Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
  • A61J1/00—Containers specially adapted for medical or pharmaceutical purposes
  • A61J1/14—Details; Accessories therefor
  • A61J1/1493—Containers with shape retaining means, e.g. to support the structure of the container during emptying or filling
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/02—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using physical processes
  • A61L2/08—Radiation
  • A61L2/081—Gamma radiation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/16—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using chemical substances
  • A61L2/18—Liquid substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2103/00—Materials or objects being the target of disinfection or sterilisation
  • A61L2103/05—Living organisms or biological materials

Definitions

• the disclosure herein relates to sterile aqueous choline salt compositions, their preparation, and methods of use, particularly in the context of treating conditions associated with choline deficiency.
• Choline is an essential nutrient and common component of a normal diet, typically ingested in the form of phosphatidylcholine found in eggs, meat, nuts, and vegetables (Buchman, Gastroent. , 2009, 137: S 119-S128).
• the structure of choline comprises a quaternary amine, which acts as a methyl donor in many metabolic reactions, similar to B-vitamins and folate.
• Choline is necessary for cell membrane structure (e.g ., phospholipids), triglyceride transport via very-low-density lipoprotein (VLDL) synthesis, cholesterol transport in bile, intracellular messaging, brain development and function (e.g., acetylcholine).
• VLDL very-low-density lipoprotein
• Choline is essential for cell health and survival: hepatocytes die from apoptosis in choline-deprived medium; increased DNA damage and apoptosis has been observed in lymphocytes in choline-deficient vs normal humans, consistent with increased liver cancer rates in rodents after long-term choline depletion (Shin et al., J. Cell. Biochem., 1997; 64:196-208; and da Costa et al., Am. J. Clin. Nutr., 2006; 84:88-94).
• PC phosphatidylcholine
• VLDL phosphatidylcholine
• ER e.g. ER, glolgi, cell membrane
• Low PC levels inhibit VLDL packaging and secretion (Vance, J. E., and Vance, D. E., 1985, Can. J. Biochem. Cell Biol., 263, 12, 5898-5909).
• Exogenous choline is required to maintain adequate PC stores. Multiple PC synthesis pathways exist but only the de novo pathway adequately replenishes PC (Boyer, 2013, Compr. Physiol., 3:3).
• the de novo pathway to PC is CDP-Choline which requires plasma free choline as the root substrate and is ubiquitous in mammalian tissues (Vance, D. E., 2002, in Biochemistry of Lipids, Lipoproteins, and Membranes (Vance, D. E., and Vance, J. E., eds) pp. 205-232).
• the liver has a unique phosphatidylethanolamine methyltransferase (PEMT) activity which provides an alternative pathway for PC synthesis (Noga and Vance D.E., 2003, J. Biol. Chem ., 278, 24, 21851-21859).
• PEMT phosphatidylethanolamine methyltransferase
• PEMT itself is partly reliant 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).
• Choline deficiency resulting in diminished levels of phosphatidylcholine adversely affects multiple hepatobiliary functions and can lead to steatosis, cholestasis, and/or hepatic cell death.
• Steatosis, or fatty liver is a broad term that describes the buildup of fats in the liver. Cholestasis is a liver disease that occurs when the flow of bile from the liver is reduced or blocked. When bile flow is altered, it can lead to a buildup of bilirubin.
• PC comprises approximately 40% of bile’s organic matter (Schmitz MGJ, Renooij W., Gastroent., 1990, 99:1292-1296).
• Choline deficiency induces fragmentation of DNA in hepatocytes in culture (Albright, et ah, 1996, FASEBJ , 10, 510-516). Further, hepatocytes die via apoptosis in choline deficient media.
• Choline deficiency results in liver injury in animals and healthy adults.
• An experimental choline deficient diet caused rapid-onset liver abnormalities (e.g ., increased liver fat as shown by MRI), which were reversed by a normalized diet (Zeisel, et ak, FASEBJ , 1991, 5:2093-2098; and Fischer, et ah, Am. J. Clin. Nutr., 2007, 85: 1275-1285).
• These findings are Consistent with 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 reviewed in Buchman, Nutr. Clin. Pract ., 2003, 18:353-358).
• Intestinal failure occurs when gut function is reduced below the minimum necessary for the absorption of macronutrients and/or water and electrolytes, such that intravenous supplementation is required to maintain health and/or growth. Often due to surgical removal of bowel (short bowel syndrome) or diseased nonfunctioning bowel.
• T-l transient, usually post-operative, and fully reversible
• T-2 which is due to severe illness and requires parenteral nutrition (PN) for weeks or months
• PN parenteral nutrition
• T-3 which requires long-term PN for survival.
• the underlying etiology of T-3 stems from diseases such as cancer, Crohn’s disease, vascular disease, AIDS, radiation enteritis, and others (Bakker, H. et al., Clin. Nutr.,
• GI gastrointestinal
• IFAD intestinal failure associated liver disease
• IFALD Liver disease in PN dependent adults and children has been widely observed for decades. Now termed IFALD, it is the complication in IF patients with the greatest risk of death (Pironi et al., Clin. Nutr., 2012, 31:831-45). 47-65% of adult PN patients have chronic cholestasis (Cavicchi, et al., Ann. Intern. Med., 2000, 132:525-532; and Salvino et al., JPEN, 2006, 30:3, 202-208).
• liver disease i.e., extensive portal fibrosis or cirrhosis, bilirubin > 3.5 mg/dl for > lmonth, ascites, portal HTN, hepatic encephalopathy or factor V ⁇ 50%
• liver disease i.e., extensive portal fibrosis or cirrhosis, bilirubin > 3.5 mg/dl for > lmonth, ascites, portal HTN, hepatic encephalopathy or factor V ⁇ 50%
• PN toxicity lipids
• infectious lipids
• nutritional deficiencies including choline deficiency
• IV choline administration for IFALD has shown promise in reducing steatosis (Buchman, et al., Hepatol ., 1995, 22:1399-1403). IV choline administration in patients > 16 years old requiring > 80% PN has shown reversal of steatosis and improved cholestasis (Buchman, et al., J. Parent. Ent. Nutr., 2001, 25:260-268). Further, IV choline administration is well-tolerated in patients (Buchman, et al., Clin. Pharm. Ther., 1994, 55:277-83). Administering a nutrient solution containing a choline salt to a patient parenterally, as a method to inhibit fatty liver disease in a human patient has been described (U.S. Patent No. 5,567,736).
