Sterile aqueous choline salt compositions

Abstract

Sterile aqueous choline salt compositions, methods for their preparation and use are disclosed. Also disclosed are methods for treating choline deficiency, intestinal failure associated liver disease (IFALD) and fatty liver disease. Additionally, methods for the synthesis of choline salts are disclosed.

Description

[Technical field]

[0001] The present disclosure relates to sterile aqueous choline salt compositions, their manufacture and methods of use, particularly in connection with the treatment of conditions associated with choline deficiency. [Background technology]

[0002] Choline is an essential nutrient and a common component of the normal diet, typically ingested in the form of phosphatidylcholine, found in eggs, meat, nuts, and vegetables (Buchman, Gastroent., 2009, 137:S119-S128). The structure of choline contains a quaternary amine, which acts as a methyl donor in many metabolic reactions, similar to vitamin B and folic acid. Choline is required for cell membrane structure (e.g., phospholipids), triglyceride transport through very low density lipoprotein (VLDL) synthesis, cholesterol transport in bile, intracellular messaging, and brain development and function (e.g., acetylcholine). Choline is essential for cell health and survival: hepatocytes die by apoptosis in choline-deprived medium; increased DNA damage and apoptosis has been observed in lymphocytes from choline-deficient compared to normal humans, consistent with increased rates of liver cancer in rodents after long-term choline deficiency (Shin et al., J. Cell. Biochem., 1997; 64:196-208; and da Costa et al., Am. J. Clin. Nutr., 2006; 84:88-94).

[0003] 95% of choline in the body is in the form of phosphatidylcholine (PC). PC is the major lipid of the VLDL particle surface monolayer and membranes of important secretory vesicles (e.g., ER, Golgi apparatus, plasma membrane). Low PC levels inhibit VLDL packaging and secretion (Vance, JE, and Vance, DE, 1985, Can. J. Biochem. Cell Biol., 263, 12, 5898-5909). Exogenous choline is required to maintain adequate PC stores. Although multiple pathways for PC synthesis exist, only the de novo pathway adequately replenishes PC (Boyer, 2013, Compr. Physiol., 3:3). The de novo pathway to PC requires plasma free choline as a source substrate, CDP-choline, which is ubiquitous in mammalian tissues (Vance, DE, 2002, in Biochemistry of Lipids, Lipoproteins, and Membranes (Vance, DE, and Vance, JE, eds) pp. 205-232). In addition to the CDP-choline pathway for PC synthesis, the liver has intrinsic phosphatidylethanolamine methyltransferase (PEMT) activity that provides an alternative pathway for PC synthesis (Noga and Vance DE, 2003, J. Biol. Chem., 278, 24, 21851-21859). However, PEMT itself is partially dependent on choline as betaine, a metabolite of choline (Sunden, S., Renduchintala, M., Park, E., Miklasz, S., & Garrow, T., 1997 Arch. Biochem. Biophys., 345, 171-174). Other choline and PC salvage and resynthesis pathways exist, but they can only recycle PC, not generate it de novo (Boyer, 2013, Compr. Physiol., 3:3).

[0004] Choline deficiency resulting in reduced phosphatidylcholine levels can adversely affect multiple hepatobiliary functions and cause steatosis, cholestasis and / or hepatocyte death. Steatosis or fatty liver is a broad term describing the accumulation of fat in the liver. Cholestasis is a liver disease that occurs when bile flow from the liver is reduced or blocked. When bile flow is altered, bilirubin can accumulate. PC comprises approximately 40% of the organic matter in bile (Schmitz MGJ, Renooij W., Gastroent., 1990, 99:1292-1296). Insufficient PC in bile reduces vesicle / mixed micelle formation with cholesterol and increases free bile salts (Barrios and Lichtenberger, 2000, Gastroent., 118:1179-1186). Free bile salts exert detergent activity on cholangiocytes, restricting bile flow (De Vree, et al., Proc. Natl. Acad. Sci. USA, 1998, 95:282-287). Choline is also an important source of intracellular signaling intermediates (Albright, et al., (2005) Cell. Physiol. Biochem., 15(1-4):59-68). Choline deficiency induces DNA fragmentation in hepatocytes in culture (Albright, et al., 1996, FASEBJ, 10, 510-516). Moreover, hepatocytes die by apoptosis in choline-deficient medium.

[0005] Choline deficiency causes liver damage in animals and healthy adults. Experimental choline-deficient diets cause rapid onset liver abnormalities (e.g., increased liver fat as shown by MRI), which are reversed by normal diet (Zeisel, et al., FASEBJ, 1991, 5:2093-2098; and Fischer, et al., Am. J. Clin. Nutr., 2007, 85:1275-1285). These findings are consistent with the findings in several animal species that choline deficiency causes hepatic steatosis and cirrhosis, skeletal muscle and other organ abnormalities (Patek, et al., Proc. Soc. Exp. Biol. Med., 1975, 148:370-374; and Buchman, Nutr. Clin. Pract., 2003, 18:353-358).

[0006] Intestinal failure (IF) occurs when intestinal function declines below the minimum required for absorption of macronutrients and / or water and electrolytes, necessitating intravenous supplementation to maintain health and / or growth. It is often due to surgical resection of the intestine (short bowel syndrome) or disease-related intestinal dysfunction. IF is classified into three types: (T-1) transient, usually postoperative, and fully reversible; (T-2) due to severe illness, requiring parenteral nutrition (PN) for weeks or months; (T-3) requiring long-term PN for survival. The underlying etiology of T-3 stems from diseases such as cancer, Crohn's disease, vascular disease, AIDS, and radiation enteritis (Bakker, H. et al., Clin. Nutr., 1999, 18:135-140). Also, after overcoming gastrointestinal (GI) disease, a significant proportion of patients with PN-dependent IF develop progressive liver disease or intestinal failure-associated liver disease (IFALD).

[0007] PN-dependent liver disease in adults and children has been widely observed for decades. Now called IFALD, it is the complication with the highest risk of mortality in IF patients (Pironi et al., Clin. Nutr., 2012, 31:831-45). Chronic cholestasis is present in 47-65% of adult PN patients (Cavicchi, et al., Ann. Intern. Med., 2000, 132:525-532; and Salvino et al., JPEN, 2006, 30:3, 202-208). Forty-two percent of all adult PN patients develop complex liver disease (i.e., extensive portal fibrosis or cirrhosis, bilirubin > 3.5 mg / dl for > 1 month, ascites, portal HTN, hepatic encephalopathy, or factor V < 50%), and liver disease accounts for 22% of PN patient deaths (Cavicchi, et al., Ann. Intern. Med., 2000, 132:525-532). The etiology is presumed to be multifactorial, with PN toxicity (lipid), infection, and nutritional deficiencies including choline deficiency all suggested in the literature.

[0008] Choline deficiency in patients with PN is common and is associated with liver damage (Buchman, et al., Clin. Nutr., 1993, 12:33-37; Buchman, Gastroent., 2009, 137:S119-128; Chawla, et al., Gastroent., 1989, 97:1514-20; and Burt et al., Lancet, 1980, Sep 20, 638-9). PN products contain only trace amounts of choline (as an emulsifier), de novo synthesis and the alternative pathway are impaired, and choline deficiency leads to impaired VLDL fat transport and pathological fat accumulation in hepatocytes (hpatoxyce) resulting in the accumulation of toxic bile salts (Noga, et al., J. Biol. Chem., 2003, 278:21851-21859; Lombardi, et al., J. Lipid Res., 1968, 9:437-446; Yao, et al., J. Biol. Chem., 1988, 263:2998-3004; and De Vree, et al., Proc. Natl. Acad. Sci. USA, 1998, 95:282-287). Choline is not present in sufficient amounts in PN products and is recognized as necessary by the American Society for Parenteral and Enteral Nutrition (ASPEN) but is not available in commercially available PN products (Vanek et al., Nutr. Clin. Pract., 2012, 27(4), 440-491).

[0009] Intravenous (IV) administration of choline for IFALD has shown promise in reducing steatosis (Buchman, et al., Hepatol., 1995, 22:1399-1403). IV administration of choline to patients over 16 years of age who required more than 80% PN has shown reversal of steatosis and improvement of cholestasis (Buchman, et al., J. Parent. Ent. Nutr., 2001, 25:260-268). IV administration of choline is also well tolerated by patients (Buchman, et al., Clin. Pharm. Ther., 1994, 55:277-83). Parenteral administration of choline salt-containing nutritional solutions to patients has been described as a method of suppressing fatty liver disease in human patients (U.S. Patent No. 5,567,736).

[0010] The Food and Drug Administration (FDA) requires that all injectable drug products be sterilized by terminal sterilization or aseptic processing methods. Terminal sterilization is the preferred sterilization method for injectable drug products and is performed after the drug product is placed in the primary packaging. Terminal sterilization of drug products that are aqueous solutions may involve heating or irradiation. Selecting the appropriate sterilization method requires an in-depth understanding of the physicochemical properties of the drug substance and the properties of the final drug product.

[0011] The gamma irradiation process uses cobalt-60, which emits gamma rays measured in kilograys (kGy) during radioactive decay. High-energy gamma rays interact with substances to form ion pairs with the release of electrons, which leads to the formation and excitation of free radicals. Free radicals are highly reactive and can participate in several types of reactions, including the release of gases, the formation and cleavage of double bonds, exchange reactions, electron transfer or cross-linking. In microorganisms, radiation-induced damage can cause biological changes that lead to cell death. Although cleavage of covalent bonds in bacterial DNA is considered the main pathway of cell damage, membrane damage can also contribute significantly to reproductive cell death. In solution, molecules may receive energy directly from the incident radiation ("direct effect") or, in aqueous solutions such as parenteral preparations, due to energy transfer from the radiolysis products of water (e.g., hydrogen, hydroxyl radicals, hydrated electrons) to the solute molecules ("indirect effect").

