Very long-chain polyunsaturated fatty acids (VLCPUFAs) for improving retinal / cognitive function and atherosclerosis

Abstract

The present invention relates to a method for treating macular degeneration, atherosclerosis, fatty liver, obesity, and cognitive ability, and more particularly to a method for treating macular degeneration, atherosclerosis, fatty liver, obesity, and cognitive ability using very long chain polyunsaturated fatty acids having 24 to 40 carbon atoms.

Description

Technical Field

[0001] The present invention relates to a method for treating macular degeneration, atherosclerosis, fatty liver, obesity, and cognitive ability, and more particularly to a method for treating macular degeneration, atherosclerosis, fatty liver, obesity, and cognitive ability using very long chain polyunsaturated fatty acids having 24 to 40 carbon atoms.

[0002] (Cross - reference to related applications) This application claims priority and the benefit of U.S. Provisional Patent Application No. 63 / 359,222, filed on July 8, 2022, which is hereby incorporated by reference in its entirety.

[0003] (Statement regarding government support) This invention was made, in part, with government support from the National Institutes of Health, U.S. Department of Health and Human Services. The U.S. government has certain rights in this invention.

Background Art

[0004] The CDC reported in 2019 that an estimated 96 million adults aged 18 and older had prediabetes. In 2018, a total of 8.25 million hospital discharges were reported among U.S. adults aged 18 and older with diabetes as some recorded diagnosis. These hospital discharges included 1.87 million for major cardiovascular diseases, including 440,000 for ischemic heart disease (17.5 per 1,000 diabetic adults). More than 46 million seniors aged 65 and older live in the United States, and it is predicted that the number will increase to nearly 90 million by 2050. The world population aged 60 and older will increase from 1 billion in 2020 to 1.4 billion by 2050.

[0005] Common health conditions associated with aging include diabetes, atherosclerosis, problems related to vision and hearing (including cataracts and age-related macular degeneration), cognitive decline, obesity, etc. According to the CDC, 50 percent of adults over 65 are prediabetic and 25 percent have diabetes. These conditions are usually caused by a number of factors including insulin resistance as a result of obesity and inactivity, decreased insulin production in the pancreas, and loss of muscle mass. People with prediabetes are more likely to develop type 2 diabetes and to have a heart attack or stroke.

[0006] Aging is also a major risk factor for the formation of clinically significant atherosclerotic lesions. Furthermore, age-related macular degeneration is the most common cause of significant loss of vision among people over 50. However, these health problems can also greatly affect the younger groups in the population.

[0007] Very long-chain fatty acids (VLCFAs) have a structurally unique long hydrocarbon chain (C 24 ~C 40 ). Although present in extremely small amounts, VLCFAs are essential lipids that play important roles in many species and organs (such as the testis, retina, brain, and sperm) and in certain biological systems that cannot be performed by more common shorter-chain C 16 ~C 18 fatty acids. Due to their very long-chain structure, some VLCFAs can spread and exist within both leaflets of the lipid bilayer, thereby conferring stability to highly curved cell membranes, such as those surrounding the nuclear pore complex. In photoreceptors, VLC-polyunsaturated FAs (VLCPUFAs) are known to be associated with rhodopsin and function in the control of the light signal transduction cascade. The absence of these VLCPUFAs appears to contribute to macular degeneration in autosomal dominant Stargardt disease macular dystrophy (STGD3).

[0008] The health benefits of diets containing fish and fish oils have received significant scientific attention since the 1970s, when epidemiological studies proposed the cardiac benefits of n-3 polyunsaturated fatty acids (PUFAs) derived from fish oil, namely eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). Reduction of n-3 PUFAs in the retina in mammalian models greatly disrupts normal nerve function and visual signal transduction pathways, leading to visual impairment, and epidemiological studies have shown that diets rich in n-3 PUFAs and fatty fish are associated with a reduced risk of developing age-related macular degeneration (AMD). However, supplementation with EPA+DHA has not shown a benefit on AMD progression in some large-scale randomized controlled trials.

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0009] Many groundbreaking drugs and treatments have been developed in medicine to help treat the above conditions. However, there continues to be a need to address these problems and find new methods and compositions for their treatment. The present invention is considered to meet that need.

MEANS FOR SOLVING THE PROBLEMS

[0010] In one aspect, the present invention is a method of treating a condition corresponding to age-related macular degeneration or reducing the risk of developing a condition corresponding to age-related macular degeneration in a subject, the method comprising administering to the subject a therapeutically effective amount of a very long chain polyunsaturated fatty acid (VLCPUFA) having 24 to 28 carbon atoms.

[0011] In another aspect, the present invention is a method of treating a disease or condition selected from hyperlipidemia, hypercholesterolemia, or hypertriglyceridemia, or a combination thereof, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a very long chain polyunsaturated fatty acid (VLCPUFA) having 24 to 40 carbon atoms.

[0012] In another aspect, the present invention relates to a method for treating hepatic steatosis, the method comprising administering to a subject in need thereof a therapeutically effective amount of a very long chain polyunsaturated fatty acid (VLCPUFA) having 24 to 40 carbon atoms.

[0013] In another aspect, the present invention relates to a method for treating an elevated plasma glucose concentration in a subject in need thereof, the method comprising administering to the subject a composition comprising an effective amount of a very long chain polyunsaturated fatty acid (VLCPUFA) having 24 to 40 carbon atoms.

[0014] In another aspect, the present invention relates to a method for treating atherosclerosis or ameliorating one or more symptoms of atherosclerosis in a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a very long chain polyunsaturated fatty acid (VLCPUFA) having 24 to 40 carbon atoms.

[0015] In another aspect, the present invention relates to a method for treating an adipofascial disorder or ameliorating one or more symptoms of an adipofascial disorder in a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a very long chain polyunsaturated fatty acid (VLCPUFA) having 24 to 40 carbon atoms.

[0016] In another aspect, the present invention relates to a method for treating cognitive impairment or ameliorating one or more symptoms of cognitive impairment in a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a very long chain polyunsaturated fatty acid (VLCPUFA) having 24 to 40 carbon atoms.

[0017] In some of the above aspects, the very long chain polyunsaturated fatty acid is preferably C24:5 n-3, C26:6 n-3 or C28:8 n-3, or a mixture thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

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Mode for Carrying Out the Invention

[0019] [Chemical Descriptions and Technical Terms] Compounds are described using their official names. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Each compound name includes the free acid or free base form of the compound, as well as the pharmaceutically acceptable salts of the compound, provided that the context does not clearly indicate a contradiction.

