Very long chain fatty acids for the treatment and alleviation of disease

Abstract

The present invention relates to methods and compositions for the treatment and alleviation of diseases. In particular, the present invention provides compositions containing very long chain fatty acids for use in treating subjects with deficiencies or abnormalities in the concentration of VLCFAs present in specific tissues that play a role in the disease. In particular, the present invention provides methods and compositions for the treatment of subjects with reduced capacity for endogenous fatty acid synthesis.

Description

[Technical Field]

[0001] The present invention relates to methods and compositions for the treatment and alleviation of diseases. In particular, the present invention provides methods for treating diseases associated with a reduced capacity for endogenous fatty acid synthesis. [Background technology]

[0002] Among long-chain polyunsaturated fatty acids (LCPUFA), especially long-chain omega-3 fatty acids (LCn3), fatty acids with chain lengths of C20 to C22 have received the most attention in the literature. The acronyms EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) have become familiar names to describe valuable omega-3 acids from fish oils and other sources. Products rich in alpha-linolenic acid (ALA) from plant sources are also available on the market.

[0003] More recently, long-chain monounsaturated fatty acids (LCMUFAs) with chain lengths of C20 to C22 have become the focus of scientific interest. See, for example, U.S. Patent No. 9,409,851 B2 to Breivik and Vojnovic, entitled "Long-Chain Monounsaturated Fatty Acid Compositions and Methods for Making Same."

[0004] In this regard, lipids are described by the formula X:YnZ, where X is the number of carbon atoms in their alkyl chain, Y is the number of double bonds in that chain, and "nZ" is the number of carbon atoms from the methyl end group to the first double bond. In the natural state, all double bonds are in the cis configuration. In polyunsaturated fatty acids, each double bond is separated from the next by a single methylene (-CH2) group. Using this nomenclature, EPA is C20:5n3, DHA is C22:6n3, and ALA is C18:3n3. Furthermore, natural sources of omega-3 fatty acids, such as fish oil, also contain fatty acids shorter and longer than C20-C22.

[0005] To produce marine omega-3 concentrates rich in EPA and DHA, conventional industrial processes are designed to concentrate the C20-C22 fraction, removing both short-chain fatty acids and molecules longer than C22. Examples of such processes include molecular / short-path distillation, urea fractionation, extraction, and chromatography, all of which can be used to concentrate the C20-C22 fraction of marine fatty acids and similar materials from other sources. These procedures are reviewed in Breivik H, "Concentrates" (2007), and in "Long-Chain Omega-3 Specialty Oils," edited by Breivik H, and published by The Oily Press, PJ Barnes & Associates, Bridgwater, UK, pp. 111-140. In addition to omega-3 acids, the polyunsaturated fatty acids in marine oils may contain small amounts of omega-6 fatty acids.

[0006] In important fish sources, such as North Atlantic herring and mackerel, the C20-C22 fatty acid fraction also contains significant amounts of C20-C22 MUFAs (monounsaturated fatty acids) in addition to omega-3 acids such as EPA and DHA. A procedure for separating C20-C22 MUFAs and PUFAs (polyunsaturated fatty acids) is disclosed in U.S. Patent No. 9,409,851 B2.

[0007] Omega-3 acids are highly susceptible to oxidation. To comply with pharmacopoeias and voluntary standards that impose upper limits on oligomeric / polymeric oxidation products, it is common to remove components with chain lengths longer than DHA, for example, by distillation, extraction, and similar procedures. Furthermore, such high molecular weight components of marine oils are typically associated with the oil's undesirable unsaponifiable components, including cholesterol, as well as organic contaminants such as brominated diphenyl ethers.

[0008] Omega-3 fatty acids, and in particular the LCPUFAs EPA, DHA, and n3DPA (n3 docosapentaenoic acid, C22:5n3), are known to have a wide range of beneficial health effects and are therefore known for a variety of uses. These LC ω-3 fatty acids are naturally found in fish and other marine organisms. They can also be derived endogenously from ALA, an ω-3 fatty acid found in certain plant- and animal-based oils. However, the body alone is insufficient to convert ALA to LC ω-3 acids. For this reason, LC ω-3 acids are often referred to as "essential" fatty acids. Fatty acids are taken up by cells, where they can serve as precursors in the synthesis of other compounds, as fuel for energy production, and as substrates for the synthesis of ketone bodies. In addition, some cells synthesize fatty acids for storage or export. Fatty acids ingested by a subject, such as from dietary sources, are often modified in vivo. Such modifications include chain elongation to produce longer fatty acids and / or desaturation, resulting in unsaturated fatty acids.

[0009] It is well known that some subjects suffer from disorders of fatty acid metabolism, which can be described as, for example, hypertriglyceridemia (excessively high levels of triglycerides) or other types of hyperlipidemia. These disorders can be familial or acquired. These disorders can be described as fat oxidation disorders or lipid storage disorders, and are any of several inborn errors of metabolism resulting from enzyme deficiencies that affect the body's ability to oxidize fatty acids to generate energy in muscle, liver, and other cell types. In addition to disorders related to fatty acid metabolism, some subjects may also have a reduced ability to endogenously synthesize fatty acids, for example, by synthesizing longer fatty acids from shorter ones. Thus, these subjects may have a reduced ability to endogenously synthesize long-chain fatty acids from shorter fatty acids. This reduced ability to endogenous synthesis may occur in specific tissues where these fatty acids are needed to maintain optimal health in the subject. This reduced ability may develop with age or may already exist at a young age. In the latter case, in particular, the reduced ability to endogenously synthesize longer fatty acids may also be due to a genetic disorder.

[0010] Nutritional supplements containing concentrates of traditional C20-C22 omega-3 fatty acids are often recommended to treat or alleviate symptoms of various diseases. Additionally, diseases and conditions such as age-related macular degeneration (AMD), dry eye disease (DED), declining mental health, and decreased sperm quality in male subjects have been treated with traditional C20-C22 omega-3 fatty acids, such as fatty acids containing high concentrations of EPA and / or DHA. However, not all subjects respond satisfactorily to this treatment, and results can appear contradictory depending on whether subjects ingest omega-3 fatty acids by eating a fish-rich diet or by consuming traditional C20-C22 concentrates. As an example, a recent publication (Gorusupudi A, Liu A, Hageman GS and Bernstein P (2016) Associations of human retinal very long-chain polyunsaturated fatty acids with dietary lipid biomarkers. Journal of Lipid Research 57: 499-508) presents the following unresolved contradiction: although epidemiological studies have shown that diets rich in n3 LCPUFA are associated with a lower risk of AMD, two clinical trials of 3-5 years of "fish oil" supplementation failed to produce any effect on the progression of advanced AMD.

[0011] One explanation for this discrepancy may be based on the incorrect assumption that very long-chain polyunsaturated fatty acids (VLCPUFA) are not typically consumed in the human diet. As shown in International Patent Publication WO 2016 / 182452 to Breivik and Svensen, oils derived from wild fish contain VLCPUFA with chain lengths of C24 and above. On the other hand, dietary "fish oil" omega-3 supplements are most often manufactured by concentrating valuable long-chain marine omega-3 fatty acids, thereby reducing the amount of fatty acids with chain lengths shorter than EPA (C20) and longer than n3DPA and DHA (C22).

[0012] Similar to the aforementioned inconsistencies stemming from Gorusupudi et al.'s publication on AMD, supplementation with omega-3 fatty acids in patients with dry eye disease (DED) has also produced conflicting results. DED, also known as keratoconjunctivitis sicca (KCS), is a common chronic condition characterized by ocular discomfort and visual impairment that reduces quality of life. As recently described by the Dry Eye Assessment and Management (DREAM) Research and Development Group (New England Journal of Medicine, April 13, 2018, DOI: 10.1056 / NEJMoa1709691), many clinicians recommend the use of omega-3 fatty acids to alleviate DED symptoms. However, the large DREAM study concluded that among patients with DED, those who took a nutritional supplement as omega-3 concentrate (3000 mg of n3 fatty acids taken daily as triglycerides, 2000 mg of EPA and 1000 mg of DHA) for 12 months did not have significantly better outcomes than those receiving a placebo.

[0013] In contrast, other studies have shown a positive effect of fish oil on DED. For example, in an article listed in the bibliography of the DREAM research report, Deinema et al. (Randomized, double-masked, placebo-controlled clinical trial of two forms of omega-3 supplementation to treat dry eye disease: Ophthalmology 2017; 124: 43-52) showed a significant positive effect on DED when using non-concentrated fish oil and krill oil as sources of omega-3.

[0014] The DREAM study noted that many clinicians recommend omega-3 fatty acid supplements because they have anti-inflammatory activity and are not associated with substantial side effects.