[0012] Each new compound must be tested to assess radiation stability. Additionally, for formulated drugs, the stability of individual components may change when irradiated as part of the product (Jacobs GP, Pharmaceutical Technology, 2007 Supplement, Iss. 2). Aqueous parenteral pharmaceutical products present additional challenges in sterilization by gamma irradiation compared to their solid counterparts due to the "indirect effect." For example, the hydroxyl radical is the most potent known oxidizing species and is the primary cause of radiation-induced damage of solutes in irradiated aqueous solutions (Sharma et al. Advances in Pharmaceutical Product Development and Research, 2020, Ch. 21, 789-848).

[0013] Thus, there is a need for sterile aqueous choline salt compositions and methods for their preparation for treating conditions associated with choline deficiency. Summary of the Invention

[0014] The present disclosure provides sterile compositions of choline salts in aqueous media, methods of making and using same. The present disclosure further provides methods of treating choline deficiency, intestinal failure associated liver disease (IFALD) and fatty liver disease. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows the steam survival curve of G. stearothermophilus spores suspended in a 50% choline chloride composition (R2=0.9895, -1 / slope of line=D121 value=34 min). [Diagram 2] FIG. 2 shows the serial dilution scheme used in Example 5. [Diagram 3] FIG. 3 plots the pH results of the "neat" samples for pH testing of a 50% w / v solution of choline chloride in water for injection (WFI) before and after terminal sterilization by heat. [Figure 4] FIG. 4 plots the pH results of the "1:5 diluted" samples for pH testing of a 50% w / v choline chloride solution in WFI before and after terminal sterilization by heat. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] In the following description, certain specific details are given to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the present invention can be practiced without these details. In other instances, well-known structures are not shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless otherwise indicated by context, throughout the following specification and claims, the word "comprise" and variations thereof, such as "comprises" and "comprising", should be interpreted in an open and inclusive sense, i.e., "including but not limited to".

[0017] References throughout this specification to "in one embodiment" or "in an embodiment" mean that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures or characteristics can be combined in any suitable manner in one or more embodiments. Also, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in the sense of including "and / or" unless the content clearly dictates otherwise. The term "about" is interpreted to mean plus or minus 10%. By way of example, "about 5" means 5±0.5. Terms not specifically defined herein should be given the meaning that would be given by one of ordinary skill in the art in light of this disclosure and the context.

[0018] As used herein, the following terms have the following meanings, unless otherwise stated.

[0019] "Sterility" is defined as the absence of viable microorganisms. "Sterilization" is defined as a validated process used to render a product free of viable microorganisms. The presence of microorganisms is expressed as a probability. Probability can be reduced to very low numbers, but cannot be reduced to zero with certainty. Hence, the term "Sterility Assurance Level (SAL)" is used as a measure of sterility. "Sterility Assurance Level (SAL)" refers to the probability of viable microorganisms being present on a product after sterilization, and is usually defined as 10 -nThe SAL can also be used to describe the number of microbial populations destroyed by a sterilization process, although this is not the same as a probabilistic definition. What is often called a "log reduction" (technically a one order of magnitude reduction) represents a 90% reduction in the microbial population. Thus, a "6 log reduction" (10 -6 The process of achieving sterility, theoretically, reduces an initial population of one million organisms to very nearly zero. Sterility can also be expressed by the presence of "colony forming units (CFU)", where CFU is used to describe the visible growth of microorganisms arising from a single cell or multiple cells.

[0020] "Choline salts" refers to a class of quaternary ammonium salts containing an N,N,N-trimethylethanolammonium cation and a corresponding counteranion, which can be represented by the general formula: - represents the corresponding counter anion: [ka]

[0021] Suitable counter anions are halides, e.g. chloride, Cl. -, and bitartrate ((2R,3R)-2,3,4-trihydroxy-4-oxobutanoate). Choline salts can be made from inorganic or organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Suitable organic acids may be selected from the aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, hippuric acid, malonic acid, oxalic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, beta-hydroxybutyric acid, salicylic acid, galactaric acid, galacturonic acid.

[0022] An "aqueous medium" is defined as a liquid that contains greater than about 50% water.

[0023] "Weight / volume percent" is a way of expressing the concentration of a solution.

number

[0024] "Water for Injection (WFI)" is a sterile, non-pyrogenic preparation of water for injection that does not contain bacteriostatic, antibacterial or buffering agents.

[0025] "Bacteriostatic" refers to a substance that inhibits the growth of bacteria without killing the bacteria.

[0026] "Antimicrobial agent" refers to a natural or synthetic substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, and algae.

[0027] "Preservative" refers to an antimicrobial component added to a composition to help maintain the safety of the composition by inhibiting the growth of, or reducing the amount of, microbial contaminants, or both.

[0028] An "amino acid" is a simple organic compound that contains both a carboxyl (-COOH) group and an amino (-NH2) group. The term "amino acid" can refer to naturally occurring or synthetic (i.e., artificial) amino acids.

[0029] "Vitamin" refers to any of a group of organic compounds that are essential for normal growth and nutrition and cannot be synthesized by the body and therefore are required in small amounts in the diet. Examples of vitamins may include, but are not limited to, vitamin A (such as all-trans retinol, all-trans retinyl esters, all-trans beta-carotene, and other provitamin A carotenoids), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B9 (folic acid or folate), vitamin B12 (cobalamin), vitamin C (ascorbic acid), vitamin D (calciferols), vitamin E (tocopherols and tocotrienols), and vitamin K (quinones).

[0030] "Fatty acid" refers to any of the carboxylic acids consisting of a hydrocarbon chain and a terminal carboxyl group, especially those present as esters in fats and oils. Fatty acids can include, but are not limited to, alpha-linolenic acid, linoleic acid, docosahexaenoic acid, and gamma-linolenic acid.

[0031] "Ionic strength" is the total ion concentration in a solution. Ionic strength is calculated by Equation I:

number

[0032] "Substantially free of microorganisms, bacteria or specific bacterial strains, such as Staphylococcus aureus (S. aureus) or Geobacillus stearothermophilus (G. stearothermophilus)" means that low (i.e., ≦1 colony forming unit, CFU / mL) levels of bacteria are present, but the bacterial levels are clinically acceptable. Microorganisms include viruses, bacteria, archaea, fungi, plants such as algae, and protozoa.

[0033] "Resistance to microbial growth" means that the composition meets the standards set forth by the Food and Drug Administration and the United States Pharmacopeia for products manufactured using, for example, an aqueous base or vehicle. For example, for bacteria, resistance to microbial growth can mean a 1.0 log or greater reduction in the number initially calculated on day 7, a 3.0 log or greater reduction on day 14, and no increase on day 28 from the number on day 14. For example, for yeasts and molds, resistance to microbial growth can mean no increase on days 7, 14, and 28 from the number initially calculated.

[0034] A "D value" or decimal reduction time (or decimal reduction dose) is the time (or dose) required to achieve a log reduction, i.e., kill 90% (1 log or more) of the relevant microorganisms, at a given condition (e.g., temperature) or set of conditions.

[0035] "Chemically stable" refers to the resistance of a substance to decomposition into known or unknown decomposition products. For example, trimethylamine is a known decomposition product of choline chloride. Acceptable levels of trimethylamine may be up to 0.2%.

[0036] "Pharmaceutical product" refers to a finished dosage form, e.g., a tablet, capsule, liquid, etc., that contains an active pharmaceutical ingredient generally, although not necessarily, in association with inactive ingredients.

[0037] In certain embodiments of the present invention, pH adjusting agent can be added to the composition.The selection of pH adjusting agent can affect the resistance to microbial attack and / or the stability of choline salt, as measured by the reduction of analyzable decomposition products.The pH adjusting agent can include an acid, such as malic acid, citric acid, acetic acid, boric acid, lactic acid, hydrochloric acid, phosphoric acid, sulfuric acid, sulfonic acid or nitric acid.The pH adjusting agent can include a base, such as acetanilide, ammonia, calcium hydroxide, potassium bicarbonate, potassium hydroxide, sodium bicarbonate, sodium dihydrogen phosphate, sodium citrate, sodium tartrate, sodium carbonate, sodium hydroxide, thiourea or urea.Any pH adjusting agent disclosed herein or known to those skilled in the art is contemplated herein.

[0038] As used herein, "pharmaceutical composition" is synonymous with composition.

[0039] "Pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other undesirable reactions when administered to a subject. As used herein, "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and substances for pharmaceutically active substances is well known in the art. To the extent that a conventional media or substance is incompatible with the active ingredient, its use in a therapeutic composition is not appropriate. A supplementary compatible active ingredient can be incorporated into the composition. For human administration, the preparation should meet the standards of sterility, pyrogenicity, general safety and purity required by the Food and Drug Administration (FDA). Conventional procedures and ingredients for the selection and preparation of suitable compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, 21 stEd., Gennaro, Ed., Lippencott Williams & Wilkins (2005) and the United States Pharmacopeia: The National Formulary (USP 36 NF31), published in 2013.

[0040] "Additive" refers to certain embodiments that are diluents (where "diluent" refers to a substance used to dilute something) or more or less inert substances added as vehicles or to provide form or viscosity. Additives may also increase resistance to microbial growth and thus function as preservatives. Such additives include, but are not limited to, xylitol, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, cellulose derivatives, magnesium carbonate, and the like.

[0041] The term "effective amount" as used herein refers to the amount of a particular drug that is sufficient to achieve the desired effect in the subject being treated with that drug.Ideally, the effective amount of a drug is sufficient to inhibit or treat disease without causing substantial toxicity in the subject.The effective amount of a drug depends on the subject being treated, the severity of the disease, and the method of administration of the pharmaceutical composition.The method of determining the effective amount of the disclosed drug that is sufficient to achieve the desired effect in the subject is understood by those skilled in the art in light of this disclosure.

