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

Abstract

The present invention relates to methods for treating macular degeneration, atherosclerosis, fatty liver, obesity and cognitive ability, and more particularly to methods 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

[0001] Citations of Related Applications

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 359,222, filed on July 8, 2022, the entire contents of which are incorporated herein by reference.

[0003] Government Support Statement

[0004] This invention was made in part with government support from the U.S. Department of Health and Human Services, National Institutes of Health. The U.S. Government has certain rights in this invention. Technical Field

[0005] The present invention relates to a method for treating macular degeneration, atherosclerosis, fatty liver, obesity and cognitive ability (cognitive function), and more specifically 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. Background Art

[0006] The CDC reports that an estimated 96 million adults 18 years and older had prediabetes in 2019. In 2018, there were 8.25 million hospital discharges with any listed diagnosis reported for U.S. adults 18 years and older. Of these discharges, 1.87 million had major cardiovascular disease (74.4 per 1,000 adults with diabetes), including 440,000 with ischemic heart disease (17.5 per 1,000 adults with diabetes). There are more than 46 million people 65 years and older in the U.S., a number that is expected to grow to nearly 90 million by 2050. Globally, the population of people 60 years and older will increase from 1 billion in 2020 to 1.4 billion in 2050.

[0007] Common health conditions associated with aging include diabetes, atherosclerosis, vision and hearing problems (including cataracts and age-related macular degeneration), cognitive decline, obesity, and more. According to the CDC, 50% of adults over 65 have prediabetes and 25% have diabetes. These conditions are caused by a variety of factors, including insulin resistance, which is often the result of obesity and lack of exercise; decreased insulin production in the pancreas; and decreased muscle mass. People with prediabetes have a greater chance of developing type 2 diabetes and having a heart attack or stroke.

[0008] Aging is also a major risk factor for the development of clinically significant atherosclerotic lesions. In addition, age-related macular degeneration is the most common cause of severe vision loss in people over the age of 50. However, these health problems also have a significant impact on younger groups in the population.

[0009] Very long chain fatty acids (VLCFAs) have unusually long hydrocarbon chains (C 24 -C 40 Although present in extremely low amounts, VLCFAs are found in many species and organs (e.g., testis, retina, brain, and sperm) and are essential lipids that play important roles in certain biological systems, whereas the more common short-chain C 16 -C 18 Fatty acids are unable to fulfill these roles. Due to their extremely long chain structure, some VLCFAs are able to span and reside within both leaflets of the lipid bilayer, providing stability to highly curved cell membranes, such as those surrounding the nuclear pore complex. In photoreceptors, VLC-polyunsaturated fatty acids (VLCPUFAs) are known to be associated with rhodopsin and play a role in regulating the phototransduction cascade. The lack of these VLCPUFAs appears to cause macular degeneration in autosomal dominant Stargardt macular dystrophy (STGD3).

[0010] The health benefits of fish diets and fish oils have attracted considerable scientific attention since the 1970s, when epidemiological studies suggested that fish oil-derived n-3 polyunsaturated fatty acids (PUFAs), namely eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), were beneficial for the heart. Depletion of retinal n-3 PUFAs in mammalian models significantly disrupts normal neural function and visual signaling pathways, leading to impaired vision, and epidemiological studies have shown that diets rich in n-3 PUFAs and oily fish are associated with a lower risk of advanced AMD. However, in several large randomized controlled trials, supplementation with EPA+DHA has not shown a benefit on AMD progression.

[0011] Many breakthrough drugs and treatments have been developed in medical practice to help treat the above conditions. However, there is still a need to solve these problems and find new treatments and compositions for them. The present invention is believed to meet this need. Summary of the invention

[0012] In one aspect, the invention relates to a method of treating a condition responsive to age-related macular degeneration in a subject or reducing the risk of a subject developing a condition responsive to age-related macular degeneration, comprising administering to the subject a therapeutically effective amount of a 24-carbon to 28-carbon very long chain polyunsaturated fatty acid (VLCPUFA).

[0013] In another aspect, the invention relates to a method for treating a disease or condition selected from hyperlipidemia, hypercholesterolemia or hypertriglyceridemia, or a combination thereof, comprising administering to a subject in need of treatment a therapeutically effective amount of 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

[0014] In another aspect, the invention relates to a method for treating hepatic steatosis comprising administering to a subject in need thereof a therapeutically effective amount of 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

[0015] In another aspect, the invention relates to a method for treating elevated plasma glucose concentration in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

[0016] In another aspect, the invention relates to a method of treating atherosclerosis in a subject or ameliorating (alleviating) one or more symptoms of atherosclerosis in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

[0017] In another aspect, the invention relates to a method of treating an adipofascial disorder in a subject or ameliorating one or more symptoms of an adipofascial disorder in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

[0018] In another aspect, the present invention is directed to a method of treating impaired cognitive function in a subject or ameliorating one or more symptoms of impaired cognitive function in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

[0019] 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

[0020] Figure 1 Shown are the changes in body weight of C57BL / 6J mice. Seven-month-old mice were fed a standard diet supplemented with 1% (w / w) or 5% VLCPUFA concentrated oil, or without supplementation (control) for 8 weeks. Data are presented as mean ± SE, n = 8 per group.

[0021] Figure 2 shows changes in retinal VLCPUFA composition in C57BL / 6J mice. 8-week-old mice were gavaged with VLCPUFA oil. At 0 hours, 2 hours, 4 hours, 8 hours and 24 hours after gavage, (A) C24 VLCPUFA, (B) C26 VLCPUFA, (C) C28 VLCPUFA, (D) C30 VLCPUFA, (E) C32 VLCPUFA, (F) C34 VLCPUFA, (G) total n-3 and n-6 ​​VLCPUFA, (H) plasma levels of n-3 / n-6 VLCPUFA ratio (percentage of total plasma fatty acids). Data are expressed as mean ± SE, n = 6 per group, *P < 0.05 compared with 0 hours.

[0022] Figure 3 shows that dietary VLCPUFA improves photopic electroretinographic responses in C57BL / 6J mice. The electroretinogram (ERG) test is used to measure the electrical responses of light-sensitive cells in the eye. Eight-week-old C57BL / 6J mice were orally gavaged with VLCPUFA oil or vehicle (control) daily for 2 weeks. Scotopic ERG a- and b-waves ( Figure 3A and 3B ) and photopic ERG a- and b-waves ( Figure 3C and 3D ) were plotted against the flash intensity. Data are presented as mean ± SE, n = 6 per group. *P < 0.05, ***P < 0.001 compared with the control.

[0023] Figure 4A and 4B It was shown that dietary VLCPUFA dose-dependently improved the scotopic and photopic electroretinogram responses of ApoE-KO mice. 9-month-old ApoE-KO mice were fed a standard diet supplemented with 1% (w / w) or 3% VLCPUFA oil or no supplementation (ApoE-KO mouse control) for 8 weeks. Age-matched C57BL / 6J mice were used as wild-type mouse controls. Data are expressed as mean ± SE, n = 8 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to wild-type mouse controls. #P < 0.05, ##P < 0.01, ###P < 0.001 compared to ApoE-KO mouse controls.

[0024] Figure 5 Dietary VLCPUFAs were shown to improve photopic and scotopic visual acuity in C57BL / 6J mice. Visual acuity test was used to assess visual discrimination and acuity. Eight-week-old mice were orally gavaged with VLCPUFA oil or vehicle (control) daily for 2 weeks. Data are presented as mean ± SE, n = 6 per group. *P < 0.05, ***P < 0.001 compared to control.

[0025] FIG6 shows that dietary VLCPUFA increases (decreases) lipid and glucose metabolism biomarkers in C57BL / 6J mice. (A) Plasma phospholipid and cholesterol FPLC profiles of pooled plasma, (B) plasma triglyceride and cholesterol levels, and (C) plasma glucose and insulin levels. 7-month-old mice were fed a standard diet supplemented with 1% (w / w) or 5% VLCPUFA oil, or without supplementation (control) for 8 weeks. Data are expressed as mean ± SE, n = 8 per group. **P < 0.01 compared to the control.