[0015] In a recently published meta-analysis of the efficacy of omega-3 fatty acid supplementation for the treatment of dry eye disease (DED), Giannaccare et al., "Efficacy of omega-3 fatty acid supplementation for treatment of dry eye disease: A meta-analysis of randomized clinical trials," Cornea 38 (5) 565-573 (2019), the authors state in the discussion section that the effects of both dietary intake and omega-3 fatty acid supplementation on the signs and symptoms of DED remain questionable. However, based on a review including 17 randomized clinical studies involving 3,363 patients, the authors conclude that omega-3 fatty acid supplementation improves dry eye symptoms, tear film stability, and tear production in patients with DED. However, the authors note that considerable heterogeneity was observed for all of these outcome variables, indicating inconsistent results across studies.

[0016] As disclosed herein, the reason for the lack of response to treatment with C20-C22 omega-3 fatty acids in some diseases may be that subjects have a reduced ability to endogenously synthesize longer fatty acids, for example from EPA and DHA, and therefore are unable to synthesize very long chain omega-3 fatty acids in sufficient amounts across the range of chain lengths and degrees of unsaturation required for optimal health.

[0017] Similar to the above-mentioned content regarding LCPUFA, VLCPUFA are also sometimes called essential fatty acids. Unfortunately, when VLCPUFA are prepared by chemical synthesis, these syntheses only produce a limited number of VLCPUFA compared to those present in major body tissues. Furthermore, it has generally been believed that VLCPUFA are synthesized in appropriate tissues and cannot be obtained from diet. Therefore, suitable compositions containing various fatty acids, including VLCPUFA, are not commercially available.

[0018] Based on the foregoing, there exists a need for new and alternative treatments for diseases and conditions in subjects, particularly subjects with a reduced capacity for endogenous synthesis of fatty acids. Summary of the Invention

[0019] It is therefore an object of the present invention to provide methods and compositions useful for the treatment and alleviation of diseases, conditions, and pathologies associated with a reduced capacity for endogenous synthesis of fatty acids, e.g., deficiencies in one or more elongation enzyme systems.

[0020] The present invention further provides compositions comprising very long chain fatty acids (VLCFAs) for use in treating diseases, conditions, and pathologies that can be ameliorated by increasing the concentration of VLCFAs in specific tissues. In one embodiment, a subject has deficient or abnormal levels of VLCFAs present in specific tissues that play a role in the disease.

[0021] Applicants contemplate that deficiencies in one or more elongation enzyme systems, and / or other enzyme systems, can be alleviated by administration of naturally occurring very long chain fatty acids (VLCFAs). [Brief explanation of the drawings]

[0022] [Figure 1] 1 shows the content of various fatty acids in eye (pupil) tissue from mice fed various test diets. [Figure 2] 1 shows the content of various fatty acids in eye (pupil) tissue from mice fed various test diets. [Figure 3] 1 shows the content of various fatty acids in eye (pupil) tissue from mice fed various test diets. [Figure 4] 1 shows the content of various fatty acids in eye (pupil) tissue from mice fed various test diets. [Figure 5] 1 shows the content of various fatty acids in eye (pupil) tissue from mice fed various test diets. [Figure 6] 1 shows the content of various fatty acids in eye (pupil) tissue from mice fed various test diets. [Figure 7] 1 shows the content of various fatty acids in eye (pupil) tissue from mice fed various test diets. [Figure 8] 1 shows the content of various fatty acids in eye (pupil) tissue from mice fed various test diets. [Figure 9] The contents (μg / g tissue) of various fatty acids identified in plasma from mice fed test diets 1, 2, and 3 are shown. [Figure 10] The contents (μg / g tissue) of various fatty acids identified in plasma from mice fed test diets 1, 2, and 3 are shown. [Figure 11] The contents (μg / g tissue) of various fatty acids identified in plasma from mice fed test diets 1, 2, and 3 are shown. [Figure 12] The contents (μg / g tissue) of various fatty acids identified in plasma from mice fed test diets 1, 2, and 3 are shown. [Figure 13] The contents (μg / g tissue) of various fatty acids identified in plasma from mice fed test diets 1, 2, and 3 are shown. [Figure 14] The contents (μg / g tissue) of various fatty acids identified in plasma from mice fed test diets 1, 2, and 3 are shown. [Figure 15] The contents (μg / g tissue) of various fatty acids identified in plasma from mice fed test diets 1, 2, and 3 are shown. [Figure 16] The contents (μg / g tissue) of various fatty acids identified in plasma from mice fed test diets 1, 2, and 3 are shown. [Figure 17] The contents of various fatty acids (mg / g tissue) identified in pupil tissue from Atlantic salmon (Salmo salar) fed five different test diets are shown. [Figure 18] The contents of various fatty acids (mg / g tissue) identified in pupil tissue from Atlantic salmon (Salmo salar) fed five different test diets are shown. [Figure 19]The contents of various fatty acids (mg / g tissue) identified in pupil tissue from Atlantic salmon (Salmo salar) fed five different test diets are shown. [Figure 20] The contents of various fatty acids (mg / g tissue) identified in pupil tissue from Atlantic salmon (Salmo salar) fed five different test diets are shown. [Figure 21] The contents of various fatty acids (mg / g tissue) identified in pupil tissue from Atlantic salmon (Salmo salar) fed five different test diets are shown. [Figure 22] The contents of various fatty acids (mg / g tissue) identified in pupil tissue from Atlantic salmon (Salmo salar) fed five different test diets are shown. [Figure 23] The contents of various fatty acids (mg / g tissue) identified in pupil tissue from Atlantic salmon (Salmo salar) fed five different test diets are shown. [Figure 24] The contents of various fatty acids (mg / g tissue) identified in pupil tissue from Atlantic salmon (Salmo salar) fed five different test diets are shown. [Figure 25] Concentrations of VLCPUFAs identified in brain, eye, and skin tissues from rats fed three different diets: vegetable oil, fish oil, or vegetable oil / fish oil are shown. [Figure 26] Figure 1 shows VLCPUFAs identified in phospholipids from the brain, eye, and skin of Atlantic salmon fed two fish oils at three different concentrations each. [Figure 27] Fluorescence images of ATCC human fibroblasts supplemented with 4 μM lipid composition A in culture medium were shown, in which a scratch wound was created and cell migration into the scratch wound / wound closure was followed over time at various concentrations of lipid composition A. [Figure 28] 1 shows measurements of cell proliferation of a dermal fibroblast cell line after culturing with lipid composition B to approximately 50% confluence. [Figure 29]The effect of Lipid Composition B on the rate of closure of scratch wounds was demonstrated in this study, in which human ATCC dermal fibroblasts were cultured with Lipid Composition B, the cells were scratched, and cell migration was followed at various time points. [Figure 30] Cell migration from salmon skins was demonstrated in this study, where skins were placed in wells containing culture medium, treated with two different concentrations of lipid composition B, and examined for cell migration the following day. [Figure 31] 1 shows the contents of some major VLC fatty acids in skin tissue of mice fed different diets. [Figure 32] 1 shows the contents of some major VLC fatty acids in skin tissue of mice fed different diets. [Figure 33] 1 shows the contents of some major VLC fatty acids in skin tissue of mice fed different diets. [Figure 34] 1 shows the content of major VLC fatty acids in brain tissue of mice fed different diets. [Figure 35] 1 shows the content of major VLC fatty acids in brain tissue of mice fed different diets. [Figure 36] 1 shows the content of major VLC fatty acids in brain tissue of mice fed different diets. [Figure 37] 1 shows the content of major VLC fatty acids in brain tissue of mice fed different diets. [Figure 38] 1 shows the content of major VLC fatty acids in testicular tissue of mice fed different diets. [Figure 39] 1 shows the content of major VLC fatty acids in testicular tissue of mice fed different diets. [Figure 40] 1 shows the content of major VLC fatty acids in testicular tissue of mice fed different diets. [Figure 41] 1 shows the content of major VLC fatty acids in testicular tissue of mice fed different diets. [Figure 42] 1 shows the contents of some major VLC fatty acids in the PL fraction of liver tissue from mice fed different diets. [Figure 43]1 shows the contents of some major VLC fatty acids in the PL fraction of liver tissue from mice fed different diets. [Figure 44] 1 shows the contents of some major VLC fatty acids in the TAG fraction of liver tissue from mice fed different diets. [Figure 45] 1 shows the contents of some major VLC fatty acids in the TAG fraction of liver tissue from mice fed different diets. [Figure 46] 1 shows the contents of some major VLC fatty acids in the TAG fraction of liver tissue from mice fed different diets. [Figure 47] 1 shows the contents of some major VLC fatty acids in the PL fraction of cardiac tissue from mice fed different diets. [Figure 48] 1 shows the contents of some major VLC fatty acids in the PL fraction of cardiac tissue from mice fed different diets. [Figure 49] 1 shows the content of some major VLC fatty acids in the TAG fraction of cardiac tissue from mice fed different diets. [Figure 50] 1 shows the content of some major VLC fatty acids in the TAG fraction of cardiac tissue from mice fed different diets. [Figure 51] 1 shows the content of some major VLC fatty acids in the TAG fraction of cardiac tissue from mice fed different diets. [Figure 52] 1 shows the contents of some major VLC fatty acids in skin tissue of salmon fed different diets. [Figure 53] 1 shows the contents of some major VLC fatty acids in skin tissue of salmon fed different diets. [Figure 54] 1 shows the contents of some major VLC fatty acids in skin tissue of salmon fed different diets. [Figure 55] 1 shows the contents of some major VLC fatty acids in brain tissue of salmon fed different diets. [Figure 56] 1 shows the contents of some major VLC fatty acids in brain tissue of salmon fed different diets. [Figure 57] 1 shows the contents of some major VLC fatty acids in the PL fraction of liver tissue from salmon fed different diets. [Figure 58] 1 shows the contents of some major VLC fatty acids in the PL fraction of liver tissue from salmon fed different diets. [Figure 59] 1 shows the contents of some major VLC fatty acids in the PL fraction of liver tissue from salmon fed different diets. [Figure 60] 1 shows the contents of some major VLC fatty acids in the TAG fraction of liver tissue from salmon fed different diets. [Figure 61] 1 shows the contents of some major VLC fatty acids in the TAG fraction of liver tissue from salmon fed different diets. [Figure 62] 1 shows the contents of some major VLC fatty acids in the TAG fraction of liver tissue from salmon fed different diets. [Figure 63] 1 shows the contents of some major VLC fatty acids in the PL fraction of cardiac tissue from salmon fed different diets. [Figure 64] 1 shows the contents of some major VLC fatty acids in the PL fraction of cardiac tissue from salmon fed different diets. [Figure 65] 1 shows the contents of some major VLC fatty acids in the PL fraction of cardiac tissue from salmon fed different diets. [Figure 66] 1 shows the content of some major VLC fatty acids in the TAG fraction of cardiac tissue from salmon fed different diets. [Figure 67] 1 shows the content of some major VLC fatty acids in the TAG fraction of cardiac tissue from salmon fed different diets. [Figure 68] 1 shows the content of some major VLC fatty acids in the TAG fraction of cardiac tissue from salmon fed different diets. [Figure 69] 1 shows the microstructure of skin from Atlantic salmon showing the different layers. [Figure 70] Measurements of salmon skin microstructure, including mucosal cell counts, epidermal and dermal thickness, as well as an assessment of scale development, are presented. [Figure 71] Shows the progression of the fish's epidermal thickness over time, indicating more mature scales over time. [Figure 72] 1 shows measurements of epidermal thickness in fish fed diets containing various concentrations of VLCPUFA. [Figure 73] 1 shows the content of two VLCMUFAs in skin tissue from mice fed three different test diets. [Figure 74] 1 shows the content of two VLCMUFAs in skin tissue from mice fed three different test diets. [Figure 75] 1 shows the content of two VLCMUFAs in the neutral lipid fraction of skin tissue from mice fed various test diets. [Figure 76] 1 shows the content of two VLCMUFAs in the neutral lipid fraction of skin tissue from mice fed various test diets. [Figure 77] 1 shows the content of C24:1 in plasma from mice fed various test diets. DETAILED DESCRIPTION OF THE INVENTION

