditions, inflammatory conditions, nervous system conditions, Epstein-Barr virus-associated growth, and one or more conditions associated with fat accumulation and inflammation.

[0532] The exemplary 25HC3S organic salt disclosed in the present invention is the organic amine triethylammonium salt of 25HC3S. The preparation method of the 25HC3S triethylammonium salt is shown in Example 41.

[0533] The preparation of the 25HC3S triethylammonium salt can be accomplished, for example, by passing a mixture of triethylammonium chloride and triethylamine through a column and treating with a solvent (e.g., an alcohol) to a neutral pH. Alternatively, crystalline 25HC3S sodium can be dissolved in a solvent (e.g., an alcohol). This solution is then passed through the same column that was previously exposed to triethylamine and combined with the triethylammonium solution. The resulting solid is isolated (e.g., under vacuum or by drying) to yield the crystalline 25HC3S triethylammonium salt, which can be homogenized using, for example, a mortar and pestle. Suitable alcohols for this method include methanol.

[0534] The preparation method of 25HC3S choline (including crystalline 25HC3S choline) is as follows: starting from sodium 25HC3S, converting it to a second salt of 25HC3S (e.g., triethylammonium salt), and then converting the second salt of 25HC3S to 25HC3S choline (including crystalline 25HC3S choline). The preparation of crystalline 25HC3S choline can be achieved by preparing a suspension of the 25HC3S triethylammonium salt in a suitable solvent (e.g., acetonitrile), and then treating it with a choline source (e.g., aqueous choline hydroxide solution) to form 25HC3S choline (including crystalline 25HC3S choline). The 25HC3S choline can be purified, for example, by washing with a suitable solvent. Additional treatments, such as vacuum drying or other treatments, may also be performed. The present application also includes crystalline 25HC3S choline prepared by the methods described herein.

[0535] Preparation method of 25-hydroxy-cholest-5-ene-3-sulfate (25HC3S)

[0536] Methods for preparing 25-hydroxy-cholest-5-ene-3-sulfate (e.g., 25-hydroxy-3β-cholest-5-ene-3-sulfate (25HC3S)) are described herein. Although many of the teachings herein relate to sulfates at the 3β position, the teachings of the present application are also generally applicable to sulfates at the 3α position. In the methods for preparing 25-hydroxy-3β-cholest-5-ene-3-sulfate described herein, the components used in each step can be purified compositions or crude compositions as needed. The term "purified" is used in its conventional sense to refer to a composition that has at least undergone some separation or purification process, for example, filtering or aqueous post-treatment of the reaction mixture. In some cases, purification includes at least one of liquid chromatography, recrystallization, distillation (e.g., azeotropic distillation), and other types of compound purification. For example, the compounds described herein can be purified by chromatographic methods, 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 phase, reverse phase, and ionic resins. The mobile phase can be selected from polar solvents and non-polar solvents. In some cases, the mobile phase comprises a polar solvent. In some cases, the polar solvent is selected from chloroform, dichloromethane, tetrahydrofuran, dichloroethane, acetone, dioxane, ethyl acetate, dimethyl sulfoxide, aniline, diethylamine, nitromethane, acetonitrile, pyridine, isopropanol, ethanol, methanol, ethylene glycol, acetic acid and water. In some cases, the mobile phase comprises a non-polar solvent. In some cases, the non-polar solvent is selected from ether, toluene, benzene, pentane, hexane, cyclohexane, petroleum ether and carbon tetrachloride. For example, see Introduction to Modern Liquid Chromatography, 2nd edition, LRSnyder and JJKirkland, ed., John Wiley and Sons, 1979; and Thin Layer Chromatography, E.Stahl, ed., Springer-Verlag, New York, 1969.

[0537] In some cases, the reaction mixture is used in the subsequent steps of the methods described herein as a crude mixture without purification or other post-processing of the reaction mixture. In some cases, the crude mixture contains the target compound in sufficient purity to be purified, for example, by chromatography (e.g., HPLC or SFC), nuclear magnetic resonance spectroscopy (e.g., 1 H NMR or 13The reaction mixture contains the target compound in a purity of 70% or more, such as 75% or more, such as 80% or more, such as 85% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more, such as 99.5% or more, such as 99.9% or more, such as 99.99% or more, and including 99.999% or more, relative to the crude reaction mixture (excluding solvent when present), as determined by C NMR or a combination thereof. In some cases, the target compound is present in the reaction mixture in an amount of 30% or more, such as 40% or more, such as 50% or more, such as 60% or more, such as 70% or more, such as 75% or more, such as by weight, relative to the crude reaction mixture (excluding solvent when present). % or more, such as 80 wt % or more, for example 85 wt % or more, such as 90 wt % or more, such as 95 wt % or more, such as 97 wt % or more, such as 99 wt % or more, such as 99.5 wt % or more, such as 99.9 wt % or more, such as 99.99 wt % or more and including 99.999 wt % or more, and can range from 5 wt % to 99.999 wt %, such as 30 wt % to 99.99 wt %, 40 wt % to 99.9 wt %, 50 wt % to 99 wt %, 70 wt % to 95 wt %, 75 wt % to 90 wt %, 80 wt % to 99 wt %, or 80 wt % to 95 wt %. In some cases, the target compound is present in the crude reaction mixture (excluding solvent when present) in an amount of 30 mol% or more, such as 40 mol% or more, such as 50 mol% or more, such as 60 mol% or more, such as 70 mol% or more, such as 75 mol% or more, such as 80 mol% or more, such as 85 mol% or more, such as 90 mol% or more, such as 95 mol% or more, such as 97 mol% or more, such as 99 mol% or more, such as 99.5 mol% or more, such as 99.9 mol% or more, such as 99.99 mol% or more and including 99.999 mol% or more, and can 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%.

[0538] The method for preparing 25-hydroxy-3β-cholest-5-ene-3-sulfate ([(3S,10R,13R,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3-yl]sulfate metal salt) according to the present invention comprises: contacting 25-hydroxy-(3β)-cholest-5-ene-3-ol with a sulfating agent to produce an organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate; and contacting the organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate with at least one metal salt to obtain a metal salt of 5-cholestene-3β,25-diol 3-sulfate, as shown in Scheme Ia.

[0539] Plan Ia

[0540]

[0541] Plan IA1

[0542]

[0543] 25-hydroxy-(3β)-cholest-5-ene-3-ol can be sulfated by contact with a sulfating agent (Scheme IA1). In some cases, the sulfating agent is selected from sulfur trioxide complexes, sulfuric acid compounds, sulfonic acid compounds and sulfonate compounds. In some cases, the sulfating agent is selected from sulfur trioxide dimethylformamide, sulfur trioxide triethylamine and sulfur trioxide trimethylamine. In some cases, the sulfating agent includes sulfuric acid, acetic anhydride and pyridine. In some cases, the sulfating agent includes sulfur trioxide triethylamine and pyridine. In some cases, the sulfating agent is selected from 1) chlorosulfonic acid and pyridine and 2) chlorosulfonic acid and 2,6-lutidine. In some cases, the sulfating agent is ethyl chlorosulfonate.

[0544] 25-Hydroxy-(3β)-cholest-5-en-3-ol can be sulfated at a temperature ranging from -10°C to 50°C, for example, from -5°C to 45°C, for example, from -4°C to 40°C, for example, from -3°C to 35°C, for example, from -2°C to 30°C, for example, from -1°C to 25°C, and including 0°C to 20°C. The reaction can be carried out for a duration of 0.1 hour to 72 hours, for example, from 0.2 hour to 48 hours, for example, from 0.3 hour to 24 hours, for example, from 0.4 hour to 21 hour, for example, from 0.5 hour to 20 hour, for example, from 0.6 hour to 19 hour, for example, from 0.7 hour to 18 hour, for example, from 0.8 hour to 17 hour, for example, from 0.9 hour to 16 hour, including from 1 hour to 15 hours. The amount of the sulfating agent used relative to 25-hydroxy-(3β)-cholest-5-en-3-ol may vary and may be 0.001 equivalents or more, such as 0.01 equivalents or more, 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 2. 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 can 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 1 to 10 equivalents, 0.9 to 8 equivalents, 0.9 to 5 equivalents, 1.3 to 10 equivalents, 1.3 to 8 equivalents, 1.3 to 5 equivalents, 1.5 to 10 equivalents, 1.5 to 8 equivalents, 1.5 to 5 equivalents, 2 to 10 equivalents, 2 to 8 equivalents, 2 to 5 equivalents, or 1 to 2 equivalents, 1 to 1.5 equivalents, or 1.1 to 1.2 equivalents.

[0545] In some cases, the method comprises sulfating 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent, wherein the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product exhibits low solubility. In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in at least one solvent, wherein the 25-hydroxy-(3β)-cholest-5-ene-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, such as 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 includes sulfated 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent, wherein the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product exhibits a solubility of 5 mmol / L or less. In some cases, 25-hydroxy-(3β)-cholest-5-ene-3-ol is sulfated in at least one solvent, wherein the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product precipitates after formation. In some cases, the at least one solvent is selected from chloroform, dichloromethane, acetone, acetonitrile, toluene, tetrahydrofuran, and methyltetrahydrofuran.

[0546] In some cases, the method comprises sulfating 25-hydroxy-(3β)-cholest-5-en-3-ol in a manner sufficient to reduce or eliminate disulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated and a bissulfate product (i.e., 5-cholestene-3β-25-diol disulfate, Structure IA) is formed in an amount 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, of the reaction product formed by contacting 25-hydroxy-(3β)-cholest-5-en-3-ol with a sulfating agent, and including methods wherein 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated and a bissulfate product (i.e., 5-cholestene-3β-25-diol disulfate, Structure IA) is formed in an amount 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, of the reaction product formed by contacting 25-hydroxy-(3β)-cholest-5-en-3-ol And the amount of the bisulphate product formed is 0.0001 wt % or less, and can range from 10 wt % to 0.001 wt %, for example, 10 wt % to 0.1 wt %, 10 wt % to 1 wt %, 10 wt % to 2 wt %, 8 wt % to 0.001 wt %, 8 wt % to 0.1 wt %, 8 wt % to 1 wt %, 8 wt % to 2 wt %, 6 wt % to 0.001 wt %, 6 wt % to 0.1 wt %, 6 wt % to 1 wt %, 6 wt % to 2 wt %, 4% wt % to 0.001 wt %, 4 wt % to 0.1 wt %, 4 wt % to 1 wt %, 4 wt % to 2 wt %, 3 wt % to 0.001 wt %, 3 wt % to 0.1 wt %, 3 wt % to 1 wt %, 2 wt % to 0.001 wt %, 2 wt % to 0.1 wt %, or 2 wt % to 1 wt %.

[0547] In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to 5-cholestene-3β-25-diol disulfate formed is 10:1 or higher, for example 25:1 or higher, for example 50:1 or higher, for example 100:1 or higher, for example 250:1 or higher, for example 500:1 or higher, for example 1000:1 or higher, for example 2500:1 or higher, for example 5000:1 or higher, for example 10,000:1 or higher, for example 25,000:1 or higher, for example 50,000:1 or higher, for example 100,000:1 or higher, for example 10 6 :1 or higher, such as 10 7 :1 or higher, such as 10 8:1 or higher, and including the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to 5-cholestene-3β-25-diol disulfate formed therein is 10 9 :1 or greater, and can range from 10:1 by weight to 10:1 by weight. 9 :1, for example, a weight ratio of 10:1 to a weight ratio of 10 6 :1, weight ratio 10:1 to weight ratio 10 3 :1, weight ratio 10:1 to weight ratio 100:1, weight ratio 100:1 to weight ratio 10 9 :1, weight ratio 100:1 to weight ratio 10 6 :1, weight ratio 100:1 to weight ratio 10 3 :1, weight ratio 250:1 to weight ratio 10 9 :1, weight ratio 250:1 to weight ratio 10 6 :1, weight ratio 250:1 to weight ratio 10 3 :1, weight ratio 500:1 to weight ratio 10 9 :1, weight ratio 500:1 to weight ratio 10 6 :1, weight ratio 500:1 to weight ratio 10 3 :1, weight ratio 10 3 :1 to 10 by weight 9 :1, weight ratio 10 3 :1 to 10 by weight 6 :1, or weight ratio 250:1 to weight ratio 10 3 :1.

[0548]

[0549] In some cases, the 5-cholestene-3β-25-diol disulfate formed when 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated remains dissolved in at least one solvent. In some cases, the 5-cholestene-3β-25-diol disulfate has a high solubility in at least one solvent. In some cases, the solubility of the 5-cholestene-3β-25-diol disulfate in at least one solvent is 500 mmol / L or more, 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 includes a solubility of 1 mol / L or more in at least one solvent.

[0550] In some cases, method also comprises 25-hydroxy-(3 β)-cholest-5-ene-3-sulfate product and bisulphate product (i.e. 5-cholestene-3 β-25-diol disulfate).In some cases, by vacuum filtration, 25-hydroxy-(3 β)-cholest-5-ene-3-sulfate product is separated from bisulphate product.In some cases, by 25-hydroxy-(3 β)-cholest-5-ene-3-sulfate product is carried out to recrystallization, 25-hydroxy-(3 β)-cholest-5-ene-3-sulfate product is separated from bisulphate product.In some cases, by chromatography (such as silica gel column), 25-hydroxy-(3 β)-cholest-5-ene-3-sulfate product is separated from bisulphate product.