[0026] Figure 7 shows that dietary VLCPUFA increases (decreases) lipid and glucose metabolism biomarkers in ApoE-KO mice. (A) Plasma total cholesterol, free cholesterol and triglyceride levels. (B) Plasma cholesterol FPLC profiles from mixed plasma, and (C) plasma glucose levels. 9-month-old mice were fed a standard diet supplemented with 1% (w / w) or 3% VLCPUFA oil or no supplement (ApoE-KO mouse control) for 8 weeks. Age-matched C57BL / 6J mice were used as wild-type mouse controls. Data are expressed as mean ± SE, n = 8 per group. Compared with wild-type mouse controls, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Compared with ApoE-KO mouse controls, #P<0.05, ##P<0.01.

[0027] Figure 8 shows that dietary VLCPUFA increases (decreases) hepatic lipid accumulation in C57BL / 6J mice. (A) Hematoxylin-eosin (H&E) and Oil Red O staining of liver sections of representative mice in each group; original magnification is 100 times. (B) Enzymatic determination of hepatic triglyceride (TG), phospholipid (PL) and cholesterol (TC) content. (C) LC-MS / MS analysis of free cholesterol, cholesterol ester and triglyceride content in the liver. 7-month-old mice were fed a standard diet supplemented with 1% (w / w) or 5% VLCPUFA concentrate, or without supplementation (control) for 8 weeks. Data are expressed as mean ± SE, n = 8 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control.

[0028] Figure 9 shows a 2D representation of partial least squares discriminant analysis (PLS-DA) of lipidomic composition in C57BL / 6J mice. (A) Plasma, (B) liver, (C) brain, (D) eye, and (E) testis. Seven-month-old mice were fed a standard diet supplemented with 1% (w / w) or 5% VLCPUFA concentrate, or no supplementation (control) for 8 weeks, n=8 per group.

[0029] Figure 10 shows a heat map of lipidomic classes detected in C57BL / 6J mice and changes due to a diet rich in VLCPUFAs. (A) Plasma, (B) liver, (C) eyes, (D) brain, and (E) testes. Seven-month-old mice were fed a standard diet supplemented with 1% (w / w) or 5% VLCPUFA concentrate, or no supplementation (control) for 8 weeks, n=8 per group.

[0030] Fig.11 Dietary VLCPUFAs were shown to increase plasma levels of certain VLCPUFAs. Seven-month-old mice were fed a standard diet supplemented with 1% or 5% VLCPUFA concentrates, or without supplementation (control), for 8 weeks. Data are presented as mean ± SE, n = 8 per group.

[0031] Figure 12 shows that dietary VLCPUFA improves body composition in ApoE-KO mice. (A) Body weight change, and (B) body composition based on NMR analysis. 9-month-old mice were fed a standard diet supplemented with 1% (w / w) or 3% VLCPUFA concentrated oil, or without supplementation (control) for 8 weeks. Age-matched C57BL / 6J mice were used as wild-type mouse controls. Data are expressed as mean ± SE, n = 8 per group. *P < 0.05, ****P < 0.0001 compared to wild-type mouse controls.

[0032] Figure 13 shows that dietary VLCPUFA improves (reduces) the progression of atherosclerosis in ApoE-KO mice. (A) Representative en face Sudan IV staining of the aorta, and (B) quantitative analysis of Sudan IV positive plaque area of ​​the aorta. 9-month-old mice were fed a standard diet supplemented with 1% (w / w) or 3% VLCPUFA concentrated oil, or without supplementation (control) for 8 weeks. Age-matched C57BL / 6J mice were used as wild-type mouse controls. Data are expressed as mean ± SE, n = 8 per group. *P < 0.05 compared to ApoE-KO mouse controls.

[0033] Figure 14 shows that dietary VLCPUFA improves spatial learning and memory in ApoE-KO mice. (A) Average latency for female mice to reach the platform, and (B) area under the curve of latency (days 1-6). The Morris water maze test was used to evaluate spatial learning and memory in mice. 9-month-old mice were fed a standard diet supplemented with 1% (w / w) or 3% VLCPUFA concentrated oil, or without supplementation (control) for 8 weeks. Age-matched C57BL / 6J mice were used as wild-type mouse controls. Data are expressed as mean ± SE, n = 8 per group. *P < 0.05 compared to ApoE-KO mouse controls.

[0034] Figure 15 shows that dietary VLCPUFA improves contextual learning in ApoE-KO mice. (A) Percentage of baseline freezing time, (B) Percentage of contextual freezing time, (C) Percentage of novel contextual freezing time, and (D) Percentage of auditory cue freezing time. The contextual fear learning test is used to assess the ability of mice to learn and remember the association between environmental cues and aversive experiences (unpleasant experiences). 9-month-old mice were fed a standard diet supplemented with 1% (w / w) or 3% VLCPUFA concentrated oil, or without supplementation (control) for 8 weeks. Age-matched C57BL / 6J mice were used as wild-type mouse controls. Data are presented as mean ± SE, n = 8 per group. *P < 0.05 compared to the corresponding controls.

[0035] Figure 16 shows that VLCPUFA exhibits dose-dependent agonist activity with (A) PPARα and (B) PPARγ in CHO cells. In the assay of expressing receptor hybrids, CHO cells were used as reporter cells in which the native N-terminal DNA binding domain (DBD) had been replaced by the binding domain of the yeast Gal4 DBD. The reporter gene firefly luciferase is functionally associated with the Gal4 upstream activation sequence (UAS). Before the luciferase assay, cells were treated with VLCPUFA in serum containing BSA for 24 hours. Data are expressed as mean ± SE. All treatment concentrations were repeated three times. Compared with the vehicle, **P<0.01, ****P<0.0001.

[0036] Fig.17A and 17B The effect of dietary VLCPUFA on liver histology in C57BL / 6J mice is shown. Seven-month-old mice were fed a standard diet supplemented with 1% or 5% VLCPUFA concentrate, or no supplementation (control), for 8 weeks. Liver sections of mice fed each diet were subjected to (A) H&E staining and (B) Oil Red O staining, n=8.

[0037] Fig.18Shown are the effects of dietary VLCPUFA on renal histology in C57BL / 6J mice. 7 month old mice were fed a standard chow supplemented with 1% or 5% VLCPUFA concentrate, or no supplementation (control), for 8 weeks. H&E staining of kidney sections from mice fed each diet, n=8.

[0038] Fig.19 Shown is the effect of dietary VLCPUFA on spleen histology in C57BL / 6J mice. 7 month old mice were fed a standard chow supplemented with 1% or 5% VLCPUFA concentrate, or no supplementation (control), for 8 weeks. H&E staining of spleen sections from mice fed each diet, n=8.

[0039] Fig. 20 Shown are the effects of dietary VLCPUFA on cardiac histology in C57BL / 6J mice. 7 month old mice were fed a standard chow supplemented with 1% or 5% VLCPUFA concentrate, or no supplementation (control), for 8 weeks. H&E staining of heart sections from mice fed each diet, n=8.

[0040] Fig.21 Shown are the effects of dietary VLCPUFA on skeletal muscle histology in C57BL / 6J mice. 7 month old mice were fed a standard chow supplemented with 1% or 5% VLCPUFA concentrate, or no supplementation (control), for 8 weeks. H&E staining of skeletal muscle sections from mice fed each diet, n=8.

[0041] Fig. 22 Shown is the effect of dietary VLCPUFA on small intestinal histology in C57BL / 6J mice. 7 month old mice were fed a standard chow supplemented with 1% or 5% VLCPUFA concentrate, or no supplementation (control), for 8 weeks. H&E staining of small intestinal sections of mice fed each diet, n=8.

[0042] Fig.23 Shown are the effects of dietary VLCPUFA on brain histology in C57BL / 6J mice. 7 month old mice were fed a standard chow supplemented with 1% or 5% VLCPUFA concentrate, or no supplementation (control), for 8 weeks. H&E staining of brain sections from mice fed each diet, n=8.