[0023] Therefore, some subjects may experience a reduced ability of endogenous synthesis of fatty acids, such as a reduced ability to synthesize longer fatty acids from shorter fatty acids.Therefore, these subjects may have a reduced ability to endogenously synthesize longer fatty acids, such as fatty acids with chain lengths longer than C22, from shorter fatty acids.This reduced ability of endogenous synthesis may occur in specific tissues where these fatty acids are required to maintain optimal health of the subject.This reduced ability may develop with age, or may already exist at a young age.In particular, in the latter case, the reduced ability of endogenous synthesis of very long-chain fatty acids may be caused by genetic disease.Therefore, diseases associated with insufficient endogenous synthesis may be familial or acquired.

[0024] Compositions containing concentrates of C20-C22 omega-3 fatty acids are often recommended for treating or alleviating the symptoms of various diseases, and such fatty acids are found in both pharmaceuticals and nutritional supplements. However, not all subjects respond satisfactorily to treatment with, for example, high concentrations of EPA and DHA. This may be because the subject's body is unable to adequately metabolize or utilize the administered fatty acids to produce longer fatty acids in vivo. An insufficient elongation enzyme system or other enzyme system may be the reason for a reduced response to traditional C20-C22 omega-3 fatty acid treatment. Accordingly, the present applicant has realized that in some instances, it is actually the presence of very long-chain fatty acids (VLCFAs), i.e., VLCFAs with a chain length of at least C24, that provides the beneficial effect. Therefore, it is the VLCFAs that are normally produced in vivo from the administered long-chain fatty acids that provide the beneficial effect, and subjects with reduced enzyme systems, such as the elongation enzyme system, may be unable to produce optimal amounts of beneficial VLCFAs from the administered fatty acids.

[0025] Thus, biologically beneficial PUFAs, including omega-3 fatty acids, are not limited to long-chain fatty acids such as EPA, DHA, and n3DPA. As Breivik and Svensen disclose in International Patent Publication WO 2016 / 182452, only small amounts of VLCn3 are present in natural oils, such as fish oil, and these and other very long-chain fatty acids are typically substantially removed during the production of conventional marine omega-3 concentrates, where the objective is to further concentrate omega-3 fatty acids containing C20-C22 chain lengths. Thus, in conventional omega-3 fatty acid supplements, any very long-chain fatty acids are substantially removed, and such supplements are not suitable for obtaining very long-chain (VLC) omega-3 fatty acids.

[0026] Unfortunately, however, when VLCFAs are isolated from natural oils, such as those from fish, crustaceans, and other organisms, marine oils such as algae, or higher plant oils, the fatty acid chain length of the VLCFAs is often shorter than that of many VLCPUFAs known to exhibit positive biological effects in tissues related to, for example, healthy eyes, male fertility, skin, epidermis, and mucosal tissues (including the lungs and airways), the brain, and the nervous system. Nevertheless, VLCFAs from natural oils have now been found to be beneficial in the treatment of various diseases associated with these tissues, with surprisingly excellent effects, as described below. For example, VLCPUFAs are found in tissues associated with high expression of genes such as ELOVL4.

[0027] The present applicant has discovered that deficiencies in one or more of the elongation enzyme systems described below or other enzyme systems can be alleviated by administering very long-chain fatty acids (VLCFAs) from natural oils, such as marine oils. Without wishing to be limited to a specific explanation, as described in more detail below, various elongation enzyme systems throughout the body (and, in particular, desaturase and β-oxidation reactions) are involved in the in vivo synthesis of numerous fatty acids. This synthesis includes fatty acids with chain lengths up to C22, as well as VLCn3, VLCn6, VLCMUFAs, such as n9 MUFAs, and VLCSFAs, which cause competition between fatty acids for these enzymes. If one or more of these in vivo systems in a subject exhibits reduced efficiency compared to a subject with a normal presentation system, one or more "bottlenecks" in the in vivo synthesis of VLCFAs may be present.

[0028] As a simplified example, consider the fatty acid C22:5n3 (n3DPA) as an intermediate for the in vivo synthesis of C24:5n3 via a reduced efficiency elongation enzyme system. The reduced efficiency of the elongation enzyme system, combined with competition from numerous other fatty acids for the same system, can create a "bottleneck," leading to reduced synthesis of C24:5n3 compared to in vivo synthesis in subjects with a normally efficient elongation enzyme system. To obtain a VLCPUFA with the structural formula C(24+2x):5n3, a reduced (compared to normal) concentration of C24:5n3 must compete x new passages through the "bottleneck" elongation enzyme system. Competition from other fatty acids requiring this same reduced efficiency elongation enzyme system would further reduce the relative concentrations of successive intermediates of the VLC fatty acid C(24+2x):5n3 with each passage compared to subjects with an optimal elongation enzyme system. For illustrative purposes only, if we assume that the efficiency of the elongation enzyme system is reduced by 50% compared to the optimal system in each of the (x + 1) passes from C22:5n3 (n3DPA) through C24:5n3 to VLCPUFA with the structural formula C(24 + 2x):5n3, then the elongated fatty acid with the structure C32:5n3 (x = 4; x + 1 = 5) will be reduced in vivo by (0.5) compared to the optimal ratio. 5 , i.e., 3%. Similarly, if the efficiency were to drop to 80%, the C32:5n3 in the example calculation would be (0.8) compared to the optimal system. 5 , i.e., a ratio of 33% produced in vivo, and if the efficiency were to drop to 20%, the C32:5n3 in the exemplary calculation would be (0.2) compared to the optimal ratio. 5 , i.e., only 0.03% of the optimal ratio. However, by way of example, if the body is supplemented with adequate amounts of C28:5n3, C28:5n3 can serve as a starting material for the in vivo synthesis of C32:5n3 (x=2), so that the same relative ratios compared to the optimal ratio are (0.5), respectively. 2 i.e. 25%, (0.8) 2 i.e. 64%, and (0.2) 2 That would be 4%.