[0020] The terms "a" and "an" do not mean a limitation of quantity, but rather mean the presence of at least one of the referenced thing. The term "or" means "and / or". The open-ended transitional phrase "comprising" encompasses the intermediate transitional phrase "consisting essentially of" and the closed-ended phrase "consisting of". Claims that recite one of these three transitional phrases, or alternative transitional phrases such as "containing" or "including", may be recited by any other transitional phrase as long as it is not clearly excluded by the context or the illustration. The recitation of a range of values is intended to function only as a convenient method of referring individually to each separate value that falls within the range, and each separate value is incorporated into the specification as if it were individually recited herein. All endpoints of a range are included within the range and are combinable independently. All methods described herein can be performed in a suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any example and all examples, or words of illustration (such as "such as"), is merely intended to better explain the invention and does not result in a limitation of the scope of the invention unless otherwise claimed. In this specification, no word should be construed as indicating that any unclaimed component is essential to the practice of the fatty acid synthesis method or deuterated fatty acid disclosed herein. Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0021] The terms "or / and" and "and / or" mean, when referring to two elements, "either ~ or ~, or both ~ and ~", and when referring to three or more elements, "~, ~, or either ~, or any combination or all of them". As an example, the phrase "A or / and B" means "either A or B, or both A and B", and the phrase "A, B, or / and C" means "A, B, or C, or any combination or all of them".

[0022] The term "about" or "approximately" means the allowable error of a specific value determined by those skilled in the art, and depends to some extent on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within one standard deviation. In some embodiments, when no specific limit of error (e.g., standard deviation with respect to the average value given in a data chart or table) is indicated, the term "about" or "approximately" means the range encompassing the indicated value, and similarly the range included by rounding up or down to the indicated value, taking significant figures into account. In certain embodiments, the term "about" or "approximately" means within 10% or 5% of a specific value. Whenever the term "about" or "approximately" precedes the first numerical value of a series of two or more numerical values or a series of two or more numerical ranges, the term "about" or "approximately" applies to each of the numerical values in that series of numerical values or that series of numerical ranges.

[0023] A dash ("-") that is not between two characters or symbols is used to indicate the bonding position of a substituent.

[0024] "Halo" or "halogen" means fluorine, chlorine, bromine, or iodine.

[0025] The term "compound" encompasses salts, solvates, hydrates, inclusion compounds, and polymorphs of the compound or salts thereof. A "solvate" of a compound contains a stoichiometric or non-stoichiometric amount of a solvent (e.g., water, acetone, or an alcohol [e.g., ethanol]) non-covalently bound to the compound. A "hydrate" of a compound contains a stoichiometric or non-stoichiometric amount of water non-covalently bound to the compound. An "inclusion compound" of a compound contains molecules of a substance (e.g., a solvent) entrapped within the crystal structure of the compound. A "polymorph" of a compound is a crystalline form of the compound. Specific descriptions of "salts", "solvates", "hydrates", "inclusion compounds", or "polymorphs" for the compounds in certain cases of the present disclosure should not be construed as an intention to omit any of these forms in other cases of the present disclosure where the term "compound", etc. is used without description of any of these forms.

[0026] A "pharmaceutical composition" is a composition comprising at least one active agent, e.g., a VLCPUFA or a salt thereof, and at least one other substance, e.g., a carrier or an excipient. The pharmaceutical composition optionally contains one or more additional active agents. In certain cases, the pharmaceutical composition meets the US FDA's GMP standards (standards for manufacturing control and quality control) for human or non-human drugs. A "pharmaceutical composition" is a combination of at least two active agents that may be combined in a single dosage form or may be supplied together in separate dosage forms along with instructions for using the active agents together for treating a disease, e.g., hepatitis C.

[0027] "Pharmaceutically acceptable salts" include derivatives of the disclosed compounds in which the parent compound is modified by making those inorganic and organic non-toxic acid addition salts or base addition salts. The salts of the parent compounds can be synthesized from the parent compounds containing basic or acidic moieties by conventional chemical methods. Usually, such salts can be prepared by reacting these compounds in the free acid form with a stoichiometric amount of an appropriate base (e.g., hydroxides, carbonates, bicarbonates of Na, Ca, Mg, or K, etc.) or reacting these compounds in the free base form with a stoichiometric amount of an appropriate acid. Such reactions are typically carried out in water, an organic solvent, or a mixture of the two. Usually, when feasible, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is preferred. The salts of the parent compounds further include solvates and hydrates of the compounds and compound salts.

[0028] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include conventional non-toxic salts and quaternary ammonium salts of the parent compounds formed from, for example, non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts are those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc., and organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, mesylic acid, esylic acid, besylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, HOOC-(CH2) where n is 0 - 4 n -COOH, etc. A further list of suitable salts may be found, for example, in Remington’s Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pennsylvania, page 1418 (1985).

[0029] As used in the pharmaceutical composition / combination of the present invention, the term "carrier" refers to a diluent, excipient, or vehicle with which an active compound is provided.

[0030] "Pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and not otherwise undesirable, whether biologically or not, and that is useful for preparing a pharmaceutical composition / combination, and includes excipients that are acceptable for use in animals and for pharmaceutical use in humans. As used in this application, "pharmaceutically acceptable excipient" includes both one and two or more of such excipients.

[0031] A "patient" is a subject in need of medical treatment. The subject or patient can be a mammalian or non-mammalian animal, such as a pet (e.g., dog or cat) or a farm animal (e.g., cow, pig, horse, or sheep). In some embodiments, the subject or patient is a human. Medical treatment can include treatment of an existing condition (e.g., disease or disorder), prophylactic or preventative treatment, or diagnostic treatment.

[0032] As used herein, "treatment" includes, as the sole active agent or in combination with at least one additional active agent, (a) preventing the occurrence of a disease or symptoms of a disease in a patient who may be susceptible to, but has not yet been diagnosed with, the disease (e.g., age-related macular degeneration, including age-related macular degeneration in a patient having an autosomal dominant mutation associated with Stargardt disease macular dystrophy, a genetic mutation that predisposes to age-related macular degeneration), (b) arresting the disease, i.e., stopping its development, and (c) alleviating the disease, i.e., causing regression of the disease, including supplying a therapeutically effective amount of a VLCPUFA or a salt thereof. "Treating" and "treatment" also include supplying a therapeutically effective amount of a VLCPUFA or a salt thereof, as the sole active agent or in combination with at least one additional active agent, to a patient who is or is likely to become in a state in which a very long chain fatty acid is known to function.

[0033] To "prevent" a disease or disorder means to bring about a statistically significant decrease in the likelihood of developing the disease or disorder, or a statistically significant delay in the onset of symptoms, or a reduction in the severity of symptoms, in a patient at risk of developing the disease or disorder.

[0034] The "therapeutically effective amount" of the pharmaceutical composition / combination of the present invention means an amount effective to provide therapeutic utility, such as improvement of symptoms, for example an amount effective to reduce symptoms, when administered to a patient.

[0035] As used herein, the term "administer" means to give a pharmaceutical or a pharmaceutical composition to a subject and includes, but is not limited to, administration by a medical professional and self-administration.

[0036] The fatty acids discussed herein are defined using the following conventional numbering system.

Chemical formula

[0037] In different aspects, methods of treatment using compositions of very long chain polyunsaturated fatty acids (VLCPUFAs) for treating or ameliorating various clinical conditions including atherosclerosis, cognitive dysfunction, elevated plasma glucose concentration, age-related macular degeneration, and other conditions are described.