[0551] In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in a reaction mixture having a pH range of 5.0 to 8.0, such as pH 5.1 to 7.9, such as pH 5.2 to 7.8, such as pH 5.3 to 7.7, such as pH 5.4 to 7.6, such as pH 5.5 to 7.5, such as pH 5.6 to 7.4, such as pH 5.7 to 7.3, such as pH 5.8 to 7.2, such as pH 5.9 to 7.1, and including sulfated in a reaction mixture having a pH of 6.0 to 7.0.

[0552] In some cases, 25-hydroxy-(3β)-cholest-5-ene-3-ol is sulfated in the presence of an organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate. In some cases, the organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is present in particulate form (e.g., seed crystals of an organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate produced in a previous reaction or purification reaction batch). In some cases, sulfation of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt in the presence of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt (e.g., as particles) is sufficient to reduce the solubility of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt produced by the reaction of the sulfating agent with 25-hydroxy-(3β)-cholest-5-ene-3-ol (compared to the solubility in the absence of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt). In some cases, the solubility of the prepared 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt in the reaction mixture is reduced 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, compared to the solubility in the absence of the added 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt, and includes reducing the solubility of the prepared 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt by 99% or more. The particle size of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt added to the reaction mixture can vary and can have a dimension (e.g., length, width, or diameter) of 0.01 mm or greater, such as 0.025 mm or greater, such as 0.05 mm or greater, such as 0.075 mm or greater, such as 0.1 mm or greater, such as 0.25 mm or greater, such as 0.5 mm or greater, such as 0.75 mm or greater, such as 1 mm or greater, such as 2 mm or greater, such as 3 mm or greater, such as 4 mm or greater, and including 5 mm or greater. In some cases, the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt particles are added to the reaction mixture immediately after the sulfating agent is contacted with 25-hydroxy-(3β)-cholest-5-ene-3-ol.In some cases, particles of an organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate are added to the reaction mixture 1 minute or more after the sulfating agent is contacted with 25-hydroxy-(3β)-cholest-5-en-3-ol, for example 5 minutes or more, for example 10 minutes or more, for example 15 minutes or more, for example 20 minutes or more, for example 30 minutes or more, for example 40 minutes or more, for example 50 minutes or more, and including adding particles of an organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the reaction mixture 60 minutes or more after the sulfating agent is contacted with 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0553] In some cases, the sulfating agent is characterized prior to contacting with 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, characterizing the sulfating agent comprises determining the extent of degradation of the sulfating agent prior to contacting with 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, determining the extent of degradation of the sulfating agent comprises determining the level of impurities in the sulfating agent prior to contacting with 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0554] In some cases, degradation of the sulfating agent can be monitored by proton nuclear magnetic resonance spectroscopy ( 1 The proton NMR spectrum of the sulfating agent can be measured in at least one deuterated solvent. In some cases, the at least one deuterated solvent is deuterated acetone ((CD3)2CO). In some cases, the at least one deuterated solvent is not deuterated benzene (CD6). In some cases, the at least one deuterated solvent is not deuterated acetonitrile (CD3CN). In some cases, the at least one deuterated solvent is not deuterated chloroform (CD3Cl).

[0555] In some cases, methods for determining the extent of degradation include 1 Integrate one or more peaks with a chemical shift between 9.2 ppm and 9.3 ppm in the H-NMR spectrum and calculate the impurity level of the sulfating agent based on the integrated peaks. In some cases, the method for determining the degree of degradation includes: 1The impurity level of the sulfating agent is calculated based on the integrated peaks. In some cases, when the impurity level of the sulfating agent is below a predetermined threshold, for example, when the impurity level is 25% or less (as determined by integrating one or more peaks with a chemical shift of 9.2 ppm to 9.3 ppm in the proton NMR spectrum), for example, 24% or less, for example, 23% or less, for example, 22% or less, for example, 21% or less, for example, 20% or less, for example, 19% or less, for example, 18% or less, for example, 17% or less, for example, 16% or less, for example, 15% or less, for example, 14% or less, for example, 16% or less, for example, 17% or less, for example, 18% or less, for example, 19% or less, for example, 18 ... 3% 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 wherein the impurity level is 1% or less (as determined by integration of one or more peaks at a chemical shift of 9.2 ppm to 9.3 ppm in a proton NMR spectrum). In some cases, the sulfating agent is not contacted with 25-hydroxy-(3β)-cholest-5-en-3-ol when the impurity level is above a predetermined threshold, for example, when the impurity level is 25% or more (as determined by integration of one or more peaks in a proton NMR spectrum with a chemical shift of 9.2 ppm to 9.3 ppm), for example, 26% or more, for example, 27% or more, for example, 28% or more, for example, 29% or more, for example, 30% or more, for example, 31% or more, for example, 32% or more, for example, 33% or more, for example, 34% or more, including when the impurity level is 35% or more (as determined by integration of one or more peaks in a proton NMR spectrum with a chemical shift of 9.2 ppm to 9.3 ppm).

[0556] In some cases, the resulting 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product contains one or more byproducts. In some cases, the byproduct is 5-cholestene-3β-25-diol disulfate. In some cases, the 5-cholestene-3β-25-diol-disulfate byproduct is present in the composition produced by the sulfation of 25-hydroxy-(3β)-cholest-5-ene-3-ol in an amount relative to 25-hydroxy-(3β)-cholest-5-ene-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, For example, 1 wt % or less, such as 0.5 wt % or less, such as 0.1 wt % or less, such as 0.01 wt % or less, such as 0.001 wt % or less, and including wherein the amount of 5-cholestene-3β-25-diol disulfate by-product present in the composition resulting from the sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol is 0.001 wt % or less, and can range from 0.1 wt % to 50 wt %, such as 0.5 wt % to 20 wt % or 1 wt % to 12 wt %. In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the formed 5-cholestene-3β-25-diol-disulfate by-product is 10:1 or higher, for example 25:1 or higher, for example 50:1 or higher, for example 100:1 or higher, for example 250:1 or higher, for example 500:1 or higher, for example 1000:1 or higher, for example 2500:1 or higher, for example 5000:1 or higher, for example 10,000:1 or higher, for example 25,000:1 or higher, for example 50,000:1 or higher, for example 100,000:1 or higher, for example 10 6 :1 or higher, such as 10 7 :1 or higher, such as 10 8 :1 or higher, and including a weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to 5-cholestene-3β-25-diol disulfate formed therein of 10 9 In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to 5-cholestene-3β-25-diol disulfate is in the range of 10:1 to 10:1. 9 :1, for example 100:1 to 10 8 :1, for example 1000:1 to 10 7 :1, and including 10000:1 to 10 6 :1.

[0557] Aspects of the present application also include compositions having 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and 5-cholestene-3β-25-diol disulfate, wherein the 5-cholestene-3β-25-diol disulfate is present in the composition at an amount of 10 wt % or less, such as 9 wt % or less, such as 8 wt % or less, such as 7 wt % or less, such as 6 wt % or less, such as 5 wt % or less, such as 4 wt % or less, such as 3 wt % or less, such as 2 wt % or less, such as 1 wt % or less, such as 0.5 wt % or less, such as 0.1 wt % or less, such as 0.01 wt % or less, such as 0.001 wt % or less, and including 0.001 wt % or less. % or less and can range from 10 wt % to 0.001 wt %, for example, 10 wt % to 0.1 wt %, 10 wt % to 1 wt %, 10 wt % to 2 wt %, 8 wt % to 0.001 wt %, 8 wt % to 0.1 wt %, 8 wt % to 1 wt %, 8 wt % to 2 wt %, 6 wt % to 0.001 wt %, 6 wt % to 0.1 wt %, 6 wt % to 1 wt %, 6 wt % to 2 wt %, 4 wt % to 0.001 wt %, 4 wt % to 0.1 wt %, 4 wt % to 1 wt %, 4 wt % to 2 wt %, 3 wt % to 0.001 wt %, 3 wt % to 0.1 wt %, 3 wt % to 1 wt %, 2 wt % to 0.001 wt %, 2 wt % to 0.1 wt %, or 2 wt % to 1 wt %.

[0558] In some cases, the composition includes 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and 5-cholestene-3β-25-diol disulfate in a weight ratio of 10:1 or higher, such as 25:1 or higher, such as 50:1 or higher, such as 100:1 or higher, such as 250:1 or higher, such as 500:1 or higher, such as 1000:1 or higher, such as 2500:1 or higher, such as 5000:1 or higher,

[0559] For example, 10,000:1 or higher, for example, 25,000:1 or higher, for example, 50,000:1 or higher, for example, 100,000:1 or higher, for example, 10 6 :1 or higher, such as 10 7 :1 or higher, such as 10 8 :1 or higher, and comprising a weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to 5-cholestene-3β-25-diol disulfate in the composition of 10 9In some cases, the composition includes a weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to 5-cholestene-3β-25-diol disulfate ranging from 10:1 to 10:1. 9 :1, for example 100:1 to 10 8 :1, for example 1000:1 to 10 7 :1, including 10000:1 to 10 6 :1.

[0560] In some cases, the byproduct is a sulfated chain sterol (Structure IB).

[0561]

[0562] In some cases, the sulfated chain sterol ([(3S,8S,9S,10R,13R,14S,17R)-17-[(1R)-1,5-dimethylhex-4-enyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3-yl] sulfate) is present in the composition produced by sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol in an amount 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, relative to 25-hydroxy-(3β)-cholest-5-en-3-sulfate. % or less, such as 5 wt % or less, such as 4 wt % or less, such as 3 wt % or less, such as 2 wt % or less, such as 1 wt % or less, such as 0.5 wt % or less, such as 0.1 wt % or less, such as 0.01 wt % or less, such as 0.001 wt % or less, and including compositions wherein the amount of sulfated chain sterol present in the composition produced by sulfation of 25-hydroxy-(3β)-cholest-5-ene-3-ol is 0.001 wt % or less relative to 25-hydroxy-(3β)-cholest-5-ene-3-sulfate, and can range from 0.1 wt % to 10 wt %, such as 0.2 wt % to 5 wt %, or 0.3 wt % to 3 wt %. In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the sulfated chain sterol formed is 10:1 or higher, such as 25:1 or higher, such as 50:1 or higher, such as 100:1 or higher, such as 250:1 or higher, such as 500:1 or higher, such as 1000:1 or higher, such as 2500:1 or higher, such as 5000:1 or higher, such as 10,000:1 or higher, such as 25,000:1 or higher, such as 50,000:1 or higher, such as 100,000:1 or higher, such as 10 6 :1 or higher, such as 107 :1 or higher, such as 10 8 :1 or higher, and including wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the sulfated chain sterol formed is 10 9 In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the sulfated chain sterol formed ranges from 10:1 to 10:1. 9 :1, for example 100:1 to 10 8 :1, for example 1000:1 to 10 7 :1, and including 10000:1 to 10 6 :1.

[0563] Aspects of the present application also include compositions having 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and a sulfated chain sterol, wherein the chain sterol is present in the composition in an amount of 10 wt % or less, such as 9 wt % or less, such as 8 wt % or less, such as 7 wt % or less, such as 6 wt % or less, such as 5 wt % or less, such as 4 wt % or less, such as 3 wt % or less, such as 2 wt % or less, such as 1 wt % or less, such as 0.5 wt % or less, such as 0.1 wt % or less, such as 0.01 wt % or less, such as 0.001 wt % or less, relative to the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate, and including 0.001 wt % or less, relative to the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate. % w / w or less and can range from 10 wt % to 0.001 wt %, for example, 10 wt % to 0.1 wt %, 10 wt % to 1 wt %, 10 wt % to 2 wt %, 8 wt % to 0.001 wt %, 8 wt % to 0.1 wt %, 8 wt % to 1 wt %, 8 wt % to 2 wt %, 6 wt % to 0.001 wt %, 6 wt % to 0.1 wt %, 6 wt % to 1 wt %, 6 wt % to 2 wt %, 4 wt % to 0.001 wt %, 4 wt % to 0.1 wt %, 4 wt % to 1 wt %, 4 wt % to 2 wt %, 3 wt % to 0.001 wt %, 3 wt % to 0.1 wt %, 3 wt % to 1 wt %, 2 wt % to 0.001 wt %, 2 wt % to 0.1 wt %, or 2 wt % to 1 wt %.

[0564] In some cases, the composition comprises a combination of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and a sulfated chain sterol in a weight ratio of 10:1 or greater, for example, 25:1 or greater, for example, 50:1 or greater, for example, 100:1 or greater, for example, 250:1 or greater, for example, 500:1 or greater, for example, 1000:1 or greater, for example, 2500:1 or greater, for example, 5000:1 or greater, for example, 10,000:1 or greater, for example, 25,000:1 or greater, for example, 50,000:1 or greater, for example, 100,000:1 or greater, for example, 10 6 :1 or higher, such as 10 7 :1 or higher, such as 10 8 :1 or higher, and including wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to sulfated chain sterol in the composition is 10 9 In some cases, the composition comprises a weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to sulfated chain sterol ranging from 10:1 to 10:1. 9 :1, for example 100:1 to 10 8 :1, for example 1000:1 to 10 7 :1, including 10000:1 to 10 6 :1.