• Terminal sterilization is the preferred method of sterilization for injectable drug products, and takes place after the drug product has been placed into its primary packaging. Terminal sterilization of drug products that are aqueous solutions may involve heat or irradiation. Selection of an appropriate sterilization method requires an in-depth understanding of the physicochemical properties of the drug substance and the characteristics of the final formulated product.
• the gamma irradiation process uses Cobalt 60, which emits gamma rays, measured in units of kiloGrays (kGy), during radioactive decay.
• High-energy gamma radiation interacts with matter to form ion pairs by ejecting electrons, which leads to free radical formation and excitation.
• Free-radicals are highly reactive and may participate in several types of reactions including gas liberation, double-bond formation and scission, exchange reactions, electron migration or cross-linking.
• damage induced by radiation may result in biological changes that result in cell death. Although breaking the covalent bonds of bacterial DNA is considered the major route of cellular damage, membrane damage also may contribute significantly to reproductive-cell death.
• a molecule may receive energy directly from the incident radiation (the "direct effect") or, in aqueous solutions such as parenterals, by the transfer of energy from the radiolysis products of water (e.g ., hydrogen and hydroxyl radicals and the hydrated electron) to the solute molecule (the "indirect effect").
• the direct effect energy directly from the incident radiation
• aqueous solutions such as parenterals
• the present disclosure provides sterile compositions of choline salts in aqueous media, their preparation, and methods of use.
• the disclosure further provides methods of treating choline deficiency, intestinal failure associated liver disease (IFALD), and fatty liver disease.
• IFAD intestinal failure associated liver disease
• Figure 2 depicts a serial dilution scheme as employed in Example 5.
• Figure 3 plots the pH results for “As is” samples of a pH study of choline chloride solution 50% w/v in water for injection (WFI) before and after terminal sterilization by heat.
• Figure 4 plots the pH results for “1:5 dilution” samples of a pH study of choline chloride solution 50% w/v in WFI before and after terminal sterilization by heat.
• Step is defined as free from viable microorganisms.
• Secondary is defined as a validated process used to render a product free from viable microorganisms. The presence of microorganisms is expressed as a probability. While the probability can be reduced to a very low number, it can never with certainty be reduced to zero. Therefore, the term “Sterility Assurance Level (SAL)” is used as a measure of sterility. “Sterility Assurance Level (SAL)” refers to the probability of a viable microorganism being present on a product after sterilization, and is normally expressed as 10 n . SALs can also be used to describe the microbial population that was destroyed by the sterilization process, though this is not the same as the probabilistic definition.
• CFU Coldy Forming Unit
• Choline salt refers to a class of quaternary ammonium salts containing the N,N,N-trimethylethanolammonium cation and corresponding counter anion, which may be represented by the following general formula wherein X denotes the corresponding counter anion:
• Suitable counter anions include, but are not limited to, halides, such as chloride Cl-, and bitartrate ((2R,3R)-2,3,4-trihydroxy-4-oxobutanoate).
• Choline salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids.
• organic acids may be selected from aliphatic, cycloaliphatic, aromatic, aromatic aliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, hippuric, malonic, oxalic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, panthothenic, trifluoromethanesulfonic, 2-hydroxy ethanesulfonic, / oluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stea
• Aqueous media(um) is defined as a liquid comprising more than about 50% water.
• Weight/volume % is a way of expressing the concentration of a solution, wherein
• Water for Injection is a sterile, nonpyrogenic preparation of water for injection which contains no bacteriostat, antimicrobial agent, or added buffer.
• Bacteriostat refers to a substance that prevents the multiplying of bacteria without destroying them.
• Antimicrobial agent refers to a natural or synthetic substance that kills or inhibits the growth of microorganisms such as bacteria, fungi and algae.
• Preservative refers to antimicrobial ingredients that are added to compositions to help maintain the safety of the composition by inhibiting the growth of, or reducing the amount of microbial contaminants, or both.
• amino acid(s) are simple organic compound containing both a carboxyl ( — COOH) and an amino ( — NEh) group.
• amino acid(s) may refer to naturally occurring or synthetic (i.e., man-made) amino acid(s).
• “Vitamin” refers to any of a group of organic compounds which are essential for normal growth and nutrition and are required in small quantities in the diet because they cannot be synthesized by the body.
• vitamins may include, but are not limited to, vitamin A (as all-trans-retinol, all-trans-retinyl-esters, as well as all-trans- beta-carotene and other provitamin A carotenoids), vitamin B 1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B9 (folic acid or folate), vitamin B 12 (cobalamins), vitamin C (ascorbic acid), vitamin D (calciferols), vitamin E (tocopherols and tocotrienols), and vitamin K (quinones).
• vitamin A as all-trans-retinol, all-trans-retinyl-esters, as well as all-trans- beta-carotene and other provitamin A carotenoids
• vitamin B 1 thiamine
• vitamin B2 riboflavin
• vitamin B3 niacin
• “Fatty acid” refers to a carboxylic acid consisting of a hydrocarbon chain and a terminal carboxyl group, especially any of those occurring as esters in fats and oils.
• Fatty acids may include, but are not limited to, alpha-linolenic acid, linoleic acid, docosahexaenoic acid, and gamma-linolenic acid.
• zi is the charge number of that ion, and the sum is taken over all ions in solution.
• a 1:1 electrolyte such as choline chloride, where each ion is singly-charged, the ionic strength is equal to the sum of the concentration of each ion (choline +1 , chloride x ).
• Substantially free of microbes, bacteria, or specific strains of bacteria, such as Staphylococcus aureus ( S . aureus) or Geobacillus stearothermophilus ( G . stearothermophilus )” means having a low (i.e., ⁇ 1 colony -forming unit, CFU/mL) but clinically acceptable level of bacteria.
• Microbes include viruses, bacteria, archaea, fungi, plants like algae, and protozoa.
• “Resistant to microbial growth” means that the composition(s) meet the criteria set forth by, for example, the Food and Drug Administration and the U.S. Pharmacopeia for products made with aqueous bases or vehicles.
• resistant to microbial growth may mean not less than 1.0 log reduction for the initial calculated count at 7 days, not less than 3.0 log reduction at 14 days, and no increase from the 14 days' count at 28 days.
• resistant to microbial growth may mean no increase from the initial calculated count at 7, 14, and 28 days.
• decimal reduction time is the time (or dose) required, at a given condition (e.g., temperature) or set of conditions, to achieve a log reduction, that is, to kill 90% (or 1 log or more) of relevant microorganisms.
• “Chemically stable” refers to the resistance of a substance to degrade into its known or unknown degradation products.