[0043] Fig.24 Effects of dietary VLCPUFA on testicular histology in C57BL / 6J mice are shown. 7 month old mice were fed a standard chow supplemented with 1% or 5% VLCPUFA concentrate, or no supplementation (control) for 8 weeks. H&E staining of testicular sections from mice fed each diet, n=4. DETAILED DESCRIPTION

[0044] Chemical Description and Terminology

[0045] The compounds are described using standard nomenclature. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Unless the context clearly prohibits, each compound name includes the free acid or free base form of the compound and all pharmaceutically acceptable salts of the compound.

[0046] The terms "a" and "an" do not represent a quantitative limitation, but rather represent the presence of at least one of the items mentioned. The term "or" means "and / or". The open transition phrase "comprising" encompasses the intermediate transition phrase "consisting essentially of" and the closed phrase "consisting of". Unless otherwise specified by the context or the art, a claim stating one of these three transition phrases or with an alternative transition phrase such as "containing" or "including" may be written in any other transition phrase. Unless otherwise specified herein, the statement of a numerical range is intended only to be used as a shorthand method for referring to each individual value falling within the range, respectively, and each individual value is incorporated into the specification as if it were stated individually herein. The endpoints of all ranges are included within the range and may be independently combined. Unless otherwise specified herein or otherwise clearly contradictory to the context, all methods described herein may be performed in a suitable order. Unless otherwise required, the use of any and all examples or exemplary language (e.g., "such as") is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention. The language in the specification should not be interpreted as indicating that any unclaimed element is essential for implementing 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 those skilled in the art to which the present disclosure belongs.

[0047] The terms “or / and” and “and / or” mean “either ... or ..., or ... and ...” when referring to two elements, and mean “..., ... or ..., or any combination or all of them” when referring to three or more elements. 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”.

[0048] The term "about" or "approximately" means within an acceptable error range of a particular value determined by one of ordinary skill in the art, which depends in part 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 a specific error range (e.g., the standard deviation of the mean value given in a data chart or table) is not cited, the term "about" or "approximately" means a range that includes the cited value, as well as a range that is rounded up or down to the cited value, taking into account significant figures. In certain embodiments, the term "about" or "approximately" means within 10% or 5% of a specified value. Whenever the term "about" or "approximately" appears before the first value in a series of two or more numerical values ​​or two or more numerical ranges, the term "about" or "approximately" applies to each of the values ​​in the series of numerical values ​​or the series of numerical ranges.

[0049] A dash ("-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent.

[0050] "Halo" or "halogen" means fluoro, chloro, bromo or iodo.

[0051] The term "compound" includes salts, solvates, hydrates, inclusion compounds and polymorphs of the compound or salts of the compound. A "solvate" of a compound includes a stoichiometric or non-stoichiometric amount of a solvent (e.g., water, acetone or alcohol [e.g., ethanol]) that is non-covalently bound to the compound. A "hydrate" of a compound includes a stoichiometric or non-stoichiometric amount of water that is non-covalently bound to the compound. A "clathrate" of a compound contains molecules of a substance (such as a solvent) encapsulated in the crystal structure of the compound. A "polymorph" of a compound is a crystalline form of a compound. In certain cases of the present disclosure, specific expressions of "salts," "solvates," "hydrates," "clathrates," or "polymorphs" of a compound should not be interpreted as intentionally omitting any of these forms in other cases of the present disclosure, in which cases the term "compound" etc. is used without mentioning any of these forms.

[0052] A "pharmaceutical composition" is a composition comprising at least one active agent (such as a VLCPUFA or a salt thereof) and at least one other substance (such as a carrier or excipient). A pharmaceutical composition optionally contains one or more additional active agents. If specified, a pharmaceutical composition complies with the U.S. Food and Drug Administration's GMP (Good Manufacturing Practice) standards for human or non-human drugs. A "pharmaceutical combination" is a combination of at least two active agents, which can be combined into a single dosage form or provided together in separate dosage forms with an indication that the active agents will be used together to treat a condition, such as hepatitis C.

[0053] "Pharmaceutically acceptable salts" include derivatives of disclosed compounds, wherein the parent compound is modified by preparing its inorganic and organic, non-toxic, acid or base addition salts. The salts of the compounds of the present invention can be synthesized by the parent compound containing an alkaline or acidic part by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base (such as Na, Ca, Mg or K hydroxide, carbonate, bicarbonate, etc.), or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid. This reaction is usually carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred where feasible. The salts of the present compounds further include solvates and hydrates of compounds and compound salts.

[0054] Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids, etc. Pharmaceutically acceptable salts include conventional non-toxic salts and quaternary ammonium salts of the parent compound, such as those formed from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, etc.; and those derived from 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, methanesulfonic acid, ethanesulfonic acid, phenylethanesulfonic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, HOOC-(CH2) n -COOH (wherein n is 0-4) etc. Lists of additional suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, Pennsylvania, page 1418 (1985).

[0055] The term "carrier" as applied to the pharmaceutical composition / combination of the present invention refers to a diluent, excipient or vehicle with which the active compound is provided.

[0056] "Pharmaceutically acceptable excipient" means an excipient that can be used to prepare a pharmaceutical composition / combination, which excipient is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use. "Pharmaceutically acceptable excipient" as used in this application includes one and more than one such excipient.

[0057] A "patient" is a subject in need of medical treatment. The subject or patient can be a mammal or a non-mammal, such as a companion animal (such as a dog or cat) or a livestock animal (such as a cow, pig, horse, or sheep). In some embodiments, the subject or patient is a human. Medical treatment can include treatment of an existing condition (such as a disease or disorder), prophylactic or preventative treatment, or diagnostic treatment.

[0058] As used herein, "treating" includes providing a VLCPUFA or a salt thereof, as the sole active agent or in combination with at least one additional active agent, sufficient to: (a) prevent the disease or disease symptoms from occurring in a patient who may be susceptible to the disease but has not yet been diagnosed as having the disease (e.g., including macular degeneration in patients with a genetic mutation that predisposes to macular degeneration, such as the autosomal dominant mutation associated with Stargardt muscular dystrophy); (b) inhibit the disease, i.e., prevent its development; and (c) alleviate the disease, i.e., cause regression of the disease. "Treating" and "treatment" also include providing 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 suffering from or susceptible to a condition in which very long chain fatty acids are known to play a role.

[0059] "Preventing" a disease or condition means reducing the likelihood of developing the disease or condition in a patient at risk of developing the disease or condition, delaying the onset of symptoms, or reducing the severity of symptoms in a patient at risk of developing the disease or condition.

[0060] A "therapeutically effective amount" of a pharmaceutical composition / combination of the present invention means an amount effective to provide a therapeutic benefit (such as improvement of symptoms) when administered to a patient, for example, an amount effective to alleviate symptoms.

[0061] As used herein, the term "administering" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration.

[0062] The fatty acids discussed in this article are identified using the following general numbering system:

[0063]

[0064] In various aspects, therapeutic methods are described using compositions of very long chain polyunsaturated fatty acids (VLCPUFAs) for treating or ameliorating various clinical conditions, including atherosclerosis, impaired cognitive function, elevated plasma glucose concentrations, age-related macular degeneration, and other conditions.

[0065] In one aspect, very long chain polyunsaturated fatty acids (VLCPUFA) have 24 to 40 carbon chain lengths. In one aspect, very long chain polyunsaturated fatty acids (VLCPUFA) have one or more cis or trans configuration double bonds. In one aspect, fatty acids described herein are identified by alphanumeric names, such as C24:5 n-3. The letter "C" represents carbon, the number before the colon represents the number of carbon atoms, and the number after the colon represents the number of double bonds. The position of the terminal double bond is represented in the form of nx, where n represents the position of the methyl end of the molecule, and x represents the position of the double bond closest to the methyl end of the molecule, or the number of carbon atoms from the methyl end of the molecule to the nearest double bond:

[0066]

[0067] Pharmaceutical compositions and methods of treatment

[0068] Provided herein is a pharmaceutical composition comprising at least one very long chain polyunsaturated fatty acid (VLCPUFA) and a pharmaceutically acceptable carrier or excipient for treating the medical condition described herein. The pharmaceutical composition may comprise a single VLCPUFA or a mixture of VLCPUFA. Similarly, the medical condition described herein may be treated by administering a single VLCPUFA or a mixture of VLCPUFA. In some embodiments, the mixture of VLCPUFA is a mixture of VLCPUFA derived from fish oil, for example, VLCPUFA concentrated oil derived from fish oil with PUFA having less than 24 carbon atoms may also be contained. In a further embodiment, VLCPUFA is C24:5n-3, C26:6 n-3 or C28:8 n-3 or any combination thereof or all thereof.