[0565] In some cases, the 25-hydroxy-(3 β)-cholest-5-ene-3-ol sulfation by-product present in 25-hydroxy-(3 β)-cholest-5-ene-3-sulfate composition is a thermal degradation product. In some cases, when separating the component of 25-hydroxy-(3 β)-cholest-5-ene-3-sulfate composition by liquid chromatography (such as HPLC), by-product can be determined by relative retention time. In some cases, by-product is sulfated chain sterol, when the component of 25-hydroxy-(3 β)-cholest-5-ene-3-sulfate composition by running at about 45 DEG C, using the HPLC separation of C8 stationary phase, the retention time of the compound is about 18.3 minutes, and the first mobile phase comprising buffer (such as the aqueous buffer of sodium phosphate) and the second mobile phase separation component comprising one or more organic solvents (see, for example, Table 13 and 14 below) are used. In some cases, the first mobile phase is an aqueous buffer. In some cases, the first mobile phase comprises sodium phosphate. In some cases, the second mobile phase is selected from one or more of methoxypropyl acetate, acetonitrile and methanol. In some cases, the flow velocity of the first mobile phase is about 1.0 ml / min. In some cases, the flow velocity of the second mobile phase is about 1.0 ml / min or higher. In some cases, under identical HPLC conditions, the retention time of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is about 7.7 minutes. In some cases, when the components of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate composition are separated by HPLC running at about 45 ° C, using C8 stationary phase, the by-product is a compound with a retention time of about 37.7 minutes, and the components of the composition are separated using the first mobile phase comprising a buffer solution (such as an aqueous buffer solution of sodium phosphate) and the second mobile phase comprising one or more organic solvents (for example, referring to Tables 13 and 14 below). Although not wishing to be bound by theory, it is believed that the compound with a retention time of about 37.7 minutes is a chain sterol. In some cases, the first mobile phase is an aqueous buffer solution. In some cases, the first mobile phase comprises sodium phosphate. In some cases, the second mobile phase is selected from one or more of methoxypropyl acetate, acetonitrile and methanol. In some cases, the flow velocity of the first mobile phase is about 1.0 ml / min. In some cases, the flow velocity of the second mobile phase is about 1.0 ml / min or higher. In some cases, the retention time of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate under identical HPLC conditions is about 7.7 minutes, so the relative retention time of sulfated chain sterols is about 2.4 (=18.3 / 7.7), and the relative retention time of the compound considered to be chain sterols is about 4.9 (=37.7 / 7.7).

[0566] Aspects of the present application also include compositions having 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and one or more byproducts of the sulfation of 25-hydroxy-(3β)-cholest-5-ene-3-ol. In some cases, the one or more by-products are present in the composition in an amount of 10% by weight or less, such as 9% by weight or less, 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 can range from 0.1% to 5% by weight, such as 0.2% to 10% by weight, or 0.3% to 15% by weight, relative to 2,5-hydroxy-(3β)-cholest-5-ene-3-sulfate. In some cases, the composition includes 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and one or more by-products in an amount of 0.0001% to 10% by weight, such as 0.005% to 9.5% by weight, such as 0.001% to 9.0% by weight, such as 0.05% to 8.5% by weight, such as 0.1% to 8.0% by weight, such as 0.5% to 7.5% by weight, such as 1% to 7% by weight, such as 1.5% to 6.5% by weight, and including 2% to 6% by weight, relative to the amount of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate.

[0567] In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the one or more by-products formed is 10:1 or higher, for example 25:1 or higher, for example 50:1 or higher, for example 100:1 or higher, for example 250:1 or higher, for example 500:1 or higher, for example 1000:1 or higher, for example 2500:1 or higher, for example 5000:1 or higher, for example 10,000:1 or higher, for example 25,000:1 or higher, for example 50,000:1 or higher, for example 100,000:1 or higher, for example 10 6 :1 or higher, such as 10 7 :1 or higher, such as 10 8 :1 or higher, and including wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the one or more by-products formed is 10 9 In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the one or more by-products formed ranges from 10:1 to 10:1. 9:1, for example 100:1 to 10 8 :1, for example 1000:1 to 10 7 :1, and including 10000:1 to 10 6 :1.

[0568] In some cases, the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is 25-hydroxy-(3β)-cholest-5-ene-3-sulfate pyridinium salt (Scheme IA2).

[0569] Plan IA2

[0570]

[0571] In some cases, the sulfating agent is contacted with an anhydride prior to contacting with 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, the anhydride is selected from acetic anhydride, trifluoroacetic anhydride, and trifluoromethanesulfonic anhydride. The amount of anhydride relative to 25-hydroxy-(3β)-cholest-5-en-3-ol can vary and can 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 relative to 25-hydroxy-(3β)-cholest-5-en-3-ol, the range can be 0.001 equivalents to 10 equivalents, for example 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 5 equivalents, 0.5 equivalents to 10 equivalents, 0.5 equivalents to 8 equivalents, 0.5 equivalents to 5 equivalents, 0.9 equivalents to 10 equivalents, 0.9 equivalents to 10 equivalents, 0.9 equivalents to 10 equivalents, 0.9 equivalents to 10 equivalents, 0.9 equivalents to 10 equivalents, 0.9 equivalents to 10 equivalents, 0.9 equivalents to 10 equivalents, 0.9 equivalents to 10 equivalents, 0.1 equivalents to 5 equivalents, 0.1 equivalents to 5 equivalents, 0.1 equivalents to 10 ... 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, 0.1 equivalents to 1.5 equivalents, 0.5 equivalents to 1.1 equivalents, or 0.1 equivalents to 1 equivalent.

[0572] In some cases, method is included in after preparing 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt, quenches (i.e., deactivates) unreacted sulfating agent.In some cases, quenching sulfating agent comprises adding water to reaction mixture.Relative to the amount of sulfating agent contacted with 25-hydroxy-(3β)-cholest-5-ene-3-ol, the amount of water added to reaction mixture can change, and can be 1 equivalent or higher, such as 2 equivalents or higher, such as 3 equivalents or higher, such as 4 equivalents or higher, such as 5 equivalents or higher, such as 6 equivalents or higher, such as 7 equivalents or higher, such as 8 equivalents or higher, such as 9 equivalents or higher, such as 10 equivalents or higher, such as 15 equivalents or higher, such as 20 equivalents or higher and including 25 equivalents or higher.

[0573] In some cases, the reactivity of quenching unreacted sulfating agent includes adding water to the reaction mixture, and then adding at least one alkali. In some cases, the at least one alkali is a trialkylamine, such as trimethylamine or triethylamine. In some cases, the at least one alkali is 2,6-lutidine. In some cases, the at least one alkali is pyridine. Pyridine can be added to the reaction mixture 1 minute or longer (for example, water is added to the reaction mixture after 5 minutes or longer, such as 10 minutes or longer, such as 15 minutes or longer, such as 30 minutes or longer, such as 45 minutes or longer, such as 60 minutes or longer, such as 90 minutes or longer, such as 120 minutes or longer, such as 150 minutes or longer, such as 180 minutes or longer, such as 210 minutes or longer, including 240 minutes or longer) after adding water. In some cases, pyridine is added to the reaction mixture after adding water 60 minutes. The amount of pyridine added to the reaction mixture relative to the amount of sulfating agent can vary and can 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.

[0574] In some cases, unreacted sulfating agent in the reaction mixture is quenched under slow stirring. In some cases, quenching unreacted sulfating agent under slow stirring includes stirring the reaction mixture in a manner sufficient to maintain agglomerates of unreacted sulfating agent in the reaction mixture. In some cases, slowly stirring the reaction mixture is sufficient to reduce the size of agglomerates of unreacted sulfating agent during the quenching process by 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, such as 1% or less, and includes wherein slowly stirring the reaction mixture is sufficient to reduce the size of agglomerates of unreacted sulfating agent during the quenching process by 0.1% or less. In some cases, slowly stirring the reaction mixture is sufficient to retain agglomerates of unreacted sulfating agent at the bottom of the reaction flask during the quenching process. In some cases, slowly stirring the reaction mixture is sufficient to cause little or no aggregation of unreacted sulfating agent to occur in the stirring vortex of the stirred reaction mixture.

[0575] In some cases, the method includes purifying the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt prior to contacting the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt with at least one metal salt. In some cases, the purified 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt has a purity of 97% or greater, such as a purity of 98% or greater, such as a purity of 99% or greater, such as a purity of 99.5% or greater, such as a purity of 99.7% or greater, such as a purity of 99.9% or greater, including a purity of 99.99% or greater. In some cases, the purified 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt has one or more sulfated byproducts (e.g., byproducts from sulfated 25-hydroxy-(3β)-cholest-5-ene-3-ol), wherein the one or more byproducts are present in an amount of 5% w / w or less, 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 0. 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, including 0.001% w / w or less relative to the amount of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt present. In some cases, the disulfated product (i.e., 5-cholestene-3β-25-diol-disulfate) is present in the purified 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt composition in an amount of 1% w / w or less, such as 0.9% w / w or less, such as 0.8% w / w or less, such as 0.7% w / w or more, relative to the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt. 0.01% w / w or less, including 0.001% w / w or less relative to the amount of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt present.

[0576] In some cases, 25-hydroxy-(3 β)-cholest-5-ene-3-sulfate organic cation salt is purified by liquid chromatography. In some cases, purification of 25-hydroxy-(3 β)-cholest-5-ene-3-sulfate organic cation salt includes the liquid chromatography using silica gel stationary phase (for example, silica gel packed column, ≥5 mass equivalents). In some cases, 25-hydroxy-(3 β)-cholest-5-ene-3-sulfate organic cation salt uses silica gel stationary phase and the mobile phase containing pyridine to purify. In some cases, mobile phase includes dichloromethane, methanol and pyridine. In some cases, mobile phase includes the mixture of dichloromethane-methanol (85:15) and pyridine (1%).

[0577] In some cases, one or more fractions collected from the stationary phase can be combined. In some cases, the combined fractions can be concentrated. In some cases, the combined fractions can be concentrated by distillation. In some cases, the combined fractions can be concentrated under vacuum. In some cases, the combined fractions can be concentrated by vacuum distillation.

[0578] In some cases, the combined fractions are contacted with one or more 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt particles (e.g., particles from a previously purified 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt sample). In some cases, contacting the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt particles with the combined fractions is sufficient to precipitate the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt in the combined fractions. In some cases, contacting the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt particles with the combined fractions includes adding particles during the distillation of the combined fractions. In some cases, the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt particles are added to the combined fractions before the combined fractions are distilled. In some cases, particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt are added to the combined fractions while the combined fractions are being distilled, for example, 1 minute or more, for example 5 minutes or more, for example 10 minutes or more, for example 15 minutes or more, for example 20 minutes or more, for example 30 minutes or more, for example 40 minutes or more, for example 50 minutes or more, and even including adding particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt to the combined fractions 60 minutes or more after starting distillation. In some cases, the combined fractions are distilled at a constant pressure, for example, wherein the pressure variation is 10% or less, for example 9% or less, for example 8% or less, for example 7% or less, for example 6% or less, for example 5% or less, for example 4% or less, for example 3% or less, for example 2% or less, for example 1% or less, and including 0.1% or less. In some cases, the pressure variation during distillation is 10 inches of mercury (inHg) or less, for example 9 inHg or less, for example 8 inHg or less, for example 7 inHg or less, for example 6 inHg or less, for example 5 inHg or less, for example 4 inHg or less, for example 3 inHg or less, for example 2 inHg or less, for example 1 inHg or less, for example 0.5 inHg or less, for example 0.1 inHg or less, for example 0.05 inHg or less, and including 0.01 inHg or less. In some cases, the combined fractions are distilled under reduced pressure, wherein the pressure is maintained between 15 inHg and 30 inHg, such as 17.5 inHg and 27.5 inHg, such as 20 inHg and 25 inHg, such as 21 inHg and 24 inHg, and including maintaining a pressure of 22 inHg to 23 inHg.

[0579] In some cases, the combined fractions are concentrated under vacuum and the concentrated combined fractions are contacted with a composition containing particles of an organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate. In some cases, the concentrated combined fractions are contacted with a composition containing particles of an organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and at least one solvent. In some cases, the at least one solvent is selected from tetrahydrofuran, such as 2-methyltetrahydrofuran. The concentrated combined fractions can be contacted with a composition containing particles of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt for a duration of 0.001 minute or longer, such as 0.005 minute or longer, such as 0.01 minute or longer, such as 0.05 minute or longer, such as 0.1 minute or longer, such as 0.5 minute or longer, such as 1 minute or longer, such as 2 minutes or longer, such as 3 minutes or longer, such as 4 minutes or longer, such as 5 minutes or longer, such as 10 minutes or longer, such as 15 minutes or longer, such as 30 minutes or longer, such as 45 minutes or longer, including 60 minutes or longer. In some cases, the combined fractions are added dropwise to a composition containing 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt in 2-methyltetrahydrofuran.

[0580] In some cases, a 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt is contacted with a metal salt to produce a 25-hydroxy-(3β)-cholest-5-ene-3-sulfate metal salt (Scheme IB1).

[0581] Scheme IB1

[0582]

[0583] In some cases, the method for preparing a metal salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate comprises contacting a 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt with at least one sodium salt. In some cases, the at least one sodium salt is selected from sodium acetate, sodium iodide, sodium chloride, sodium hydroxide, and sodium methoxide. The metal salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt can be contacted with the metal salt at a temperature of -10°C to 75°C, for example, -5°C to 70°C, for example, -4°C to 65°C, for example, -3°C to 60°C, for example, -2°C to 55°C, for example, -1°C to 50°C, for example, 0°C to 45°C, for example, 5°C to 40°C, and including 10°C to 35°C.