• trimethylamine is a known degradation product of choline chloride.
• the level of trimethylamine that is acceptable can be up to 0.2%.
• Drug product means a finished dosage form, for example, tablet, capsule, solution, etc., that contains an active drug ingredient generally, but not necessarily, in association with inactive ingredients.
• a pH-adjusting agent may be added to the composition.
• the choice of a pH adjusting agent may affect the resistance to microbial challenge and/or the stability of the choline salt, as measured by the reduction in assayable degradation products.
• a pH adjusting agent may include acids such as, malic acid, citric acid, acetic acid, boric acid, lactic acid, hydrochloric acid, phosphoric acid, sulfuric acid, sulfonic acid, or nitric acid.
• a pH adjusting gent may also include bases such as, acetanilide, ammonia, calcium hydroxide, potassium bicarbonate, potassium hydroxide, sodium bicarbonate, sodium dihydrogen phosphate, sodium citrate, sodium taitrate, sodium carbonate, sodium hydroxide, thiourea, or urea. Any pH adjusting agent disclosed herein or as would be known to one of ordinary skill in the art is contemplated herein.
• composition as recited herein is synonymous with composition.
• “Pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a subject.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is not appropriate. Supplementary compatible active ingredients can be incorporated into the compositions. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the Food and Drug Administration (FDA).
• FDA Food and Drug Administration
• excipient refers to certain embodiments which are more or less inert substances added as diluents (wherein “diluent” refers to a substance used to dilute something) or vehicles, or to provide form or consistency. Excipients may also enhance resistance to microbial growth, and thus act as a preservative. Such excipients include, but are not limited to, xylitol, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, cellulose derivatives, magnesium carbonate and the like.
• an effective amount refers to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent.
• an effective amount of an agent is an amount sufficient to inhibit or treat the disease without causing substantial toxicity in the subject.
• the effective amount of an agent will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the pharmaceutical composition. Methods of determining an effective amount of the disclosed agent sufficient to achieve a desired effect in a subject will be understood by those of skill in the art in light of this disclosure.
• chronic refers to a medical disorder or condition that persists over time or is frequently recurring.
• Dosage forms can be administered once a day, or more than once a day, such as twice or thrice daily. Alternatively, dosage forms can be administered less frequently than daily, such as every other day, or weekly, if found to be advisable by a prescribing physician or drug’s prescribing information.
• Dosing regimens include, for example, dose titration to the extent necessary or useful for the indication to be treated, thus allowing the patient’s body to adapt to the treatment, to minimize or avoid unwanted side effects associated with the treatment, and/or to maximize the therapeutic effect of the treatment. Suitable dosage regimens and/or forms include those set out, for example, in the latest edition of the Physicians' Desk Reference, incorporated herein by reference.
• Direct injection refers to an intravenous injection wherein a substance is injected directly into a vein.
• an indirect injection refers to an intravenous injection wherein a substance is introduced into another source prior to injection directly into a vein.
• an indirect injection comprises introducing a substance into an IV bag, wherein the contents of the IV bag are then administered to a subject via injection into a vein as the terminal point of travel (e.g., the contents of the IV bag including, or not including, the substance to be introduced may flow through or be contained in other channels or apparatuses prior to eventual injection into the vein).
• kits refers to a combination of any number of item(s).
• a kit may comprise a composition and a syringe for injection of the composition.
• a kit may also comprise a pre-loaded syringe for injection.
• “Gamma irradiation” refers to subjecting a material to gamma radiation, wherein high-energy photons are emitted from an isotope source (e.g., Cobalt 60).
• an isotope source e.g., Cobalt 60.
• High-energy gamma radiation produces electron disruptions (ionization) in any material that it encounters. In living cells, these disruptions result in damage to the DNA and other cellular structures.
• These photon-induced changes at the molecular level may cause the death of the organism or render the organism incapable of reproduction. This effect is useful in killing bacteria, insects, or other living contaminants, which may exist in, or on, a material.
• Choline deficient refers to a clinically determined deficiency in choline. Choline is an essential nutrient for humans and is necessary for the normal function of all cells. As a critical component of the cell membrane, it ensures the structural integrity and signaling functions of the cell. Choline is also used for neurotransmission (as the metabolite, acetylcholine), is a major source of methyl donors, and is required for lipid transport from the liver. Considering these many diverse roles, choline deficiency can cause disorders in many bodily systems, including liver, muscle, and lymphocytes in humans and, additionally, the kidney, pancreas, and developing brain and nervous system in animals. Choline deficiency may be characterized as ⁇ 7 nmol free choline.
• PN Parenteral nutrition
• Parenteral support includes administration of parenteral fluids alone or in combination with parenteral nutrition solutions.
• IF Intestinal failure
• IFALD Intestinal failure associated liver disease
• IFALD refers to liver disease associated with intestinal failure, which may arise in PN-dependent subjects.
• IFALD develops in patients on long-term parenteral nutrition for chronic intestinal failure, and may be characterized by liver steatosis and/or cholestasis, and may be accompanied by one or more other signs of liver injury including but not limited to, elevated liver function test(s), fibrosis, cirrhosis, and end stage liver disease (ESLD).
• ESLD end stage liver disease
• IFALD may previously have been referred to as PN associated liver disease (PNALD).
• “Fatty liver disease” refers to a condition characterized by excessive accumulation of lipids (fat) in the liver.
• fat lipids
• the build-up of fat in the liver results in a range of clinical manifestations and progresses in stages.
• each stage can be characterized as non-alcoholic or alcoholic.
• the progression begins with simple fatty liver, or steatosis.
• This stage generally regarded as benign, is characterized by the increased appearance of fat in the liver.
• Fatty liver can be characterized as non alcoholic (NAFL) or alcoholic (AFL).
• NAFL non alcoholic
• AFL alcoholic
• the next stage of a fatty liver disease is a form of hepatitis known as steatohepatitis, characterized by further fat accumulation and liver tissue inflammation.
• Steatohepatitis can be non-alcoholic (NASH) or alcoholic (ASH). Both NASH and ASH can lead to the next stage of fatty liver disease, NASH-associated or ASH-associated fibrosis, respectively, which is 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.
• Type 1 glass refers to a borosilicate glass with good chemical resistance. Type 1 glass is used for pharmaceuticals requiring the least reactive containers. Exemplary products include, but are not limited to, glass vials, filled syringes, cartridges, and ampoules.
• “Delamination controlled (DC) glass” refers to glass that is resistant to delamination (i.e., degradation of surface glass).