[0069] In addition, the pharmaceutical composition may include one or more polyunsaturated fatty acids (PUFAs) having less than 24 carbon atoms (e.g., 20 or 22 carbon atoms), and the medical conditions described herein may be treated by administering one or more polyunsaturated fatty acids (PUFAs) having less than 24 carbon atoms (e.g., 20 or 22 carbon atoms). 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 thereof, or all thereof.

[0070] 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, an injectable dosage form (e.g., intravenous, subcutaneous or intramuscular), a topical dosage form (e.g., ophthalmic 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 pre-prepared solution in a vial or syringe, a lyophilized solid or a lyophilized solid that needs to be redissolved (reconstructed) before administration. The pharmaceutical composition can be an eye preparation, such as a liquid topical preparation. The pharmaceutical composition can be an oral dosage form such as a tablet or capsule form. In a preferred embodiment, VLCFA is formulated into any oral dosage form, including solid, semisolid, liquid, powder, granules (small medicine bag, sachet) etc. Solid oral dosage forms can include, for example, tablets, capsules (hard capsules or soft capsules) or subunits (subunit) etc. "Subunits" include mini-tablets, beads, spheres, microspheres, seeds (seed-like dosage forms, seeds), pills, caplets, microcapsules, granules, particles, etc., which can be provided as oral dosage forms alone or in combination with other subunits. Exemplary semisolid or liquid dosage forms include suspensions, solutions, emulsions, etc. Solid oral dosage forms can also include orally dissolving / disintegrating dosage forms (ODT). Exemplary ODTs include orally dissolving / disintegrating tablets, orally dissolving films, and dosage forms intended for sublingual / tongue / buccal delivery, such as rapidly dissolving / disintegrating sublingual tablets and films.

[0071] Oral dosage forms used in the methods and compositions can be formulated for a particular release profile, including immediate-release, controlled-release, sustained-release, or extended-release.

[0072] The present disclosure includes methods and compositions in which one or more compounds are a mixture or otherwise combined with one or more compounds and can be used in the presence or absence of conventional 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 carboxymethyl cellulose, polyvinyl pyrrolidone, etc.; iii) lubricants, such as stearic acid, talc, silicon dioxide, polyethylene glycol, polypropylene glycol, etc.; iv) absorbents, colorants, sweeteners, etc.; v) disintegrants (e.g., calcium carbonate and sodium bicarbonate) such as effervescent mixtures, etc.; vi) excipients (e.g., cyclodextrins, etc.); vii) surfactants (e.g., cetyl alcohol and glyceryl monostearate), adsorption carriers (e.g., kaolin and bentonite), emulsifiers, etc. Examples of carriers include, but are not limited to, any liquid, liquid crystal, solid, and semisolid suitable for use in the composition, such as water and saline, gels, creams, salves, solvents, diluents, fluid ointment bases, ointments, pastes, implants, liposomes, micelles, giant micelles, etc. 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.

[0073] The present disclosure includes ophthalmic compositions. The disclosed compositions can be emulsions, solutions, suspensions, gels, ointments, occlusive films or sustained-release films, which can be preservative preparations or non-preservative preparations. The compositions can be formulated as eye drops, creams, ointments and films applicable to the eyes. The preparations can be applied to the eyes, upper eyelids, lower eyelids or a combination thereof.

[0074] Ophthalmic compositions may include polymeric emulsifiers such as castor oil, squalene, isostearate and isopropyl myristate; lipophilic components such as mineral oil, silicone oil and caprylic / capric triglyceride; and / or alcohols such as cetyl alcohol and stearyl alcohol. The ophthalmic compositions of the present disclosure may also contain diethylene glycol monoethyl ether, propylene glycol and / or dipropylene glycol; cosolvents (cosolvents) such as dimethyl ether, diethylene glycol and dipropylene glycol; and / or buffers and pH adjusters such as sodium citrate dihydrate, boric acid, monosodium phosphate (sodium dihydrogen phosphate), dibasic heptahydrate (disodium hydrogen phosphate heptahydrate, dibasicheptahydrate) and sodium dihydrogen phosphate monohydrate (sodium phosphate monobasic monohydrate). The ophthalmic compositions may contain cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxyethylcellulose, PEG 300, PEG 400, povidone, glycerol, propylene glycol or hydroxypropyl methylcellulose, or any combination thereof. Suitable preservatives include, for example, benzalkonium chloride. The ophthalmic composition may contain a plasticizer or a film former. The ophthalmic composition typically contains a solvent, such as water or an isotonic solvent system (e.g., phosphate buffered saline), wherein the concentration of the active compound (e.g., VLCPUFA) is about 0.01-90%, 0.1-50%, 0.1-30%, 0.5-20% or 1-10% wt / vol.

[0075] The present disclosure includes methods using conventional mixing, granulation or coating methods and compositions prepared using conventional mixing, granulation or coating methods, and the compositions can contain 0.01% to 90% of active ingredients. The resulting composition (preparation) can exist in unit dosage form and can be prepared by methods known in the pharmaceutical field. All methods include the behavior of combining (one or more) active ingredients with the carrier constituting one or more ingredients. Therefore, the composition (preparation) can be prepared by blending (one or more) active ingredients with, for example, liquid carriers and / or finely divided solid carriers, and then shaping the product into a desired preparation when necessary.

[0076] Certain compositions and methods of the present disclosure contain a compound described herein (e.g., a VLCPUFA) in an amount of about 90 wt % to about 80 wt %, about 80 wt % to about 70 wt %, about 70 wt % to about 60 wt %, about 60 wt % to about 50 wt %, about 50 wt % to about 40 wt %, about 40 wt % to about 30 wt %, about 30 wt % to 20 wt %, about 20 wt % to about 10 wt %, about 10 wt % to about 4 wt %, about 4 wt % to about 2 wt %, about 2 wt % to about 1 wt %, or about 1 wt % to about 0.01 wt %.

[0077] Exposure to light, oxygen or heat can lead to oxidation of VLCPUFAs, which can be prevented or minimized by methods known in the art. For example, storage of tightly closed or sealed containers or pharmaceutical compositions containing VLCPUFAs in the dark and / or at reduced temperatures (e.g., in a refrigerator) can minimize oxidation.

[0078] The therapeutically effective amount of a compound of the present disclosure (e.g., VLCPUFA) for treating the medical conditions described herein and the frequency of administration, as well as the length of treatment, may depend on a variety of factors, including the severity of the condition, the potency of the compound, the route of administration, the age, weight, general health, sex, and diet of the subject, and the subject's response to treatment, and can be determined by the treating physician.

[0079] In some embodiments, the effective daily dose of a compound 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., ophthalmic or transdermal) composition) is about 0.1 to 100 milligrams (mg) per kilogram (kg) of subject body weight, which can be provided in a single dose or divided doses (e.g., administered two, three or four times a day to provide a total daily dose). In certain embodiments, the daily dose of a compound of the present disclosure (e.g., VLCPUFA) is about 0.0001 mg / kg to 0.1 mg / kg (e.g., for diagnostic monitoring), 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 a compound of the 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, an effective daily dose of a compound of the present disclosure (e.g., a VLCPUFA), whether in the form of an oral, parenteral (e.g., intravenous, subcutaneous or intramuscular), or topical (e.g., ophthalmic or transdermal) composition, is from about 10 mg to about 3 g, from about 50 mg to about 2 g, or from about 100 mg to about 2 g, or from about 100-500 mg, from about 500-1000 mg, from about 1-1.5 g, or from about 1.5-2 g, which can be provided in a single dose or in divided doses (e.g., administered two, three, or four times daily to provide a total daily dose).