[0029] The above calculations indicate that VLCPUFA compositions, for example as disclosed herein, can significantly improve the in vivo synthesis of biologically active VLCPUFAs in the body compared to traditional long-chain omega-3 concentrates from marine oils.

[0030] For example, if a subject ingests little or no marine omega-3 fatty acids from food, the predominant omega-3 fatty acid in the diet can be expected to be C18:3n3 (ALA), which requires two additional in vivo elongation steps to obtain a fatty acid with the structure C(24+2x):5n3. Furthermore, compared to the three double bonds in C18:3n3, two desaturase steps are required to reach the five double bonds in C(24+2x):5n3. Therefore, if a subject does not ingest marine omega-3 fatty acids, for example, due to allergies, dietary problems, or preferences, LCPUFAs have less opportunity to be available in the relevant tissues for further elongation. Therefore, the subject may be deficient in LCPUFAs. Such subjects may benefit from supplementation with fatty acids from the disclosed compositions containing VLCFAs, even if they have a normal ability to endogenously synthesize VLCFAs. Thus, the present invention provides a composition comprising VLCFAs for use in treating a subject's disease or condition that can be improved by increasing the concentration of VLCFAs in a particular tissue. When the VLCFA composition is administered, the fatty acids are taken up by the target body tissue, where the VLCFAs are responsible for normal tissue function. Thus, the composition is used for preventing or treating disease by administering VLCFAs, and the VLCFAs are transported to the target body tissue where they are responsible for normal tissue function.

[0031] Compositions of VLCFAs according to the present application can be administered to subjects to overcome similar "obstacles" as those described above, particularly in subjects in whom one or more of the body's systems for fatty acid synthesis exhibit reduced efficiency. Even in situations where the VLCFAs administered according to the present application have a shorter chain length and / or contain a different number of double bonds than the VLCPUFAs that confer the desired positive health benefits, surprisingly high improvements in the patient's health can be achieved.

[0032] VLCPUFAs are typically found in specific body tissues, including various epidermal and mucosal tissues, including the eye (eyeball, retina, and meibum from the meibomian glands in the eyelids), sperm and testes, the brain and nervous system, and the lungs and airways. Sebaceous glands are tiny exocrine glands in the skin that secrete an oily or waxy substance called sebum to lubricate and waterproof mammalian skin and hair. In humans, these sebaceous glands are highly abundant on the face, scalp, and all areas of the skin except the palms of the hands. The meibomian glands in the eyelids are a type of sebaceous gland that secrete a special type of sebum into tears. There is growing evidence that sebaceous fatty acids play a role in maintaining the integrity of the skin barrier. As will be seen in this application, mucous membranes are membranes that line various cavities in the body and coat the surfaces of internal organs. Mucous membranes consist of one or more layers of epithelial cells overlying a layer of loose connective tissue. They are primarily endodermal in origin and are continuous with the skin at various body openings, including the eyes, ears, lining of the nose, lining of the mouth, lips, vagina, urethral opening, and anus. Some mucous membranes secrete mucus, a highly concentrated, protective fluid. The function of this membrane is to prevent pathogens and dirt from entering the body and to prevent dehydration of body tissues. Therefore, VLCFAs are normally present in various tissues and function there. Future research will likely provide more knowledge about VLCFA biosynthesis—in which tissues such synthesis occurs and which tissues and bodily functions benefit from VLCFAs.

[0033] As reported in the examples below, VLCPUFAs and VLCMUFAs administered to a subject are taken up by specific body tissues of the subject to provide positive health benefits. More specifically, the administered VLCFAs are transported to specific tissues that play a role in a disease or pathology and are taken up into the tissues where VLCFAs are normally present. Thus, the present invention provides compositions containing VLCFAs for use in treating diseases that can be improved by increasing the concentration of VLCFAs in specific tissues. Specific tissues in which uptake occurs include, for example, the eye (meibum from the meibomian glands in the eyeball, retina, and eyelids), sperm and testes, the brain and nervous system, various epidermal and mucosal tissues / mucosa, including the lungs and airways, tissues of the cardiovascular system, and tissues of the bladder, urinary system, and digestive system.

[0034] Surprisingly, deficiencies in one or more enzyme systems, such as the elongation enzyme system, can be alleviated by the administration of very long-chain fatty acids (VLCFAs). Furthermore, the administered VLCFAs are taken up by the relevant tissues. The term "VLCFA" includes VLCPUFAs, as well as VLCn3, VLCMUFA, VLCSA, and VLCn6. Also as used herein, the term "very long chain fatty acid (or VLCFA)" is intended to mean a fatty acid (FA) having a chain length of more than 22 carbon atoms, i.e., a fatty acid having a chain length of at least C24; the term "very long chain polyunsaturated fatty acid (VLCPUFA)" is intended to mean a polyunsaturated fatty acid (PUFA) having a chain length of more than 22 carbon atoms; the term "very long chain monounsaturated fatty acid (VLCMUFA)" is intended to mean a monounsaturated fatty acid (MUFA) having a chain length of more than 22 carbon atoms; and the term "VLCn3" is intended to refer to polyunsaturated omega-3 fatty acids having a chain length of more than 22 carbon atoms, VLCn3 being known to represent a subgroup of VLCPUFA. Similarly, the term "VLCn6" is intended to refer to polyunsaturated omega-6 fatty acids having a chain length of more than 22 carbon atoms. The term "very long chain saturated fatty acid (VLCSA)" is intended to refer to saturated fatty acids having a chain length of more than 22 carbon atoms. Thus, VLCFAs as used herein have chain lengths of C24 to C40, such as C24 to C38, and preferably C24 to C32. VLCFAs as used herein have chain lengths of C24 to C38, preferably C24 to C40, such as C24 to C32. In some embodiments, VLCFAs as used herein have chain lengths of C26 to C38, preferably C26 to C40, such as C26 to C32. In some embodiments, VLCFAs as used herein are VLCn3 fatty acids having a length of C28 to C32, having more than six double bonds, preferably seven or eight double bonds, and even more preferably having seven or eight double bonds.

[0035] Very long-chain fatty acids confer functional diversity to cells by modifying their chain length and degree of unsaturation. In vivo, fatty acid elongation occurs in three intracellular compartments: the cytosol, mitochondria, and the endoplasmic reticulum (microsomes). In the cytosol, fatty acid elongation is part of de novo lipogenesis and involves acetyl-CoA carboxylase and fatty acid synthase. Fatty acid synthase utilizes acetyl-CoA and malonyl-CoA to elongate fatty acids by two carbons. Microsomal fatty acid elongation represents the primary pathway determining the chain length of saturated, monounsaturated, and polyunsaturated fatty acids in cellular lipids. The overall fatty acid elongation reaction involves a four-enzyme elongation system, which utilizes malonyl-CoA, NADPH, and fatty acyl-CoA as substrates. This pathway includes a class of enzymes involved in the first step of the reaction, the condensation reaction. Seven fatty acid elongase subtypes (ELOVL #1–7) have been identified in the mouse, rat, and human genomes. These enzymes determine the overall fatty acid elongation rate. Furthermore, these enzymes also display different substrate specificities, tissue distributions, and regulations, making them important regulators of cellular lipid composition and specific cellular functions. Methods for measuring elongation enzyme activity, analyzing elongation products, and altering cellular elongation enzyme expression are described by Jump, D., Methods Mol Biol. 2009; 579, 375-389.

[0036] Thus, VLCPUFAs are produced in vivo from shorter fatty acids by fatty acid chain elongation and, for certain fatty acids, by desaturation, saturation, and β- and ω-oxidation reactions, among others.

[0037] As noted above, fatty acid elongation occurs in a complex reaction that results in the addition of two carbon atoms to the carbonyl end of the fatty acid. In the nomenclature used here, this means that ω-3 acids remain ω-3 acids after elongation; i.e., fatty acid C20:5n3 (EPA) can be elongated to C22:5n3 (n3DPA), which can further elongate to C24:5n3, etc. Similar biological reactions occur for ω-6 PUFAs, other PUFAs, MUFAs, and SFAs.