• Exemplary products include, but are not limited to, glass vials, filled syringes, cartridges, and ampoules.
• “Pure ethanol” refers to 200 proof (100%) ethanol.
• Substantially no(t) detectable when referring to either 2-chloroethanol or aluminum, refers to an amount of either substance that is within 10% of the limit of detection.
• the present invention provides a sterile composition comprising a choline salt in an aqueous medium.
• the composition contains 25-75% choline salt by weight/volume %. In specific embodiments, the composition contains 50% choline salt by weight/volume %.
• the choline salt is choline chloride. In other embodiments, the choline salt is choline bitartrate.
• 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 %.
• 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 %.
• 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 %.
• the aqueous medium is water for injection.
• the composition does not contain a preservative. In other embodiments, the composition contains a preservative. In some embodiments, the composition contains at least one amino acid, at least one vitamin, and/or at least one fatty acid. In some embodiments, the composition contains at least one amino acid. In still other embodiments, the composition contains at least one vitamin. In certain embodiments, the composition contains at least one fatty acid. In other embodiments, the composition contains a pharmaceutically acceptable carrier, diluent, or excipient.
• the composition has an ionic strength of at least 0.3 Molar. In certain embodiments, the composition has an ionic strength of 0.3-7 Molar.
• the composition has an ionic strength of about 7 Molar.
• the composition has a pH of about 4-7.
• the composition is substantially free of microbes. In some embodiments, the composition is substantially free of bacteria.
• the composition is substantially free of S. aureus, G. stearothermophilus , and/or B. pumilus. In certain embodiments, the composition contains less than 10 1 CFU/mL of S. aureus, G. stearothermophilus , and/or . pumilus. In certain embodiments, the composition contains less than 10 2 CFU/mL of S. aureus, G. stearothermophilus , and/or B. pumilus. In certain embodiments, the composition contains less than 10 3 CFU/mL of S. aureus, G. stearothermophilus , and/or . pumilus.
• the composition contains less than 10 4 CFU/mL of S. aureus, G. stearothermophilus , and/or B. pumilus. In certain embodiments, the composition contains less than 10 5 CFU/mL of S. aureus, G. stearothermophilus , and/or . pumilus. In certain embodiments, the composition contains less than 10 6 CFU/mL of S. aureus, G. stearothermophilus , and/or B. pumilus.
• the composition is substantially free of S. aureus. In certain embodiments, the composition contains less than 10 1 CFU/mL of S. aureus. In certain embodiments, the composition contains less than 10 2 CFU/mL of S. aureus. In certain embodiments, the composition contains less than 10 3 CFU/mL of S. aureus. In certain embodiments, the composition contains less than 10 4 CFU/mL of S. aureus. In certain embodiments, the composition contains less than 10 5 CFU/mL of S. aureus. In certain embodiments, the composition contains less than 10 6 CFU/mL of S. aureus.
• the composition is substantially free of G. stearothermophilus. In certain embodiments, the composition contains less than 10 1 CFU/mL of G. stearothermophilus. In certain embodiments, the composition contains less than 10 2 CFU/mL of G. stearothermophilus . In certain embodiments, the composition contains less than 10 3 CFU/mL of G. stearothermophilus . In certain embodiments, the composition contains less than 10 4 CFU/mL of G. stearothermophilus. In certain embodiments, the composition contains less than 10 5 CFU/mL of G. stearothermophilus. In certain embodiments, the composition contains less than 10 6 CFU/mL of G. stearothermophilus .
• the composition is substantially free of B. pumilus. In certain embodiments, the composition contains less than 10 1 CFU/mL of B. pumilus.
• the composition contains less than 10 2 CFU/mL of B. pumilus. In certain embodiments, the composition contains less than 10 3 CFU/mL of B. pumilus. In certain embodiments, the composition contains less than 10 4 CFU/mL of B. pumilus. In certain embodiments, the composition contains less than 10 5 CFU/mL of B. pumilus. In certain embodiments, the composition contains less than 10 6 CFU/mL of B. pumilus. In some embodiments, the composition has a sterility assurance level of at least 10 3 to 10 6 . In certain embodiments, the composition has a sterility assurance level of at least 10 3 .
• the composition has a sterility assurance level of at least 10 4 . In other embodiments, the composition has a sterility assurance level of at least 10 5 . In still other embodiments, the composition has a sterility assurance level of at least 10 6 .
• 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.
• the composition is suitable for administration via indirect injection or via direct injection. In specific embodiments, the composition is suitable for administration via direct injection. In other specific embodiments, the composition is suitable for administered via indirect injection.
• the composition is resistant to microbial growth. In some embodiments, the composition is chemically stable.
• the present invention provides a method of producing a composition comprising combining a choline salt with an aqueous medium, and adjusting the concentration of the choline salt in the aqueous medium.
• the pH is also adjusted.
• the pH adjusting agent is an acid.
• the acid is 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 is a base.
• the base is acetanilide, ammonia, calcium hydroxide, potassium bicarbonate, potassium hydroxide, sodium bicarbonate, sodium dihydrogen phosphate, sodium citrate, sodium taitrate, sodium carbonate, sodium hydroxide, thiourea, or urea.
• the method comprises sterilizing the composition by the ionic strength of the composition.
• the method comprises sterilizing the composition by gamma irradiation.
• the method comprises sterilizing the composition by a combination of ionic strength and gamma irradiation.
• 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.
• the present invention provides a method of producing a composition as described herein, comprising combining choline chloride with water for injection to produce a 50%(w/v) solution, that has an ionic strength of about 7M and a pH between about 4-7, and exposing the solution to gamma irradiation to produce a sterility assurance level of at least 10 6 .
• 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.
• the present invention provides a composition as described herein, produced by a method as described herein.
• 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 further sterilizing the composition by exposure to gamma irradiation.
• the composition contains 50% choline chloride by weight/volume %.
• 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 micron filter. In some embodiments, the composition is filtered through two 0.2 micron filters in series. In some embodiments, the composition is filtered through a 0.45 micron filter. In some embodiments, the composition is filtered through two 0.45 micron filters in series. In some embodiments, the present invention provides a method of treating choline deficiency in a subject, comprising administering to the subject an effective amount of a composition as described herein.
• the present invention provides a method of providing parenteral support to a subject, comprising administering to the subject an effective amount of a composition as described herein.
• the present invention provides a method of providing parenteral nutrition to a subject, comprising administering to the subject an effective amount of a composition as described herein.