[0042] As used herein, the term "chronic" refers to a medical disorder or condition that persists for a long period of time or that recurs frequently.

[0043] The dosage form can be administered once a day, or more than once a day, for example, twice or three times a day. Alternatively, if deemed desirable by the prescribing physician or by the prescription information of the drug, the dosage form can be administered less frequently than once a day, for example, once every two days or once a week. The administration regimen includes, for example, a dose escalation to the extent necessary or useful for the indication being treated, so that the patient's body can adapt to the treatment, minimize or avoid undesirable side effects associated with the treatment, and / or maximize the therapeutic effect of the treatment. Suitable administration regimens and / or administration forms include, for example, those described in the latest edition of the Physicians' Desk Reference, which is incorporated herein by reference.

[0044] "Direct injection" refers to intravenous injection, where a substance is injected directly into a vein.

[0045] "Indirect injection" refers to intravenous injection in which a substance is introduced into another source before being directly injected into a vein. For example, without limiting the possible routes of indirect injection, indirect injection includes introducing a substance into an IV bag and then administering the contents of the IV bag to a subject by injection into a vein as the end point of travel (e.g., the contents of the IV bag, with or without the substance introduced, may flow through or be contained within other channels or devices before finally being injected into a vein).

[0046] As used herein, a "kit" refers to a combination of any number of items. For example, a kit can include a composition and a syringe for injection of the composition. A kit can also include a pre-filled syringe for injection.

[0047] "Gamma irradiation" refers to subjecting a material to gamma radiation, in which high-energy photons are emitted from an isotopic source (e.g., Cobalt-60). High-energy gamma radiation causes the disruption (ionization) of electrons in any material it encounters. In living cells, these disruptions result in damage to DNA and other cellular structures. These photon-induced changes at the molecular level can cause the death of an organism or render it infertile. This effect is useful for killing bacteria, insects, or other live contaminants that may be present in or on the material.

[0048] "Choline deficiency" refers to clinically determined choline deficiency. Choline is an essential nutrient in humans and is required for the normal function of all cells. As a key component of cell membranes, it ensures the structural integrity and signaling function of cells. Choline is also used in neurotransmission (as the metabolite acetylcholine), is a major source of methyl donors, and is required for lipid transport from the liver. Given these many diverse roles, choline deficiency can cause damage to many body systems, including the liver, muscle, and lymphocytes in humans, and the kidneys, pancreas, and developing brain and nervous system in animals. Choline deficiency can be characterized by less than 7 nmol of free choline.

[0049] "Parenteral nutrition (PN)" can refer to the intravenous administration of nutrients, which can include proteins, carbohydrates, fats, minerals and electrolytes, vitamins, and other trace elements, for patients who cannot ingest or absorb enough food, either through a feeding tube or by mouth, to maintain good nutritional status.

[0050] "Parenteral support" includes the administration of parenteral liquids alone or in combination with parenteral nutritional solutions.

[0051] "Intestinal failure (IF)" refers to a decrease in intestinal function below the minimum required for absorption of macronutrients and / or water and electrolytes, necessitating intravenous supplementation to maintain health and / or growth. In some instances, IF may be due to surgical resection of the intestine (short bowel syndrome) or pathological intestinal dysfunction.

[0052] "Intestinal failure associated liver disease (IFALD)" refers to liver disease associated with intestinal failure that may occur in PN-dependent subjects. In some instances, IFALD develops in patients receiving long-term parenteral nutrition for chronic intestinal failure and may be characterized by hepatic steatosis and / or cholestasis, and may be accompanied by one or more other signs of liver damage, including but not limited to elevated liver function tests, fibrosis, cirrhosis, and end-stage liver disease (ESLD). In some instances, IFALD may have previously been referred to as PN-associated liver disease (PNALD).

[0053] "Fatty liver disease" refers to a condition characterized by excess accumulation of lipids (fat) in the liver. Accumulation of fat in the liver leads to various clinical signs and a stepwise progression. Depending on the etiology, each stage can be characterized as nonalcoholic or alcoholic. The progression begins with simple steatosis or steatosis. This stage is generally considered benign and is characterized by an increasing appearance of fat in the liver. Fatty liver can be characterized as nonalcoholic (NAFL) or alcoholic (AFL). The next stage of fatty liver disease is a type of hepatitis known as steatohepatitis, which is characterized by further accumulation of fat and inflammation of liver tissue. Steatohepatitis can be nonalcoholic (NASH) or alcoholic (ASH). Both NASH and ASH can lead to the next stage of fatty liver disease, NASH-associated or ASH-associated fibrosis, characterized by scarring of the liver. Finally, fibrosis can progress to cirrhosis, which causes irreversible damage to the liver and is the most severe stage. Cirrhosis can be non-alcoholic or alcoholic.

[0054] "Type 1 glass" refers to borosilicate glass that has good chemical resistance. Type 1 glass is used for pharmaceutical products that require the least reactive containers. Exemplary products include, but are not limited to, glass vials, prefilled syringes, cartridges, and ampoules.

[0055] "Delamination Control (DC) Glass" refers to glass that is resistant to delamination (i.e., degradation of the surface glass). Exemplary products include, but are not limited to, glass vials, pre-filled syringes, cartridges, and ampoules.

[0056] "Pure ethanol" refers to 200 proof (100%) ethanol.

[0057] When referring to either 2-chloroethanol or aluminum, "substantially undetectable" refers to an amount of either substance that is within 10% of the detection limit.

[0058] In some embodiments, the present invention provides a sterile composition comprising a choline salt in an aqueous medium.

[0059] In certain embodiments, the composition comprises 25-75% by weight / volume of a choline salt, hi a specific embodiment, the composition comprises 50% by weight / volume of a choline salt.

[0060] In some embodiments, the choline salt is choline chloride. In other embodiments, the choline salt is choline bitartrate.

[0061] In some embodiments, the present invention provides a sterile composition for intravenous injection comprising choline chloride in an aqueous medium, wherein the choline chloride is present in the composition at a level of 25-75% choline chloride salt by weight / volume.

[0062] In some embodiments, the present invention provides a sterile composition for intravenous injection comprising choline chloride in an aqueous medium, wherein the choline chloride is present in the composition at a level of 50% choline chloride salt by weight / volume.

[0063] In some embodiments, the present invention provides a sterile composition for intravenous injection consisting of choline chloride in an aqueous medium, wherein the choline chloride is present in the composition at a level of 50% choline chloride by weight / volume.

[0064] In some embodiments, the aqueous medium is water for injection.

[0065] In certain embodiments, the composition does not contain a preservative. In other embodiments, the composition includes a preservative. In some embodiments, the composition includes at least one amino acid, at least one vitamin and / or at least one fatty acid. In some embodiments, the composition includes at least one amino acid. In yet other embodiments, the composition includes at least one vitamin. In certain embodiments, the composition includes at least one fatty acid. In other embodiments, the composition includes a pharma- ceutically acceptable carrier, diluent or additive.

[0066] In some embodiments, the ionic strength of the composition is at least 0.3 M. In certain embodiments, the ionic strength of the composition is 0.3-7 M. In certain specific embodiments, the ionic strength of the composition is about 7 M.

[0067] In some embodiments, the pH of the composition is about 4-7.

[0068] In some embodiments, the composition is substantially free of microorganisms, hi some embodiments, the composition is substantially free of bacteria.

[0069] In some embodiments, the composition is substantially free of S. aureus, G. stearothermophilus, and / or B. pumilus. -1 In certain embodiments, the composition comprises less than 10 CFU / mL of S. aureus, G. stearothermophilus, and / or B. pumilus. -2 In certain embodiments, the composition comprises less than 10 CFU / mL of S. aureus, G. stearothermophilus, and / or B. pumilus. -3 In certain embodiments, the composition comprises less than 10 CFU / mL of S. aureus, G. stearothermophilus, and / or B. pumilus. -4 In certain embodiments, the composition comprises less than 10 CFU / mL of S. aureus, G. stearothermophilus, and / or B. pumilus. -5 In certain embodiments, the composition comprises less than 10 CFU / mL of S. aureus, G. stearothermophilus, and / or B. pumilus. -6 Contains less than CFU / mL of S. aureus, G. stearothermophilus and / or B. pumilus.

[0070] In some embodiments, the composition is substantially free of S. aureus. -1 In certain embodiments, the composition contains less than 10 CFU / mL of S. aureus. -2 In certain embodiments, the composition contains less than 10 CFU / mL of S. aureus. -3 In certain embodiments, the composition contains less than 10 CFU / mL of S. aureus. -4 In certain embodiments, the composition contains less than 10 CFU / mL of S. aureus. -5 In certain embodiments, the composition contains less than 10 CFU / mL of S. aureus. -6 Contains S. aureus less than CFU / mL.

[0071] In some embodiments, the composition is substantially free of G. stearothermophilus. -1 In certain embodiments, the composition contains less than 10 CFU / mL of G. stearothermophilus. -2 In certain embodiments, the composition contains less than 10 CFU / mL of G. stearothermophilus. -3 In certain embodiments, the composition contains less than 10 CFU / mL of G. stearothermophilus. -4 In certain embodiments, the composition contains less than 10 CFU / mL of G. stearothermophilus. -5 In certain embodiments, the composition contains less than 10 CFU / mL of G. stearothermophilus. -6 Contains less than CFU / mL of G. stearothermophilus.

[0072] In some embodiments, the composition is substantially free of B. pumilus. -1 In certain embodiments, the composition contains less than 10 CFU / mL of B. pumilus. -2 In certain embodiments, the composition contains less than 10 CFU / mL of B. pumilus. -3 In certain embodiments, the composition contains less than 10 CFU / mL of B. pumilus. -4 In certain embodiments, the composition contains less than 10 CFU / mL of B. pumilus. -5 In certain embodiments, the composition contains less than 10 CFU / mL of B. pumilus. -6 Contains B. pumilus less than CFU / mL.