[0038] In one aspect, very long-chain polyunsaturated fatty acids (VLCPUFAs) have a carbon chain length between 24 and 40. In one aspect, very long-chain polyunsaturated fatty acids (VLCPUFAs) have one or more double bonds in a cis or trans configuration. In one aspect, the fatty acids described herein are defined by a letter-number designation, such as C24:5 n-3. The letter "C" means carbon, the number before the colon specifies the number of carbon atoms, and the number after the colon specifies the number of double bonds. The position of the terminal double bond is indicated in the form of n-x, where n represents the locant of the methyl terminus of the molecule, and x refers to the locant of the double bond closest to the methyl terminus of the molecule, or the number of carbon atoms from the methyl terminus of the molecule to the double bond closest thereto. [Chemical Formula]

[0039] [Pharmaceutical Compositions and Methods of Treatment] Provided herein are pharmaceutical compositions comprising at least one very long-chain polyunsaturated fatty acid (VLCPUFA) described herein, together with a pharmaceutically acceptable carrier or excipient, for use in treating the medical conditions described herein. The pharmaceutical composition can comprise an individual VLCPUFA or a mixture of VLCPUFAs. Similarly, the medical conditions described herein can be treated by administering an individual VLCPUFA or a mixture of VLCPUFAs. In some embodiments, the mixture of VLCPUFAs is a fish oil-derived VLCPUFA concentrated oil that can also contain a mixture of VLCPUFAs derived from fish oil, such as PUFAs having less than 24 carbon atoms. In further embodiments, the VLCPUFA is C24:5 n-3, C26:6 n-3, or C28:8 n-3, or any combination or all of them.

[0040] Furthermore, the pharmaceutical composition can include one or more polyunsaturated fatty acids (PUFAs) having less than 24 carbon atoms, such as 20 or 22 carbon atoms, and the medical conditions described herein can be treated by administering them. In some embodiments, the one or more PUFAs are a mixture of PUFAs derived from fish oil. In further embodiments, the one or more PUFAs are C20:2 n-6, C20:4 n-6, C22:4 n-6, or C22:6 n-3, or any combination or all thereof.

[0041] The pharmaceutical composition can have any suitable form and can be a tablet, capsule, lyophilized solid, solution, suspension, or a combination thereof. The pharmaceutical composition can be, for example, injectable (e.g., intravenous, subcutaneous, or intramuscular), topical (e.g., ocular or transdermal), or an oral dosage form. The pharmaceutical composition can be a dosage form intended for parenteral (e.g., intravenous, subcutaneous, or intramuscular) administration, such as a solution pre-prepared in a vial or syringe, a lyophilized solid that requires reconstitution prior to administration, or a reconstituted solution of a lyophilized solid. The pharmaceutical composition can be an ophthalmic formulation, such as a liquid topical formulation. The pharmaceutical composition can be, for example, an oral dosage form in the form of a tablet or capsule. In a preferred embodiment, the VLCFA is formulated in any oral dosage form including solids, semi-solids, liquids, powders, sachets, etc. Solid oral dosage forms can include, for example, tablets, (hard or soft) capsules, or sub-units, etc. A "sub-unit" includes mini-tablets, beads, spheroids, microspheres, seeds, pills, caplets, microcapsules, granules, particles, etc., that can provide an oral dosage form alone or in combination with other sub-units. Typical semi-solid or liquid dosage forms include suspensions, solutions, emulsions, etc. Solid oral dosage forms can also include an orally dissolving / disintegrating dosage (ODT) form. Typical ODTs include orally dissolving / disintegrating tablets, orally dissolving films, and dosage forms intended for sublingual / lingual / buccal delivery, such as fast-dissolving / disintegrating sublingual tablets and films.

[0042] Oral dosage forms used in methods and compositions can be formulated for a particular type of release, including short-acting, controlled-release, sustained-release, or extended-release.

[0043] The present disclosure relates to methods and compositions in which one or more compounds are either a mixture or alternatively are combined with one or more compounds, in the presence or absence of commonly used excipients (or "pharmaceutically acceptable carriers"), such as, but not limited to, i) diluents and carriers such as starch, mannitol, lactose, dextrose, sucrose, sorbitol, cellulose, etc., ii) binders such as starch paste, gelatin, magnesium aluminum silicate, methylcellulose, alginates, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone, etc., iii) lubricants such as stearic acid, talc, silica, polyethylene glycol, polypropylene glycol, etc., iv) adsorbents, colorants, sweeteners, etc., v) disintegrants (such as calcium carbonate and sodium bicarbonate), such as foaming mixtures, etc., vi) excipients (such as cyclodextrin, etc.), vii) surfactants (such as cetyl alcohol and glycerol monostearate), adsorptive carriers (such as kaolin and bentonite), emulsifiers, etc. Examples of carriers include any liquid, liquid crystal, solid, and semi-solid suitable for use in the composition, such as water and physiological saline, gels, creams, ointments, solvents, diluents, fluid ointment bases, ointments, pastes, implants, liposomes, micelles, macromicelles, etc., but are not limited thereto. The disclosure of this paragraph also applies to pharmaceutical compositions comprising one or more VLCPUFAs, optionally in combination with one or more additional therapeutic agents.

[0044] The present disclosure includes ophthalmic compositions. The disclosed compositions can be emulsions, solutions, suspensions, gels, ointments, sealed films, or sustained-release films, and they can be either preservative formulations or non-preservative formulations. The compositions can be formulated as eye drops, creams, ointments, and films that can be applied to the eye. The formulations can be administered to the eye, upper eyelid, lower eyelid, or combinations thereof.

[0045] The ophthalmic composition can include a polymeric emulsifier such as castor oil, squalene, isostearate, and isopropyl myristate; a lipophilic component such as mineral oil, silicone oil, and glyceryl tricaprylate / caprate; and / or an alcohol such as cetyl alcohol and stearyl alcohol. The ophthalmic composition of the present disclosure can also include diethylene glycol monoethyl ether, propylene glycol, and / or dipropylene glycol; a co-solvent such as dimethyl ether, diethylene glycol, and dipropylene glycol; and / or a buffer and a pH adjuster such as sodium citrate dihydrate, boric acid, monosodium phosphate, dibasic heptahydrate, and monobasic sodium phosphate monohydrate. The ophthalmic composition can include cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxyethyl cellulose, PEG300, PEG400, povidone, glycerin, propylene glycol, or hydroxypropylmethyl cellulose, or any combination thereof. Suitable preservatives include, for example, benzalkonium chloride. The ophthalmic composition can include a plasticizer or a film-forming agent. The ophthalmic composition typically includes a vehicle such as water or an isotonic solvent system (e.g., phosphate buffered saline), and the active compound (e.g., VLCPUFA) has a concentration of about 0.01 - 90%, 0.1 - 50%, 0.1 - 30%, 0.5 - 20%, or 1 - 10% wt / vol.

[0046] The present disclosure includes methods using conventional mixing, granulation, or coating methods and compositions prepared using these, which may contain 0.01 - 90% of the active ingredient. The resulting composition (formulation) may be presented in unit dosage form and may be prepared by methods known in the pharmaceutical art. All methods include the act of associating the active ingredient with a carrier that constitutes one or more components. Thus, the composition (formulation) may be prepared by mixing the active ingredient with, for example, a liquid carrier and / or a micronized solid carrier, and then shaping the product into the desired formulation, if necessary.