• the present invention provides a method of treating liver cholestasis in a subject, comprising administering to the subject an effective amount of a composition as described herein.
• the present invention provides a method of treating liver steatosis in a subject, comprising administering to the subject an effective amount of a composition as described herein.
• 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 as described herein.
• IAVD intestinal failure associated liver disease
• the present invention provides a method of treating a fatty liver disease in a subject, comprising administering to the subject an effective amount of a composition as described herein.
• the fatty liver disease is AFL, ASH, NAFL, NASH, NASH-associated liver fibrosis or ASH- associated liver fibrosis.
• the fatty liver disease is alcoholic fatty liver (AFL).
• the fatty liver disease is alcoholic steatohepatitis (ASH).
• the fatty liver disease is non-alcoholic fatty liver (NAFL).
• the fatty liver disease is non-alcoholic steatohepatitis (NASH).
• the fatty liver disease is NASH-associated liver fibrosis.
• the fatty liver disease is ASH-associated liver fibrosis.
• the treatment comprises administering a composition as described herein to a subject as parenteral support.
• Parenteral support includes administration of parenteral fluids alone or in combination with parenteral nutrition solutions.
• a solution comprising a composition as described herein may be provided through a Y-line administration as a stand alone when just fluid is administered.
• the treatment comprises administering a composition as described herein to a subject as part of a parenteral support solution, wherein administration occurs at least once per day or as determined by the treating physician.
• the treatment comprises administering a composition as described herein to a subject as part of a parenteral support solution, wherein administration occurs at least once per day or on a schedule to obtain normal plasma levels of choline as determined by the treating physician.
• infusion time may be several hours (e.g ., 10-14 hours), which may be administered continuously or may be broken up in one or more administration intervals.
• the treatment comprises administering a composition as described herein to a subject as part of a parenteral nutrition solution. In some embodiments, the treatment comprises administering a composition as described herein to a subject as part of a parenteral nutrition solution, wherein administration occurs at least once per day or as determined by the treating physician. In some embodiments, the treatment comprises administering a composition as described herein to a subject as part of a parenteral nutrition solution, wherein administration occurs at least once per day or on a schedule to obtain normal plasma levels of choline as determined by the treating physician. For example, infusion time may be several hours (e.g., 10-14 hours), which may be administered continuously or may be broken up in one or more administration intervals.
• the composition as described herein is administered to a subject via direct injection. In other specific embodiments, the composition as described herein, is administered to a subject via indirect injection.
• the present invention provides a choline salt composition, wherein the composition has been sterilized in liquid form by gamma irradiation of at least 25 kGy and is the irradiation product of a composition comprising a 25-75% choline salt by weight/volume % in an aqueous medium, wherein the composition has a: (a) pH of about 4-7; (b) ionic strength > 0.3 M; and (c) a sterility assurance level of at least 10 3 to 10 6 .
• the ionic strength of the composition is about 7M.
• the sterility assurance level of the composition is at least 10 6 M.
• the aqueous medium is water for injection.
• the choline salt is choline chloride.
• the composition comprises 50% choline salt by weight/volume%.
• the composition is for use in a method of treating choline deficiency in a subject comprising administering to the subject an effective amount of a composition as 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 as described herein.
• 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 as described herein.
• the composition is for use in a method of treating liver steatosis in a subject comprising administering to the subject an effective amount of a composition as described herein.
• the composition is for use in a method of treating liver cholestasis in a subject comprising administering to the subject an effective amount of a composition as described herein.
• 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 as described herein.
• IFAD intestinal failure associated liver disease
• the composition is for use in a method of treating a fatty liver disease in a subject comprising administering to the subject an effective amount of a composition as described herein.
• the fatty liver disease is AFL, ASH, NAFL, NASH, NASH-associated liver fibrosis or ASH-associated liver fibrosis.
• the present invention provides a composition as described herein, wherein the composition is packaged in the form of a vial for injection.
• the composition is packaged in a glass container.
• the composition is packaged in a Type 1 glass container.
• the composition is packaged in a delamination controlled (DC) glass container.
• DC delamination controlled
• the present invention provides a composition as described herein, wherein the composition is packaged in the form of an IV bag.
• the present invention provides a composition as described herein, wherein the composition is packaged in the form of a pre-loaded syringe.
• 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.
• the kit comprises the composition packaged in a glass container.
• the kit comprises the composition packaged in a Type 1 glass container.
• the kit comprises the composition packaged in a delamination controlled (DC) glass container.
• the kit comprises the composition packaged in an IV bag.
• the kit comprises the composition packaged as a pre- loaded syringe.
• the present invention provides a composition and packaging as described herein, wherein the composition and packaging are sterilized by gamma irradiation.
• the present invention provides a composition and packaging as described herein, wherein the composition is sterilized by both ionic strength and gamma irradiation and the packaging is sterilized by gamma irradiation.
• the composition and packaging are sterilized by gamma irradiation of at least 20 kGy. In some embodiments, the composition and packaging are sterilized by gamma irradiation of 18-25 kGy.
• the composition and packaging are sterilized by gamma irradiation of 25-33 kGy. In some embodiments, the composition and packaging are sterilized by the gamma irradiation of 45-59 kGy.
• the present invention provides a sterile, ready to use pharmaceutical composition of choline chloride comprising: a primary packaging container containing a sterile aqueous choline chloride solution having a concentration of 25-75% choline chloride by weight/volume % in an aqueous medium; and a seal sealing the primary packaging container; wherein the choline chloride solution is free of viable microbial contamination in accordance with a sterility assurance level of at least 10 3 to 10 6 .
• the aqueous choline chloride solution has a concentration of 50% choline chloride by weight/volume %. In specific embodiments, the sterility assurance level is at least 10 6 . In certain embodiments, the aqueous medium is water for injection.
• 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 still other embodiments, the primary packaging is a in an IV bag. In further embodiments, the primary packaging is a pre-loaded syringe.
• the ready to use pharmaceutical composition is sterilized by gamma irradiation. In specific embodiments, ready to use pharmaceutical composition is sterilized by gamma irradiation of at least 20 kGy. In other specific embodiments, ready to use pharmaceutical composition is sterilized by gamma irradiation of 25-33 kGy. In other specific embodiments, ready to use pharmaceutical composition is sterilized by gamma irradiation of 45-59 kGy.
• the ready to use pharmaceutical composition is suitable for administration via indirect injection. In other embodiments, the ready to use pharmaceutical is suitable for administration via direct injection. In further embodiments, the ready to use pharmaceutical is stable in the primary packaging for at least 3 months.