[0073] In some embodiments, the sterility assurance level of the composition is at least 10 -3 ~10 -6 In certain embodiments, the sterility assurance level of the composition is at least 10 -3 In another embodiment, the sterility assurance level of the composition is at least 10 -4In another embodiment, the sterility assurance level of the composition is at least 10 -5 In yet another embodiment, the sterility assurance level of the composition is at least 10 -6 It is.

[0074] In some embodiments, the composition is sterilized by the ionic strength of the composition. In some embodiments, the composition is sterilized by gamma irradiation. In further embodiments, the composition is sterilized by a combination of ionic strength and gamma irradiation. In some embodiments, the gamma irradiation is at least 20 kGy. In some embodiments, the gamma irradiation is 18-25 kGy. In some embodiments, the gamma irradiation is 25-33 kGy. In some embodiments, the gamma irradiation is 45-59 kGy.

[0075] In some embodiments, the composition is suitable for administration by indirect injection or direct injection. In a specific embodiment, the composition is suitable for administration by direct injection. In another specific embodiment, the composition is suitable for administration by indirect injection.

[0076] In some embodiments, the composition is resistant to microbial growth, hi some embodiments, the composition is chemically stable.

[0077] In some embodiments, the present invention provides a method of making a composition comprising the steps of combining a choline salt with an aqueous medium and adjusting the concentration of the choline salt in the aqueous medium.

[0078] In some embodiments, pH is also adjusted.In certain embodiments, pH adjusting agent is acid.In specific embodiments, acid is malic acid, citric acid, acetic acid, boric acid, lactic acid, hydrochloric acid, phosphoric acid, sulfuric acid, sulfonic acid or nitric acid.

[0079] In other embodiments, the pH adjusting agent is a base.In other specific embodiments, the base is acetanilide, ammonia, calcium hydroxide, potassium bicarbonate, potassium hydroxide, sodium bicarbonate, sodium dihydrogen phosphate, sodium citrate, sodium tartrate, sodium carbonate, sodium hydroxide, thiourea or urea.

[0080] In some embodiments, the method comprises sterilizing the composition by ionic strength of the composition. In some embodiments, the method comprises sterilizing the composition by gamma irradiation. In further embodiments, the method comprises sterilizing the composition by a combination of ionic strength and gamma irradiation.

[0081] In some embodiments, the gamma irradiation is at least 20 kGy. In some embodiments, the gamma irradiation is 18-25 kGy. In some embodiments, the gamma irradiation is 25-33 kGy. In some embodiments, the gamma irradiation is 45-59 kGy.

[0082] In some embodiments, the present invention provides a method for the treatment of a choline chloride-induced hypercalcemia by the steps of: formulating choline chloride in water for injection to produce a 50% (w / v) solution having an ionic strength of about 7M and a pH of about 4-7; and exposing the solution to gamma irradiation to produce a 50% (w / v) solution having an ionic strength of about 7M and a pH of about 4-7. -6 In accordance with another aspect of the present invention, there is provided a method for producing a composition as described herein, comprising the step of obtaining a sterility assurance level of

[0083] In some embodiments, the gamma irradiation is at least 20 kGy. In some embodiments, the gamma irradiation is 18-25 kGy. In some embodiments, the gamma irradiation is 25-33 kGy. In some embodiments, the gamma irradiation is 45-59 kGy.

[0084] In some embodiments, the present invention provides a composition described herein, produced by a method described herein.

[0085] In some embodiments, the present invention provides a composition produced by combining a choline salt and water for injection such that the ionic strength of the composition promotes sterilization of the composition, and sterilizing the composition by exposure to gamma radiation.In a specific embodiment, the composition comprises 50% choline chloride by weight / volume.

[0086] In some embodiments, the composition is filtered through a micron filter. In some embodiments, the composition is filtered through two micron filters in series. In some embodiments, the composition is filtered through a 0.2 μm filter. In some embodiments, the composition is filtered through two 0.2 μm filters in series. In some embodiments, the composition is filtered through a 0.45 μm filter. In some embodiments, the composition is filtered through two 0.45 μm filters in series.

[0087] In some embodiments, the present invention provides a method of treating a choline deficiency in a subject, comprising administering to the subject an effective amount of a composition described herein.

[0088] In some embodiments, the present invention provides a method of providing parenteral support to a subject, comprising administering to the subject an effective amount of a composition described herein.

[0089] In some embodiments, the present invention provides a method of providing parenteral nutrition to a subject, comprising administering to the subject an effective amount of a composition described herein.

[0090] In some embodiments, the present invention provides a method of treating hepatic cholestasis in a subject, comprising administering to the subject an effective amount of a composition described herein.

[0091] In some embodiments, the present invention provides a method of treating hepatic steatosis in a subject, comprising administering to the subject an effective amount of a composition described herein.

[0092] In some embodiments, the present invention provides a method of treating intestinal failure associated liver disease (IFALD) in a subject, comprising administering to the subject an effective amount of a composition described herein.

[0093] In some embodiments, the present invention provides a method for treating fatty liver disease in a subject, comprising administering to the subject an effective amount of a composition described herein.In some embodiments, the fatty liver disease is AFL, ASH, NAFL, NASH, NASH-related liver fibrosis or ASH-related liver fibrosis.In some embodiments, the fatty liver disease is alcoholic fatty liver (AFL).In some embodiments, the fatty liver disease is alcoholic steatohepatitis (ASH).In some embodiments, the fatty liver disease is non-alcoholic fatty liver (NAFL).In some embodiments, the fatty liver disease is non-alcoholic steatohepatitis (NASH).In some embodiments, the fatty liver disease is NASH-related liver fibrosis.In some embodiments, the fatty liver disease is ASH-related liver fibrosis.

[0094] In some embodiments, the treatment comprises administering to the subject a composition described herein as parenteral support. Parenteral support includes administration of parenteral liquid alone or in combination with a parenteral nutrition solution. For example, a solution containing a composition described herein can be provided by Y-line administration as a stand-alone when only liquid is administered. In some embodiments, the treatment comprises administering to the subject a composition described herein as part of a parenteral support solution, where administration is performed at least once a day, or as determined by the treating physician. In some embodiments, the treatment comprises administering to the subject a composition described herein as part of a parenteral support solution, where administration is performed at least once a day, or according to a schedule that obtains normal plasma levels of choline, as determined by the treating physician. For example, the infusion time can be several hours (e.g., 10-14 hours) and can be administered continuously or can be divided into one or more dosing intervals.

[0095] In some embodiments, the treatment comprises administering to the subject a composition described herein as part of a parenteral nutrition solution. In some embodiments, the treatment comprises administering to the subject a composition described herein as part of a parenteral nutrition solution, where administration occurs at least once a day, or as determined by the treating physician. In some embodiments, the treatment comprises administering to the subject a composition described herein as part of a parenteral nutrition solution, where administration occurs at least once a day, or according to a schedule that obtains normal plasma levels of choline, as determined by the treating physician. For example, the infusion time can be several hours (e.g., 10-14 hours) and can be administered continuously or can be divided into one or more administration periods.

[0096] In specific embodiments, the compositions described herein are administered to a subject by direct injection. In other specific embodiments, the compositions described herein are administered to a subject by indirect injection.

[0097] In some embodiments, the invention provides a choline salt composition, the composition being an irradiation product of a composition that has been sterilized in liquid form by gamma irradiation of at least 25 kGy and that comprises 25-75% choline salt by weight / volume in an aqueous medium, the composition having (a) a pH of about 4-7; (b) an ionic strength of >0.3M; and (c) an ionic strength of at least 10 -3 ~10 -6 In a specific embodiment, the ionic strength of the composition is about 7 M. In a specific embodiment, the sterility assurance level of the composition is at least 10 -6 In a specific embodiment, the aqueous medium is water for injection. In a specific embodiment, the choline salt is choline chloride. In a specific embodiment, the composition comprises 50% choline salt by weight / volume.

[0098] In some embodiments, the composition is for use in a method of treating a choline deficiency in a subject comprising administering to the subject an effective amount of a composition described herein. In some embodiments, the composition is for use in a method of treating a deficiency in parenteral support in a subject comprising administering to the subject an effective amount of a composition described herein. In some embodiments, the composition is for use in a method of treating a deficiency in parenteral nutrition in a subject comprising administering to the subject an effective amount of a composition described herein. In some embodiments, the composition is for use in a method of treating hepatic steatosis in a subject comprising administering to the subject an effective amount of a composition described herein. In some embodiments, the composition is for use in a method of treating hepatic cholestasis in a subject comprising administering to the subject an effective amount of a composition described herein. In some embodiments, the composition is for use in a method of treating intestinal failure associated liver disease (IFALD) in a subject comprising administering to the subject an effective amount of a composition described herein. In some embodiments, the composition is for use in a method of treating fatty liver disease in a subject comprising administering to the subject an effective amount of a composition described herein. In some embodiments, the fatty liver disease is AFL, ASH, NAFL, NASH, NASH-associated liver fibrosis or ASH-associated liver fibrosis.

[0099] In some embodiments, the present invention provides a composition as described herein, wherein the composition is packaged in the form of an injection vial.

[0100] In some embodiments, the composition is packaged in a glass container. In some embodiments, the composition is packaged in a Type 1 glass container. In other embodiments, the composition is packaged in a delamination-controlled (DC) glass container.

[0101] In some embodiments, the present invention provides a composition described herein, wherein the composition is packaged in the form of an IV bag.

[0102] In some embodiments, the present invention provides a composition as described herein, wherein the composition is packaged in the form of a pre-filled syringe.