[0080] The frequency of administration of the compounds of the present disclosure (e.g., VLCPUFA) can be, for example, once or more times per day (e.g., 1, 2, 3, 4 or more times per day), once every two days or three times per week, once every three days or twice per week, once per week, once every two weeks or once per month. For example, an ophthalmic solution containing a compound of the present disclosure (e.g., VLCPUFA) can be administered once, 2, 3, 4 or more times per day via eye drops, with 1, 2, 3, 4 or more drops of solution applied to each eye at each administration. The frequency of administration can be reduced (e.g., once per week, once every two weeks or once per month) by controlled or sustained release of the compound (e.g., via micelles or polymer nanoparticles or via delayed release tablets or capsules).

[0081] Treatment with the compounds of the present disclosure (e.g., VLCPUFAs) can be continued, for example, until the condition being treated resolves, such as substantially alleviating or eliminating one or more symptoms, complications, or causes of the condition. In some embodiments, the symptoms or complications of the condition are substantially alleviated if the severity, frequency, or duration of the condition is reduced by at least about 30%, 50%, 75%, or 90%. Thus, the length of time for treatment with the compounds of the present disclosure (e.g., VLCPUFAs) can be, for example, at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks, or at least about 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or at least about 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years.

[0082] The compounds of the present disclosure (e.g., VLCPUFA) can be administered by any suitable route to treat medical conditions. The route of administration includes, but is not limited to, oral, parenteral (e.g., intravenous, subcutaneous, subdermal, intradermal, intramuscular, organ lumen or parenchymal administration, intraperitoneal (intraperitoneal), intracavitary administration, intrauterine and topical administration), local (e.g., ocular, transdermal, buccal, sublingual, intranasal, pulmonary, anal / rectal and vaginal) and surgical administration. Topical administration of active agents (e.g., VLCPUFA) can deliver active agents to diseased tissues with minimal systemic distribution. The compounds of the present disclosure (e.g., VLCPUFA) can be administered by any suitable means. The modes of administration include, but are not limited to, tablets, capsules, powders, injections, implants, oral or nasal inhalation, 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.

[0083] The present disclosure also provides a kit or package containing a pharmaceutical composition, the 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, which can be provided in, for example, a prefilled syringe; an ophthalmic solution provided in, for example, an eye drop bottle; or a composition provided in a transdermal patch.

[0084] VLCFA (for example, VLCPUFA) carboxylic acid or salt form of alternatives, VLCFA (for example, VLCPUFA) can be in the form of esters, which can serve as prodrugs. Esterases present in blood and other body fluids, tissues and cells can quickly hydrolyze the ester of VLCFA (for example, VLCPUFA) to carboxylic acid or its salt. Therefore, pharmaceutical compositions can contain esters of VLCFA (for example, VLCPUFA) and pharmaceutically acceptable excipients or carriers, and the esters of VLCFA (for example, VLCPUFA) can be used to treat any medical condition described herein. Therefore, the entire disclosure of the present application is also applicable to the esters of VLCFA (for example, VLCPUFA). When the ester of VLCFA (for example, 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.

[0085] Therapeutic Uses

[0086] Age-related macular degeneration (AMD) affects central vision, which in turn affects the ability to see fine detail. In AMD, the part of the retina called the macula is damaged. Dry AMD is associated with the slow breakdown of light-sensitive cells in the macula. Wet AMD is associated with abnormal growth of blood vessels under the retina. The blood vessels leak fluid and blood (hence the name wet AMD), which creates a large blind spot in the center of the visual field.

[0087] The aging retina is susceptible to developing degenerative diseases such as AMD. Changes in corneal toricity (curvature) can lead to changes in refraction in the elderly, usually from "with the rule" to "against the rule" astigmatism. Other aging-related observations include increased thickness of Descemet's membrane, cornea farinata, white limbus girdle, mosaic degeneration, deep crocodile shagreen, Hassall-Henle bodies, arcus senilis, etc.

[0088] Hyperlipidemia is associated with high blood lipid (or fat) levels such as cholesterol and triglycerides. One type of hyperlipidemia, hypercholesterolemia, is associated with high levels of non-HDL cholesterol and LDL ("bad") cholesterol in the blood. Hypertriglyceridemia is a condition in which triglyceride levels are elevated in the blood. It is associated with or exacerbated by uncontrolled diabetes, obesity, and other conditions.

[0089] Hepatic steatosis is defined as intrahepatic fat of at least 5% of liver weight. Simple accumulation of triacylglycerols in the liver may have a hepatoprotective effect. However, prolonged hepatic lipid storage may lead to hepatic metabolic dysfunction, inflammation, and advanced nonalcoholic fatty liver disease (NAFLD). Nonalcoholic hepatic steatosis is associated with obesity, type 2 diabetes, and dyslipidemia.

[0090] Hyperglycemia, or high blood sugar, occurs when the level of sugar in the blood is too high. For example, this can occur when insulin (a hormone that promotes the absorption of glucose from the blood into the liver, fat, and skeletal muscle cells) is low, or when it occurs in cases of insulin insensitivity / resistance. Insulin resistance means that the liver, fat, and skeletal muscle cells do not respond well to insulin and cannot use glucose in the blood as energy. Hyperglycemia is most often associated with diabetes. Broadly speaking, hyperglycemia is associated with blood sugar levels above 125 mg / dL (milligrams per deciliter) when fasting (at least 8 hours without eating), and people with fasting blood sugar levels above 125 mg / dL have diabetes. In one aspect, for example, a human subject has impaired glucose tolerance or prediabetes and has a fasting blood sugar of 100 mg / dL to 125 mg / dL.

[0091] In one aspect, for example, a human subject has hyperglycemia if his blood glucose is above 180 mg / dL one to two hours after eating. Postprandial means after a meal is consumed. Postprandial hyperglycemia is characterized by high blood glucose peaks that induce oxidative stress.

[0092] 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, high blood sugar, excess body fat around the waist and abnormal cholesterol or triglyceride levels.

[0093] Obesity is a complex disease condition involving excess 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.

[0094] Subcutaneous adipose tissue diseases involving adipose tissue and its fascia, also referred to as adipofascial disorders, represent variations in the spectrum of obesity. Adipofascial disorders can be local or systemic and include common conditions that primarily affect women, i.e., lipedema, and four rare diseases, i.e., familial multiple lipomatosis, angiolipomatosis, Dercum disease, and multiple symmetric lipomatosis. The fat in adipofascial disorders is difficult to reduce by standard weight loss methods, including lifestyle (diet and exercise), medication, and even bariatric surgery, in part due to tissue fibrosis.

[0095] Atherosclerosis is the thickening or hardening of arteries caused by a buildup of plaque in the lining of the arteries. Plaque deposits include fatty materials, cholesterol, cellular waste products, calcium, and fibrin. Plaque buildup causes the artery walls to thicken and stiffen. Atherosclerosis is a slowly progressive disease that may begin as early as childhood but may also develop rapidly.

[0096] Cognitive health or function is the ability to think clearly, learn and remember, an important part of carrying out daily activities. Cognitive impairment is associated with difficulty remembering, learning new things, concentrating or making decisions that affect daily life. Cognitive impairment ranges from mild to severe. Subjects with mild cognitive impairment may begin to notice changes in cognitive function but still be able to carry out daily activities. Severe cognitive impairment may lead to a loss of the ability to understand the meaning or significance of things, as well as the ability to speak or write, resulting in an inability to live independently.

[0097] Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily, including PPARα, PPARγ, and PPARβ / δ isoforms. They are involved in regulating glucose and lipid homeostasis, inflammation, proliferation, and differentiation. VLCPUFAs can act as agonists or otherwise activate signaling pathways of PPARs such as PPARα and PPARγ.