[0584] The reaction may be carried out for a duration of 0.1 hour to 72 hours, such as 0.2 hour to 48 hours, such as 0.3 hour to 24 hours, such as 0.4 hour to 21 hour, such as 0.5 hour to 20 hours, such as 0.6 hour to 19 hours, such as 0.7 hour to 18 hours, such as 0.8 hour to 17 hours, such as 0.9 hour to 16 hours, and including 1 hour to 15 hours. The amount of the metal salt used relative to the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt can vary and can 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 can 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 equivalent to 10 equivalents, 1 equivalent to 8 equivalents , 1 equivalent to 6 equivalents, 1 equivalent to 4 equivalents, 1 equivalent to 3 equivalents, 1.5 equivalents to 10 equivalents, 1.5 equivalents to 8 equivalents, 1.5 equivalents to 6 equivalents, 1.5 equivalents to 4 equivalents, 1.5 equivalents to 3 equivalents, 2 equivalents to 10 equivalents, 2 equivalents to 8 equivalents, 2 equivalents to 6 equivalents, 2 equivalents to 4 equivalents, or 2 equivalents to 3 equivalents, 1 equivalent to 100 equivalents, 1 equivalent to 5 equivalents, 1 equivalent to 2 equivalents.

[0585] In some cases, the method comprises contacting 25-hydroxy-(3β)-cholest-5-ene-3-sulfate pyridinium salt with sodium iodide to provide 25-hydroxy-(3β)-cholest-5-ene-3-sulfate sodium salt (Scheme IB2).

[0586] Scheme IB2

[0587]

[0588] In some cases, the method for preparing 25-hydroxy-3β-cholest-5-ene-3-sulfate comprises contacting 25-hydroxy-(3β)-cholest-5-ene-3-ol with a sulfur trioxide-pyridine complex to form 25-hydroxy-(3β)-cholest-5-ene-3-sulfate pyridinium salt; and contacting the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate pyridinium salt with a sodium salt to produce 5-cholest-3β,25-diol 3-sulfate sodium salt (Scheme Ib).

[0589] Plan I

[0590]

[0591] In some cases, the method for preparing 25-hydroxy-3β-cholest-5-ene-3-sulfate comprises contacting (3β)-cholest-5-ene-3-ol with a sulfating agent to produce a first (3β)-cholest-5-ene-3-sulfate organic cationic salt; contacting the first (3β)-cholest-5-ene-3-sulfate organic cationic salt with an organic base to produce a second (3β)-cholest-5-ene-3-sulfate organic cationic salt; oxidizing the second (3β)-cholest-5-ene-3-sulfate organic cationic salt in the presence of at least one surfactant. ion salt to produce 25-hydroxy-(3β)-cholester-(5,6-epoxy)-3-sulfate organic cation salt; 25-hydroxy-(3β)-cholester-(5,6-epoxy)-3-sulfate organic cation salt is deoxygenated to produce 25-hydroxy-(3β)-cholester-5-ene-3-sulfate organic cation salt; and 25-hydroxy-(3β)-cholester-5-ene-3-sulfate organic cation salt is contacted with at least one metal salt to produce 5-cholester-3β,25-diol 3-sulfate metal salt (Scheme IIa).

[0592] Scheme IIa

[0593]

[0594] In some cases, cholesterol is sulfated with a sulfating agent (Scheme IIA1). In some cases, the sulfating agent is selected from sulfur trioxide complexes, sulfuric acid compounds, sulfonic acid compounds, and sulfonate / ester compounds. In some cases, the sulfating agent is a sulfur trioxide-pyridine complex. In some cases, the sulfating agent is selected from sulfur trioxide dimethylformamide, sulfur trioxide triethylamine, and sulfur trioxide trimethylamine. In some cases, the sulfating agent is sulfuric acid, acetic anhydride, and pyridine. In some cases, the sulfating agent is selected from chlorosulfonic acid and pyridine. In some cases, the sulfating agent is selected from chlorosulfonic acid and 2,6-lutidine. In some cases, the sulfating agent is selected from ethyl chlorosulfonate.

[0595] The temperature range for cholesterol sulfation is 0° C. to 100° C., for example 5° C. to 95° C., for example 10° C. to 90° C., for example 15° C. to 85° C., for example 20° C. to 80° C., for example 25° C. to 75° C., including 30° C. to 70° C. The reaction is carried out for a duration ranging from 0.1 hour to 72 hours, for example 0.2 hour to 48 hours, for example 0.3 hour to 24 hours, for example 0.4 hour to 21 hour, for example 0.5 hour to 20 hours, for example 0.6 hour to 19 hours, including 0.7 hour to 18 hours. The amount of sulfating agent used relative to cholesterol can vary and can 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 can 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 equivalent to 10 equivalents, 1 equivalent to 8 equivalents, 1 equivalent to 6 equivalents, 1 equivalent to 4 equivalents, 1 equivalent to 3 equivalents, 1.5 equivalents to 10 equivalents, 1.5 equivalents to 8 equivalents, 1.5 equivalents to 6 equivalents, 1.5 equivalents to 4 equivalents, 1.5 equivalents to 3 equivalents, 2 equivalents to 10 equivalents, 2 equivalents to 8 equivalents, 2 equivalents to 6 equivalents, 2 equivalents to 4 equivalents, 2 equivalents to 3 equivalents, 1 equivalent to 30 equivalents, 1 equivalent to 5 equivalents, or 1 equivalent to 2 equivalents.

[0596] Scheme IIA1

[0597]

[0598] In some cases, the first (3β)-cholest-5-ene-3-sulfate organic cationic salt is (3β)-cholest-5-ene-3-sulfate pyridinium salt (Scheme IIA2).

[0599] Scheme IIA2

[0600]

[0601] In some cases, a first (3β)-cholest-5-ene-3-sulfate organic cationic salt (Structure IIA) is contacted with an organic base to produce a second (3β)-cholest-5-ene-3-sulfate organic cationic salt (Structure IIB) (Scheme IIB1).

[0602] Solution VB1

[0603]

[0604] In some cases, the organic base contacted with the first (3β)-cholest-5-ene-3-sulfate organic cation is selected from a hydroxide base. In some cases, the hydroxide base is selected from tetraethylammonium hydroxide, tetrabutylammonium hydroxide, tetrapropylammonium hydroxide and tetramethylammonium hydroxide. In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is selected from tetraethylammonium cation salt, tetrabutylammonium cation salt, tetrapropylammonium cation salt and tetramethylammonium cation salt. In some cases, the organic base is contacted with the first (3β)-cholest-5-ene-3-sulfate organic cation salt at a temperature of -10°C to 75°C, for example, -5°C to 70°C, for example, -4°C to 65°C, for example, -3°C to 60°C, for example, -2°C to 55°C, for example, -1°C to 50°C and including 0°C to 15°C. The reaction may be carried out for a duration ranging from 0.1 hour to 72 hours, such as 0.2 hour to 48 hours, such as 0.3 hour to 24 hours, such as 0.4 hour to 21 hour, such as 0.5 hour to 20 hours, such as 0.6 hour to 19 hours, such as 0.7 hour to 18 hours, such as 0.8 hour to 17 hours, such as 0.9 hour to 16 hours, and including 1 hour to 15 hours. The amount of the organic base used relative to the first (3β)-cholest-5-ene-3-sulfate organic cation can vary and can 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. The amount of the compound may be 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 equivalent to 10 equivalents, 1 equivalent to 8 equivalents , 1 equivalent to 6 equivalents, 1 equivalent to 4 equivalents, 1 equivalent to 3 equivalents, 1.5 equivalents to 10 equivalents, 1.5 equivalents to 8 equivalents, 1.5 equivalents to 6 equivalents, 1.5 equivalents to 4 equivalents, 1.5 equivalents to 3 equivalents, 2 equivalents to 10 equivalents, 2 equivalents to 8 equivalents, 2 equivalents to 6 equivalents, 2 equivalents to 4 equivalents, 2 equivalents to 3 equivalents, 1 equivalent to 10 equivalents, 1 equivalent to 5 equivalents or 1 equivalent to 2 equivalents.

[0605] In some cases, the method comprises contacting a first (3β)-cholest-5-ene-3-sulfate organic cation with tetrabutylammonium hydroxide to produce a (3β)-cholest-5-ene-3-sulfate tetrabutylammonium cation salt (Structure IIB1) (Scheme IIB2).

[0606] Solution VB1

[0607]

[0608] In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is oxidized.

[0609] 25-Hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organocation salt (Structure IIC) is generated (Scheme IIC1).

[0610] Scheme IIC1

[0611]

[0612] In some cases, oxidizing the second (3β)-cholest-5-ene-3-sulfate organic cationic salt comprises contacting the second (3β)-cholest-5-ene-3-sulfate organic cationic salt with a composition comprising an oxidizing agent and at least one surfactant.

[0613] In some cases, at least one surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants and zwitterionic surfactants.Nonionic surfactants can be selected from block copolymers of polyoxyethylene glycol ethers (for example, polyoxyethylene glycol octylphenol ether), polyoxyethylene glycol dehydrated sorbitan alkyl esters, dehydrated sorbitan alkyl esters, polyethylene glycol and polypropylene glycol etc.Anionic surfactants can be selected from surfactants with anionic functional head groups, for example surfactants containing sulfonate, phosphate, sulfate or carboxylate head groups.For example, anionic surfactants can be selected from alkyl sulfates, for example lauryl ammonium sulfate, dioctyl sodium sulfosuccinate, perfluorooctane sulfonate, perfluorononanoate, perfluorooctanoate, linear alkylbenzene sulfonate, alkyl aryl ether phosphate, sodium lauryl ether sulfate, lignin sulfonate or sodium stearate etc.Cationic surfactants can be selected from surfactants with cationic functional head groups, for example pyridinium or quaternary ammonium salt head groups. For example, the cationic surfactant can be selected from hexadecyltrimethylammonium hydrogen sulfate, tetrabutylammonium hydrogen sulfate, cetyltrimethylammonium bromide, tetrabutylammonium bromide, tetrabutylammonium iodide, tetrabutylphosphonium bromide, tetraoctylammonium bromide, tetraoctylammonium iodide, benzyltriethylammonium chloride, benzyltriethylammonium bromide, benzylcetyldimethylammonium chloride or benzylcetyldimethylammonium bromide. Zwitterionic surfactants contain both cationic and anionic centers, such as sulfobetaines (e.g., 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate) or betaines (e.g., cocamidopropyl betaine). In some cases, at least one surfactant is Extran laboratory soap, La Parisienne soap or DL-α-tocopheryl methoxypolyethylene glycol succinate (e.g., TPGS-750-M-2).

[0614] The amount of surfactant used can vary relative to the second (3β)-cholest-5-ene-3-sulfate organic cation, wherein in some cases 0.0001 equivalents or more of surfactant is used, for example 0.001 equivalents or more, for example 0.01 equivalents or more, for example 0.1 equivalents or more, for example 0.2 equivalents or more, for example 0.3 equivalents or more, for example 0.4 equivalents or more, for example 0.5 equivalents or more, for example 0.6 equivalents or more, for example 0.7 equivalents or more, for example 0.8 equivalents or more, for example 0.9 equivalents or more, for example 1 equivalent or more, for example 1.1 equivalents or more, for example 1.2 equivalents or more, for example 1.3 equivalents or more, for example 1.4 equivalents or more, for example 1.5 equivalents or more, for example 1.6 equivalents or more, for example 1.7 equivalents or more, for example 1.8 equivalents or more, for example 1.9 equivalents or more. The amount of surfactant may be 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and includes 10 equivalents or more of a surfactant, and may be in the range of 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 equivalent to 10 equivalents, 1 equivalent to 8 equivalents, 1 equivalent to 6 equivalents, 1 equivalent to 4 equivalents, 1 equivalent to 3 equivalents, 1.5 equivalents to 10 equivalents, 1.5 equivalents to 8 equivalents, 1.5 equivalents to 6 equivalents, 1.5 equivalents to 4 equivalents, 1.5 equivalents to 3 equivalents, 2 equivalents to 10 equivalents, 2 equivalents to 8 equivalents, 2 equivalents to 6 equivalents, 2 equivalents to 4 equivalents, 2 equivalents to 3 equivalents, 0.1 equivalents to 5 equivalents, 0.15 equivalents to 1 equivalent, or 0.2 equivalents to 0.3 equivalents.

[0615] In some cases, oxidizing the second (3β)-cholest-5-ene-3-sulfate organic cationic salt includes contacting the second (3β)-cholest-5-ene-3-sulfate organic cationic salt with an oxidizing agent and at least one ketone in the presence of at least one surfactant.

[0616] In some cases, the at least one ketone is selected from tetrahydrothiopyran-4-one 1,1-dioxide and halogenated ketone. In some cases, the halogenated ketone is selected from 1,1,1-trifluoro-2-butanone, 4,4-difluorocyclohexanone, 2-2-2-4'-tetrafluoroacetophenone and 1,1,1-trifluoroacetone. In some cases, the at least one ketone is 1,1,1-trifluoro-2-butanone. The amount of ketone used can vary relative to the oxidant in the reaction being carried out, and can 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, 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 ketone, and can range from 1 equivalent to 50 equivalents. The amount of the compound to be used may be, for example, 1 to 35 equivalents, 1 to 25 equivalents, 1 to 15 equivalents, 1 to 10 equivalents, 1 to 8 equivalents, 1 to 5 equivalents, 2 to 50 equivalents, 2 to 35 equivalents, 2 to 25 equivalents, 2 to 15 equivalents, 2 to 10 equivalents, 2 to 8 equivalents, 2 to 5 equivalents, 4 to 50 equivalents, 4 to 35 equivalents, 4 to 25 equivalents, 4 to 15 equivalents, 4 to 10 equivalents, 4 to 8 equivalents, 1 to 50 equivalents, 2 to 25 equivalents, or 5 to 10 equivalents.