• the present invention provides a sterile aqueous choline chloride drug product produced by the process 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; (v) sterilizing the drug product using gamma irradiation.
• the sterile aqueous choline chloride drug product is filtered through a 0.2 micron filter.
• step (ii) the sterile aqueous choline chloride drug product is filtered through a 0.45 micron filter. In certain embodiments, in step (ii) the sterile aqueous choline chloride drug product is filtered through two micron filters in series.
• the present invention provides a sterile aqueous choline chloride drug product produced by the process 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 micron filter; (iii) transferring the solution to a glass vial; (iv) sealing the vial; (v) sterilizing the drug product using gamma irradiation.
• the sterile aqueous choline chloride drug product contains 50% choline chloride by weight/volume %. In certain embodiments, the sterile aqueous choline chloride drug product is filtered through two 0.2 micron filters in series. In certain embodiments, the sterile aqueous choline chloride drug product is filtered through two 0.45 micron filters in series. In further embodiments, the sterile aqueous choline chloride drug product is sterilized by gamma irradiation of at least 20 kGy. In further specific embodiments, the sterile aqueous choline chloride drug product is sterilized by gamma irradiation of 25-33 kGy. In further specific embodiments, the sterile aqueous choline chloride drug product is sterilized by gamma irradiation of 45- 59 kGy.
• the sterile aqueous choline chloride drug product is transferred to a vial, wherein the vial is a glass container. In some embodiments, the sterile aqueous choline chloride drug product is transferred to a vial, wherein the vial is a Type 1 glass container. In some embodiments, the sterile aqueous choline chloride drug product is transferred to a vial, wherein the vial is a DC glass container. In specific embodiments, the sterile aqueous choline chloride drug product is sealed in a vial, wherein the vial is sealed with a rubber stopper.
• the sterile aqueous choline chloride drug product is sealed in a vial, wherein the vial is further sealed with an aluminum crimp.
• the drug product does not contain a preservative.
• the drug product contains a preservative.
• the drug product contains at least one amino acid, at least one vitamin, and/or at least one fatty acid.
• the drug product contains at least one amino acid.
• the drug product contains at least one vitamin.
• the drug product contains at least one fatty acid.
• the drug product contains a pharmaceutically acceptable carrier, diluent, or excipient.
• the drug product has an ionic strength of at least 0.3 Molar. In certain embodiments, the drug product has an ionic strength of 0.3-7 Molar. In certain specific embodiments, the drug product has an ionic strength of about 7 Molar.
• the drug product has a pH of about 4-7.
• the drug product is substantially free of microbes. In some embodiments, the drug product is substantially free of bacteria.
• the drug product is substantially free of S. aureus, G. stearothermophilus , and/or B. pumilus. In certain embodiments, the drug product contains less than 10 1 CFU/mL of S. aureus, G. stearothermophilus , and/or . pumilus. In certain embodiments, the drug product contains less than 10 2 CFU/mL of S. aureus, G. stearothermophilus , and/or B. pumilus. In certain embodiments, the drug product contains less than 10 3 CFU/mL of S. aureus, G. stearothermophilus , and/or . pumilus.
• the drug product contains less than 10 4 CFU/mL of S. aureus, G. stearothermophilus , and/or B. pumilus. In certain embodiments, the drug product contains less than 10 5 CFU/mL of S. aureus, G. stearothermophilus , and/or . pumilus. In certain embodiments, the drug product contains less than 10 6 CFU/mL of S. aureus, G. stearothermophilus , and/or B. pumilus.
• the drug product is substantially free of S. aureus. In certain embodiments, the drug product contains less than 10 1 CFU/mL of S. aureus. In certain embodiments, the drug product contains less than 10 2 CFU/mL of S. aureus. In certain embodiments, the drug product contains less than 10 3 CFU/mL of S. aureus. In certain embodiments, the drug product contains less than 10 4 CFU/mL of S. aureus. In certain embodiments, the drug product contains less than 10 5 CFU/mL of S. aureus. In certain embodiments, the drug product contains less than 10 6 CFU/mL of S. aureus.
• the drug product is substantially free of G. stearothermophilus. In certain embodiments, the drug product contains less than 10 1 CFU/mL of G. stearothermophilus. In certain embodiments, the drug product contains less than 10 2 CFU/mL of G. stearothermophilus . In certain embodiments, the drug product contains less than 10 3 CFU/mL of G. stearothermophilus. In certain embodiments, the drug product contains less than 10 4 CFU/mL of G. stearothermophilus. In certain embodiments, the drug product contains less than 10 5 CFU/mL of G. stearothermophilus. In certain embodiments, the drug product contains less than 10 6 CFU/mL of G. stearothermophilus .
• the drug product is substantially free of B. pumilus. In certain embodiments, the drug product contains less than 10 1 CFU/mL of B. pumilus.
• the drug product contains less than 10 2 CFU/mL of B. pumilus. In certain embodiments, the drug product contains less than 10 3 CFU/mL of B. pumilus. In certain embodiments, the drug product contains less than 10 4 CFU/mL of B. pumilus. In certain embodiments, the drug product contains less than 10 5 CFU/mL of B. pumilus. In certain embodiments, the drug product contains less than 10 6 CFU/mL of B. pumilus.
• the drug product has a sterility assurance level of at least 10 3 to 10 6 . In certain embodiments, the drug product has a sterility assurance level of at least 10 3 . In other embodiments, the drug product has a sterility assurance level of at least 10 4 . In other embodiments, the drug product has a sterility assurance level of at least 10 5 . In still other embodiments, the drug product has a sterility assurance level of at least 10 6 .
• the drug product is resistant to microbial growth. In some embodiments, the drug product is chemically stable. In specific embodiments, the drug product as described herein, is administered to a subject via direct injection. In other specific embodiments, the drug product as described herein, is administered to a subject via indirect injection.
• the present invention provides a drug product as described herein, wherein the drug product is packaged in the form of a kit comprising the drug product and a syringe.
• the present invention provides a drug product and packaging as described herein, wherein the drug product and packaging are sterilized by gamma irradiation. In some embodiments, the present invention provides a drug product and packaging as described herein, wherein the drug product is sterilized by both ionic strength and gamma irradiation and the packaging is sterilized by gamma irradiation. In specific embodiments, the drug product and packaging are sterilized by gamma irradiation of at least 20 kGy. In some embodiments, the drug product and packaging are sterilized by gamma irradiation of 18-25 kGy.