[0047] Certain compounds and methods of the present disclosure comprise from about 90 wt% to about 80 wt%, from about 80 wt% to about 70 wt%, from about 70 wt% to about 60 wt%, from about 60 wt% to about 50 wt%, from about 50 wt% to about 40 wt%, from about 40 wt% to about 30 wt%, from about 30 wt% to 20 wt%, from about 20 wt% to about 10 wt%, from about 10 wt% to about 4 wt%, from about 4 wt% to about 2 wt%, from about 2 wt% to about 1 wt%, or from about 1 wt% to about 0.01 wt% of a compound described herein (e.g., VLCPUFA).

[0048] Exposure to light, oxygen, or heat can cause oxidation of VLCPUFA and can be prevented or minimized by methods known in the art. For example, storage of a sealed or capped container or pharmaceutical composition containing VLCPUFA in the dark and / or at low temperature (e.g., in a refrigerator) minimizes oxidation.

[0049] The therapeutically effective amount and frequency of administration, and treatment duration of a compound of the present disclosure (e.g., VLCPUFA) for treating a medical condition described herein may depend on various factors including the severity of the condition, the efficacy of the compound, the route of administration, the age, weight, general health, sex, and diet of the subject, and the response of the subject to the treatment, and may be determined by the physician treating the subject.

[0050] In some embodiments, the effective daily dose of the compounds of the present disclosure (e.g., VLCPUFA), whether in the form of an oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), or topical (e.g., ocular or transdermal) composition, is about 0.1 to 100 milligrams (mg) per kilogram (kg) of the subject's body weight, which may be administered as a single dose or in divided doses (e.g., administered 2, 3, or 4 times a day to provide the total daily dose). In certain embodiments, the daily dose of the compounds of the present disclosure (e.g., VLCPUFA) is about 0.0001 mg / kg to 0.1 mg / kg (e.g., for diagnostic observation), about 0.1 mg / kg to 2 mg / kg, or about 2 mg / kg to 5 mg / kg. In other embodiments, the daily dose of the compounds of the present disclosure (e.g., VLCPUFA) is about 5 mg / kg to 10 mg / kg, about 10 mg / kg to 20 mg / kg, about 20 mg / kg to 30 mg / kg, about 30 mg / kg to 40 mg / kg, about 40 mg / kg to 50 mg / kg, about 50 mg / kg to 75 mg / kg, or about 75 mg / kg to 100 mg / kg. In further embodiments, the effective daily dose of the compounds of the present disclosure (e.g., VLCPUFA), whether in the form of an oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), or topical (e.g., ocular or transdermal) composition, is about 10 mg to about 3 g, about 50 mg to about 2 g, or about 100 mg to about 2 g, or about 100 to 500 mg, about 500 to 1000 mg, about 1 to 1.5 g, or about 1.5 to 2 g, which may be administered as a single dose or in divided doses (e.g., administered 2, 3, or 4 times a day to provide the total daily dose).

[0051] The dosing frequency of the compounds of the present disclosure (e.g., VLCPUFA) can be, for example, more than once a day (e.g., once, twice, three times, four or more times a day), once every two days or three times a week, once every three days or twice a week, once a week, every other week, or once a month. For example, an ophthalmic solution containing a compound of the present disclosure (e.g., VLCPUFA) can be administered by eye drops once, twice, three times, four or more times a day, where each administration applies a solution of 1, 2, 3, 4 or more drops per eye. A decrease in dosing frequency (e.g., once a week, every other week, or once a month) can be achieved by, for example, controlling the release or sustained release of the compound by micelles or polymeric nanoparticles, or by sustained-release tablets or capsules.

[0052] Treatment with the compounds of the present disclosure (e.g., VLCPUFA) can continue, for example, until resolution of the condition being treated, such as substantial alleviation or elimination of one or more symptoms, complications, or causes of the condition. In some embodiments, symptoms or complications of a condition are substantially alleviated when their severity, frequency, or duration is reduced by at least about 30%, 50%, 75%, or 90%. Thus, the length of treatment with the compounds of the present disclosure (e.g., VLCPUFA) can be, for example, at least about 1, 2, 3, 4, 5 or 6 weeks, or at least about 1, 2, 3, 4, 5 or 6 months, or at least about 1, 2, 3, 4, 5 or 10 years.

[0053] The compounds of the present disclosure (e.g., VLCPUFA) can be administered by any suitable route for treating a medical condition. Routes of administration include, but are not limited to, oral, parenteral (e.g., intravenous, subcutaneous, subdermal, intradermal, intramuscular, administration into the lumen or parenchyma of an organ, intraperitoneal, administration into a body cavity, intrauterine, and topical), topical (e.g., ocular, transdermal, buccal, sublingual, intranasal, intrapulmonary, anal / rectal, vaginal), and surgical administration. Topical administration of the active agent (e.g., VLCPUFA) may deliver the active agent to the diseased tissue with minimal systemic distribution. The compounds of the present disclosure (e.g., VLCPUFA) can be administered by any suitable means. Means of administration include, but are not limited to, tablets, capsules, powders, injections, implants, oral or nasal inhalants, transdermal delivery devices (e.g., patches), suppositories (e.g., rectal and vaginal suppositories), solutions, suspensions, emulsions, creams, gels, ointments, pastes, sprays, aerosols, particles, nanoparticles, microparticles, microspheres, and liposomes.

[0054] The present disclosure further provides a kit or package comprising a pharmaceutical composition comprising a compound of the present disclosure (e.g., VLCPUFA) and instructions for administering and using the compound to treat a medical condition. The composition can be, for example, a solid oral dosage form, such as a tablet or capsule, a sterile solution for parenteral (e.g., intravenous, subcutaneous, or intramuscular) administration supplied in a pre-filled syringe, an ophthalmic solution supplied in an eye drop bottle, or a composition supplied in a transdermal patch.

[0055] Instead of being in the form of a carboxylic acid or salt of a VLCFA (e.g., VLCPUFA), the VLCFA (e.g., VLCPUFA) can be in the form of an ester that can function as a prodrug. Esterases present in blood or other body fluids, tissues and cells can rapidly hydrolyze the ester of the VLCFA (e.g., VLCPUFA) to its carboxylic acid or salt. Thus, a pharmaceutical composition can include an ester of a VLCFA (e.g., VLCPUFA) and a pharmaceutically acceptable excipient or carrier, and the ester of the VLCFA (e.g., VLCPUFA) can be used to treat any medical condition described herein. Thus, the entire disclosure of this application also applies to esters of VLCFA (e.g., VLCPUFA). When the ester of VLCFA (e.g., VLCPUFA) has the general formula RC(=O)OR’, in some embodiments, R’ is C1-C6 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, or n-hexyl.

[0056] [Therapeutic Use] Age-related macular degeneration (AMD) affects the central vision, thereby affecting the ability to see details. In AMD, a part of the retina called the macula is damaged. Dry AMD is associated with the slowly decaying photoreceptor cells of the macula. Wet AMD is associated with the growth of abnormal blood vessels under the retina. The blood vessels can exude fluid and blood (thus the name wet AMD), and can create a large blind spot in the center of the visual field.