[0098] The present disclosure provides a method for preventing or treating a medical condition responsive to treatment of very long chain fatty acids (VLCFA, such as VLCPUFA) in a patient, comprising administering VLCFA (e.g., VLCPUFA) or a salt thereof to the patient. Conditions responsive to treatment of VLCFA (e.g., non-deuterated or deuterated VLCPUFA) or a salt thereof include, but are not limited to, impaired retinal function, macular degeneration (e.g., AMD), hyperlipidemia (e.g., hypercholesterolemia and hypertriglyceridemia), obesity, overweight, adipose fascia disorders, hepatic steatosis, fatty liver disease (e.g., NAFLD), hyperglycemia (e.g., fasting hyperglycemia and postprandial hyperglycemia, impaired glucose tolerance, insulin resistance, prediabetes, diabetes (e.g., type 1 and type 2 diabetes), metabolic syndrome, cardiovascular disease (e.g., coronary artery disease and atherosclerosis), hypertension, and impaired cognitive function.

[0099] Example

[0100] Animals and experimental design

[0101] All experiments were conducted in accordance with the guidelines provided by the NIH Guide for the Care and Use of Laboratory Animals. Nippon Suisan Kaisha, Ltd. produces fish oil-derived very long-chain polyunsaturated fatty acids (VLCPUFA) concentrated oils rich in PUFAs with carbon chain lengths greater than C22. VLCPUFA oils concentrated in C24:5 n-3, C26:6 n-3, and C28:8 n-3 were produced from sardine oil. In the first step, cholesterol and environmental pollutants (such as dioxins) were removed from sardine oil by short-path distillation. In a further reaction step, ethyl esters were obtained by transesterification with sodium ethoxide, and the bleaching of the fish oil was completed by treating the oil with bleaching clay (process yield percentage: 100%). The obtained fish oil ethyl esters were refined by molecular distillation, and fatty acids with carbon chain lengths less than 20 were removed (process yield percentage: 18.3%-19.7%). The ethyl esters were further purified by thin film distillation to remove as much as possible the ethyl esters of fatty acids with a carbon chain length of 22, polymers and diacylglycerols (process yield percentage: 7%). The ethyl ester oil was further purified by high performance liquid chromatography (HPLC) using methanol as the mobile phase and further removing the ethyl esters of fatty acids with a carbon chain length of 22 (process yield percentage: 0.6%). After the final silica gel purification process, the peroxides were removed to obtain a VLCPUFA concentrated oil (process yield percentage: 94.4%). The contents of the major fatty acids in the VLCPUFA concentrated oil are shown in Table 1.

[0102] Experiments with mice were approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute and the University of Utah. Black C57BL / 6J mice were purchased from Taconic Biosciences (Germantown, New York), and ApoE-KO mice were purchased from Jackson Lab (Bar Harbor, Maine). Animals were housed in a 12 / 12 light-dark cycle with free access to food and water. Twenty-four 7-month-old C57BL / 6J mice were fed a standard diet AIN-93G (Harlan Teklad) supplemented with 1% (w / w) VLCPUFA oil, 5% (w / w) VLCPUFA oil, or no supplementation (control) for 8 weeks (n=8, 4 males and 4 females per group). In another study, 24 9-month-old ApoE-KO mice were fed a standard diet AIN-93G (Harlan Teklad) supplemented with 1% (w / w) VLCPUFA oil, 3% (w / w) VLCPUFA oil, or no supplement (control) for 8 weeks (n=8, 4 males and 4 females per group), and age-matched C57BL / 6J mice were used as wild-type controls. Body weight was monitored every 2-3 weeks in animals fasted for 5 hours. After 8 weeks of food intake, mice were euthanized by intraperitoneal injection of tribromoethanol (avertin). Blood samples were collected through the retro-orbital plexus (retro-orbital plexus), and plasma was separated by centrifugation. Tissue samples were quickly frozen and stored at -80°C until further use. Another aliquot of liver, brain, kidney, spleen, heart, skeletal muscle, small intestine, and testicles (male) were fixed in 10% formalin. In a single-dose gavage experiment, 25 3-month-old male mice were orally gavaged with 100 μL of VLCPUFA oil prepared with a liposome kit (Sigma-Aldrich). α-Tocopherol (0.025%) was added to prevent VLCPUFA oxidation. A VLCPUFA dose of 6 mg / day / mouse (250 mg / kg body weight) was used, which is comparable to the dose of purified C32:6 n-3 in previous studies [Gorusupudi et al., Proc. Natl. Acad. Sci. USA, 118 (6): e2017739118 (2021)]. The animals were euthanized at 0 hours, 2 hours, 4 hours, 8 hours, and 24 hours (n=5 at each time point), and the retinas were dissected and isolated from the eyes under a microscope. Lipids were extracted from one pair of retina / mouse for further GC-MS analysis as described previously [Liu et al., J. Lipid Res., 51(11):3217-3229 (2010)].In a repeated dose oral gavage experiment, 3-month-old male mice were randomly divided into the following two treatment groups: VLCPUFA concentrated oil (n=6) and vehicle (control) (n=6). Once a day, the mice were administered liposomes (control) and VLCPUFA oil (80 mg / [kg·d]) by oral gavage. After the experimental period of 15 days, the mice fasted for 6 hours were sacrificed to collect eye tissues, and the retina and retinal pigment epithelium (RPE) were isolated for GC-MS analysis.

[0103] The half-maximal effective concentration (EC 50 ) is the concentration of a drug, antibody or toxicant that induces a response halfway between baseline and maximum after a specified exposure time.

[0104] Electroretinogram and visual behavioral tests

[0105] As previously described, retinal function in mice was assessed using electroretinography (ERG) and visual behavioral examinations after 15 consecutive days of oral gavage of VLCPUFA oil [Gorusupudi et al., Proc. Natl. Acad. Sci. USA, 118(6): e2017739118 (2021)]. Briefly, mice were dark-adapted overnight before ERG recording and surgery was performed in a light-proof room with the help of dim red light. After anesthesia and mydriasis induction, 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 ERG a-wave and b-wave amplitudes were measured and analyzed under multiple flash luminances. In addition, at the end of 15 days of repeated gavage of VLCPUFA oil or vehicle, optokinetic behavioral tests were performed to analyze the visual function of mice. Visual acuity was measured using an optokinetic testing system (OptoMotry from Cerebral Mechanics), a validated non-invasive method. Briefly, tracking responses (optokinetic reflexes) to rotating visual stimuli displayed on an LCD panel around the mouse were recorded, and visual acuity was measured at 100% contrast.

[0106] Biochemical assays

[0107] Plasma triglycerides (TG), cholesterol, glucose, bilirubin, chloride, calcium, magnesium, creatine, alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), potassium, albumin, total creatine kinase, dihydrolactic acid (lactate dehydrogenase, lactatedihydrogen), total protein, amylase, inorganic phosphorus, urea nitrogen and uric acid were measured using an ILab 600 automatic analyzer and enzyme-based kits and quality control. Plasma insulin (R&D Systems, Minneapolis, Minnesota) and adiponectin (adiponectin, adiponectin) (R&D Systems) were measured using an enzyme-linked immunosorbent assay (ELISA) kit. As previously described, liver total lipids were extracted with a mixture of chloroform and methanol (2:1) [Folch et al., J.Biol.Chem., 226:497-509 (1957)], and the content of TG and total cholesterol was measured by colorimetric determination (Wako Chemicals, Richmond, Virginia).

[0108] Fast Protein Liquid Chromatography (FPLC) Spectrum Analysis

[0109] Pooled (pooled) plasma (150 μL) from each dietary group (n=8) was injected onto 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 and phospholipid levels in FPLC samples were measured according to the manufacturer's instructions (Wako Chemicals).