• the drug product and packaging are sterilized by gamma irradiation of 25-33 kGy. In some embodiments, the drug product and packaging are sterilized by the gamma irradiation of 45-59 kGy.
• the present invention provides a method of treating choline deficiency in a subject, comprising administering to the subject an effective amount of a drug product as described herein.
• the present invention provides a method of providing parenteral support to a subject, comprising administering to the subject an effective amount of a drug product as described herein.
• the present invention provides a method of providing parenteral nutrition to a subject, comprising administering to the subject an effective amount of a drug product as described herein.
• the present invention provides a method of treating liver cholestasis in a subject, comprising administering to the subject an effective amount of a drug product as described herein. In some embodiments, the present invention provides a method of treating liver steatosis in a subject, comprising administering to the subject an effective amount of a drug product as described herein.
• 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 drug product as described herein.
• IAVD intestinal failure associated liver disease
• the present invention provides a method of treating a fatty liver disease in a subject, comprising administering to the subject an effective amount of a drug product as described herein.
• the fatty liver disease is AFL, ASH, NAFL, NASH, NASH-associated liver fibrosis or ASH- associated liver fibrosis.
• the fatty liver disease is alcoholic fatty liver (AFL).
• the fatty liver disease is alcoholic steatohepatitis (ASH).
• the fatty liver disease is non-alcoholic fatty liver (NAFL).
• the fatty liver disease is non-alcoholic steatohepatitis (NASH).
• the fatty liver disease is NASH-associated liver fibrosis.
• the fatty liver disease is ASH-associated liver fibrosis.
• 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 drug product as 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 as 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 drug product as described herein.
• 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 a subject. In some embodiments, the choline deficiency in a subject is associated with liver cholestasis or liver steatosis. In some embodiments, the choline deficiency in a subject is associated with liver cholestasis. In some embodiments, the choline deficiency in a subject is associated with liver steatosis.
• the choline deficiency in a subject is associated with intestinal failure associated liver disease (IFALD). In some embodiments, the choline deficiency in a subject is associated with a fatty liver disease. In some embodiments, the choline deficiency in a subject is associated with a 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 a subject is associated with a fatty liver disease, wherein the fatty liver disease is AFL.
• the choline deficiency in a subject is associated with a fatty liver disease, wherein the fatty liver disease is ASH. In some embodiments, the choline deficiency in a subject is associated with a fatty liver disease, wherein the fatty liver disease is NAFL. In some embodiments, the choline deficiency in a subject is associated with a fatty liver disease, wherein the fatty liver disease is NASH. In some embodiments, the choline deficiency in a subject is associated with a fatty liver disease, wherein the fatty liver disease is NASH-associated liver fibrosis. In some embodiments, the choline deficiency in a subject is associated with a fatty liver disease, wherein the fatty liver disease is ASH- associated liver fibrosis.
• the present invention provides a method of synthesizing choline chloride comprising introducing gaseous trimethylamine into a hydrogenator, under pressure, with 2-chloroethanol, in the presence of pure ethanol and m ethyl -/tvV- butyl ether, wherein the process is a non-continuous process.
• the choline chloride is produced in > 99% purity. In some embodiments, the choline chloride is produced in > 99.5% purity. In some embodiments, the choline chloride is produced in > 99.8% purity.
• the choline chloride is produced with substantially no detectable 2-chloroethanol. In some embodiments, the choline chloride is produced with substantially no detectable aluminum. In some embodiments, the choline chloride is produced with ⁇ 0.05 ug/g of aluminum. In some embodiments, the choline chloride is produced with ⁇ 0.1 % wt:wt of trimethylamine.
• TNTC too numerous to count
• kGy kilo Gray
• VLDL very-low-density lipoprotein
• PC phosphatidylcholine
• PEMT phosphatidylethanolamine methyltransferase
• DNA deoxyribonucleic acid
• IFALD intestinal failure associated liver disease
• IV intravenous ESLD: end stage liver disease
• PNALD PN associated liver disease
• NAFL non-alcoholic fatty liver
• AFL alcoholic fatty liver
• NASH non-alcoholic steatohepatitis
• TSA tryptic soy agar
• API active pharmaceutical ingredient
• ICP-MS inductively coupled plasma mass spectrometry
• Choline chloride (500g) is combined with WFI, the choline chloride is dissolved, and the total volume is brought up to 1 L, resulting in a choline chloride 50% (w/v) in WFI.
• ionic strength may be used as a method of reducing the population of bacteria in a medium, thereby sterilizing that medium.
• the ionic strength of the choline chloride 50% (w/v) in WFI is approximately 7M, which likely imparts bactericidal effects.
• the ionic strength of choline chloride 50% (w/v) in WFI is not sufficient to sterilize against organisms such as G. stearothermophilus , B. pumilus , and S. aureous.
• other methods of sterilization such as heat and gamma irradiation were investigated.
• stearothermophilus spores suspended in 50% choline chloride composition was determined using spore population data from unexposed vials and vials exposed for 30.0, 60.0, 90.0, and 120.0 minutes. Vials were exposed at 121 °C in a Joslyn/Steris Sterilizer Corp. steam B.I.E.R. Population assays were then performed to determine the spore populations in terms of colony forming units (CFUs). Four replicate units were tested in the same dilution assay at each exposure time. Thus, plate counts listed below are per assay (per four units), while the final population is listed as spores per one unit.
• the stability of G. stearothermophilus spores suspended in Choline Chloride Injection in vials was demonstrated. The final population was greater than l.OxlO 6 spores/vial.
• the Dm-value of G. stearothermophilus spores suspended in choline chloride 50% (w/v) in WFI was found to be 34.0 minutes. A Dm value of 34 minutes would result in a sterilization cycle of over 7 hours. A 7-hour sterilization cycle may result in degradation or formation of degradants. As such, terminal sterilization with steam is not feasible.
• Gamma irradiation was tested as a method of sterilization for a composition of choline chloride 50% (w/v) in WFI inoculated with either Geobacillus stearothermophilus (G. stearothermophilus) or Bacillus pumilus (B. pumilus). Vials containing the composition were inoculated with 0.1 mL of a G. stearothermophilus spore suspension which resulted in a population of 10 6 CFU per vial. In this manner, separate vials were inoculated with Bacillus pumilus (B. pumilus).
• Vials were sterilized using gamma irradiation using either 25-33 kGy or 45-59 kGy. Select vials were left unexposed to be used as positive controls (see, Table 4). All vials were then assayed to determine the spore populations in terms of CFUs (see, Table 3). Additional vials were also used for validation of the population assay test (see, Table 5).