[0103] In some embodiments, the present invention provides a composition as described herein, wherein the composition is packaged in the form of a kit comprising the composition and a syringe. In a specific embodiment, the kit comprises the composition packaged in a glass container. In a specific embodiment, the kit comprises the composition packaged in a type 1 glass container. In a specific embodiment, the kit comprises the composition packaged in a delamination control (DC) glass container. In a specific embodiment, the kit comprises the composition packaged in an IV bag. In another specific embodiment, the kit comprises the composition packaged as a pre-filled syringe.

[0104] In some embodiments, the present invention provides compositions and packaging described herein, wherein the compositions and packaging are sterilized by gamma irradiation. In some embodiments, the present invention provides compositions and packaging described herein, wherein the compositions are sterilized by both ionic strength and gamma irradiation, and the packaging is sterilized by gamma irradiation. In specific embodiments, the compositions and packaging are sterilized by gamma irradiation at least 20 kGy. In some embodiments, the compositions and packaging are sterilized by gamma irradiation at 18-25 kGy. In some embodiments, the compositions and packaging are sterilized by gamma irradiation at 25-33 kGy. In some embodiments, the compositions and packaging are sterilized by gamma irradiation at 45-59 kGy.

[0105] In some embodiments, the present invention provides a sterile, ready-to-use pharmaceutical composition of choline chloride, the pharmaceutical composition comprising: a primary packaging container containing a sterile aqueous choline chloride solution having a concentration of 25-75% choline chloride in an aqueous medium by weight / volume; and a seal sealing the primary packaging container; and the choline chloride solution is provided with at least 10 -3 ~10-6 In a specific embodiment, the aqueous choline chloride solution has a concentration of 50% choline chloride by weight / volume. In a specific embodiment, the sterility assurance level is at least 10. -6 In certain embodiments, the aqueous medium is water for injection. In some embodiments, the primary packaging is a glass container. In some embodiments, the primary packaging is a type 1 glass container. In other embodiments, the primary packaging is a DC glass container. In yet other embodiments, the primary packaging is an IV bag. In further embodiments, the primary packaging is a pre-filled syringe.

[0106] In some embodiments, the ready-to-use pharmaceutical composition is sterilized by gamma irradiation. In specific embodiments, the ready-to-use pharmaceutical composition is sterilized by gamma irradiation of at least 20 kGy. In other specific embodiments, the ready-to-use pharmaceutical composition is sterilized by gamma irradiation of 25-33 kGy. In other specific embodiments, the ready-to-use pharmaceutical composition is sterilized by gamma irradiation of 45-59 kGy.

[0107] In some embodiments, the ready-to-use pharmaceutical composition is suitable for administration by indirect injection. In other embodiments, the ready-to-use medicament is suitable for administration by direct injection. In further embodiments, the ready-to-use medicament is stable in the primary packaging for at least 3 months.

[0108] In some embodiments, the present invention provides a sterile aqueous choline chloride pharmaceutical product produced by the steps of: (i) dissolving choline chloride in water for injection to a final concentration of 40-60% choline chloride by weight / volume; (ii) filtering the solution through a micron filter; (iii) transferring the solution to a glass vial; (iv) sealing the vial; and (v) sterilizing the pharmaceutical product using gamma irradiation. In certain embodiments, in step (ii), the sterile aqueous choline chloride pharmaceutical product is filtered through a 0.2 μm filter. In certain embodiments, in step (ii), the sterile aqueous choline chloride pharmaceutical product is filtered through a 0.45 μm filter. In certain embodiments, in step (ii), the sterile aqueous choline chloride pharmaceutical product is filtered through two micron filters in series.

[0109] In some embodiments, the present invention provides a sterile aqueous choline chloride drug product prepared by the steps of: (i) dissolving choline chloride in water for injection to a final concentration of 40-60% choline chloride by weight / volume; (ii) filtering the solution through a 0.2 μm filter; (iii) transferring the solution to a glass vial; (iv) sealing the vial; and (v) sterilizing the drug product using gamma irradiation.

[0110] In a specific embodiment, the sterile aqueous choline chloride pharmaceutical comprises 50% choline chloride by weight / volume. In certain embodiments, the sterile aqueous choline chloride pharmaceutical is filtered through two 0.2 μm filters in series. In certain embodiments, the sterile aqueous choline chloride pharmaceutical is filtered through two 0.45 μm filters in series. In a further embodiment, the sterile aqueous choline chloride pharmaceutical is sterilized by gamma irradiation at least 20 kGy. In a further specific embodiment, the sterile aqueous choline chloride pharmaceutical is sterilized by gamma irradiation at 25-33 kGy. In a further specific embodiment, the sterile aqueous choline chloride pharmaceutical is sterilized by gamma irradiation at 45-59 kGy.

[0111] In some embodiments, the sterile aqueous choline chloride pharmaceutical is transferred to a vial, where the vial is a glass container. In some embodiments, the sterile aqueous choline chloride pharmaceutical is transferred to a vial, where the vial is a type 1 glass container. In some embodiments, the sterile aqueous choline chloride pharmaceutical is transferred to a vial, where the vial is a DC glass container. In a specific embodiment, the sterile aqueous choline chloride pharmaceutical is sealed in a vial, where the vial is sealed with a rubber stopper. In a further specific embodiment, the sterile aqueous choline chloride pharmaceutical is sealed in a vial, where the vial is further sealed with an aluminum crimp.

[0112] In certain embodiments, the pharmaceutical preparation does not contain a preservative. In other embodiments, the pharmaceutical preparation contains a preservative. In some embodiments, the pharmaceutical preparation contains at least one amino acid, at least one vitamin and / or at least one fatty acid. In some embodiments, the pharmaceutical preparation contains at least one amino acid. In yet other embodiments, the pharmaceutical preparation contains at least one vitamin. In certain embodiments, the pharmaceutical preparation contains at least one fatty acid. In other embodiments, the pharmaceutical preparation contains a pharmaceutically acceptable carrier, diluent or excipient.

[0113] In some embodiments, the ionic strength of the pharmaceutical agent is at least 0.3 M. In certain embodiments, the ionic strength of the pharmaceutical agent is between 0.3 and 7 M. In certain specific embodiments, the ionic strength of the pharmaceutical agent is about 7 M.

[0114] In some embodiments, the pH of the pharmaceutical preparation is about 4-7.

[0115] In some embodiments, the pharmaceutical product is substantially free of microorganisms, hi some embodiments, the pharmaceutical product is substantially free of bacteria.

[0116] In some embodiments, the pharmaceutical preparation is substantially free of S. aureus, G. stearothermophilus and / or B. pumilus. -1 In certain embodiments, the pharmaceutical agent contains less than 10 CFU / mL of S. aureus, G. stearothermophilus and / or B. pumilus. -2 In certain embodiments, the pharmaceutical agent contains less than 10 CFU / mL of S. aureus, G. stearothermophilus and / or B. pumilus. -3 In certain embodiments, the pharmaceutical agent contains less than 10 CFU / mL of S. aureus, G. stearothermophilus and / or B. pumilus. -4 In certain embodiments, the pharmaceutical agent contains less than 10 CFU / mL of S. aureus, G. stearothermophilus and / or B. pumilus. -5 In certain embodiments, the pharmaceutical agent contains less than 10 CFU / mL of S. aureus, G. stearothermophilus and / or B. pumilus. -6 Contains less than CFU / mL of S. aureus, G. stearothermophilus and / or B. pumilus.

[0117] In some embodiments, the pharmaceutical preparation is substantially free of S. aureus. -1 In certain embodiments, the pharmaceutical agent contains less than 10 -2 In certain embodiments, the pharmaceutical agent contains less than 10 -3 In certain embodiments, the pharmaceutical agent contains less than 10 -4 In certain embodiments, the pharmaceutical agent contains less than 10 -5 In certain embodiments, the pharmaceutical agent contains less than 10 -6 Contains S. aureus less than CFU / mL.

[0118] In some embodiments, the pharmaceutical preparation is substantially free of G. stearothermophilus. -1 In certain embodiments, the pharmaceutical preparation contains less than 10 -2 In certain embodiments, the pharmaceutical preparation contains less than 10 -3 In certain embodiments, the pharmaceutical preparation contains less than 10 -4 In certain embodiments, the pharmaceutical preparation contains less than 10 -5 In certain embodiments, the pharmaceutical agent contains less than 10 -6 Contains less than CFU / mL of G. stearothermophilus.

[0119] In some embodiments, the pharmaceutical preparation is substantially free of B. pumilus. -1 In certain embodiments, the pharmaceutical contains less than 10 CFU / mL of B. pumilus. -2 In certain embodiments, the pharmaceutical contains less than 10 CFU / mL of B. pumilus. -3 In certain embodiments, the pharmaceutical contains less than 10 CFU / mL of B. pumilus. -4 In certain embodiments, the pharmaceutical contains less than 10 CFU / mL of B. pumilus. -5 In certain embodiments, the pharmaceutical contains less than 10 CFU / mL of B. pumilus. -6 Contains B. pumilus less than CFU / mL.

[0120] In some embodiments, the sterility assurance level of the pharmaceutical product is at least 10 -3 ~10 -6 In certain embodiments, the sterility assurance level of the pharmaceutical product is at least 10 -3 In another embodiment, the sterility assurance level of the pharmaceutical product is at least 10 -4In another embodiment, the sterility assurance level of the pharmaceutical product is at least 10 -5 In yet another embodiment, the sterility assurance level of the drug product is at least 10 -6 It is.

[0121] In some embodiments, the pharmaceutical agent is resistant to microbial growth, hi some embodiments, the pharmaceutical agent is chemically stable.

[0122] In specific embodiments, the pharmaceutical agents described herein are administered to a subject by direct injection. In other specific embodiments, the pharmaceutical agents described herein are administered to a subject by indirect injection.