[0110] Histological analysis

[0111] After formalin fixation, liver, brain, kidney, spleen, heart, skeletal muscle, small intestine and testis (male) were embedded in paraffin and sliced ​​at 5 μm. All slices were stained with hematoxylin and eosin (H&E) for microscopic examination. In addition, Oil Red O staining was performed on fresh frozen sections (10 μm) to show liver lipid accumulation. Then, a pathologist scanned all slides with a Hamamatsu NDP scanner for histological evaluation in a blinded method.

[0112] Lipidomic analysis

[0113] By non-targeted lipidomics and metabolomics approaches, the incorporation of VLCPUFA into specific tissues was characterized using HRAM-MS and MS / MS. In order to determine the utilization of VLCPUFA by incorporation into complex lipids, global non-targeted lipidomics analysis was used to identify and quantify individual molecular species of complex lipids, including monoacylglycerol, diacylglycerol and triacylglycerol; 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, as described previously [Busik et al., Methods Mol. Biol., 579: 33-70 (2009)]. All LC-MS features were statistically analyzed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) methods.

[0114] Statistical analysis

[0115] All statistical analyses were performed using GraphPad Prism statistical software (GraphPad Software, version 6). One-way ANOVA with Tukey post hoc test was used for multiple comparisons to compare differences between multiple groups, and two-tailed Student's t-test was used for comparisons between two groups. Results are presented as mean ± SE, and the significance level was set at 0.05.

[0116] Safety of dietary VLCPUFA

[0117] In all studies, the general appearance of mice was monitored throughout the experimental period and no abnormalities were found. In a 2-month ad libitum feeding study, body weight measurements were recorded every 2 weeks and no significant changes over time were found in any of the dietary groups. There were significant differences in the results of plasma metabolic panel tests between the control and VLCPUFA diet groups, starting with alkaline phosphatase levels, with the exception of dihydrolactate levels (Table 2). In addition, H&E stained sections of the liver, kidney, spleen, heart, skeletal muscle, small intestine, brain, and testes were examined at the end of the 2-month feeding study and no abnormalities were found in either the VLCPUFA treated or control mice (Figures 17-24).

[0118] Fatty acid composition in the retina and RPE

[0119] In a single-dose oral gavage study, retinal levels of C24:5 n-3 increased by 70% (P < 0.05) and C24:6 n-3 increased by 79% (P < 0.05) 8 h after oral administration of VLCPUFA oil, and then gradually decreased to basal baseline levels at 24 h ( Figure 2AThere were no significant differences in retinal levels of n-3C26-C34 VLCPUFA at any time point ( Figure 2B-2F Retinal levels of n-6C24-C34 VLCPUFA remained largely unchanged across all time periods ( Figures 2A-2F ), except for C28:4 n-6 which increased from 0.0014% at baseline to 0.0048% at 24 h after VLCPUFA treatment. There was a trend toward a decrease in total retinal levels of n-6 VLCPUFA at 4 h compared with baseline, resulting in a 2-fold increase in the n-3 / n-6 VLCPUFA ratio from 0 to 4 h after VLCPUFA treatment.

[0120] Table 3 shows the retinal and retinal pigment epithelium (RPE) levels of short-chain (C<24) fatty acids in C57BL / 6J mice after repeated oral administration of VLCPUFA oil for 2 weeks. In addition to the retinal levels of saturated fatty acids, there were no significant differences in the retinal and RPE levels of total saturated fatty acids and monounsaturated fatty acids (MUFA) in the control group and the VLCPUFA group, although the levels of C14:0, C16:0 and C16:1 in the retina of the VLCPUFA group were significantly increased. After repeated oral administration of VLCPUFA oil for 2 weeks, the total levels of n-6PUFA in the retina and RPE were significantly reduced by 11% and 25%, respectively (P<0.05). Compared with the control, the total level of n-3PUFA in the retina was also significantly reduced, but there was no significant reduction in RPE. Therefore, compared with the control, the significant reduction in RPE n-6PUFA levels plus the lack of significant changes in RPE n-3PUFA levels resulted in a significant increase in the RPE n-3 / n-6PUFA ratio (P<0.05).

[0121] After repeated gavage of VLCPUFA oil in C57BL / 6J mice for 2 weeks, Table 4 shows that there were no significant differences in the retinal and RPE levels of individual and total n-3 and n-6 ​​C24-C34 VLCPUFAs between the control and VLCPUFA groups, except that the RPE levels of 26:5 n-6 and the retinal and RPE levels of C28:4 n-6 were significantly increased with VLCPUFA oil treatment, and the retinal and RPE levels of C26:6 n-3 and the RPE levels of 34:6 n-3 were significantly decreased with VLCPUFA oil treatment. After repeated gavage of VLCPUFA oil for 2 weeks, the levels of total n-6 C18-C34 PUFA in the retina and RPE were significantly decreased by 12% (P<0.05) and 24% (P<0.0001), respectively, the retinal level of total n-3 C18-C34 PUFA was significantly decreased by 11% (P<0.05), and the n-3 / n-6C18-C34PUFA ratio in RPE was significantly increased by 38% (P<0.001).

[0122] Effects of dietary VLCPUFA on visual function

[0123] ERG recordings showed that after repeated intragastric administration of VLCPUFA for 2 weeks, the amplitudes of the photopic a-wave and b-wave increased by an average of 78% and 52%, respectively ( Figure 3C and 3D In another 8-week intervention study, dietary VLCPUFA dose-dependently improved photopic and scotopic a- and b-waves ( Figure 4A and 4B In addition, VLCPUFA-treated mice showed a small but significant improvement in both photopic (P<0.001) and scotopic (P<0.05) visual acuity compared with controls ( Figure 5 ).

[0124] Effects of dietary VLCPUFA on plasma lipids, lipoproteins, glucose and insulin

[0125] After the 8-week intervention period, dietary VLCPUFA dose-dependently reduced plasma triglyceride and total cholesterol levels in C57BL / 6J mice compared with the control diet (P < 0.05) ( Figure 6B ). Lipoprotein profiling analysis showed that the decrease was due to a decrease in VLDL and LDL cholesterol and phospholipid levels compared with controls ( Fig. 6A ). Compared with the control group or the 1% VLCPUFA group, the plasma glucose and insulin levels in the 5% VLCPUFA group were significantly decreased (P<0.05) ( Figure 6C In ApoE-KO mice, supplementation with VLCPUFA also reduced the lipid content of VLDL and LDL ( Figure 7B ) and dose-dependently reduced plasma cholesterol and triglyceride levels ( Fig. 7A ).

[0126] Effects of dietary VLCPUFA on hepatic lipid accumulation

[0127] Reduced levels of Oil Red O staining in liver tissues and smaller and fewer lipid droplets were detected in the 1% and 5% VLCPUFA groups, and the decrease was greater in the 5% VLCPUFA group ( Fig. 8A ). The data of H&E staining confirmed the results of Oil Red O staining ( Fig. 8A ). Quantification of liver lipid content, such as by enzymatic methods ( Figure 8B ) or by LC-MS / MS( Figure 8C ) measured, liver triglyceride (TG), phospholipids (PL) and cholesterol (TC) decreased in a dose-dependent manner.

[0128] Effects of dietary VLCPUFA on tissue-specific lipidomic profiles

[0129] In the partial least squares discriminant analysis (PLS-DA) score plot, it was observed that the lipid species clusters of the VLCPUFA group were separate from those of the control diet group, and the 1% and 5% VLCPUFA groups were also clearly distinguished (Figure 9). Heat maps of metabolite abundance in plasma and various key tissues (such as liver, eyes, brain, and testes) showed that the VLCPUFA group (especially 5% VLCPUFA) had specific curves compared with the control group (Figure 10). The VLCPUFA diet caused a dose-dependent decrease in the total levels of lipid species in liver extracts of male and female mice, and the lipid species that caused these changes were triglycerides and cholesterol ( Figure 8B and 8C ). Dietary VLCPUFAs lead to remodeling of plasma lipids and incorporation of C24:5 n-3, C26:6 n-3, and C28:8 n-3 ( Fig.11 ).