[0617] In some cases, the ketone needs to be further purified before use. For example, the ketone can be purified by distillation before use. In some cases, it is necessary to test the reactivity of the ketone (e.g., by 1 H-NMR test for impurities) to determine whether purification is required.

[0618] In some cases, oxidizing the second (3β)-cholest-5-ene-3-sulfate organic cationic salt comprises contacting the second (3β)-cholest-5-ene-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 can vary and range from 0.0000001% w / v or more, 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 0.1% w / v, such as 0.05% w / v or more, such as 0.1% w / v or more of the reaction mixture. The amount of the reaction mixture may be as high as 0.5% w / v or higher, for example 1% w / v or higher, for example 5% w / v or higher, for example 10% w / v or higher, for example 15% w / v or higher, including 25% w / v or higher of the reaction mixture, and may range from 0.0000001% w / v to 25% w / v, for example 0.0000001% w / v to 15% w / v, 0.0000001% w / v to 10% w / v 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 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.

[0619] The second (3β)-cholest-5-ene-3-sulfate organic cation salt can be oxidized at a temperature in the range of -25°C to 50°C, for example, -20°C to 45°C, for example, -15°C to 40°C, for example, -10°C to 35°C, for example, -5°C to 30°C, for example, -1°C to 25°C, including 0°C to 15°C. In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is oxidized at a temperature of 0°C to 5°C. When the reaction mixture contains a certain amount of water, the reaction can be carried out at a temperature of -10°C to 50°C, for example, -5°C to 45°C, for example, 0°C to 40°C, for example, 0°C to 35°C, for example, 0°C to 30°C, for example, 0°C to 25°C, for example, 0°C to 20°C, for example, 0°C to 15°C, and including 0°C to 10°C.

[0620] The second (3β)-cholest-5-ene-3-sulfate organic cation can be oxidized at a pH in the range of 5 to 7.5, for example, a pH of 5.5 to 7.0, including a pH of 5.5 to 6.5. In some cases, when the reaction mixture contains water (for example, in a biphasic solvent system), the pH range is 5.0 to 6.0, for example, a pH of 5.0 to 5.9, for example, a pH of 5.0 to 5.8, for example, a pH of 5.0 to 5.7, for example, a pH of 5.0 to 5.6, including a pH of 5.0 to 5.5.

[0621] The reaction may be carried out for a duration ranging from 0.1 hour to 72 hours, such as 0.2 hour to 48 hours, such as 0.3 hour to 24 hours, such as 0.4 hour to 21 hour, such as 0.5 hour to 20 hours, such as 0.6 hour to 19 hours, such as 0.7 hour to 18 hours, such as 0.8 hour to 17 hours, such as 0.9 hour to 16 hours, and including 1 hour to 15 hours.

[0622] In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation is contacted in situ with a composition containing potassium peroxymonosulfate and at least one ketone in the presence of at least one surfactant. In some cases, the method includes contacting potassium peroxymonosulfate with at least one ketone in the presence of at least one surfactant to form a separate oxidation reaction mixture, and then adding the oxidation reaction mixture to the second (3β)-cholest-5-ene-3-sulfate organic cation. In these cases, potassium peroxymonosulfate can be contacted with at least one ketone in the presence of at least one surfactant for a duration of 0.1 minute or longer before contacting the oxidation reaction mixture with the second (3β)-cholest-5-ene-3-sulfate organic cation salt, for example, 1 minute or longer, for example, 2 minutes or longer, for example, 3 minutes or longer, for example, 5 minutes or longer, including 10 minutes or longer, and the time range can be 2 minutes to 180 minutes, for example, 3 minutes to 120 minutes or 4 minutes to 60 minutes. In some cases, potassium peroxymonosulfate may be contacted with at least one ketone in the presence of at least one surfactant to form a separate oxidation reaction mixture, and then the oxidation reaction mixture may be immediately contacted with the second (3β)-cholest-5-ene-3-sulfate organic cation salt. The oxidation reaction mixture may be formed at a temperature in the range of -10°C to 50°C, such as -5°C to 45°C, such as -4°C to 40°C, such as -3°C to 35°C, such as -2°C to 30°C, such as -1°C to 25°C, and including 0°C to 15°C. When the oxidation reaction mixture is not immediately contacted with the second (3β)-cholest-5-ene-3-sulfate organic cation salt, the oxidation reaction mixture may be maintained at a temperature in the range of -10°C to 50°C, such as -5°C to 45°C, such as -4°C to 40°C, such as -3°C to 35°C, such as -2°C to 30°C, such as -1°C to 25°C, and including 0°C to 15°C.

[0623] In some cases, the method further comprises adding the oxidation reaction mixture to the second (3β)-cholest-5-ene-3-sulfate organic cation salt. In some cases, the method comprises adding the oxidation reaction mixture dropwise to the second (3β)-cholest-5-ene-3-sulfate organic cation salt. In some cases, the oxidation reaction mixture is added to the second (3β)-cholest-5-ene-3-sulfate organic cation salt in a metered manner. The metered amount can be added continuously, or added according to a predetermined time interval (for example, every 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes or other time intervals). In some cases, the oxidation reaction mixture is added to the second (3β)-cholest-5-ene-3-sulfate organic cation salt by controlled addition, for example, using a mechanical or computer-controlled pump, such as a syringe pump. In some cases, the method comprises generating the oxidation reaction mixture, and adding the composition containing the second (3β)-cholest-5-ene-3-sulfate organic cation salt to the oxidation reaction mixture. In some cases, the method includes adding the second (3β)-cholest-5-ene-3-sulfate organic cation salt dropwise to the oxidation reaction mixture. In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is added to the oxidation reaction mixture in a metered manner. The metered amount can be added continuously or at predetermined time intervals (e.g., every 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, or other time intervals). In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is added to the oxidation reaction mixture by controlled addition, for example, using a mechanical or computer-controlled pump, such as a syringe pump.

[0624] In some cases, oxidizing the second (3β)-cholest-5-ene-3-sulfate organic cation salt comprises contacting the second (3β)-cholest-5-ene-3-sulfate organic cation salt with at least one oxidizing substance. In some cases, the at least one oxidizing substance is selected from dioxirane. In some cases, the dioxirane is generated in situ in the composition containing the second (3β)-cholest-5-ene-3-sulfate organic cation salt. In some cases, the dioxirane is generated separately (e.g., in a separate reaction vessel, such as a flask) and added to the composition containing the second (3β)-cholest-5-ene-3-sulfate organic cation salt.

[0625] In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation is oxidized in the presence of at least one base. In some cases, the at least one base is selected from a weak base. In some cases, the at least one base is selected from potassium bicarbonate, sodium bicarbonate, potassium phenate, sodium citrate buffer, sodium phosphate buffer, potassium formate and potassium acetate. In some cases, the at least one base is potassium bicarbonate. In some cases, the at least one base can be added to the reaction mixture over time, for example, in a metered manner, wherein the base is added at predetermined time intervals (for example, every 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes or other time intervals). In some cases, the at least one base can be a composition with water, wherein the base can be present in the composition at 0.0000001% w / v or more, 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 0.05% w / v or more, of the composition. Such as 0.1% w / v or more, for example 0.5% w / v or more, for example 1% w / v or more, for example 5% w / v or more, for example 10% w / v or more, for example 15% w / v or more, and including 25% w / v or more of the composition, and can range from 0.0000001% w / v to 25% w / v, for example 0.0000001% w / v to 15% w / v, 0.0000001% w / v 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 some cases, the at least one base can be an aqueous potassium bicarbonate composition.

[0626] In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is oxidized by contacting with oxone in the presence of hexadecyltrimethylammonium hydrogen sulfate (CTAHS), followed by the addition of trifluorobutanone and potassium hydrogen sulfate to form 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cation salt (Scheme IIC2).

[0627] Scheme IIC2

[0628]

[0629] In some cases, the method comprises forming an oxidizing species in situ with the second (3β)-cholest-5-ene-3-sulfate organic cation salt, for example, by contacting potassium peroxymonosulfate and trifluorobutanone with the second (3β)-cholest-5-ene-3-sulfate organic cation salt in the presence of hexadecyltrimethylammonium hydrogen sulfate (CTAHS) in a reaction mixture. In some cases, forming an oxidizing species in situ with the second (3β)-cholest-5-ene-3-sulfate organic cation salt comprises forming a dioxirane in situ with the second (3β)-cholest-5-ene-3-sulfate organic cation salt.

[0630] In some cases, the method includes forming a dioxirane in a separate reaction and then adding the dioxirane to the second (3β)-cholest-5-ene-3-sulfate organic cation. In these cases, potassium peroxymonosulfate can be contacted with trifluorobutanone in the presence of hexadecyltrimethylammonium hydrogen sulfate (CTAHS) for a duration of 0.1 minute or longer before contacting the reactive composition with the second (3β)-cholest-5-ene-3-sulfate organic cation (e.g., 1 minute or longer, such as 2 minutes or longer, such as 3 minutes or longer, such as 5 minutes or longer, and including 10 minutes or longer), and the time range can be 0.01 minute to 120 minutes, such as 0.1 minute to 90 minutes, or 0.5 minute to 60 minutes. In some cases, potassium peroxymonosulfate can be contacted with trifluorobutanone in the presence of cetyltrimethylammonium hydrogen sulfate (CTAHS) to form an oxidation reaction composition that is immediately contacted with a second (3β)-cholest-5-ene-3-sulfate organic cation salt.

[0631] 25-Hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cation salt is deoxygenated to generate 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt (Structure IID) (Scheme IID1).

[0632] Scheme IID1

[0633]

[0634] In some cases, generating a 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt from a 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cation salt includes deoxygenating the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cation salt by contacting the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cation salt with zinc. In some cases, the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cation salt with zinc is carried out in the presence of at least one halide and at least one acid. In some cases, at least one halide is selected from iodine and metal halides. In some cases, the metal halide is selected from sodium iodide and lithium iodide. In some cases, at least one acid is selected from a weak acid. In some cases, the at least one acid is selected from acetic acid, hydrochloric acid, citric acid, p-toluenesulfonic acid, formic acid and methanesulfonic acid.

[0635] The amount of the reagent used to deoxygenate the 25-hydroxy-(3β)-cholesterol-(5,6-epoxy)-3-sulfate organic cation salt can vary, with some cases using 0.0001 equivalents or more of the reagent relative to the 25-hydroxy-(3β)-cholesterol-(5,6-epoxy)-3-sulfate organic cation salt, 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. equivalent or more, such as 0.3 equivalent or more, such as 0.4 equivalent or more, such as 0.5 equivalent or more, such as 0.6 equivalent or more, such as 0.7 equivalent or more, such as 0.8 equivalent or more, such as 0.9 equivalent or more, such as 1 equivalent or more, such as 1.1 equivalent or more, such as 1.2 equivalent or more, such as 1.3 equivalent or more, such as 1.4 equivalent or more, such as 1.5 equivalent or more, such as 1.6 equivalent 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 can 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 equivalent to 10 equivalents, 1 equivalent to 8 equivalents, 1 equivalent to 6 equivalents, 1 equivalent to 4 equivalents, 1 equivalent to 3 equivalents, 1.5 equivalents to 10 equivalents, 1.5 equivalents to 8 equivalents, 1.5 equivalents to 6 equivalents, 1.5 equivalents to 4 equivalents, 1.5 equivalents to 3 equivalents, 2 equivalents to 10 equivalents, 2 equivalents to 8 equivalents, 2 equivalents to 6 equivalents, 2 equivalents to 4 equivalents, 2 equivalents to 3 equivalents, 1 equivalent to 20 equivalents, 1 equivalent to 10 equivalents, or 4 equivalents to 6 equivalents.

[0636] 25-Hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cation salt can be deoxygenated at a temperature in the range of -10°C to 75°C, for example, -5°C to 70°C, for example, -4°C to 65°C, for example, -3°C to 60°C, for example, -2°C to 55°C, for example, -1°C to 50°C, and including 0°C to 25°C. The reaction can be carried out for a duration ranging from 0.1 hour to 72 hours, for example, 0.2 hour to 48 hours, for example, 0.3 hour to 24 hours, for example, 0.4 hour to 21 hour, for example, 0.5 hour to 20 hours, for example, 0.6 hour to 19 hours, for example, 0.7 hour to 18 hours, for example, 0.8 hour to 17 hours, for example, 0.9 hour to 16 hours, and including 1 hour to 15 hours.

[0637] In some cases, the method comprises contacting 25-hydroxy-(3β)-cholesta-(5,6-epoxy)-3-sulfate organic cation salt with zinc in the presence of iodine and acetic acid to produce 25-hydroxy-(3β)-cholesta-5-ene-3-sulfate organic cation salt (Scheme IID2).

[0638] Scheme IID2

[0639]

[0640] In some cases, a 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt (Structure IID) is contacted with a metal salt to produce a 25-hydroxy-(3β)-cholest-5-ene-3-sulfate metal salt (Structure IIE) (Scheme IIE1).