[0038] In addition to elongation, there is also a need for in vivo desaturation reactions, particularly those that result in the creation of carbon / carbon bonds, and for chain shortening steps. For example, the aforementioned C24:5n3 can undergo a Δ6 desaturase reaction to form C24:6n3, generating a double bond, followed by the removal of two carbons via β-oxidation to generate C22:6n3 (DHA). Thus, in the biosynthesis of essential fatty acids, elongases alternate with various desaturases (e.g., Δ6 desaturases) that repeatedly insert ethyl groups and then form a double bond.

[0039] The present inventors have discovered that by utilizing a composition according to the present invention further comprising DHA (C22:6n3), the endogenous synthesis of VLCPUFAs in a subject can be enhanced, reducing or completely eliminating the need for the endogenous synthesis system to synthesize C22:6n3 from C24:6n3. Thus, C24:6n3 and / or its biological precursor C24:5n3 can be more widely utilized for the endogenous synthesis of longer-chain VLCn3s. This means that the VLCPUFA composition according to the present invention can exhibit a surprisingly enhanced effect due to the presence of DHA. In some embodiments, the VLCFA composition can effectively contain n3DPA (C22:5n3), for example, to reduce or eliminate the need for the endogenous synthesis system to synthesize 22:5n3 and / or to improve the ability of the endogenous synthesis system to synthesize 24:5n3 from 22:5n3.

[0040] The various elongase, desaturase, and β-oxidation reactions described above in DHA are also involved in the in vivo synthesis of other fatty acids, including VLCn3, VLCn6, VLCMUFAs such as n9 MUFAs, and VLCSFAs, thereby causing competition between fatty acids for these enzymes.

[0041] As mentioned above, seven VLCPUFA elongation systems / elongases (ELOVL1-7) have been identified in mammals, and each elongase exhibits distinctive substrate specificity and tissue distribution, meaning that deficiency in a particular elongation system can result in negative biological effects that cannot usually be compensated for by other elongation systems.

[0042] For example, diseases such as diabetes affect the expression levels of elongases and desaturases, and this effect on elongases is particularly strong for ELOVL4, an elongase that can elongate VLCPUFAs, VLCMUFAs, and VLCFAs.

[0043] ELOVL4 is also expressed in the thymus, a lymphoid tissue, and there are indications that it plays a role in priming the immune system and signaling molecules.

[0044] ELOVL4 is the most highly expressed elongation enzyme in the retina and produces VLCPUFA and VLCSA, which are important for a healthy eye. ELOVL4 dysfunction can be caused by aging, leading to the development of age-related macular degeneration (AMD), genetic disorders, such as Stargardt-like macular degeneration (STGD3), and metabolic diseases such as diabetes, which can lead to vision loss and retinal inflammation.

[0045] ELOVL4 also plays an important role in skin, producing VLCSA, which is incorporated into ceramides, essential for maintaining the skin's moisture barrier. The stratum corneum is the outermost layer of the epidermis and is composed of dead cells (keratinocytes). These keratinocytes are embedded in a lipid matrix composed of ceramides, cholesterol, and free fatty acids. The stratum corneum functions to form a barrier to protect underlying tissues from infection, dehydration, chemicals, and mechanical stress. During the conversion of live keratinocytes into dead keratinocytes, the cell membrane is replaced by a layer of ceramides, which become covalently bound to the structural protein envelope. This complex is thought to contribute significantly to the skin's barrier function and also plays an important role in maintaining a healthy skin appearance, preventing wrinkles, and protecting against the harmful effects of solar UV rays.

[0046] Endogenous biological systems can be utilized to transfer VLCFAs to ω-hydroxy fatty acids, including (O-acyl) ω-hydroxy FAs (OAHFAs). ELOVL4 appears to be involved in the synthesis of VLC ω-hydroxy fatty acids. Wenmei et al. (Wenmei L, Sandhoff R, Kono M, Zerfas P, Hoffmann V, Ding B CH, Proia RL and Deng CX, "Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4-deficient mice," Int. J. Biol. Sci. 2007 3(2):120-128) found that ceramides containing ω-hydroxy very long chain fatty acids (C28 or greater) are essential components of the epidermal permeability barrier and that ELOVL4 plays an essential role in the formation of very long chain fatty acids that function as building blocks of sphingolipids in the epidermal barrier. According to Wenmei et al., ceramides with fatty acids C28 or greater were absent or significantly reduced in ELOVL4-deficient mice compared with controls. The majority of epidermal VLCFAs, with chain lengths greater than 26 carbon atoms, are ω-hydroxylated and may be saturated or unsaturated (one to two double bonds). Sphingolipids containing these fatty acids are ceramides and glucosylceramides (see Sandhoff (2010) "Very long chain sphingolipids: Tissue expression, function, and synthesis," FEBS Letters 584 1907-192 3, section 1.2, paragraph 1). These molecules form an important part of the protective function of the epidermis.Endogenous biological systems other than the elongase system can be utilized to transfer LCFAs, including VLCMUFAs and VLCFAs, to beneficial (O-acyl)-ω-hydroxy FAs (OAHFAs), cholesteryl esters, ceramides, free fatty acids, phospholipids, sphingomyelin, and wax esters. Compositions according to the present invention containing VLCFAs, albeit in a form other than ω-hydroxy fatty acids, can be used to provide these critically important fatty acids to relevant tissues, particularly skin and mucous membranes / mucosal tissues. This may be particularly important for compositions according to the present invention containing VLCFAs with a chain length of C28 or greater.

[0047] Notably, Wenmei et al. found that ELOVL4-deficient mice had no desire to find and suckle their mother's nipples. The authors suspected that this reflected abnormal neurological behavior due to the absence of ELOVL4 in the brain. The composition of the present invention may offer a method for alleviating this neurological behavior by providing VLCFAs to the brain.

[0048] The following description of ELOVL1-3 and 5-7 is based largely on Sassa and Kihara (2014) "Metabolism of very long chain fatty acids: and Genes and pathophysiology," Biomol Ther 22(2): 83-92. However, future research will likely provide more knowledge regarding the in vivo synthesis of VLCFAs, i.e., in which tissues such synthesis occurs and which tissues and bodily functions benefit from VLCFAs.

[0049] ELOVL1 elongates saturated and monounsaturated C20-C26 acyl-CoAs.

[0050] ELOVL2 elongates both n3 and n6 C20-C22 polyunsaturated acyl-CoAs. ELOVL2 deficiency results in a decrease in VLCPUFAs, including C28:5n6 and C30:5n6, in the testes, potentially reducing spermatogenesis and male fertility. Mammalian testes and sperm contain both n3 and n6 VLCPUFAs.

[0051] ELOVL3 and ELOVL7 are known to elongate both saturated and unsaturated C16–C22 acyl-CoAs.

[0052] ELOVL3 is known to be expressed in the sebaceous glands and hair follicles of the skin, as well as in brown adipose tissue. Studies in mice have shown that ELOVL3 deficiency exhibits accumulation of C20:1 in the skin and is associated with impaired water repellency and sparse hair coverage. By reducing inflammation in hair follicles and through other currently unknown mechanisms, VLCFAs can prevent hair loss and improve overall hair health. Mice lacking ELOVL3 do not suffer from the rapid neonatal death caused by water loss as do ELOVL4 mice (Sandhoff 2010), indicating that the ELOVL3 elongation enzyme system exerts effects distinct from those of ELOVL4.

[0053] ELOVL5 is thought to be essential for the elongation of both n3 and n6 C18-CoA in the liver. ELOVL5 deficiency in mice is associated with hepatic steatosis. ELOVL5 encodes a multispan membrane protein that is highly expressed in the adrenal gland and testis and localized within the endoplasmic reticulum. Mutations in this gene are associated with spinocerebellar ataxia-38 (SCA38), a rare form of ataxia.

[0054] ELOVL6 elongates shorter fatty acids than other ELOVLs and has activity towards C12:0-16:0 acyl-CoA. It is expressed in the cytoplasm of several tissues, including the liver.

[0055] Decreased efficiency in one or more pathways similar to those described above may lead to impairments in the system of in vivo elongation and subsequent β-oxidation and desaturation, reactions that form VLCFAs essential for optimal health. As noted above, these impairments may occur at multiple sites in the complex in vivo synthesis, possibly not all of which are fully understood at this time.

[0056] Therefore, in individual subjects, the ability of endogenous synthesis of VLCFAs, including VLCMUFAs and VLCPUFAs, may be reduced in specific tissues where these fatty acids are needed to maintain optimal health.This reduction in ability may occur with age or may already exist at a young age.In particular, in the latter case, the reduction in the ability of endogenous synthesis of VLCFAs may be caused by a genetic disease.