[0130] Effects of dietary VLCPUFA on body composition

[0131] Supplementation of VLCPUFA for 8 weeks tended to reduce body weight in C57BL / 6J mice compared with control mice ( Figure 1 ), and judging by NMR-based body composition analysis ( FIG. 12 ), supplementation with 1% VLCPUFA significantly reduced fat mass and increased lean mass in ApoE-KO mice compared with wild-type mouse controls.

[0132] Effects of dietary VLCPUFA on the development of atherosclerosis

[0133] Sudan IV staining of open aorta sections showed that the area of ​​atherosclerotic lesions in mice in the 1% VLCPUFA diet group was reduced by an average of about 30% compared with the ApoE-KO mouse control group (p<0.05) ( FIG. 13 ).

[0134] Effects of dietary VLCPUFA on cognitive ability

[0135] In the Morris water maze test, mice rely on visual cues to navigate to a submerged escape platform. Spatial learning was assessed by daily repeated trials for 6 consecutive days. ApoE-KO mice fed a VLCPUFA diet had a significantly reduced latency to reach the hidden platform compared to ApoE-KO mouse controls (Figure 14). In the contextual learning test, baseline freezing time % and novel context freezing time % were low, and no significant differences were observed among the four groups of mice. However, contextual and auditory freezing time % were significantly increased in mice fed a VLCPUFA diet, indicating that dietary VLCPUFA improved spatial learning, memory, and contextual learning in mice (Figure 15).

[0136] Agonist activity of VLCPUFA and peroxisome proliferator-activated receptors (PPAR)

[0137] In order to evaluate the agonist activity of VLCPUFA oil with human PPARα and PPARγ receptors, reporter cells were treated with 6 concentrations of VLCPUFA oil, starting from 16 μM and continuing with 3.17-fold serial dilutions. All treatment concentrations were repeated three times. The solvent ethanol did not show agonist activity, but the VLCPUFA oil showed agonist activity with PPARα and PPARγ receptors in a dose-dependent manner (Figure 16).

[0138] Table 1. Levels of major fatty acids in fish oil-derived VLCPUFA concentrates

[0139]

[0140]

[0141] Values ​​correspond to the mean of three individual samples processed independently.

[0142] *Other VLCPUFA isomers are present in trace amounts, including C24:4 n-3 and C25:5 n-3.

[0143] Table 2. Plasma clinical biochemistry in C57BL / 6J mice during an 8-week feeding study on VLCPUFA oil

[0144] Comparison 1% VLCPUFA 5% VLCPUFA Total bilirubin (mg / dL) <0.1 <0.1 <0.1 Direct bilirubin (mg / dL) <0.2 <0.2 <0.2 Chloride (mmol / L) <60 <60 <60 Calcium (mmol / L) <0.20 <0.20 <0.20 Magnesium (mmol / L) <0.10 <0.10 <0.10 Creatinine (mg / dL) <0.1 <0.1 <0.1 Alkaline phosphatase (U / L) 12.97±1.95 10±1.09 8.62±0.75 ALT / GPT(U / L) 36.56±10.38 22.25±2.14 37±5.44 AST / GOT(U / L) 74.59±0.17 60.5±0.12 67.88±0.07 Potassium (mmol / L) 3.76±0.17 3.39±0.12 3.5±0.07 Albumin (g / dL) 2.14±0.07 1.96±0.07 2.04±0.04 Total creatine kinase (U / L) 339.47±107.61 418±147.38 628.75±95.46 Dihydrolactic acid (U / L) 260.44±20.27 265.37±29.09 281±31.54 Total protein (g / dL) 3.09±0.11 2.76±0.1 2.84±0.05 Amylase (U / L) 1546.59±106.63 1215.88±85.52 1138.25±49.27 Inorganic phosphorus (mg / dL) 3.22±0.09 3.09±0.14 3.89±0.26 Urea nitrogen (mg / dL) 9.47±0.2 8.38±0.5*** 5.87±0.3**** Uric acid (mg / dL) 0.46±0.06 0.39±0.04 0.29±0.02*

[0145] Values ​​are mean ± SEM (n = 8).

[0146] *P<0.05, ***P<0.001, ****P<0.0001 were significantly different from the control.

[0147] Table 3. Effects of repeated oral administration of VLCPUFA oil for 2 weeks on the retinal and retinal pigment epithelial (RPE) levels of fatty acids with a C chain length less than 24 in C57BL / 6J mice

[0148]

[0149] FA = fatty acids; SAT FA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids.

[0150] Values ​​are mean ± SEM (n = 6). ND = not determined.

[0151] *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 were significantly different from the control.

[0152] Table 4. Effects of repeated oral administration of VLCPUFA oil for 2 weeks on the levels of VLCPUFA (C chain length ≥ 24) in the retina and retinal pigment epithelium (RPE) of C57BL / 6J mice

[0153]

[0154]

[0155] PUFA = polyunsaturated fatty acids; VLCPUFA = very long chain polyunsaturated fatty acids.

[0156] Values ​​are mean ± SEM (n = 6). ND = not determined.

[0157] *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 were significantly different from the control.

Claims

1. A method of treating or reducing the risk of developing age-related macular degeneration in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a 24-carbon to 40-carbon very long chain polyunsaturated fatty acid (VLCPUFA).

2. The method of claim 1, wherein the VLCPUFA is C24:5n-3, C26:6n-3 or C28:8n-3, or any combination thereof or all thereof.

3. The method according to claim 1 or 2, wherein the VLCPUFA is derived from fish oil or prepared by chemical synthesis.

4. A method of treating impaired retinal function or ameliorating one or more symptoms of impaired retinal function in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

5. The method of claim 4, wherein the VLCPUFA is C24:5n-3, C26:6n-3 or C28:8n-3, or any combination thereof or all thereof.

6. The method according to claim 4 or 5, wherein the VLCPUFA is derived from fish oil or prepared by chemical synthesis.

7. The method according to any one of claims 4 to 6, wherein the subject suffers from retinal aging.

8. A method of treating hyperlipidemia, hypercholesterolemia, or hypertriglyceridemia, or any combination or all thereof, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

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

10. The method according to claim 8 or 9, wherein the VLCPUFA is derived from fish oil or prepared by chemical synthesis.

11. A method of treating metabolic syndrome, obesity or overweight in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

12. The method of claim 11, wherein the VLCPUFA is C24:5n-3, C26:6n-3 or C28:8n-3, or any combination thereof or all thereof.

13. The method according to claim 11 or 12, wherein the VLCPUFAs are derived from fish oil or prepared by chemical synthesis.

14. A method of treating hepatic steatosis or fatty liver disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

15. The method of claim 14, wherein the VLCPUFA is C24:5n-3, C26:6n-3, or C28:8n-3, or any combination thereof, or all thereof.

16. The method of claim 14 or 15, wherein the VLCPUFAs are derived from fish oil or prepared by chemical synthesis.

17. A method for treating elevated glucose concentrations in blood, plasma or serum, or impaired fasting glucose, postprandial hyperglycemia, impaired glucose tolerance, insulin resistance or diabetes in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

18. The method of claim 17, wherein the VLCPUFA is C24:5n-3, C26:6n-3, or C28:8n-3, or any combination thereof, or all thereof.

19. The method of claim 17 or 18, wherein the VLCPUFAs are derived from fish oil or are prepared by chemical synthesis.

20. A method of treating atherosclerosis or ameliorating one or more symptoms of atherosclerosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a 24-carbon to 40-carbon very long chain polyunsaturated fatty acid (VLCPUFA).

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

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

23. A method of treating adipofascial disorders or ameliorating one or more symptoms of adipofascial disorders in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

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

25. The method of claim 23 or 24, wherein the VLCPUFAs are derived from fish oil or are prepared by chemical synthesis.

26. A method of treating impaired cognitive function or ameliorating one or more symptoms of impaired cognitive function in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of 24-carbon to 40-carbon very long chain polyunsaturated fatty acids (VLCPUFAs).

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

28. The method of claim 26 or 27, wherein the VLCPUFAs are derived from fish oil or prepared by chemical synthesis.

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

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

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

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

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