[0057] The aging retina has a tendency to develop degenerative diseases, such as AMD. Annular (curvature) changes in the cornea cause refractive changes in the elderly, usually from "straight" astigmatism to "inverse" astigmatism. Other observations related to aging include increased Descemet's membrane thickness, cornea Farinata, white limbus girdle, mosaic degeneration, deep crocodile shagreen, Hassall-Henle bodies, arcus senilis, etc.

[0058] Hyperlipidemia is associated with high blood levels of lipids (or fats), such as cholesterol and triglycerides. Hypercholesterolemia, a type of hyperlipidemia, is associated with high blood levels of non-HDL cholesterol and LDL ("bad") cholesterol. Hypertriglyceridemia is a condition in which blood triglyceride concentrations are elevated. It is associated with or exacerbated by uncontrolled diabetes, obesity, and other conditions.

[0059] Hepatic steatosis is defined by at least 5% liver fat of the liver weight. Simple accumulation of triacylglycerol in the liver can be hepatoprotective. However, long-term liver lipid storage can cause liver metabolic dysfunction, inflammation, and progressive non-alcoholic fatty liver disease (NAFLD). Non-alcoholic hepatic steatosis is associated with obesity, type 2 diabetes, and dyslipidemia.

[0060] Hyperglycemia, or high blood glucose, occurs when there is too much sugar in the blood. This can occur, for example, due to low levels of insulin (a hormone that promotes the uptake of glucose from the blood into the liver, fat, and skeletal muscle cells), or when insulin non-sensitivity / resistance occurs. Insulin resistance is the case where the liver, fat, and skeletal muscle cells do not respond sufficiently to insulin and cannot use the glucose in the blood for energy. Hyperglycemia is most often associated with diabetes. Broadly, hyperglycemia is associated with blood glucose greater than 125 mg / dL (milligrams per deciliter) when fasting (not having eaten for at least 8 hours), and a person with fasting blood glucose greater than 125 mg / dL has diabetes. In one aspect, a human subject has impaired glucose tolerance, or prediabetes, for example, by fasting blood glucose of 100 mg / dL to 125 mg / dL.

[0061] In one aspect, a human subject is hyperglycemic, for example, when blood glucose is greater than 180 mg / dL one to two hours after a meal. "After a meal" means after eating. Postprandial hyperglycemia is characterized by a rapid increase to hyperglycemic levels that induces oxidative stress.

[0062] Metabolic syndrome can be defined as a cluster of conditions that occur together and increase the risk of heart disease, stroke, and type 2 diabetes. These conditions include elevated blood pressure, hyperglycemia, excess abdominal body fat, and abnormal cholesterol or triglyceride levels.

[0063] Obesity is a complex medical condition involving an excessive amount of body fat. It is a medical condition that increases the risk of other diseases and health problems, such as heart disease, diabetes, high blood pressure, and certain cancers.

[0064] Diseases of the subcutaneous adipose tissue involving adipose tissue and its fascia are also known as adipose fascia diseases and exhibit variations within the spectrum of obesity. Adipose fascia diseases can be either local or systemic and include the common disease lipedema, which mainly affects women, as well as four rare diseases: familial multiple lipomatosis, angiolipomatosis, painful fat syndrome, and multiple symmetric lipomatosis. In adipose fascia diseases, fat is difficult to reduce by standard weight loss approaches including lifestyle (diet and exercise), pharmacotherapy, and bariatric surgery, at least in part due to fibrosis of the tissue.

[0065] Atherosclerosis is the thickening or hardening of arteries caused by the buildup of plaques on the inside of arteries. Plaque deposits contain fatty substances, cholesterol, cellular waste products, calcium, and fibrin. The buildup of plaque thickens and stiffens the artery wall. Atherosclerosis is a slow, progressive disease that may start as early as childhood, but may also progress rapidly.

[0066] Cognitive health or function is the ability to think clearly, learn, and remember, and is an important component in performing daily activities. Cognitive impairment is associated with problems in remembering, learning new things, concentrating, or making decisions that affect daily life. Cognitive impairment ranges from mild to severe. At the mild level, the subject may begin to notice changes in cognitive function but can still perform daily activities. Severe-level impairment can cause loss of the ability to understand the meaning or importance of things and the ability to speak or write, and can lead to an inability to live independently.

[0067] Peroxisome proliferator-activated receptors (PPARs) are transcription factors activated by ligands of the nuclear hormone receptor superfamily, including the subtypes PPARα, PPARγ, and PPARβ / δ. They are involved in the control of glucose and lipid homeostasis, inflammation, proliferation, and differentiation. VLCPUFAs can function as agonists or alternatively activate the signaling pathways of PPARs, such as PPARα and PPARγ.

[0068] The present disclosure provides a method for preventing or treating a medical condition responsive to treatment with very long-chain fatty acids (VLCFAs, such as VLCPUFAs) in a patient, the method comprising administering to the patient a VLCFA (such as VLCPUFA) or a salt thereof. Medical conditions responsive to treatment with a VLCFA (such as non-deuterated or deuterated VLCPUFA) or a salt thereof include, but are not limited to, retinal dysfunction, macular degeneration (such as AMD), hyperlipidemia (such as hypercholesterolemia and hypertriglyceridemia), obesity, overweight, adipose fascia disease, hepatic steatosis, fatty liver disease (such as NAFLD), hyperglycemia (such as fasting hyperglycemia and postprandial hyperglycemia), glucose intolerance, insulin resistance, prediabetes, diabetes (such as type 1 and type 2 diabetes), metabolic syndrome, cardiovascular disease (such as coronary artery disease and atherosclerosis), hypertension, and cognitive impairment.

Examples

[0069] [Animals and Experimental Design] All experiments were conducted in accordance with the guidelines provided by the NIH Guide for the Care and Use of Laboratory Animals. Ultra-long-chain polyunsaturated fatty acid (VLCPUFA)-enriched oil derived from fish oil rich in PUFAs with a carbon chain length longer than C22 was manufactured by Nippon Suisan Kaisha, Ltd. VLCPUFA oil rich in C24:5 n-3, C26:6 n-3, and C28:8 n-3 was produced from sardine oil. In the first step, cholesterol and environmental contaminants (e.g., dioxins) were removed from sardine oil by short-path distillation. In a further reaction step, ethyl esters were obtained in transesterification with sodium ethoxide, and the fish oil was bleached by treating the oil with bleaching earth (percent yield in the manufacturing process: 100%). The obtained fish oil ethyl esters were purified by molecular distillation to remove fatty acids with a carbon chain length of less than 20 (percent yield in the manufacturing process: 18.3 - 19.7%). The ethyl esters were further purified by thin-film distillation to remove as much as possible fatty acid ethyl esters with a carbon chain length of 22, polymers, and diacylglycerol (percent yield in the manufacturing process: 7%). The ethyl ester oil was further purified by high-performance liquid chromatography (HPLC) using methanol as the mobile phase to remove further fatty acid ethyl esters with a carbon chain length of 22 (percent yield in the manufacturing process: 0.6%). After the final silica gel purification method, peroxides were removed to obtain the VLCPUFA-enriched oil (percent yield in the manufacturing process: 94.4%). The levels of the main fatty acids in the VLCPUFA-enriched oil are shown in Table 1.