[0057] Infants require DHA for growing tissues but do not possess fully developed enzyme systems. Applicant recognizes that infants, particularly those not breastfed, may benefit from supplementation with naturally occurring VLCPUFAs for optimal health, in addition to known DHA supplements (e.g., infant formulas, infant medical foods, etc.).

[0058] It is now known that VLCPUFAs from natural oils, as described herein, administered to a subject can be absorbed by the subject's body, and that deficiencies in one or more elongation enzyme systems and / or desaturase enzyme systems and / or β-oxidation systems can be alleviated by administering VLCFAs with chain lengths of C24 to C40, such as C24 to C32 (including VLCn3, VLCn6, VLCMUFA, and VLCSA).Furthermore, as described below and shown in the Examples, supplemented VLCFAs are taken up by various body tissues where they can perform their functions.

[0059] In accordance with the present invention, the various groups of VLCFAs described above can be administered simultaneously, as detailed below, and in certain embodiments. It has further been found that in certain other embodiments, one or more subgroups of VLCFAs, i.e., one or more of VLCn3, VLCn6, VLCMUFA, and VLCSA, with chain lengths of C24 to C32, can be enriched relative to the others to enhance the effectiveness of the VLCFA composition. Because VLCFAs confer functional diversity to cells through alterations in their chain length and degree of unsaturation, the administered composition, in one embodiment, can include a mixture of several different fatty acids of varying lengths and degrees of unsaturation, as disclosed below. Use of such VLCFA-enriched compositions avoids competition between fatty acids for the desired elongation enzymes, desaturases, and enzymes involved in β-oxidation reactions, thereby transferring the desired group of VLCFA "building blocks" to the final VLCFA. As used herein, the term "VLCFA" should be understood to include additional biotransformations of VLCFAs. By way of example, the term includes hydroxy derivatives of VLCFAs produced in vivo, including ω-hydroxy VLCFAs, and further biotransformations of ω-hydroxy VLCFAs.

[0060] In the body, the final VLCFAs may exist in various forms due to their beneficial effects, including but not limited to (O-acyl)-ω-hydroxy FAs, cholesteryl esters, ceramides, free fatty acids, glycerides, phospholipids, sphingomyelin, and wax esters.

[0061] A subject with a deficiency in one or more complex systems for endogenous synthesis may be unable to produce VLCFAs from short-chain and long-chain fatty acids, or may produce them at a lower level than normal. Defects in enzyme systems may include mutations or small deletions in the ELOVL gene, and such defects may be associated with disease. Diseases and disorders that would be improved by increasing the concentration of VLCFAs normally produced by elongation of fatty acids in vivo may be exacerbated in the presence of such defects. Thus, a subject may suffer from a reduced capacity for endogenous synthesis of VLCFAs, i.e., this reduced capacity may be caused, for example, by a low concentration of one of the enzymes involved in synthesis, resulting in a lower and / or slower level of fatty acid synthesis.

[0062] In one aspect, the present invention provides a method for treating a subject by administering a composition comprising VLCFA to the subject. The chain length of the VLCFA is C24-C40, e.g., C24-C38 or C24-C32. Similarly, the present invention provides a composition comprising VLCFA for use in treating a subject. Related treatable diseases and related compositions are disclosed herein. In one embodiment, the disease is associated with a deficiency in one or more endogenous systems and / or a reduced capacity for endogenous synthesis of VLCFA. In one embodiment, the subject has insufficient or abnormal concentrations of VLCFA present in specific tissues that play a role in the disease. Examples demonstrate the uptake of administered VLCFA into various tissues. Furthermore, positive effects of administered VLCFA on skin and other tissues are demonstrated. This novel finding combines the knowledge that VLCFAs are normally present in various tissues with the knowledge of disease-promoting decreases in enzyme activity. See below for a discussion of endogenous and exogenous factors that may influence aging patterns and are also associated with other diseases and conditions. In one embodiment, the present invention provides a composition comprising at least 5% by weight of VLCFAs for use in treating a subject, wherein the composition is administered to treat the subject, and the subject has insufficient or abnormal levels of VLCFAs present in specific tissues that play a role in the disease.

[0063] In one embodiment, the present invention provides a composition comprising at least 5% by weight of VLCFAs for use in treating a subject associated with a deficiency in one or more endogenous elongation enzyme systems and / or associated with a reduced capacity for endogenous synthesis of VLCFAs.

[0064] In one embodiment, the present invention provides a composition comprising at least 5% by weight of VLCFAs for use in treating a disease in a subject, the composition being administered to the subject. In one embodiment, the disease is associated with a deficiency in one or more endogenous elongation enzyme systems and / or a reduced capacity for endogenous synthesis of VLCFAs.

[0065] Thus, in one embodiment, the present invention provides a composition comprising at least 5% by weight of VLCFAs having a chain length of more than 22 carbon atoms and isolated from a natural oil for use in treating a subject, wherein the composition is administered to the subject for treatment of, or for the prevention or treatment of, a disease associated with a deficiency in one or more endogenous elongation enzyme systems and / or a reduced capacity for endogenous synthesis of VLCFAs, and the administered VLCFAs are transported to target body tissues where they are responsible for normal tissue function.

[0066] The term "disease" as used herein refers to any of a disease, condition, disorder, or illness. In particular, the methods and compositions of the present invention are useful for treating diseases associated with or involving specific tissues that typically contain VLCFAs. The associated tissues are selected from the following non-limiting group of tissues: eye (ocular, retinal, or meibum), sperm and testes, brain and nervous system, tissues of the skin, epidermis and mucous membranes / mucosal tissues, including tissues of the lungs and airways, tissues of the cardiovascular system, and tissues of the bladder, urinary system, and digestive system.

[0067] In particular, this treatment may be aimed at supplying VLCFAs to tissues to maintain normal tissue function, and the administered VLCFAs can help maintain good function in tissues where VLCFAs are normally found. For example, adding VLCFAs to various tissues can contribute to inducing, modifying, or improving cell membrane fluidity. Such treatments, including the treatment of diseases by administering the compositions used, include or are related to eye health, male fertility, skin and / or endothelial and mucosal tissues / mucosa, brain and nervous tissue, and cardiovascular diseases. Diseases of skin and / or endothelial and mucosal tissues / mucosa include, for example, diseases of the urinary system and digestive system, as well as lung diseases such as eczema, allergies, and asthma.

[0068] The cardiovascular system is meant to include the organ system consisting of arterial, capillary, and venous components that transport blood through vessels to and from all parts of the body, including the pulmonary and systemic circulation. It therefore includes vascular tissue and myocardial tissue, as well as diseases associated with these. Cardiovascular diseases of the heart and blood vessels, whether congenital or acquired, are all relevant for treatment with the compositions used in the present invention. The most important of these include atherosclerosis, rheumatic heart disease, and vascular inflammation.

[0069] The composition can be used to treat eye diseases adversely affected by a decrease in the amount of VLCFA. These include age-related macular degeneration (AMD), diseases caused by diabetic inflammation of the eye, and dominant Stargardt macular degeneration (STGD3). These are typically caused by mutations in the ELOVL4 gene. The latter, STGD3, usually occurs during childhood or adolescence. Dry eye disease (DED) and meibomianitis are eye-related diseases.

[0070] In AMD, the macula experiences a progressive accumulation of distinctive yellow deposits called drusen (extracellular protein and lipid accumulations). Studies have shown that AMD-associated drusen have a molecular composition similar to that of beta-amyloid (βA) plaques and deposits in other age-related diseases, such as Alzheimer's disease and atherosclerosis. This suggests that similar pathways may be involved in the pathogenesis of AMD and other age-related diseases.

[0071] Diseases associated with the brain and nervous tissue, including diseases of the central nervous system, that may be treated by the compositions of the present invention include at least the following: mental health decline and demyelinating diseases such as multiple sclerosis, Parkinson's disease, schizophrenia, dementia, Alzheimer's disease, cognitive impairment, migraine, seizures, and epilepsy.

[0072] For the treatment of male fertility, the use of VLCFA compositions can enhance sperm function and / or viability or increase the quantity of mature sperm cells.