[0641] Scheme IIE1

[0642]

[0643] In some cases, the method for preparing a 25-hydroxy-(3β)-cholest-5-ene-3-sulfate metal salt comprises contacting a 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation with at least one sodium salt. In some cases, the at least one sodium salt is selected from sodium acetate, sodium iodide, sodium chloride, sodium hydroxide, and sodium methoxide. The 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt can be contacted with the metal salt at a temperature in the range of -10°C to 75°C, for example, -5°C to 70°C, for example, -4°C to 65°C, for example, -3°C to 60°C, for example, -2°C to 55°C, for example, -1°C to 50°C, for example, 0°C to 45°C, for example, 5°C to 40°C, and including 10°C to 35°C.

[0644] The reaction may be carried out for a duration ranging from 0.1 hour to 72 hours, such as 0.2 hour to 48 hours, such as 0.3 hour to 24 hours, such as 0.4 hour to 21 hour, such as 0.5 hour to 20 hours, such as 0.6 hour to 19 hours, such as 0.7 hour to 18 hours, such as 0.8 hour to 17 hours, such as 0.9 hour to 16 hours, and including 1 hour to 15 hours. The amount of metal salt used relative to the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt can vary and can 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 can 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 equivalent to 10 equivalents, 1 equivalent to 8 equivalents , 1 equivalent to 6 equivalents, 1 equivalent to 4 equivalents, 1 equivalent to 3 equivalents, 1.5 equivalents to 10 equivalents, 1.5 equivalents to 8 equivalents, 1.5 equivalents to 6 equivalents, 1.5 equivalents to 4 equivalents, 1.5 equivalents to 3 equivalents, 2 equivalents to 10 equivalents, 2 equivalents to 8 equivalents, 2 equivalents to 6 equivalents, 2 equivalents to 4 equivalents, 2 equivalents to 3 equivalents, 1 equivalent to 20 equivalents, 1 equivalent to 10 equivalents, or 1 equivalent to 7 equivalents.

[0645] In some cases, the method comprises contacting 25-hydroxy-(3β)-cholest-5-ene-3-sulfate pyridinium salt with sodium iodide to provide 25-hydroxy-(3β)-cholest-5-ene-3-sulfate sodium salt (Scheme IIE2).

[0646] Scheme IIE2

[0647]

[0648] In some embodiments, salts of 25HC3S (including crystalline salts of 25HC3S) have relatively high solubility, which can be used to prepare concentrated solutions, such as for intravenous administration. For example, salts of 25HC3S with relatively high solubility in water, saline, glucose, and / or ethanol can 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 glucose; and crystalline 25HC3S zinc, which has relatively high solubility in ethanol.

[0649] In some embodiments, salts of 25HC3S have relatively high solubility, which can be used to prepare concentrated solutions, such as for topical administration. For example, crystalline zinc 25HC3S has relatively high solubility in ethanol, a well-known topical penetration enhancer that can be used in transdermal delivery systems.

[0650] In some embodiments, salts of 25HC3S have relatively high solubility, which can be used to prepare concentrated solutions, such as for oral administration. For example, crystalline zinc 25HC3S has relatively high solubility in ethanol.

[0651] In some embodiments, salts of 25HC3S have relatively low solubility, which can be used, for example, in controlled-release formulations, such as injectable or oral controlled-release formulations. As shown in the Examples, the following salts of 25HC3S can be used in controlled-release formulations at least due to their low solubility in saline: crystalline 25HC3S potassium, crystalline 25HC3S calcium, crystalline 25HC3S zinc, crystalline 25HC3S choline, crystalline 25HC3S meglumine, crystalline 25HC3S trihydroxymethylmethonium, crystalline 25HC3S benzathine, crystalline 25HC3S diethanolamine, and crystalline 25HC3S magnesium. The following salts of 25HC3S may be used in controlled-release formulations at least due to 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 used in controlled-release formulations due to its low solubility in fasted state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF).

[0652] 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 tert-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.

[0653] In some embodiments, the salt of 25HC3S has a high solubility in a solvent useful for synthesis. For example, as shown in the Examples, crystalline 25HC3S diethylammonium has a relatively high solubility in isopropyl alcohol (IPA). Crystalline 25HC3S diethylammonium and crystalline 25HC3S magnesium have a relatively high solubility in methanol, which is useful for synthesis and can also be used to spray-dry drug-polymer dispersions to prepare amorphous formulations of the drug substance, thereby improving oral bioavailability. Crystalline 25HC3S hydroxyethylpyrrolidinium has a relatively high solubility in acetonitrile (ACN).

[0654] In some embodiments, the salts of 25HC3S are non-hygroscopic, which facilitates handling of the drug substance under ambient conditions and avoids the need for special precautions, such as handling under low humidity conditions, handling in a dry environment, or storing in sealed containers. Weighing these drug substance salts under ambient conditions presents no problem in the preparation process because there is no need to worry about weight variations due to moisture absorption on the balance. Furthermore, containers of these salts can be opened and closed multiple times under ambient conditions without worrying about changes in the powder composition due to moisture absorption. The non-hygroscopic nature of these salts also enables the preparation of wet granules for oral tablet and capsule products and minimizes the potential for polymorphic or other solid form transformations (e.g., hydrate formation). For example, as shown in the Examples, the crystalline 25HC3S tert-butylammonium, crystalline 25HC3S benzathine, and crystalline 25HC3S choline salts of 25HC3S absorb less than 0.5% water at 95% relative humidity. Furthermore, as shown by the DVS isotherms, crystalline 25HC3S tert-butylammonium, crystalline 25HC3S benzathine, and crystalline 25HC3S choline salt absorbed less water when the relative humidity was increased to 95%, but they reversibly lost all that water when the relative humidity was decreased to 5%.

[0655] In some embodiments, salts of 25HC3S are highly crystalline, which can have advantages, for example, in processing. Crystalline 25HC3S hydroxyethylpyrrolidinium salt, crystalline 25HC3S diethylammonium salt, crystalline 25HC3S diethanolamine salt, crystalline 25HC3S tert-butylammonium salt, crystalline 25HC3S benzathine salt, and crystalline 25HC3S choline salt all exhibit high crystallinity. XRPD patterns have been successfully indexed using single unit cells, and preliminary crystallographic unit cell parameters provide a reliable description of the crystalline form. The molecular formula unit volumes in the indexed results are consistent with the anhydrous form and the expected salt stoichiometry.

[0656] In some embodiments, salts of 25HC3S have relatively high DSC (differential scanning calorimetry) endothermic transitions (indicative of thermal degradation or solid-state transitions). While not wishing to be bound by theory, this property may allow for dry heat sterilization of the drug substance (e.g., 160°C for 2 hours), thereby facilitating the preparation of sterile dosage forms. For example, as shown in the Examples, the first significant endothermic transition temperatures for potassium, lysine, diethanolamine, tert-butylammonium, benzathine, and choline are near 198°C, 186°C, 181°C, 201°C, 211°C, and 198°C, respectively, suggesting that these salts of 25HC3S can be sterilized by dry heat treatment.

[0657] In some embodiments, the powder of the salt of 25HC3S has relatively good flowability, which may be useful in the production process. For example, as shown in the Examples, the crystalline hydroxyethylammonium salt of 25HC3S and the crystalline lysine salt of 25HC3S have relatively good flowability.

[0658] In some embodiments, the salt of 25HC3S has good temperature stability. As shown in the examples, the following salts of 25HC3S have good stress stability at 80°C: crystalline 25HC3S diethylammonium, crystalline 25HC3S tert-butylammonium, crystalline 25HC3S choline, crystalline 25HC3S diethanolamine, crystalline 25HC3S trishydroxymethylmethylammonium, and crystalline 25HC3S lysine.

[0659] In some embodiments, the counterion of 25HC3S can have a beneficial effect in vivo. For example, choline salts can be beneficial because choline deficiency is also associated with conditions related to fat accumulation and inflammation, and choline supplementation is considered to be a potential need for treating and / or managing such conditions (see, for example, Zeisel et al. Nutr Rev. 2009 Nov; 67(11): 615-623, Corbin et al. Curr Opin Gastroenterol. 2012 Mar; 28(2): 159-165). Zinc salts of 25HC3S can 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 for such conditions, such as those related to the skin and gastrointestinal tract; the brain and central nervous system, the immune system, the skeletal system, and the reproductive system (see, for example, Himoto et al. Nutrients 2018, 10, 88; Grüngreiff et al. Annals of Hepatology 2016 15 (1) 7-16; Mohammad et al. Nutr Clin Pract 20012 27 (1) 8-20). Magnesium deficiency is also seen in many pathological conditions, including those for which 25HC3S is a potential therapeutic agent. Magnesium supplementation may be beneficial for conditions such as celiac disease, Crohn's disease, type 2 diabetes, and conditions associated with alcohol consumption (see, for example, Agus et al. J Am Soc Nephrol, 10: 1616-1622 (1999) and Martin et al. J Am Soc Nephrol, 20: 2291-2295 (2009)).

[0660] In some embodiments, the salt of 25HC3S is a salt having a beneficial combination of two or more such properties, for example, which makes it particularly suitable as an active pharmaceutical ingredient in a pharmaceutical composition for a specific clinical application. For example, such a combination can include two or more (e.g., at least three, at least four, or at least five) properties selected from the group consisting of: high solubility in a specific solvent (e.g., the solvents studied in the Examples), low solubility in a specific solvent (e.g., the solvents studied in the Examples), low hygroscopicity, high crystallinity, a high DSC endothermic transition (allowing dry heat sterilization), high flowability, high temperature stability, and beneficial in vivo effects of the 25HC3S counterion (particularly when these in vivo effects are beneficial in subjects suffering from a specific condition for which the dosage form incorporating the salt of 25HC3S has been designed).

[0661] Terms

[0662] Item 1. A salt of 25HC3S that is not (i) an alkali metal salt or (ii) an ammonium salt.

[0663] Item 2. A salt of 25HC3S that is not (i) an alkali metal salt or (ii) an ammonium salt, wherein the ammonium salt is not a choline salt.

[0664] Item 3. A crystalline salt of 25HC3S, which is not the crystalline sodium salt of 25HC3S.

[0665] Item 4. The crystalline salt of Item 3, which is not the crystalline choline salt of 25HC3S.

[0666] Item 5. The crystalline salt of Item 3, which is a crystalline 25HC3S metal salt.

[0667] Item 6. The crystalline salt of Item 5, wherein the metal is in the +1 oxidation state.

[0668] Item 7. The salt of any one of Items 1 to 5, which is a 25HC3S metal salt, wherein the metal is in the +2 oxidation state or the +3 oxidation state.

[0669] Item 8. The crystalline metal salt of Item 7, wherein the metal is an alkaline earth metal.

[0670] Item 9. The crystalline metal salt of Item 6, wherein the metal is selected from potassium, lithium, and rubidium.

[0671] Item 10. A substantially pure salt of 25HC3S according to any one of Items 1 to 9.

[0672] Item 11. Crystalline potassium 25HC3S.

[0673] Item 12. The crystalline potassium 25HC3S of Item 11, having an x-ray powder diffraction pattern comprising a peak at about 2.2° 2θ.

[0674] Item 13. The crystalline potassium 25HC3S of Item 11 or 12, which has an x-ray powder diffraction pattern comprising a peak at about 2.3° 2θ.

[0675] Item 14. The crystalline potassium 25HC3S of any one of Items 11 to 13, having an x-ray powder diffraction pattern comprising a peak at about 8.8° 2θ.

[0676] Item 15. The crystalline potassium 25HC3S of any one of Items 11 to 14, having an x-ray powder diffraction pattern comprising a peak at about 9.3° 2θ.

[0677] Item 16. The crystalline potassium 25HC3S of any one of Items 11 to 15, having an x-ray powder diffraction pattern comprising a peak at about 15.3° 2θ.

[0678] Item 17. The crystalline potassium 25HC3S of any one of Items 11 to 16, having an x-ray powder diffraction pattern comprising a peak at about 4.6° 2θ.

[0679] Item 18. The crystalline potassium 25HC3S of any one of Items 11 to 17, having an x-ray powder diffraction pattern comprising a peak at about 14.7° 2θ.

[0680] Item 19. The crystalline potassium 25HC3S of any one of Items 11 to 18, having an x-ray powder diffraction pattern comprising a peak at about 14.9° 2θ.

[0681] Item 20. The crystalline potassium 25HC3S of any one of Items 11 to 19, having an x-ray powder diffraction pattern comprising a peak at about 16.1° 2θ.

[0682] Item 21. The crystalline potassium 25HC3S of any one of Items 11 to 20, having an x-ray powder diffraction pattern comprising two peaks at about 2.2° 2θ to about 2.3° 2θ, wherein the two peaks (a) do not overlap; (b) partially overlap; or (c) superimpose so as to appear as a single peak.

[0683] Item 22. The crystalline potassium 25HC3S of Item 11, having an x-ray powder diffraction pattern substantially the same as that of FIG. 2 or FIG. 2A .

[0684] Item 23. Crystallized calcium 25HC3S.

[0685] Item 24. The crystalline calcium 25HC3S of Item 23, having an x-ray powder diffraction pattern comprising a peak at about 2.2° 2θ.

[0686] Item 25. The crystalline calcium 25HC3S of Item 23 or 24, having an x-ray powder diffraction pattern comprising a peak at about 4.5° 2θ.

[0687] Item 26. The crystalline calcium 25HC3S of any one of Items 23 to 25, having an x-ray powder diffraction pattern comprising a peak at about 9.0° 2θ.