[0070] The mouse experiments were approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute and at the University of Utah. Black C57BL / 6J mice were obtained from Taconic Biosciences (Germantown, New York), and ApoE-KO mice were obtained from Jackson Lab (Bar Harbor, Maine). The animals were housed under a 12 / 12 light / dark cycle and had free access to food and water. Twenty-four 7-month-old C57BL / 6J mice were fed for 8 weeks with either a diet AIN-93G (Harlan Teklad) supplemented with 1% (w / w) VLCPUFA oil, 5% (w / w) VLCPUFA oil, or not supplemented (control) (n = 8 per group, 4 males and 4 females). In a separate study, twenty-four 9-month-old ApoE-KO mice were fed for 8 weeks with either a diet AIN-93G (Harlan Teklad) supplemented with 1% (w / w) VLCPUFA oil, 3% (w / w) VLCPUFA oil, or not supplemented (control) (n = 8 per group, 4 males and 4 females), and age-matched C57BL / 6J mice were used as wild-type controls. Body weight was observed every 2 - 3 weeks in 5-hour fasted animals. After 8 weeks of diet consumption, the mice were euthanized by intraperitoneal injection of tribromoethanol (Avertin). Blood samples were collected from the retroorbital plexus, and plasma was separated by centrifugation. Tissue samples were snap-frozen and stored at -80 °C until further use. Additional aliquots of liver, brain, kidney, spleen, heart, skeletal muscle, small intestine, and testis (males) were fixed in 10% formalin. In a single-dose forced oral administration study, 25 3-month-old male mice were force-fed 100 μL of VLCPUFA oil prepared with a liposome kit (Sigma-Aldrich). α-Tocopherol (0.025%) was added to prevent oxidation of VLCPUFA. A dose of 6 mg / day / mouse (250 mg / kg body weight) of VLCPUFA was used, which is comparable to the dose of purified C32:6 n-3 administered in a previous study [Gorusupudi et al., Proc. Natl. Acad. Sci. USA, 118(6):e2017739118 (2021)]. The animals were euthanized at 0, 2, 4, 8, and 24 hours (n = 5 at each time point), and the retina was dissected from the enucleated eyes under a microscope.Lipids were extracted from a pair of retinas / mice for further GC-MS analysis as previously described [Liu et al., J. Lipid Res., 51(11):3217-3229 (2010)]. In the repeated-dose forced oral administration experiment, 3-month-old male mice were randomly divided into two following treatment groups: VLCPUFA-enriched oil (n = 6) and vehicle (control) (n = 6). Liposomes (control) and VLCPUFA oil (80 mg / [kg·d]) were administered to the mice once a day by forced oral administration. After a 15-day experimental period, 6-hour fasted mice were sacrificed to collect eye tissues, and the retinas and retinal pigment epithelium (RPE) were separated for GC-MS analysis.

[0071] Half maximal effective concentration (EC 50 ) refers to the concentration of a drug, antibody, or toxic substance that induces a response midway between the baseline and the maximum after a specific exposure time.

[0072] [Electroretinogram and visual behavior tests] The electroretinogram (ERG) and visual behavior tests were used to evaluate the retinal function of mice after 15 consecutive days of forced oral administration of VLCPUFA oil as previously described [Gorusupudi et al., Proc. Natl. Acad. Sci. USA, 118(6):e2017739118 (2021)]. Briefly, mice were dark adapted overnight before ERG recording, and the procedure was performed in a light-shielded room using a dim red light. Anesthesia and mydriasis were induced, and the mice were placed on a thermostatically controlled heating pad, and a gold ERG electrode was placed in the center of the cornea. Stimulus-response functions were obtained under dark-adapted and then light-adapted conditions, and the amplitudes of ERG a-waves and b-waves at multiple flash luminances were measured and analyzed. Furthermore, at the end of 15 days of repeated forced oral administration of VLCPUFA oil or vehicle, a optomotor behavior test was performed to analyze the visual function of the mice. Visual acuity was measured using a valid non-invasive method, the optomotor test system (OptoMotry from Cerebral Mechanics). Briefly, the tracking response (optomotor reflex) to a rotating visual stimulus displayed on an LCD panel around the mouse was recorded, and visual acuity was measured at 100% contrast.

[0073] [Biochemical assay] Plasma triglyceride (TG), cholesterol, glucose, bilirubin, chloride, calcium, magnesium, creatinine, alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), potassium, albumin, total creatine kinase, lactate dihydrogen, total protein, amylase, inorganic phosphorus, urea nitrogen, and uric acid were measured using an ILab 600 autoanalyzer, enzyme-based kits, and quality control. Plasma insulin (R&D Systems, Minneapolis, Minnesota) and adiponectin (R&D Systems) were determined by enzyme-linked immunosorbent assay (ELISA) kits. Total liver lipids were extracted with a mixture of chloroform and methanol (2:1) as previously described [Folch et al., J. Biol. Chem., 226:497-509 (1957)], and the contents of TG and total cholesterol were measured by colorimetric analysis (Wako Chemicals, Richmond, Virginia).

[0074] [High-performance protein liquid chromatography (FPLC) profile analysis] Pooled plasma (150 μL) from each feed group (n = 8) was injected into a Superose FPLC column (GE Healthcare). Lipoproteins were eluted with elution buffer, and 0.5 mL fractions were collected at a flow rate of 0.5 mL / min. Total cholesterol levels and phospholipid levels in the FPLC samples were measured according to the manufacturer's instructions (Wako Chemicals).

[0075] [Histological analysis] After formalin fixation, the liver, brain, kidney, spleen, heart, skeletal muscle, small intestine, and testis (male) were embedded in paraffin and sectioned into 5-μm slices. All slices were stained with hematoxylin and eosin (H&E) for microscopic examination. Additionally, Oil Red O staining was performed on fresh frozen sections (10 μm) to account for lipid accumulation in the liver. Subsequently, all slides were scanned with a Hamamatsu NDP scanner by a pathologist in a blinded fashion for tissue structure evaluation.

[0076] [Lipidomics analysis] Characterization of the uptake of VLCPUFAs into specific tissues was performed using HRAM-MS and MS / MS by non-targeted lipidomics and metabolomics approaches. To determine the utilization of VLCPUFAs by incorporation into complex lipids, as previously described [Busik et al. Methods Mol. Biol., 579:33-70 (2009)], comprehensive non-targeted lipidomics analysis was used to identify and quantify individual molecular species of complex lipids including mono-, di-, and triglycerides, phospholipid classes PA, PC, PE, PG, PI, PS, and their lyso and ether-linked subclasses, free fatty acids, free and esterified cholesterol, and major sphingolipids sphingomyelin, ceramide, hexosylceramide, and lactosylceramide. Statistical analysis was performed for all LC-MS features by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) approaches.

[0077] [Statistical analysis] All statistical analyses were performed using GraphPad Prism statistical software (GraphPad Software, version 6.). One-way ANOVA with Tukey's post hoc test for multiple comparisons was used to compare differences among multiple groups. A two-sided Student's t-test was used for comparisons between two groups. Results were expressed as mean ± SE, and the significance level was set at 0.05.