[0123] In some embodiments, the present invention provides a pharmaceutical agent as described herein, wherein the pharmaceutical agent is packaged in the form of a kit comprising the pharmaceutical agent and a syringe.

[0124] In some embodiments, the present invention provides a pharmaceutical product and packaging as described herein, wherein the pharmaceutical product and packaging are sterilized by gamma irradiation. In some embodiments, the present invention provides a pharmaceutical product and packaging as described herein, wherein the pharmaceutical product is sterilized by both ionic strength and gamma irradiation, and the packaging is sterilized by gamma irradiation. In a specific embodiment, the pharmaceutical product and packaging are sterilized by gamma irradiation at least 20 kGy. In some embodiments, the pharmaceutical product and packaging are sterilized by gamma irradiation at 18-25 kGy. In some embodiments, the pharmaceutical product and packaging are sterilized by gamma irradiation at 25-33 kGy. In some embodiments, the pharmaceutical product and packaging are sterilized by gamma irradiation at 45-59 kGy.

[0125] In some embodiments, the present invention provides a method of treating a choline deficiency in a subject, comprising administering to the subject an effective amount of a pharmaceutical agent described herein.

[0126] In some embodiments, the present invention provides a method of providing parenteral support to a subject, comprising administering to the subject an effective amount of a pharmaceutical agent described herein.

[0127] In some embodiments, the present invention provides a method of providing parenteral nutrition to a subject, comprising administering to the subject an effective amount of a pharmaceutical agent described herein.

[0128] In some embodiments, the present invention provides a method of treating hepatic cholestasis in a subject, comprising administering to the subject an effective amount of a pharmaceutical agent described herein.

[0129] In some embodiments, the present invention provides a method of treating hepatic steatosis in a subject, comprising administering to the subject an effective amount of a pharmaceutical agent described herein.

[0130] In some embodiments, the present invention provides a method of treating intestinal failure associated liver disease (IFALD) in a subject, comprising administering to the subject an effective amount of a pharmaceutical agent described herein.

[0131] In some embodiments, the present invention provides a method for treating fatty liver disease in a subject, comprising administering to the subject an effective amount of a pharmaceutical agent described herein.In some embodiments, the fatty liver disease is AFL, ASH, NAFL, NASH, NASH-associated liver fibrosis or ASH-associated liver fibrosis.In some embodiments, the fatty liver disease is alcoholic fatty liver (AFL).In some embodiments, the fatty liver disease is alcoholic steatohepatitis (ASH).In some embodiments, the fatty liver disease is non-alcoholic fatty liver (NAFL).In some embodiments, the fatty liver disease is non-alcoholic steatohepatitis (NASH).In some embodiments, the fatty liver disease is NASH-associated liver fibrosis.In some embodiments, the fatty liver disease is ASH-associated liver fibrosis.

[0132] In some embodiments, the present invention provides a method for treating choline deficiency in a subject, comprising administering to the subject an effective amount of a composition or pharmaceutical preparation described herein.In some embodiments, the present invention provides a method for treating choline deficiency in a subject, comprising administering to the subject an effective amount of a composition described herein.In some embodiments, the present invention provides a method for treating choline deficiency in a subject, comprising administering to the subject an effective amount of a pharmaceutical preparation described herein.

[0133] In some embodiments, the method for treating choline deficiency in a subject further comprises providing parenteral support or parenteral nutrition to the subject.In some embodiments, the method for treating choline deficiency in a subject further comprises providing parenteral support to the subject.In some embodiments, the method for treating choline deficiency in a subject further comprises providing parenteral nutrition to the subject.

[0134] In some embodiments, the choline deficiency in the subject is associated with hepatic cholestasis or hepatic steatosis. In some embodiments, the choline deficiency in the subject is associated with hepatic cholestasis. In some embodiments, the choline deficiency in the subject is associated with hepatic steatosis. In some embodiments, the choline deficiency in the subject is associated with intestinal failure associated liver disease (IFALD). In some embodiments, the choline deficiency in the subject is associated with fatty liver disease. In some embodiments, the choline deficiency in the subject is associated with fatty liver disease, wherein the fatty liver disease is AFL, ASH, NAFL, NASH, NASH-associated liver fibrosis or ASH-associated liver fibrosis. In some embodiments, the choline deficiency in the subject is associated with fatty liver disease, wherein the fatty liver disease is AFL. In some embodiments, the choline deficiency in the subject is associated with fatty liver disease, wherein the fatty liver disease is ASH. In some embodiments, the choline deficiency in the subject is associated with fatty liver disease, wherein the fatty liver disease is NAFL. In some embodiments, the choline deficiency in subject is related to fatty liver disease, and wherein fatty liver disease is NASH.In some embodiments, the choline deficiency in subject is related to fatty liver disease, and wherein fatty liver disease is NASH-related liver fibrosis.In some embodiments, the choline deficiency in subject is related to fatty liver disease, and wherein fatty liver disease is ASH-related liver fibrosis.

[0135] In some embodiments, the present invention provides a method for synthesizing choline chloride by introducing gaseous trimethylamine into a hydrogenation unit under pressure together with 2-chloroethanol in the presence of pure ethanol and methyl-tert-butyl ether, wherein the process is a discontinuous process.

[0136] In some embodiments, choline chloride is produced with a purity of more than 99%. In some embodiments, choline chloride is produced with a purity of more than 99.5%. In some embodiments, choline chloride is produced with a purity of more than 99.8%.

[0137] In some embodiments, choline chloride is produced with substantially no detectable 2-chloroethanol. In some embodiments, choline chloride is produced with substantially no detectable aluminum. In some embodiments, choline chloride is produced with 0.05 μg / g or less aluminum. In some embodiments, choline chloride is produced with 0.1% wt:wt or less trimethylamine.

[0138] (abbreviation) The following abbreviations are used in the examples; other abbreviations have their usual meaning in the art: MTBE: Methyl tert-butyl ether WFI: Water for injection g: grams L: Liters mL: milliliter mol: mole min:minutes h or hr: hours M: mol / L or "molarity" ℃: Celsius temperature CFU: colony forming unit TNTC: Too many to count kGy: kilogray USP: United States Pharmacopoeia RH: Relative humidity VLDL: very low density lipoprotein PC: phosphatidylcholine PEMT: phosphatidylethanolamine methyltransferase DNA: Deoxyribonucleic acid MRI: Magnetic Resonance Imaging IF: Intestinal failure PN: Parenteral nutrition IFALD: Intestinal failure associated liver disease IV: Intravenous ESLD: End-stage liver disease PNALD: PN-associated liver disease NAFL: Nonalcoholic fatty liver AFL: Alcoholic fatty liver NASH: Nonalcoholic steatohepatitis ASH: Alcoholic steatohepatitis DC: Delamination control TSB: Tryptic soy broth TSA: Tryptic soy agar NLT: or above NMT: Below RRT: Relative retention time RH: Relative humidity HIAC: High accuracy LC: Liquid chromatography TS: Terminal sterilization API: Active Pharmaceutical Ingredient FTIR: Fourier transform infrared spectroscopy GC-MS: Gas chromatography mass spectrometry ICP-MS: Inductively coupled plasma mass spectrometry IC: Ion chromatography QL: Quantitation limit EXAMPLES

[0139] Embodiments of the present invention are further illustrated by the following examples, which are non-limiting and merely representative of various aspects of embodiments of the present invention.

[0140] Example 1 Preparation of a Choline Salt Composition Combine choline chloride (500 g) with WFI and dissolve the choline chloride to a total volume of 1 L to give 50% (w / v) choline chloride in WFI.

[0141] Example 2 Sterilization of Choline Salt Compositions by Ionic Strength Most unicellular organisms maintain a normal ionic strength within the cell of about 0.3M. Cells in any medium with an ionic strength higher than 0.3M will pull water from inside the bacteria until both the inside and both sides are equal. Bacteria cannot survive normally with little or no water in the cell membrane. Therefore, ionic strength can be used as a method to reduce the bacterial population in a medium and thereby sterilize the medium. Here, choline chloride 50% (w / v) in WFI has an ionic strength of about 7M, which is believed to provide a bactericidal effect. However, the ionic strength of choline chloride 50% (w / v) in WFI is not sufficient to sterilize organisms such as G. stearothermophilus, B. pumilus, and S. aureus. Therefore, other sterilization methods such as heat and gamma irradiation were considered.

[0142] Example 3 Sterilization of Choline Salt Compositions by Heat D 121 Values ​​were determined for G. stearothermophilus spores suspended in a 50% choline chloride composition. Vials containing 50% (w / v) choline chloride in WFI were dosed with 10 6 The tubes were inoculated with G. stearothermophilus spores and sealed. Microbial assays were performed at the beginning of the test (initial population = 1.3031 x 10 6 spores) and at completion (final population = 1.1813 × 10 6 These assays confirmed that the spore concentration in the vials remained stable over the duration of the study.

[0143] D of G. stearothermophilus spores suspended in a 50% choline chloride composition 121The D value was determined using spore population data from unexposed vials and vials exposed for 30.0, 60.0, 90.0 and 120.0 minutes. The vials were exposed to 121°C in a Joslyn / Steris Sterilizer Corp. steam BIER. A population assay was then performed to determine the spore population in terms of colony forming units (CFU). Four replicate units were tested in the same dilution assay at each exposure time. Therefore, the plate counts shown below are per assay (per 4 units) and the final population is shown as spores per unit. The spore population per vial corresponding to each exposure time (see Table 1, the bold numbers are used to calculate the final population following USP section 55 guidance) was used to generate a survival curve and D 121 Values ​​were calculated according to the survival curve method (see Figure 1). [Table 1]

[0144] The stability of G. stearothermophilus spores suspended in choline chloride injection in vials was demonstrated. The final population was 1.0 × 10 6 The D of G. stearothermophilus spores suspended in 50% (w / v) choline chloride in WFI was > 100 spores / vial. 121 The value was found to be 34.0 min. 121 A value of 31 minutes results in a sterilization cycle of more than 7 hours. A sterilization cycle of 7 hours may result in degradation or the formation of degradation products. Therefore, terminal sterilization with steam cannot be performed.