[0688] Item 27. The crystalline calcium 25HC3S of any one of Items 23 to 26, having an x-ray powder diffraction pattern comprising a peak at about 10.0° 2θ.

[0689] Item 28. The crystalline calcium 25HC3S of any one of Items 23 to 27, having an x-ray powder diffraction pattern comprising a peak at about 15.0° 2θ.

[0690] Item 29. The crystalline calcium 25HC3S of any one of Items 23 to 28, having an x-ray powder diffraction pattern comprising a peak at about 15.1° 2θ.

[0691] Item 30. The crystalline calcium 25HC3S of any one of Items 23 to 29, having an x-ray powder diffraction pattern comprising a peak at about 15.7° 2θ.

[0692] Item 31. The crystalline calcium 25HC3S of any one of Items 23 to 30, having an x-ray powder diffraction pattern comprising a peak at about 15.4° 2θ.

[0693] Item 32. The crystalline calcium 25HC3S of any one of Items 23 to 31, having an x-ray powder diffraction pattern comprising a peak at about 16.5° 2θ.

[0694] Item 33. The crystalline calcium 25HC3S of any one of Items 23 to 32, having an x-ray powder diffraction pattern comprising a peak at about 18.0° 2θ.

[0695] Item 34. The crystalline calcium 25HC3S of any one of Items 23 to 33, having an x-ray powder diffraction pattern comprising a peak at about 18.1° 2θ.

[0696] Item 35. The crystalline calcium 25HC3S of any one of Items 23 to 34, having an x-ray powder diffraction pattern comprising a peak at about 18.4° 2θ.

[0697] Item 36. The crystalline calcium 25HC3S of any one of Items 23 to 35, having an x-ray powder diffraction pattern comprising a peak at about 19.2° 2θ.

[0698] Item 37. The crystalline calcium 25HC3S of any one of Items 23 to 36, having an x-ray powder diffraction pattern comprising two peaks at about 15.0° 2θ to about 15.1° 2θ, wherein the two peaks: (a) do not overlap; (b) partially overlap; or (c) superimpose so as to appear as a single peak.

[0699] Item 38. The crystalline calcium 25HC3S of Item 23, having an x-ray powder diffraction pattern substantially the same as that of FIG. 4 or FIG. 4A .

[0700] Item 39. Crystalline 25HC3S zinc.

[0701] Item 40. The crystalline 25HC3S zinc of Item 39, having an x-ray powder diffraction pattern comprising a peak at about 2.1° 2θ.

[0702] Item 41. The crystalline 25HC3S zinc of Item 39 or 40, having an x-ray powder diffraction pattern comprising a peak at about 2.3° 2θ.

[0703] Item 42. The crystalline 25HC3S zinc of any one of Items 39 to 41, having an x-ray powder diffraction pattern comprising a peak at about 6.0° 2θ.

[0704] Item 43. The crystalline 25HC3S zinc of any one of Items 39 to 42, having an x-ray powder diffraction pattern comprising a peak at about 8.6° 2θ.

[0705] Item 44. The crystalline 25HC3S zinc of any one of Items 39 to 43, having an x-ray powder diffraction pattern comprising a peak at about 8.9° 2θ.

[0706] Item 45. The crystalline 25HC3S zinc of any one of Items 39 to 44, having an x-ray powder diffraction pattern comprising a peak at about 9.3° 2θ.

[0707] Item 46. The crystalline 25HC3S zinc of any one of Items 39 to 45, having an x-ray powder diffraction pattern comprising a peak at about 15.1° 2θ.

[0708] Item 47. The crystalline 25HC3S zinc of any one of Items 39 to 46, having an x-ray powder diffraction pattern comprising a peak at about 18.3° 2θ.

[0709] Item 48. The crystalline 25HC3S zinc of any one of Items 39 to 47, having an x-ray powder diffraction pattern comprising a peak at about 18.8° 2θ.

[0710] Item 49. The crystalline 25HC3S zinc of any one of Items 39 to 48, having an x-ray powder diffraction pattern comprising two peaks at about 2.1° 2θ to about 2.3° 2θ, wherein the two peaks: (a) do not overlap; (b) partially overlap; or (c) superimpose so as to appear as a single peak.

[0711] Item 50. The crystalline 25HC3S zinc of Item 40, further comprising an x-ray powder diffraction pattern comprising a peak at about 6.0° 2θ.

[0712] Item 51. The crystalline 25HC3S zinc of Item 40, which has an x-ray powder diffraction pattern further comprising a peak at about 8.6° 2θ.

[0713] Item 52. The crystalline 25HC3S zinc of Item 40, which has an x-ray powder diffraction pattern further comprising a peak at about 8.9° 2θ.

[0714] Item 53. The crystalline 25HC3S zinc of Item 40, further comprising an x-ray powder diffraction pattern comprising a peak at about 9.3° 2θ.

[0715] Item 54. The crystalline 25HC3S zinc of Item 40, which has an x-ray powder diffraction pattern further comprising a peak at about 15.1° 2θ.

[0716] Item 55. The crystalline 25HC3S zinc of Item 40, which has an x-ray powder diffraction pattern further comprising a peak at about 18.3° 2θ.

[0717] Item 56. The crystalline 25HC3S zinc of Item 40, which has an x-ray powder diffraction pattern further comprising a peak at about 18.8° 2θ.

[0718] Item 57. The crystalline 25HC3S zinc of Item 50, which has an x-ray powder diffraction pattern further comprising a peak at about 8.6° 2θ.

[0719] Item 58. The crystalline 25HC3S zinc of Item 50, which has an x-ray powder diffraction pattern further comprising a peak at about 8.9° 2θ.

[0720] Item 59. The crystalline 25HC3S zinc of Item 50, which has an x-ray powder diffraction pattern further comprising a peak at about 9.3° 2θ.

[0721] Item 60. The crystalline 25HC3S zinc of Item 50, further comprising an x-ray powder diffraction pattern comprising a peak at about 15.1° 2θ.

[0722] Item 61. The crystalline 25HC3S zinc of Item 50, which has an x-ray powder diffraction pattern further comprising a peak at about 18.3° 2θ.

[0723] Item 62. The crystalline 25HC3S zinc of Item 50, which has an x-ray powder diffraction pattern further comprising a peak at about 18.8° 2θ.

[0724] Item 63. The crystalline 25HC3S zinc of Item 57, which has an x-ray powder diffraction pattern further comprising a peak at about 8.9° 2θ.

[0725] Item 64. The crystalline 25HC3S zinc of Item 57, which has an x-ray powder diffraction pattern further comprising a peak at about 9.3° 2θ.

[0726] Item 65. The crystalline 25HC3S zinc of Item 57, which has an x-ray powder diffraction pattern further comprising a peak at about 15.1° 2θ.

[0727] Item 66. The crystalline 25HC3S zinc of Item 57, which has an x-ray powder diffraction pattern further comprising a peak at about 18.3° 2θ.

[0728] Item 67. The crystalline 25HC3S zinc of Item 57, which has an x-ray powder diffraction pattern further comprising a peak at about 18.8° 2θ.

[0729] Item 68. The crystalline 25HC3S zinc of Item 63, which has an x-ray powder diffraction pattern further comprising a peak at about 9.3° 2θ.

[0730] Item 69. The crystalline 25HC3S zinc of Item 63, which has an x-ray powder diffraction pattern further comprising a peak at about 15.1° 2θ.

[0731] Item 70. The crystalline 25HC3S zinc of Item 63, which has an x-ray powder diffraction pattern further comprising a peak at about 18.3° 2θ.

[0732] Item 71. The crystalline 25HC3S zinc of Item 63, which has an x-ray powder diffraction pattern further comprising a peak at about 18.8° 2θ.

[0733] Item 72. The crystalline 25HC3S zinc of Item 68, which has an x-ray powder diffraction pattern further comprising a peak at about 15.1° 2θ.

[0734] Item 73. The crystalline 25HC3S zinc of Item 68, which has an x-ray powder diffraction pattern further comprising a peak at about 18.3° 2θ.

[0735] Item 74. The crystalline 25HC3S zinc of Item 68, which has an x-ray powder diffraction pattern further comprising a peak at about 18.8° 2θ.

[0736] Item 75. The crystalline 25HC3S zinc of Item 72, which has an x-ray powder diffraction pattern further comprising a peak at about 18.3° 2θ.

[0737] Item 76. The crystalline 25HC3S zinc of Item 72, which has an x-ray powder diffraction pattern further comprising a peak at about 18.8° 2θ.

[0738] Item 77. The crystalline 25HC3S zinc of Item 39, having an x-ray powder diffraction pattern comprising one or more peaks selected from the group consisting of 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.

[0739] Item 78. The crystalline 25HC3S zinc of Item 39, having an x-ray powder diffraction pattern comprising one or more peaks selected from the group consisting of 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.

[0740] Item 79. The crystalline 25HC3S zinc of Item 39, having an x-ray powder diffraction pattern comprising one or more peaks selected from the group consisting of 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.

[0741] Item 80. The crystalline 25HC3S zinc of Item 39, having an x-ray powder diffraction pattern comprising one or more peaks selected from the group consisting of 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.

[0742] Item 81. The crystalline 25HC3S zinc of Item 39, having an x-ray powder diffraction pattern comprising one or more peaks selected from the group consisting of about 8.9° 2θ, about 9.3° 2θ, about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0743] Item 82. The crystalline 25HC3S zinc of Item 39, having an x-ray powder diffraction pattern comprising one or more peaks selected from the group consisting of about 9.3° 2θ, about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0744] Item 83. The crystalline 25HC3S zinc of Item 39, having an x-ray powder diffraction pattern comprising one or more peaks selected from the group consisting of about 15.1° 2θ, about 18.3° 2θ, and about 18.8° 2θ.

[0745] Item 84. The crystalline 25HC3S zinc of Item 39, having an x-ray powder diffraction pattern comprising one or more peaks selected from about 18.3° 2θ and about 18.8° 2θ.

[0746] Item 85. The crystalline 25HC3S zinc of Item 39, having an x-ray powder diffraction pattern comprising a peak at about 18.8° 2θ.

[0747] Item 86. The crystalline 25HC3S zinc of Item 39, having an x-ray powder diffraction pattern substantially the same as Figure 85 or Figure 86.

[0748] Item 87. Crystalline 25HC3S magnesium.

[0749] Item 88. The crystalline 25HC3S magnesium of Item 87, having an x-ray powder diffraction pattern comprising a peak at about 2.2° 2θ.

[0750] Item 89. The crystalline 25HC3S magnesium of Item 87 or 88, having an x-ray powder diffraction pattern comprising a peak at about 4.4° 2θ.

[0751] Item 90. The crystalline 25HC3S magnesium of any one of Items 87 to 89, having an x-ray powder diffraction pattern comprising a peak at about 6.6° 2θ.

[0752] Item 91. The crystalline 25HC3S magnesium of any one of Items 87 to 90, having an x-ray powder diffraction pattern comprising a peak at about 8.9° 2θ.

[0753] Item 92. The crystalline 25HC3S magnesium of any one of Items 87 to 91, having an x-ray powder diffraction pattern comprising a peak at about 15.1° 2θ.

[0754] Item 93. The crystalline 25HC3S magnesium of any one of Items 87 to 92, having an x-ray powder diffraction pattern comprising a peak at about 15.6° 2θ.

[0755] Item 94. The crystalline 25HC3S magnesium of any one of Items 87 to 93, having an x-ray powder diffraction pattern comprising a peak at about 16.4° 2θ.

[0756] Item 95. The crystalline 25HC3S magnesium of any one of Items 87 to 94, having an x-ray powder diffraction pattern comprising a peak at about 17.6° 2θ.

[0757] Item 96. The crystalline 25HC3S magnesium of any one of Items 87 to 95, having an x-ray powder diffraction pattern comprising a peak at about 17.8° 2θ.

[0758] Item 97. The crystalline 25HC3S magnesium of Item 87, having an x-ray powder diffraction pattern substantially the same as that of Figure 6 or Figure 6A.

[0759] Item 98. An organic salt of 25HC3S that is not the ammonium salt of 25HC3S.

[0760] Item 99.25 Crystalline organic salts of HC3S.

[0761] Clause 100. The organic salt of Clause 98 or 99, wherein the organic salt is an amine salt of 25HC3S.

[0762] Item 101. The crystalline salt of 25HC3S of Item 100.

[0763] Clause 102. The salt of 25HC3S of Clause 100 or 101, wherein the amine is aliphatic, cyclic, aromatic, or a combination thereof.

[0764] 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.

[0765] Clause 104. The salt of any one of Clauses 100 to 103, wherein the amine is substituted.

[0766] Clause 105. The salt of Clause 104, wherein the substitution is one or more alcohol, alkyl, aryl, or another amine group.

[0767] Clause 106. The salt of Clause 105, wherein the substitution is one or more alcohol groups.

[0768] Clause 107. The salt of Clause 106, wherein the one or more alcohol groups are selected from primary, secondary, and tertiary alcohol groups.

[0769] 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 attached to the nitrogen of the amine group.

[0770] Clause 109. The salt of Clause 108, wherein the attached carbon atom comprises a cyclic alkyl group.

[0771] Clause 110. The organic salt of any one of clauses 100 to 109, wherein the amine is an amino acid.

[0772] Clause 111. The salt of Clause 110, wherein the amino acid is a naturally occurring amino acid, such as a proteinogenic amino acid.