• the sterilized vials were tested for sterility using a filtration method. After vortexing, the contents of each vial was removed using a sterile needle and syringe, and filtered through its own filter (e.g., a 0.2 or 0.45 pm (micron) filter). Using the same needle, syringe, and filter per vial, the vials were then rinsed, vortexed, and the contents were filtered as described above. The process was then repeated to ensure all residual product was removed from each vial. After filtration of the product and vial rinsate, each filter was then rinsed and plated to tryptic soy agar (TSA).
• TSA tryptic soy agar
• Test positive controls were performed for each organism type by rinsing a filter three times and spiking the last aliquot with 0.1 mL of the spore suspension used for inoculation. All plates were incubated according to the organism type, with G. stearothermophilus at 55-60 °C and B. pumilus at 30-35 °C for no less than 48 hours (see, Table 3). Table 3
• Vials used for positive controls were tested for population and purity using a pour plate method utilizing a serial dilution scheme (see, Figure 2).
• 1 mL of each vial i.e., vial containing choline chloride solution and either 10 5 CFU/mL, 10 4 CFU/mL, 10 3 CFU/mL, or 10 2 CFU/mL
• the test tube was heat shocked (e.g ., 95 - 100 °C for 15 minutes for G. stearothermophilus and 65 - 75 °C for 15 minutes for B.
• Vials exposed to gamma irradiation using either 25-33 kGy or 45-59 kGy showed no growth of bacteria (CFUs 0 for all samples). These data show the effectiveness of gamma irradiation as a sterilization method for choline chloride 50% (w/v) in WFI compositions.
• the unexposed positive control samples showed a lower population recovered than what was initially inoculated within the vials.
• Vials that were tested for validation of the population assay test resulted in lower CFUs than anticipated which could indicate that organisms while in solution could decrease over time with varying results/conditions.
• Microbial retention was determined as follows. In brief, a stock solution of S. aureus was made using 10-100 CFU of organism into 15 mL of tryptic soy broth (TSB) and incubated for approximately 72 hours at 30-35 °C, creating a culture of approximately 10 9 CFU/mL (approximated using a number 4 McFarland Standard). A dilutions series of the stock culture was made in duplicate to test for population verification by plating 10 3 and 10 2 dilutions in duplicate to TSA and incubated at 30-35 °C for no more than 24 hours. 5 mL product vials were spiked with 0.1 mL of the prepared stock culture.
• TSA tryptic soy broth
• Serial dilutions of the spiked product vials were also made in duplicate using 0.1 mL of the spiked product into 9.9 mL of sterile water, then 1 mL of that dilution into 9 mL of sterile water. Both dilutions were plated in duplicate using 0.1 mL to TSA and incubated at 30-35 °C for no more than 24 hours. Serial dilutions of the same spiked product vials were tested as above in time increments of 24 hours, 72 hours, and 7 days. Microbial retention results can be seen in Table 6.
• Sterility results for S. aureus were obtained as follows.
• a stock culture of Staphylococcus aureus was made using 10-100 CFU of organism into 15 mL of TSB and incubated for approximately 72 hours in 30-35 °C, creating a culture of approximately 10 9 CFU/mL (approximated using a number 4 McFarland Standard).
• a dilution series of the stock culture was made in duplicate to test for population verification by plating 10 3 and 10 2 dilutions in duplicate to TSA and incubated at 30-35 °C for no more than 24 hours.
• Three 5 mL product vials were spiked with 0.1 mL of the prepared stock culture.
• gamma irradiation is an effective sterilization method for choline chloride 50% (w/v) in WFI compositions against organisms such as S. aureus.
• the combination of high ionic strength and gamma irradiation has been shown to be very effective for sterilization of choline chloride aqueous compositions.
• Each forced degradation solution was prepared by transferring 25 mg of the choline chloride into a 100 mL volumetric flask followed by exposure to the appropriate condition. At the end of the treatment, the acid and base treated samples were neutralized with an equivalent amount of hydrochloric acid or sodium hydroxide. After equilibration to room temperature, each degradation sample was prepared according to the test method. The treated sample preparations along with the control were analyzed (single injection) according to the method, except the run time was extended to allow for potential late-eluting degradation peaks. Mass balance was examined for each treatment.
• the results of the forced degradation study are shown in Table 9.
• the method was capable of separating known (trimethylamine) and unknown degradation products in the presence of acid, base, oxidation, light, and heat to the extent that they could accurately be quantified.
• Two unknown impurities were observed in the oxidation stressed samples (RRT 0.75 and 0.78 at approximately 0.3% each) and one unknown (RRT 1.42/1.44 at 0.14%) was observed in the acid and base stressed sample.
• Chromatographic resolution between the active and the closest-eluting peak (if present at a level of > 0.05%) was not less than (NLT) 1.2 and degradation peaks > 0.05% were resolved from each other and from choline chloride to the extent that they could be quantified (target resolution NLT 1.2).
• Choline chloride solution 50% w/v in WFI was prepared according to Example 1.
• 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.
• choline chloride solution 50% w/v in WFI (5.5 mL) was filled in each type of vial. Two filled vials of each type were retained as controls and the rest of the vials were terminally sterilized at 121 °C for 45 minutes.
• Type I tubular vials, DC and Molded vials showed an initial pH drift upwards and then flattened at the 4 week time point.
• 1:5 dilution results were higher for Type I Plus and Molded vials, and comparable for Type I Tubular and DC vials respectively.
• pH results for 1 :5 dilution for DC vials were stable as compared to other vial types.
• Choline salt compositions as described in Examples 1-7 may be used for the treatment of choline deficiency in a subject related to liver steatosis and/or cholestasis. Choline salt compositions as described in Examples 1-7 may also be used for the treatment of choline deficiency in a subject related to Intestinal Failure Associated Liver Disease (IFALD), fatty liver disease (e.g ., alcoholic fatty liver (AFL), alcoholic steatohepatitis (ASH), non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), NASH-associated liver fibrosis, or ASH-associated liver fibrosis). Choline salt compositions as described in Examples 1-7 may be used as a component of parenteral support. Choline salt compositions as described in Examples 1-7 may also be used as a component of parenteral nutrition.
• IFAD Intestinal Failure Associated Liver Disease
• AFL alcoholic fatty liver
• ASH alcoholic stea
• Choline chloride was synthesized by combining gaseous trimethylamine in a hydrogenator under pressure with 2-chloroethanol in the presence of ethanol and methyl-tertiary-butyl ether.

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