[0145] Example 4 Gamma Irradiation of Choline Salt Compositions Extremely high D 121 Because the time required for sterilization (approximately 34 minutes) makes terminal sterilization impractical, the feasibility of gamma irradiation for sterilization of choline salt compositions was evaluated using Choline Chloride Injection 50 (w / v) in WFI. The analytical results of the gamma irradiation feasibility study are summarized in Table 2.

[0146] Briefly, vials and glassware were heat treated to ensure sterility. The initial pH of a 50% (w / v) solution of choline chloride in WFI was measured. The spiked samples were incubated with G. stearothermophilus (10 6 spores) or B. pumilus (10 6 The vials were prepared by inoculation with either 1000 gamma radiation (0.01 mg / mL) or 1000 gamma radiation (0.01 mg / mL) of 1000 spores. The vials were irradiated with gamma radiation doses of either 25-33 kGy (kilograys) or 45-59 kGy. Control vials were not exposed to gamma radiation. [Table 2]

[0147] The results in Table 2 show that gamma irradiation doses of 45-59 kGy produce more impurities (both known and unknown) in the drug product compared to the control. Gamma irradiation doses of 25-33 kGy produce a slightly higher percentage of the known impurity triethylamine in the drug product compared to the control, but below the detection limit for the unknown impurity. Based on these results, it was determined that the optimal range for sterilization of Choline Chloride Injection in WFI 50 (w / v) is a gamma irradiation dose of 25-33 kGy.

[0148] Example 5 Sterilization of Choline Salt Compositions by Gamma Irradiation Gamma sterilization of IG Stearothermophilus / B. pumilus Gamma irradiation was tested as a sterilization method for compositions of choline chloride 50% (w / v) in WFI inoculated with either Geobacillus stearothermophilus (G. stearothermophilus) or Bacillus pumilus (B. pumilus). Vials containing the compositions were filled with 10 6Separate vials were inoculated with Bacillus pumilus (B. pumilus) in this manner. The vials were sterilized using gamma irradiation using either 25-33 kGy or 45-59 kGy. Selected vials were left unexposed and used as positive controls (see Table 4). All vials were then assayed to determine the spore population in terms of CFU (see Table 3). Additional vials were also used for validation of the population assay study (see Table 5).

[0149] Briefly, the sterility of the sterile vials was tested using a filtration method. After vortexing, the contents of each vial were removed using a sterile needle and syringe and filtered through its own filter (e.g., a 0.2 or 0.45 μm (micron) filter). The vial was then rinsed, vortexed, and the contents filtered as above, using the same needle, syringe, and filter for each vial. This process was then repeated to ensure that all residual formulation was removed from each vial. After filtration of the formulation and vial rinses, each filter was then rinsed and plated on tryptic soy agar (TSA). Test positive controls were performed for each organism type by rinsing the filters three times and spiking the last aliquot with 0.1 mL of the spore suspension used for inoculation. All plates were incubated according to organism type at 55-60°C for G. stearothermophilus and 30-35°C for B. pumilus for at least 48 hours (see Table 3). [Table 3]

[0150] The vials used for the positive control were tested for population and purity using the pour plate method utilizing a serial dilution scheme (see Figure 2). Briefly, after vortexing the vials, 1 mL of each vial (i.e., choline chloride solution and 10 5 CFU / mL, 10 4 CFU / mL, 10 3CFU / mL or 10 2 Vials containing either 0.1 CFU / mL of sucrose or 0.1% CFU / mL of sucrose were removed using a sterile needle and syringe and transferred to a test tube containing 9 mL of sterile water. The tubes were heat shocked (e.g., 95-100°C for 15 min for G. stearothermophilus and 65-75°C for 15 min for B. pumilus). After heat shock, each tube was cooled in an ice bath and vortexed. After cooling, 1 mL was transferred to a test tube containing 9 mL of sterile water and vortexed (in duplicate). 2 and 10 1 One mL of the dilution was plated into a sterile Petri dish and 18 mL of molten TSA was poured into the Petri dish and allowed to solidify. The above steps were completed for all three positive control vials of each organism type. All plates were incubated for at least 48 hours according to organism type at 55-60°C for G. stearothermophilus and 30-35°C for Bacillus pumilus. After incubation, plates were read and validated to show colonies as CFU / plate (see Table 4). [Table 4]

[0151] A positive control was performed to challenge the population assay procedure. One vial of formulation was spiked with 0.1 mL of G. stearothermophilus spore suspension using a sterile needle and syringe. This inoculation resulted in 10 6 CFU counts were obtained. This above process was repeated with the B. pumilus spore suspension. The above process for the population assay against the unexposed positive control vials was completed for both organism types. The results are shown in Table 5. [Table 5]

[0152] Vials exposed to gamma irradiation using either 25-33 kGy or 45-59 kGy showed no bacterial growth (CFU=0 for all samples). These data indicate the effectiveness of gamma irradiation as a sterilization method for the choline chloride 50% (w / v) in WFI composition. The non-exposed positive control sample showed lower populations recovered than those initially inoculated in the vial. The vials tested for validation of the population assay test had lower CFUs than expected, which may indicate that organisms in the solution may decrease over time as outcomes / conditions change. The population assays performed on the test controls showed much higher recovery than the positive control vials, indicating that the method used for inoculation was acceptable. All results indicate that from a microbiological standpoint, the choline chloride 50% (w / v) in WFI composition is an effective sterilization method for organisms such as G. stearothermophilus and B. pumilus.

[0153] II. Gamma sterilization of S. aureus Gamma irradiation was tested as a sterilization method for a composition of choline chloride 50% (w / v) in WFI inoculated with Staphylococcus aureus (S. aureus). A vial containing the composition (5 mL) was filled with 0.1 mL of S. aureus spore suspension (10 9 The vials were inoculated with spore populations in terms of CFU (see Table 7). Vials were sterilized using gamma irradiation at 25-33 kGy. Selected vials were left unexposed and used as positive controls. All vials were then assayed to determine the spore population in terms of CFU (see Table 7). Additional vials were also used for validation of the population assay study (see Table 6).

[0154] Microbial retention was determined as follows. Briefly, a stock solution of S. aureus was made by adding 10–100 CFU of the organism to 15 mL of tryptic soy broth (TSB) and incubating at 30–35°C for approximately 72 h to obtain a retention of approximately 10 9 Cultures were made of CFU / mL (estimated using McFarland standard no. 4).3 and 10 2 Duplicate dilution series of the stock culture were prepared and tested for population validation by plating the dilutions in duplicate onto TSA and incubating at 30-35°C for no more than 24 hours. 5 mL formulation vials were spiked with 0.1 mL of the prepared stock culture. Serial dilutions of the spiked formulation vials were also prepared in duplicate by adding 0.1 mL of the spiked formulation to 9.9 mL of sterile water, followed by adding 1 mL of that dilution to 9 mL of sterile water. 0.1 mL of both dilutions were plated in duplicate onto TSA and incubated at 30-35°C for no more than 24 hours. Serial dilutions of the same spiked formulation vials were tested as above at time increments of 24 hours, 72 hours, and 7 days. Microbial retention results are shown in Table 6. [Table 6]

[0155] Sterility results for S. aureus were obtained as follows. Stock cultures of Staphylococcus aureus were made by adding 10–100 CFU of the organism to 15 mL of TSB and incubating at 30–35°C for approximately 72 h. 9 Cultures were made of CFU / mL (estimated using McFarland standard no. 4). 3 and 10 2Duplicate dilution series of the stock culture were prepared and tested for population confirmation by plating the dilutions in duplicate onto TSA and incubating at 30-35°C for no more than 24 hours. Three 5 mL formulation vials were spiked with 0.1 mL of the prepared stock culture. Two additional formulation vials were spiked with 0.1 mL of the stock culture to serve as positive controls. These vials were sterilized using gamma irradiation at 25-33 kGy (positive and negative controls remained unexposed). After sterilization, the three exposed vials and the negative control were tested using a filtration method. The contents of each vial were filtered separately through a 0.45 μm MCE filter. The vials were rinsed and filtered in three portions of approximately 100 mL with the remaining rinse. The filters were plated onto TSA and incubated at 30-35°C for 5 days. Serial dilutions of the spiked formulation vials used for the positive control were made in duplicate by adding 0.1 mL of the spiked formulation to 9.9 mL of sterile water, then adding 1 mL of that dilution to 9 mL of sterile water in triplicate. 0.1 mL of the final three dilutions were plated in duplicate on TSA and incubated at 30-35°C for 5 days. The S. aureus sterility results are shown in Table 7. [Table 7]

[0156] Verification of populations of both stock culture inocula used for microbial retention and sterility test determinations confirms that the reported populations are correct. The inoculated formulation vials were able to demonstrate microbial retention over an extended period of time with minimal population reduction. Microbial retention testing demonstrated zero log reduction over a one week period, while the positive control tested in the sterility test determination demonstrated a 2-3 log reduction over a period of >5 weeks between inoculation and testing. Terminal sterilization utilizing gamma irradiation was proven effective with results of zero CFU recovery in all three exposed formulation vials. Results indicate that from a microbiological perspective, choline chloride 50% (w / v) in WFI composition is an effective method of sterilization against organisms such as S. aureus. The combination of high ionic strength and gamma irradiation was shown to be extremely effective in sterilizing choline chloride aqueous compositions.