[0073] Skin and hair-related diseases that can be treated (i.e., preventively treated) by the compositions for use of the present invention to protect against the adverse effects of ultraviolet radiation from the sun on the skin, on hair follicles, and on the decline of hair health, including the risk of hair loss, include at least the following: dry and wrinkled skin, rough, uncomfortable or sensitive skin, wound healing ability, and the adverse effects of ultraviolet radiation from the sun on the skin, on hair follicles, and on the decline of hair health, including the risk of hair loss. Examples of skin diseases and conditions that typically result in rough, uncomfortable skin and may benefit from treatment with the compositions used include eczema, psoriasis, acne, and rosacea (papulopustular rosacea). The compositions or methods of the present invention can be used to normalize the fatty acid composition of tissues such as the skin, for example, by compensating for abnormal sebum fatty acid composition, i.e., by compensating for the decreased concentration of endogenously synthesized very long-chain fatty acids.

[0074] It is known that infants require DHA for tissue development but do not have a fully developed enzyme system. Because DHA is an important fatty acid component of breast milk, it is common to supplement infant formulas and medicated infant nutrition with DHA. The present applicants have recognized that infants also benefit from supplementation with naturally occurring VLCPUFAs for optimal health. In one embodiment, the present invention provides a composition for supplementing infant nutrition, such as weaning foods, infant formulas, and medicated infant nutrition, including parenterally administered nutrients. According to the present invention, infants refer to infants in utero and children under the age of about 2 years, including premature infants and newborns. Therefore, the composition may be administered to pregnant women as part of nutritional supplementation to contribute to fetal development, and may be administered as an oral or parenteral formulation.

[0075] The composition used can also treat diseases associated with a weakened immune system. In particular, fatty acids strengthen the skin, epidermis, and mucosal tissue / mucosa, thereby forming a barrier that protects the underlying tissue from pathogens, including infection, inflammation, dehydration, chemicals, and mechanical stress. It has now been found that administered VLCFAs are also taken up by immune cells. Referring to the Examples section, Example 1 shows that VLCPUFAs contained in mouse diets are taken up by plasma.

[0076] Additionally, inflammation-related diseases and cardiovascular diseases can be treated by the compositions used, particularly for example atherosclerosis and rheumatoid arthritis.

[0077] As used herein, the term "treating" or "treatment" refers to 1) inhibiting a disease, e.g., inhibiting a disease, condition, or disorder in a subject experiencing or exhibiting a pathology or symptom of the disease, condition, or disorder, including preventing the disease (i.e., prophylactic treatment, preventing further progression of the pathology and / or symptom), or 2) alleviating the symptoms of the disease, or 3) ameliorating a disease, e.g., ameliorating a disease, condition, or disorder in a subject experiencing or exhibiting a pathology or symptom of the disease, condition, or disorder (i.e., reversing the pathology and / or symptom). In one particular embodiment, the composition used is for prophylactic treatment, e.g., by providing VLCFAs to tissues, to maintain normal tissue function or improve tissue function. The administered VLCFAs help maintain good function in tissues where VLCFAs are normally known to be present.

[0078] As used herein, the term "subject" encompasses both human and non-human animals, which also include fish, such as farmed fish.

[0079] In particular, the present invention provides methods for treating, and compositions for use in treating, diseases associated with one or more of eye health, male fertility, skin and endothelial tissue and mucosal tissue / mucosa, brain and nervous tissue, and cardiovascular tissue by administering lipid compositions comprising very long chain fatty acids.

[0080] In one embodiment, the present invention provides a composition for use in treating a subject with a deficiency in one or more endogenous elongation enzymes and / or other enzyme systems required for the in vivo synthesis of VLCPUFAs. The elongation enzyme system and / or other enzyme systems may be important to the health of the subject. The method includes administering to the subject a lipid composition containing VLCFAs. VLCFAs may have a direct positive health effect on the subject or may function as "building blocks" for additional long-chain fatty acids that have direct positive health effects. Thus, VLCFA-containing lipid compositions are particularly intended for use in treating subjects with reduced capacity for endogenous synthesis of VLCFAs. Furthermore, VLCFAs may also function, through certain epigenetic effects, to trigger the expression of enzymes in fatty acid elongation or desaturase enzymes.

[0081] In particular, as described above, ELOVL2 deficiency can result in decreased VLCPUFAs, including certain fatty acids C28:5n6 and C30:5n6, in the testes, potentially resulting in decreased spermatogenesis and male fertility. In one embodiment, the present invention provides a composition for use in treating a subject's ability to produce healthy sperm by administering to the subject a composition containing VLCFAs having a chain length of C24 to C32, such as a chain length of C28 to C30. More specifically, the composition is enriched in one or more fatty acids C28:5n6 and C30:5n6. In one embodiment, the composition is enriched in one or more fatty acids C28:5n3, C28:6n3, C28:7n3, C28:8n3, and C30:5n3. See Example 1A below, for mice fed a diet containing VLCPUFAs (Test Diet 2), it is shown that dietary VLCPUFAs are incorporated into the phospholipid fraction of testicular tissue. Elongation enzyme:

[0082] In one embodiment, the composition for use is for the treatment of one or more diseases associated with deficiencies in any of the elongation enzyme systems ELOVL1 to 7. Non-limiting examples of diseases associated with these enzymes are provided above.

[0083] In particular, in one embodiment, the treatment targets a deficiency in the ELOVL4 enzyme system, and the composition can be used to treat diseases associated with this deficiency, such as eye, skin, or diabetic diseases. In another embodiment, the treatment targets a deficiency in the ELOVL2 enzyme system, and the composition can be used to treat diseases associated with this deficiency, such as improving male fertility. In another embodiment, the treatment targets a deficiency in the ELOVL3 enzyme system, and the composition can be used to treat diseases associated with this deficiency, such as skin, hair, and brown adipose tissue diseases. The composition has been found to improve wound healing, particularly because VLCFAs are taken up by cells of skin, endothelial, or mucosal tissues, providing for faster cell division. Thus, wounds are likely to heal more quickly. Thus, deficiencies in ELOVL3 or ELOVL4 that result in diseases or conditions, particularly those related to the skin, can be treated in accordance with the present invention. When taken up by skin cells, such as fibroblasts, the VLCFAs of the composition contribute to a strengthened barrier protecting underlying tissues from infection, dehydration, chemicals, and mechanical stress. In one embodiment, the composition used to treat the skin further comprises VLCMUFA, particularly α-hydroxy VLCMUFA having up to 34 carbon atoms. As seen in A. Poulos (1995) "Very long chain fatty acids in higher animals - a review," Lipids, 30: 1-14, α-hydroxy VLCMUFA having up to 34 carbon atoms are found in epidermal lipids. α-Hydroxy forms of fatty acids may also be synthesized by modifying VLCMUFA from natural oils.

[0084] In one embodiment, the composition is particularly used for treating farmed fish, for example, to strengthen the skin against parasites and mechanical stress, or to promote faster wound healing and increased survival. For example, as disclosed herein, the VLCFA composition for use may be included in fish diets. Referring to the Examples, Examples 1A and 2B show the uptake of VLCPUFA in skin tissue. Examples 5 and 6, which involve fish fed a diet containing VLCPUFA, show a positive effect on wound healing, promoting thicker epidermis and improved scale development.

[0085] In another embodiment, the treatment targets a deficiency in the ELOVL5 enzyme system, and the composition can be used to treat diseases associated with this deficiency, such as liver diseases such as fatty liver or mild forms of fatty liver (non-alcoholic fatty liver disease, NAFLD). Deficiencies in any of the ELOVL1-7 enzyme systems can be compensated for by treatment according to the present invention.

[0086] Furthermore, the present invention provides a composition for improving the VLCFA concentration in tissues where this fatty acid is important for the health and well-being of a subject.The applicant has found that the very long chain fatty acid administered to a subject is absorbed by the tissue in which the fatty acid is normally present.Therefore, the applicant has found that the inability of the body to synthesize related VLCFA and provide the necessary concentration of these in various tissues can be compensated for by administering related VLCFA to the body, for example, so that the VLCFA is actually transported and taken up by related tissues.

[0087] In one embodiment, the subject suffers from a decrease in the effectiveness of one or more of the body's elongation enzyme systems. In one embodiment, the composition for use is intended for individuals who suffer from an age-related decrease in the effectiveness of one or more of the body's elongation enzyme systems. In another embodiment of the invention, the composition for use is intended for individuals who suffer from an inherited decrease in the effectiveness of one or more of the body's elongation enzyme systems. Aging is a complex process characterized by a decline in physiological function and associated with an increased risk of various diseases.