[0078] [Safety of VLCPUFA diet] When the whole body appearance of the mice was observed throughout the experimental period of all the studies, no abnormalities were found. When body weight measurements were recorded biweekly in a 2-month ad libitum feeding study, no significant changes over time were observed in any of the diet groups. In the plasma metabolic panel test results, starting from the alkaline phosphatase level, there were significant differences between the control and VLCPUFA diet groups, except for the dihydrogen lactate level (Table 2). Furthermore, when H&E-stained sections of the liver, kidney, spleen, heart, skeletal muscle, small intestine, brain, and testis were examined at the end of the 2-month feeding study, no abnormalities were found in either the VLCPUFA-treated mice or the control mice (Figures 17 - 24).

[0079] [Fatty Acid Composition of Retina and RPE] In the single-dose forced oral administration study, at 8 hours after oral administration of VLCPUFA oil, the retinal levels of C24:5 n-3 increased by 70% (P < 0.05), and the retinal levels of C24:6 n-3 increased by 79% (P < 0.05), and then gradually decreased basically to the baseline levels at 24 hours (Figure 2A). There were no significant differences in the retinal levels of n-3 C26 - C34 VLCPUFA at any time point (Figures 2B - 2F). The retinal levels of n-6 C24 - C34 VLCPUFA remained basically unchanged throughout all time points, except that the level of C28:4 n-6 increased from 0.0014% at baseline to 0.0048% at 24 hours after VLCPUFA treatment (Figures 2A - 2F). The total retinal levels of n-6 VLCPUFA tended to decrease at 4 hours compared to the baseline, resulting in a two-fold increase in the n-3 / n-6 VLCPUFA ratio from 0 hours to 4 hours after VLCPUFA treatment.

[0080] Table 3 shows the retinal and retinal pigment epithelium (RPE) levels of shorter-chain (C < 24) fatty acids after 2 weeks of repeated forced oral administration of VLCPUFA oil in C57BL / 6J mice. Except for the retinal levels of saturated fatty acids, there were no significant differences in the total retinal and RPE levels of saturated fatty acids and the total of monounsaturated fatty acids (MUFA) between the control and VLCPUFA groups, but the C14:0 level, C16:0 level, and C16:1 level were significantly increased in the retina of the VLCPUFA group. Two weeks of repeated forced oral administration of VLCPUFA oil significantly decreased the total levels of n-6 PUFA in the retina and RPE by 11% and 25% respectively (P < 0.05). The total level of n-3 PUFA also significantly decreased in the retina compared to the control, but not in the RPE. Therefore, the significant decrease in the n-6 PUFA level in the RPE, in conjunction with no significant change in the n-3 PUFA level in the RPE, resulted in a significant increase in the n-3 / n-6 PUFA ratio in the RPE compared to the control (P < 0.05).

[0081] After 2 weeks of repeated forced oral administration of VLCPUFA oil in C57BL / 6J mice, Table 4 shows a significant increase in the RPE level of 26:5 n-6 and the retinal and RPE levels of C28:4 n-6, and a significant decrease in the retinal and RPE levels of C26:6 n-3 and the RPE level of 34:6 n-3 due to VLCPUFA oil treatment. Except for these, there were no significant differences in the retinal and RPE levels of individual and total n-3 C24 - C34 VLCPUFA and total n-6 C24 - C34 VLCPUFA between the control and VLCPUFA groups. Two weeks of repeated oral administration of VLCPUFA oil significantly decreased the total levels of n-6 C18 - C34 PUFA in the retina and RPE by 12% (P < 0.05) and 24% (P < 0.0001) respectively, significantly decreased the total retinal level of n-3 C18 - C34 PUFA by 11% (P < 0.05), and significantly increased the n-3 / n-6 C18 - C34 PUFA ratio in the RPE by 38% (P < 0.001).

[0082] [Effect of VLCPUFA diet on visual function] ERG recordings showed that 2 weeks of repeated nutritional supplementation with VLCPUFA resulted in an increase of 78% and 52% in the average amplitude of the a-wave and b-wave of photopic vision, respectively (Figs. 3C and D). In another 8-week study, the VLCPUFA diet improved the a-wave and b-wave of photopic and scotopic vision in a dose-dependent manner (Figs. 4A and B). Furthermore, mice treated with VLCPUFA showed a small but significant increase in photopic (P < 0.001) and scotopic (P < 0.05) visual acuity compared to the control (Fig. 5).

[0083] [Effect of VLCPUFA diet on plasma lipids, lipoproteins, glucose, and insulin] After an 8-week investigation period, the VLCPUFA diet decreased the levels of plasma triglyceride and total cholesterol in a dose-dependent manner in C57BL / 6J mice compared to the control diet (P < 0.05) (Fig. 6B). Analysis of the lipoprotein profile revealed that the decrease was due to a decrease in the levels of VLDL and LDL cholesterol and phospholipids compared to the control (Fig. 6A). The plasma levels of glucose and insulin were significantly decreased in the 5% VLCPUFA group compared to the control group or the 1% VLCPUFA group (P < 0.05) (Fig. 6C). In ApoE-KO mice, supplementation with VLCPUFA also decreased the lipid content in VLDL and LDL (Fig. 7B) and decreased the levels of plasma cholesterol and triglyceride in a dose-dependent manner (Fig. 7A).

[0084] [Effect of VLCPUFA diet on liver lipid accumulation] Smaller and fewer lipid droplets in liver tissue, with decreased Oil Red O staining levels, were detected in the 1% and 5% VLCPUFA groups, and were more significantly decreased in the 5% VLCPUFA group (Figure 8A). H&E staining data verified the results of the Oil Red O staining (Figure 8A). Quantification of liver lipid content showed a dose-dependent decrease in liver triglyceride (TG), phospholipid (PL), and cholesterol (TC), as measured enzymatically (Figure 8B) or by LC-MS / MS (Figure 8C).

[0085] [Effect of VLCPUFA diet on tissue-specific lipidomic profiles] In the score plots of partial least squares discriminant analysis (PLS-DA), the populations of lipid species in the VLCPUFA groups were observed as distinct from those in the control diet group, and the 1% and 5% VLCPUFA groups were also clearly distinguished (Figure 9). Heatmaps of metabolite abundance in plasma and various important tissues, such as the liver, eye, brain, and testis, showed that the VLCPUFA groups, especially 5% VLCPUFA, had specific profiles compared to the control group (Figure 10). The total levels of lipid species in liver extracts decreased in a dose-dependent manner with VLCPUFA diet in both male and female mice, and the lipid species responsible for these changes were triglycerides and cholesterol (Figures 8B and 8C). VLCPUFA diet resulted in lipid remodeling in plasma incorporating C24:5 n-3, C26:6 n-3, and C28:8 n-3 (Figure 11).

[0086] [Effect of VLCPUFA diet on body composition] Eight weeks of VLCPUFA supplementation tended to decrease the body weight of C57BL / 6J mice compared to control mice (Figure 1). When evaluated by NMR-based body composition analysis, supplementation with 1% VLCPUFA significantly decreased the fat mass and increased the fat-free mass of ApoE-KO mice compared to WT control mice (Figure 12).