[0157] Example 6 Forced decomposition of choline chloride Choline chloride was exposed to stress conditions as shown in Table 8. [Table 8]

[0158] Each forced degradation solution was prepared by transferring 25 mg of choline chloride to a 100 mL volumetric flask followed by exposure to the appropriate conditions. At the end of treatment, acid and base treated samples were neutralized with an equal volume of hydrochloric acid or sodium hydroxide. After equilibration to room temperature, each degradation sample was prepared according to the test method. Treated sample preparations and controls were analyzed according to the method (single injection) except that the run time was extended to account for potential late eluting degradation peaks. Mass balances were performed for each treatment.

[0159] The results of the forced degradation tests are shown in Table 9. The method was able to separate known (trimethylamine) and unknown degradation products in the presence of acid, base, oxidation, light and heat to a degree that allowed accurate quantification. Two unknown impurities (approximately 0.3% at RRT 0.75 and 0.78, respectively) were observed in the oxidatively stressed sample, and one unknown impurity (0.14% at RRT 1.42 / 1.44) was observed in the acid and base stressed sample. The chromatographic resolution between the active elution peak and the closest elution peak (if present at a level of ≥ 0.05%) was ≥ 1.2 (NLT), and the degradation peaks ≥ 0.05% were quantifiable and separated from each other and from choline chloride (target resolution NLT 1.2). The chromatography also showed acceptable mass balances for all stress conditions. Choline chloride showed good stability under all stress conditions, with no significant degradation observed even when exposed to maximum stress conditions. Thus, no significant degradation of choline chloride is expected under the conditions of sterilization by gamma irradiation described in Example 5. [Table 9]

[0160] Example 7 Stability of Choline Salt Compositions Choline chloride solutions 50% (w / v) in WFI were prepared and transferred into Type I glass tubular vials or delamination controlled (DC) vials and stored for 6 months under storage conditions of 25 °C / 60% relative humidity (RH) or 40 °C / 75% RH. Data from the 3-month stability results are summarized in Tables 10-13. The results indicate acceptable stability of the drug product at both storage conditions, i.e., 25 °C / 60% RH and 40 °C / 75% RH. [Table 10] [Table 11] [Table 12] [Table 13]

[0161] Example 8 pH Testing of Choline Salt Compositions A pH test was performed to evaluate the pH of choline chloride solutions after terminal sterilization by heating. Choline chloride solutions 50% w / v in WFI were prepared according to Example 1. Several vial types were also evaluated, including Schott Type I glass tubular vials, Schott Type I Plus glass vials, Schott delamination controlled (DC) glass vials, and Kimble Type I glass molded vials. Briefly, choline chloride solutions 50% w / v in WFI (5.5 mL) were filled into each type of vial. Two filled vials of each type were kept as controls, and the remaining vials were terminally sterilized at 121°C for 45 minutes. 10% choline chloride active pharmaceutical ingredient (API) in water is the recommended USP method for measuring the pH of choline chloride API. Therefore, a 1:5 dilution of 50% choline chloride in WFI solution was also included in the pH test at each time point. At each time point, the contents from two vials of each vial type were pooled together, respectively. 2 mL of the pooled solution was diluted with 8 mL of WFI to prepare a 1:5 dilution sample. The remaining 9 mL solution was tested as the "undiluted" (undiluted) sample. For pre-TS (terminally sterilized) samples, two holding vials of each vial type were stored at room temperature until the terminally sterilized samples were ready to be tested. All pH checks were performed simultaneously. At each time point, the pH meter was calibrated with pH standards of 4.0, 7.0 and 10.0 and the pH of the samples was then tested (see Table 14). The pH change over time for each type of vial is plotted in Figure 3 and Figure 4. Figure 3 plots the pH results for the "undiluted" sample and Figure 4 plots the pH results for a "1:5 dilution" of choline chloride solution 50% w / v in WFI before and after terminal sterilization by heating. [Table 14]

[0162] The results show that the pH of the solutions increased during the course of this study. Compared to the starting pH measurements, the solution pH in the Type I Plus vials did not fluctuate significantly and was stable for the pH measurements of the "neat" samples without dilution. The Type I tubular vials, DC and molded vials showed an initial increase in pH that then flattened out at the 4 week time point. Compared to the "neat" pH results, the 1:5 dilution results were higher for the Type I Plus and molded vials and comparable for the Type I tubular and DC vials, respectively. The pH results of the 1:5 dilution of the DC vials were stable compared to the other vial types.

[0163] Example 9. Methods of Treatment with Choline Salt Compositions The choline salt compositions described in Examples 1-7 may be used to treat choline deficiency in subjects associated with hepatic steatosis and / or cholestasis. The choline salt compositions described in Examples 1-7 may also be used to treat choline deficiency in subjects associated with intestinal failure associated liver disease (IFALD), fatty liver disease (e.g., alcoholic fatty liver (AFL), alcoholic steatohepatitis (ASH), nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), NASH-associated liver fibrosis, or ASH-associated liver fibrosis). The choline salt compositions described in Examples 1-7 may also be used as a component of parenteral support. The choline salt compositions described in Examples 1-7 may also be used as a component of parenteral nutrition.

[0164] Example 10 Synthesis of choline chloride [ka] Choline chloride was synthesized by combining gaseous trimethylamine with 2-chloroethanol under pressure in a hydrogenation apparatus in the presence of ethanol and methyl-tert-butyl ether.

[0165] Example 11 Analysis of Choline Chloride The choline chloride synthesized in Example 10 was analyzed. The results of two different batches of choline chloride are shown in Table 15. Both batches were analyzed three months after production. [Table 15-1] [Table 15-2]

[0166] The results of the choline chloride analysis indicate that both batches were produced with high purity (99.5% and 99.8%, respectively). The amount of residual chloroethanol was below the limit of quantification (QL). The amount of aluminum detected was ≤0.05 μg per gram of choline chloride.

[0167] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent documents referenced herein and / or listed in the Application Data Sheet, including but not limited to U.S. Provisional Application No. 63 / 181,858, filed April 29, 2021, are hereby incorporated by reference in their entirety. If necessary, aspects of the embodiments can be modified to provide further embodiments using concepts from various patents, applications, and publications.

[0168] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but rather to include the full scope of all possible embodiments and equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. A sterile composition for intravenous injection comprising choline chloride in an aqueous medium, the choline chloride being present in the composition at a level of 25-75% choline chloride by weight / volume.

2. 2. The composition of claim 1, wherein the composition comprises 50% choline chloride by weight / volume.

3. 2. The composition of claim 1, wherein the aqueous medium is water for injection.

4. 10. The composition of claim 1, wherein the composition is sterilized by gamma irradiation.

5. 2. The composition of claim 1, wherein the gamma irradiation is at least 20 kGy.

6. 2. The composition of claim 1, wherein the gamma irradiation is from 25 to 33 kGy.

7. The sterility assurance level of the composition is at least 10 -6 2. The composition of claim 1, wherein

8. The composition is -1 2. The composition of claim 1, comprising less than CFU / mL of S. aureus, G. stearothermophilus and / or B. pumilus.

9. The composition of claim 1 , wherein the composition is preservative-free.

10. The composition of claim 1 , wherein the composition comprises a preservative.

11. The composition of claim 1 , wherein the composition comprises at least one amino acid, at least one vitamin and / or at least one fatty acid.

12. The composition of claim 1, wherein the composition comprises a pharma- ceutically acceptable carrier, diluent or excipient.

13. The composition of claim 1, wherein the ionic strength of the composition is from 0.3 to 7M.

14. The composition of claim 1, wherein the ionic strength of the composition is about 7M.

15. 10. The composition of claim 1, wherein the pH of the composition is about 4 to 7.

16. The composition of claim 1, wherein the composition is suitable for administration by indirect or direct injection.

17. combining choline chloride with water for injection to produce a 50% (w / v) solution having an ionic strength of about 7M and a pH of about 4-7; and exposing the solution to gamma radiation to produce a choline chloride solution having an ionic strength of about 7M and a pH of about 4-7. -6 2. A method for producing a sterile choline chloride composition, comprising the step of obtaining a sterility assurance level of

18. 18. The method of claim 17, wherein the gamma irradiation is at least 20 kGy.

19. 18. The method of claim 17, wherein the gamma irradiation is from 25 to 33 kGy.

20. The following steps: i) dissolving choline chloride in water for injection to a final concentration of 40-60% choline chloride by weight / volume; ii) filtering the solution through a 0.2 μm filter; iii) transferring the solution into a glass vial; iv) sealing the vial; and v) Sterilizing pharmaceutical products using gamma irradiation A sterile aqueous choline chloride drug product manufactured by.

21. 21. A sterile aqueous choline chloride pharmaceutical preparation according to claim 20, wherein the pharmaceutical preparation comprises 50% choline chloride by weight / volume.

22. 21. The sterile aqueous choline chloride pharmaceutical preparation of claim 20, wherein the pharmaceutical preparation has been filtered through two 0.2 μm filters in series.

23. 21. The sterile aqueous choline chloride pharmaceutical preparation of claim 20, wherein the gamma irradiation is at least 20 kGy.

24. 21. The sterile aqueous choline chloride pharmaceutical preparation of claim 20, wherein the gamma irradiation is 25-33 kGy.

25. A composition according to any one of claims 1 to 16 for treating choline deficiency in a subject.

26. 26. The composition of claim 25, further comprising providing parenteral support or nutrition to the subject.

27. 26. The composition of claim 25, wherein the choline deficiency is associated with hepatic cholestasis or hepatic steatosis.

28. 26. The composition of claim 25, wherein the choline deficiency is associated with intestinal failure associated liver disease (IFALD).

29. 26. The composition of claim 25, wherein the choline deficiency is associated with fatty liver disease.

30. 30. The composition of claim 29, wherein the fatty liver disease is AFL, ASH, NAFL, NASH, NASH-associated liver fibrosis or ASH-associated liver fibrosis.

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