[0773] Clause 112. The salt of Clause 110, wherein the amino acid is a non-naturally occurring amino acid.

[0774] Clause 113. The salt of 25HC3S of Clause 105, wherein the substitution is aryl.

[0775] Item 114. The salt of 25HC3S according to Item 113, wherein the aryl group comprises a phenyl group or a benzyl group.

[0776] Clause 115. The salt of 25HC3S of any one of clauses 100 to 114, comprising a cyclic amine.

[0777] Clause 116. The salt of any one of clauses 100 to 114, wherein the oxidation state of the amine is +1.

[0778] Clause 117. The salt of any one of clauses 100 to 114, wherein the oxidation state of the amine is +2.

[0779] Clause 118. The salt of any one of clauses 100 to 114, wherein the amine comprises one or more alcohol groups.

[0780] Item 119. The salt of Item 118, wherein the amine comprises two alcohol groups.

[0781] Item 120. The salt of Item 118, wherein the amine comprises three alcohol groups.

[0782] Clause 121. The salt of Clause 118, wherein the amine comprises four or more alcohol groups.

[0783] Item 122. The salt of Item 118, wherein the amine comprises five alcohol groups.

[0784] Clause 123. The salt of any one of clauses 118 to 122, wherein the alcohol group is a primary alcohol.

[0785] Clause 124. The salt of any one of clauses 100 to 123, wherein the amine comprises two amine groups.

[0786] Clause 125. The salt of Clause 124, wherein at least one amine group is a secondary amine group.

[0787] Item 126. The salt of Item 125, wherein the two amine groups are secondary amine groups.

[0788] Clause 127. The salt of any one of Clauses 124 to 216, further comprising at least one aryl group.

[0789] Clause 128. The salt of any one of clauses 124 to 216, further comprising at least two aryl groups.

[0790] Clause 129. The salt of Clause 108 or 109, wherein at least one amine group is bonded to one, two, or three independently substituted or unsubstituted alkyl groups.

[0791] Clause 130. The salt of Clause 129, wherein the alkyl group contains 1, 2, 3, 4, 5, or 6 carbon atoms.

[0792] Clause 131. The salt of Clause 130, wherein at least one alkyl group is substituted with an aryl group.

[0793] Clause 132. The salt of Clause 131, wherein aryl is phenyl or benzyl.

[0794] Article 133.25 HC3S hydroxyethylammonium.

[0795] Item 134. Crystalline 25HC3S hydroxyethylammonium.

[0796] Item 135. The crystalline 25HC3S hydroxyethylammonium of Item 134, having an x-ray powder diffraction pattern comprising a peak at about 2.1° 2θ.

[0797] Item 136. The crystalline 25HC3S hydroxyethylammonium of Item 135, wherein the x-ray powder diffraction pattern further comprises a peak at about 8.6° 2θ.

[0798] Item 137. The crystalline 25HC3S hydroxyethylammonium of Item 134, having an x-ray powder diffraction pattern substantially the same as that of Figure 8 or Figure 8A.

[0799] Article 138.25 HC3S tris(hydroxymethyl)methylammonium.

[0800] Item 139. Crystalline 25HC3S tris(hydroxymethyl)methylammonium.

[0801] Item 140. The crystalline 25HC3S trishydroxymethylmethylammonium salt of Item 139, having an x-ray powder diffraction pattern comprising a peak at about 1.9° 2θ.

[0802] Item 141. The crystalline 25HC3S trishydroxymethylmethylammonium salt of Item 139 or 140, having an x-ray powder diffraction pattern comprising a peak at about 2.1° 2θ.

[0803] Item 142. The crystalline 25HC3S trishydroxymethylmethylammonium salt of any one of Items 139 to 141, having an x-ray powder diffraction pattern comprising a peak at about 3.8° 2θ.

[0804] Item 143. The crystalline 25HC3S trishydroxymethylmethylammonium salt of any one of Items 139 to 142, having an x-ray powder diffraction pattern comprising peaks at about 4.2° 2θ and / or about 15.4° 2θ.

[0805] Item 144. The crystalline 25HC3S trishydroxymethylmethylammonium salt of any one of Items 139 to 143, having an x-ray powder diffraction pattern comprising two peaks at about 1.9° 2θ to about 2.1° 2θ, wherein the two peaks: (a) do not overlap; (b) partially overlap; or (c) superimpose so as to appear as a single peak.

[0806] Item 145. The crystalline 25HC3S tris(hydroxymethyl)methylammonium salt of Item 139, having an x-ray powder diffraction pattern substantially the same as that of Figure 14 or Figure 14A.

[0807] Clause 146.25 HC3S lysine.

[0808] Item 147. Crystallized 25HC3S lysine.

[0809] Item 148. The crystalline 25HC3S lysine salt of Item 147, having an x-ray powder diffraction pattern comprising a peak at about 1.5° 2θ.

[0810] Item 149. The crystalline 25HC3S lysine salt of Item 147 or 148, which has an x-ray powder diffraction pattern comprising a peak at about 7.0° 2θ.

[0811] 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.

[0812] Item 151. The crystalline 25HC3S lysine salt of any one of Items 147 to 150, having an x-ray powder diffraction pattern comprising a peak at about 11.8° 2θ.

[0813] Item 152. The crystalline 25HC3S lysine salt of any one of Items 147 to 151, having an x-ray powder diffraction pattern comprising a peak at about 16.8° 2θ.

[0814] Item 153. The crystalline 25HC3S lysine salt of any one of Items 147 to 152, having an x-ray powder diffraction pattern comprising a peak at about 3.2° 2θ.

[0815] Item 154. The crystalline 25HC3S lysine salt of any one of Items 147 to 153, having an x-ray powder diffraction pattern comprising a peak at about 10.0° 2θ.

[0816] Item 155. The crystalline 25HC3S lysine salt of any one of Items 147 to 154, having an x-ray powder diffraction pattern comprising a peak at about 12.2° 2θ.

[0817] Item 156. The crystalline 25HC3S lysine salt of any one of Items 147 to 155, having an x-ray powder diffraction pattern comprising a peak at about 15.2° 2θ.

[0818] Item 157. The crystalline 25HC3S lysine salt of Item 147, having an x-ray powder diffraction pattern substantially the same as that of Figure 24 or Figure 24A.

[0819] Article 158.25HC3S meglumine.

[0820] Item 159. Crystalline 25HC3S meglumine.

[0821] Item 160. The crystalline 25HC3S meglumine salt of Item 159, which has an x-ray powder diffraction pattern comprising a peak at about 1.7° 2θ.

[0822] Item 161. The crystalline 25HC3S meglumine salt of Item 159 or 160, which has an x-ray powder diffraction pattern comprising a peak at about 3.5° 2θ.

[0823] Item 162. The crystalline 25HC3S meglumine salt of any one of Items 159 to 161, having an x-ray powder diffraction pattern comprising a peak at about 5.2° 2θ.

[0824] 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.

[0825] 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.

[0826] 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.

[0827] Item 166. The crystalline 25HC3S meglumine salt of any one of Items 159 to 165, having an x-ray powder diffraction pattern comprising a peak at about 14.5° 2θ.

[0828] 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.

[0829] 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.

[0830] Item 169. The crystalline 25HC3S meglumine salt of any one of Items 159 to 168, having an x-ray powder diffraction pattern comprising a peak at about 18.2° 2θ.

[0831] Item 170. The crystalline 25HC3S meglumine salt of Item 159, having an x-ray powder diffraction pattern substantially the same as that of Figure 12 or Figure 12A.

[0832] Item 171.25HC3S hydroxyethylpyrrolidinium.

[0833] Item 172. Crystalline 25HC3S hydroxyethylpyrrolidinium.

[0834] Item 173. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of Item 172, having an x-ray powder diffraction pattern comprising a peak at about 3.8° 2θ.

[0835] Item 174. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of Item 172 or 173, having an x-ray powder diffraction pattern comprising a peak at about 7.5° 2θ.

[0836] Item 175. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 174, having an x-ray powder diffraction pattern comprising a peak at about 7.6° 2θ.

[0837] Item 176. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 175, having an x-ray powder diffraction pattern comprising a peak at about 8.2° 2θ.

[0838] Item 177. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 176, having an x-ray powder diffraction pattern comprising a peak at about 8.6° 2θ.

[0839] Item 178. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 177, having an x-ray powder diffraction pattern comprising a peak at about 12.4° 2θ.

[0840] Item 179. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 178, having an x-ray powder diffraction pattern comprising a peak at about 13.3° 2θ.

[0841] Item 180. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 179, having an x-ray powder diffraction pattern comprising a peak at about 15.0° 2θ.

[0842] Item 181. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 180, having an x-ray powder diffraction pattern comprising a peak at about 10.5° 2θ.

[0843] Item 182. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 181, having an x-ray powder diffraction pattern comprising a peak at about 15.3° 2θ.

[0844] Item 183. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 182, having an x-ray powder diffraction pattern comprising a peak at about 15.6° 2θ.

[0845] Item 184. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 183, having an x-ray powder diffraction pattern comprising a peak at about 16.3° 2θ.

[0846] Item 185. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 184, having an x-ray powder diffraction pattern comprising a peak at about 16.7° 2θ.

[0847] Item 186. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 185, having an x-ray powder diffraction pattern comprising a peak at about 20.9° 2θ.

[0848] Item 187. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 186, having an x-ray powder diffraction pattern comprising two peaks at about 7.5° 2θ to about 7.6° 2θ, wherein the two peaks: (a) do not overlap; (b) partially overlap; or (c) superimpose so as to appear as a single peak.

[0849] Item 188. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of any one of Items 172 to 187, having an x-ray powder diffraction pattern comprising two peaks at about 8.2° 2θ to about 8.6° 2θ, wherein the two peaks: (a) do not overlap; (b) partially overlap; or (c) superimpose so as to appear as a single peak.

[0850] Item 189. The crystalline 25HC3S hydroxyethylpyrrolidinium salt of Item 172, having an x-ray powder diffraction pattern substantially the same as that of Figure 10.

[0851] 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.

[0852] Item 191. The crystalline 25HC3S hydroxyethyl pyrrolidinium salt of Item 190, wherein the unit cell has a molecular formula volume of about / unit cell.

[0853] Article 192.25 HC3S diethylammonium.

[0854] Item 193. Crystalline 25HC3S diethylammonium.

[0855] Item 194. The crystalline 25HC3S diethylammonium of Item 193, having an x-ray powder diffraction pattern comprising a peak at about 3.8° 2θ.

[0856] Item 195. The crystalline 25HC3S diethylammonium of Item 193 or 194, having an x-ray powder diffraction pattern comprising a peak at about 7.9° 2θ.

[0857] Item 196. The crystalline 25HC3S diethylammonium of any one of Items 193 to 195, having an x-ray powder diffraction pattern comprising a peak at about 8.6° 2θ.

[0858] Item 197. The crystalline 25HC3S diethylammonium of any one of Items 193 to 196, having an x-ray powder diffraction pattern comprising a peak at about 9.6° 2θ.

[0859] Item 198. The crystalline 25HC3S diethylammonium of any one of Items 193 to 197, having an x-ray powder diffraction pattern comprising a peak at about 10.9° 2θ.

[0860] Item 199. The crystalline 25HC3S diethylammonium of any one of Items 193 to 198, having an x-ray powder diffraction pattern comprising a peak at about 12.3° 2θ.

[0861] 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.

[0862] 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.

[0863] Item 202. Crystalline 25HC3S diethylammonium as described in Item 193, which has an x-ray powder diffraction pattern substantially the same as that of Figure 18.

[0864] Clause 203. The crystalline 25HC3S diethylammonium of any one of Clauses 193 to 202, wherein the unit cell of the crystalline salt is orthorhombic.

[0865] Clause 204. The crystalline 25HC3S diethylammonium salt 203 of clause 204, wherein the molecular formula volume of the unit cell is about / unit cell.

[0866] Article 205.25 HC3S diethanolamine.

[0867] Item 206. Crystalline 25HC3S diethanolamine.

[0868] Item 207. The crystalline 25HC3S diethanolamine of Item 206, having an x-ray powder diffraction pattern comprising a peak at about 3.8° 2θ.

[0869] Item 208. The crystalline 25HC3S diethanolamine of Item 206 or 207, having an x-ray powder diffraction pattern comprising a peak at about 7.7° 2θ.

[0870] Item 209. The crystalline 25HC3S diethanolamine of any one of Items 206 to 208, having an x-ray powder diffraction pattern comprising a peak at about 8.1° 2θ.

[0871] Item 210. The crystalline 25HC3S diethanolamine of any one of Items 206 to 209, having an x-ray powder diffraction pattern comprising a peak at about 8.8° 2θ.

[0872] Item 211. The crystalline 25HC3S diethanolamine of any one of Items 206 to 210, having an x-ray powder diffraction pattern comprising a peak at about 14.6° 2θ.

[0873] Item...

Claims

1. 25 A salt of HC3S that is not (i) an alkali metal salt or (ii) an ammonium salt.

2. A salt of 25HC3S that is not (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, which is not the crystalline sodium salt of 25HC3S.

4. The crystalline salt of claim 3, which is not the 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 as claimed in any one of claims 1 to 9.

11. Crystallize potassium 25HC3S.

12. Crystallized 25HC3S zinc.

13. An organic salt of 25HC3S that is not the ammonium salt of 25HC3S. 14.25Crystalline organic salt of HC3S.

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

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

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

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

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

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