[0087] [Effect of VLCPUFA diet on the development of atherosclerosis] Positive IV staining of open aortic sections revealed that mice in the 1% VLCPUFA diet group had on average less than about 30% atherosclerotic lesion sites compared to the ApoE-KO control mouse group (p<0.05) (Figure 13).

[0088] [Effect of VLCPUFA diet on cognitive ability] In the Morris water maze test, mice relied on visual cues to reach the escape platform. Spatial learning was evaluated by daily repeated tests for 6 days. The latency to reach the hidden platform in ApoE-KO mice on the VLCPUFA diet was significantly reduced compared to ApoE-KO control mice (Figure 14). In the context learning test, the % baseline immobility time and the % immobility time in the new context were low, and no significant differences were observed among the four groups of mice. However, the % immobility time in the context and auditory cue was significantly increased in mice on the VLCPUFA diet, suggesting that the VLCPUFA diet improved spatial learning, memory, and context learning in mice (Figure 15).

[0089] [Agonist activity of VLCPUFA on peroxisome proliferator-activated receptor (PPAR)] To evaluate the agonist activity of VLCPUFA oil on human PPARα and PPARγ receptors, reporter cells were treated with six concentrations of VLCPUFA oil starting from 16 μM and continuing with serial dilutions of 3.17-fold. All treatment concentrations were performed in triplicate. Vehicle ethanol did not show agonist activity, but VLCPUFA oil showed agonist activity on PPARα and PPARγ receptors in a dose-dependent manner (Figure 16).

[0090] [Table 1]

[0091] [Table 2]

[0092]

Table 3

[0093]

Table 4

Claims

**Claim 1** A method for treating age-related macular degeneration or reducing the risk of developing age-related macular degeneration in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a very long-chain polyunsaturated fatty acid (VLCPUFA) having 24 to 40 carbon atoms. **Claim 2** The method according to claim 1, wherein the VLCPUFA is C24:5 n-3, C26:6 n-3 or C28:8 n-3, or any combination or all of them. **Claim 3** The method according to claim 1 or 2, wherein the VLCPUFA is derived from fish oil or produced by chemical synthesis. **Claim 4** A method for treating retinal dysfunction or improving one or more symptoms of retinal dysfunction in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a very long-chain polyunsaturated fatty acid (VLCPUFA) having 24 to 40 carbon atoms. **Claim 5** The method according to claim 4, wherein the VLCPUFA is C24:5 n-3, C26:6 n-3 or C28:8 n-3, or any combination or all of them. **Claim 6** The method according to claim 4 or 5, wherein the VLCPUFA is derived from fish oil or produced by chemical synthesis. **Claim 7** The method according to any one of claims 4 to 6, wherein the subject has retinal aging. **Claim 8** A method for treating hyperlipidemia, hypercholesterolemia or hypertriglyceridemia, or any combination or all of them, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a very long-chain polyunsaturated fatty acid (VLCPUFA) having 24 to 40 carbon atoms. **Claim 9** The method according to claim 8, wherein the VLCPUFA is C24:5 n-3, C26:6 n-3 or C28:8 n-3, or any combination or all of them. **Claim 10** The method according to claim 8 or 9, wherein the VLCPUFA is derived from fish oil or produced by chemical synthesis. **Claim 11** A method for treating metabolic syndrome, obesity, or overweight in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a very long chain polyunsaturated fatty acid (VLC-PUFA) having 24 to 40 carbon atoms. **Claim 12** The method according to claim 11, wherein the VLC-PUFA is C24:5 n-3, C26:6 n-3, or C28:8 n-3, or any combination or all thereof. **Claim 13** The method according to claim 11 or 12, wherein the VLC-PUFA is derived from fish oil or produced by chemical synthesis. **Claim 14** A method for treating hepatic steatosis or fatty liver disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a very long chain polyunsaturated fatty acid (VLC-PUFA) having 24 to 40 carbon atoms. **Claim 15** The method according to claim 14, wherein the VLC-PUFA is C24:5 n-3, C26:6 n-3, or C28:8 n-3, or any combination or all thereof. **Claim 16** The method according to claim 14 or 15, wherein the VLC-PUFA is derived from fish oil or produced by chemical synthesis. **Claim 17** A method for treating an increase in blood glucose concentration, or impaired fasting glucose, postprandial hyperglycemia, glucose tolerance impairment, insulin resistance, or diabetes in the blood, plasma, or serum of a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a very long chain polyunsaturated fatty acid (VLC-PUFA) having 24 to 40 carbon atoms. **Claim 18** The method according to claim 17, wherein the VLC-PUFA is C24:5 n-3, C26:6 n-3, or C28:8 n-3, or any combination or all thereof. **Claim 19** The method according to claim 17 or 18, wherein the VLC-PUFA is derived from fish oil or produced by chemical synthesis. **Claim 20** A method for treating atherosclerosis or improving one or more symptoms of atherosclerosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a very long chain polyunsaturated fatty acid (VLCPUFA) having 24 to 40 carbon atoms.

21. The method according to claim 20, wherein the VLCPUFA is C24:5 n-3, C26:6 n-3 or C28:8 n-3, or any combination or all thereof.

22. The method according to claim 20 or 21, wherein the VLCPUFA is derived from fish oil or produced by chemical synthesis.

23. A method for treating adipose fascia disease or improving one or more symptoms of adipose fascia disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a very long chain polyunsaturated fatty acid (VLCPUFA) having 24 to 40 carbon atoms.

24. The method according to claim 23, wherein the VLCPUFA is C24:5 n-3, C26:6 n-3 or C28:8 n-3, or any combination or all thereof.

25. The method according to claim 23 or 24, wherein the VLCPUFA is derived from fish oil or produced by chemical synthesis.

26. A method for treating cognitive impairment or improving one or more symptoms of cognitive impairment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a very long chain polyunsaturated fatty acid (VLCPUFA) having 24 to 40 carbon atoms.

27. The method according to claim 26, wherein the VLCPUFA is C24:5 n-3, C26:6 n-3 or C28:8 n-3, or any combination or all thereof.

28. The method according to claim 26 or 27, wherein the VLCPUFA is derived from fish oil or produced by chemical synthesis.

29. A method for activating the peroxisome proliferator-activated receptor (PPAR)-α pathway in a subject, comprising administering to the subject a therapeutically effective amount of a very long-chain polyunsaturated fatty acid (VLC-PUFA) having 24 to 40 carbon atoms.

30. The method according to claim 29, wherein the VLC-PUFA is C24:5 n-3, C26:6 n-3 or C28:8 n-3, or any combination or all thereof.

31. A method for activating the peroxisome proliferator-activated receptor (PPAR)-γ pathway in a subject, comprising administering to the subject a therapeutically effective amount of a very long-chain polyunsaturated fatty acid (VLC-PUFA) having 24 to 40 carbon atoms.

32. The method according to claim 31, wherein the VLC-PUFA is C24:5 n-3, C26:6 n-3, C28:8 n-3, or any combination or all thereof.

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