[0088] Genomic methylation is known to be a powerful and reproducible biomarker of biological aging. Methylation patterns allow for quantitative models of aging that can be used across multiple tissues and generally function as a form of "molecular clock." As an example, the human elongation gene ELOVL2 has been reported to exhibit increased methylation with age. The degree of methylation is highly correlated with age and, in a study conducted by Garagnani, P., Bacalini, MG et al. (2012) "Methylation of ELOVL2 gene as a new epigenetic marker of age." Aging Cell, 11, 1132-1134. https: / / doi.org / 10.1111 / acel.12005, shows a roughly "on-off" methylation trend, with methylation ranging from 7% to 91% across the lifespan. The elongase ELOVL2 elongates both n3 and n6 C20-C22 polyunsaturated acyl-CoAs. ELOVL2 is thought to be present in multiple tissues, including the retina, liver, and testis. Given the correlation between increased methylation of the ELOVL2 gene and decreased elongase activity, aging is thought to correspond to ELOVL2 deficiency, resulting in decreased in vivo synthesis of VLCPUFAs. This elongase deficiency, caused by age-related downregulation of ELOVL2 expression, adversely affects biological functions, particularly those related to healthy eyes, male fertility, healthy liver function, and neurological function. For example, even in healthy human individuals with optimal visual acuity, aging leads to a decline in visual function, including age-related declines in rod-driven or scotopic vision and spatial contrast sensitivity. As recognized by the present inventors, the observed age-related loss of rod vision may be related to age-induced decline in the physiological function of elongation genes, including, but not limited to, age-related methylation of the elongation gene ELOVL2, which causes a decrease in ELOVL2 elongation of C20-C22 polyunsaturated fatty acids.As described in more detail below, the inventors have discovered that the effects of age-related decline in the ability of elongation enzymes (including but not limited to ELOVL2) to carry out the in vivo synthesis of VLCFAs can be ameliorated by VLCFA supplementation in accordance with the present disclosure.

[0089] Similarly, in an individual, the effects of age-related decline in elongation enzyme activity in tissues other than the eye, particularly in skin and endothelial tissue, testes, nervous tissue, and liver, can be ameliorated by supplementation with VLCFAs in accordance with the present disclosure. Beneficial effects in an individual include, but are not limited to, improved vision and eye health, improved fertility, improved skin health (including reduced skin wrinkling), improved brain and nervous tissue function, and improved liver function.

[0090] The present inventors believe that the same beneficial effect can be obtained from various elongation enzymes, which improve the age-related decline in enzyme activity, within the group of enzymes involved in the in vivo synthesis of VLCFAs.As mentioned above, VLCFAs in the body are produced in vivo from shorter fatty acids by elongating the fatty acid chain.Furthermore, for certain VLCFAs, other enzyme systems also include enzyme systems for desaturation, saturation, and β-oxidation and ω-oxidation, among others.

[0091] DHA deficiency is known to be associated with aging, and Applicant believes this is also the case for VLCFAs, and discloses how compositions containing VLCFAs, as disclosed herein, can mitigate the consequences of these deficiency-causing aging effects.

[0092] As mentioned above, aging is associated with widespread changes in DNA methylation patterns throughout the genome. These methylation changes can be influenced by both genetic and environmental factors, in addition to aging itself. For example, exogenous environmental factors such as smoking, sun exposure, and obesity are associated with specific changes in DNA methylation patterns. Endogenous factors such as genetic background can also affect aging patterns, such as "baseline" DNA methylation levels. Therapeutic methods and compositions according to the present invention are intended to ameliorate the negative health effects of exogenous environmental and endogenous genetic factors on genomic methylation associated with enzymes for in vivo VLCPUFA synthesis and modification, including, but not limited to, the factors identified above.

[0093] In another embodiment, the composition used is intended for infants, eg, those whose bodies' enzyme systems are not fully developed. Eye Health:

[0094] In one embodiment, the present invention provides a composition for treating a disease related to ocular health in a subject by introducing a lipid composition containing VLCFA into the subject, wherein the subject has a defect in one or more endogenous elongation enzyme systems important for a healthy eye. This may result in a direct positive health effect, or the VLCFA may function as a "building block" for longer fatty acids that provide a positive health effect for a healthy eye. Thus, the present invention provides a composition containing VLCFA for use in treating ocular health in a subject, thereby increasing VLCFA concentrations in specific tissues of the eye. In one embodiment, the disease related to ocular health is selected from the group consisting of macular degeneration (AMD), eye diseases caused by diabetic inflammation, and dominant Stargardt macular degeneration (STGD3).

[0095] In one embodiment, the present invention provides a composition for treating a subject's condition related to dry eye disease or meibomianitis by administering a lipid composition containing VLCFA to the subject, where the subject has a deficiency in one or more endogenous elongation enzyme systems. This may result in a direct positive health effect, or the VLCFA may function as a "building block" for longer fatty acids that provide positive health effects for DED or meibomianitis. However, in one embodiment, the composition, used to treat a subject's condition related to ocular health, negates dry eye disease and meibomianitis. Similar to the inconsistencies discussed on page 3 of Gorusupudi et al.'s publication on AMD, omega-3 fatty acid supplementation in patients with dry eye disease (DED) has produced conflicting results. The present inventors investigated published studies included in Giannaccare et al.'s (2019) meta-analysis on the effectiveness of omega-3 fatty acid supplementation for the treatment of dry eye disease (DED) to examine and possibly identify which types of fatty acids affect dry eye symptoms. The compositions of the omega-3 fatty acid supplements used in the 17 individual studies in the meta-analysis were examined to identify the presence and concentrations of various fatty acids, including very long-chain fatty acids (VLCFAs). In summary, studies based on vegetable oils and marine omega-3 concentrates, which are assumed to be free of VLCFAs or have been substantially removed to concentrate the desired C20-C22 omega-3 acids, tend to have no or limited positive effects on DED. On the other hand, studies based on unenriched fish oil and krill oil and containing concentrates of VLCPUFAs and LCPUFAs with small amounts of VLCPUFAs (e.g., study 15 in the meta-analysis) tend to produce clear positive results for DED patients. This surprising relationship between omega-3 fatty acid source and effect was not understood by the authors of the meta-analysis, nor by the authors of the individual studies included in the meta-study. Giannaccare et al. and the authors of all individual studies remain silent about the presence or effect of VLCPUFAs / VLCn3s. It is also clear that the positive effects of VLCPUFA supplementation on DED symptoms were not clear to the scientific community.For many other indications, meta-studies on DED have focused on the potential benefits of C20-C22 omega-3 fatty acids (EPA + DHA). After meta-analysis and consideration of the compositions used, the applicant concluded that it is the VLCFAs in the composition that contribute to the therapeutic effect, and that more effective benefits for alleviating and treating DED symptoms can be obtained by administering compositions containing VLCFAs, such as VLCn3. Similar effects are expected for other ocular indications. As shown in the attached Examples 1, 2, and 3 below, dietary VLCFAs are taken up by ocular tissues. Therefore, supplemented VLCFAs beneficial to ocular health can reach ocular tissues and exert their functions there. This means that supplementation with VLCFA compositions according to the present invention can be used to treat ocular diseases, as well as diseases and conditions other than DED, such as macular degeneration (AMD), eye diseases caused by diabetic inflammation, and dominant Stargardt macular degeneration (STGD3). Male Fertility:

[0096] In another embodiment, the present invention provides a composition for treating a subject's ability to produce healthy sperm by introducing a lipid composition containing VLCFA into the subject, where the subject has a deficiency in one or more endogenous elongation enzyme systems important for healthy sperm production in men. This can provide a direct positive health benefit, or the VLCFAs function as "building blocks" for longer fatty acids that provide a direct positive effect on healthy sperm production. Accordingly, the present invention provides a composition containing VLCFA for treating a male subject's ability to produce healthy sperm, resulting in increased VLCFA concentrations in the testes and specific sperm-related tissues. This treatment can therefore enhance sperm function and / or viability or increase the quantity of mature sperm cells. Similar misconceptions, such as those presented in publications by Gorusupudi et al. (related to age-related macular degeneration) and Giannaccare et al. (related to dry eye disease), appear to exist in studies conducted to investigate the effects of omega-3 supplements on testicular function and male fertility. According to a review by Esmaeili et al. (Esmaeili, V., Shahverdi, AH, Moghadasian, MH and Alizadeh, AR (2015) "Dietary Fatty Acids Affect Semen Quality: A Review," Andrology 3: 450-461), insufficient DHA concentrations are a major cause of low-quality sperm (p. 453, column 1). In contrast to other PUFA-rich tissues such as the brain and retina, sperm are transported to the epididymis, resulting in continuous excretion of PUFAs (including DHA) from the testes. However, three published studies using DHA supplements appear to have produced conflicting results. i) Two studies utilizing high DHA concentrates derived from fish oil reported positive outcome parameters for male fertility.

[0097] Martinez-Soto JC, Domingo JC, Cordobilla B et al. (Dietary supplementation with docosahexaenoic acid (DHA) improves seminal antioxidant status and decreases sperm DNA fragmentation. Syst Biol Reprod Med. 2016;62(6): 387-395. doi:10.1080 / 19396368.2016.1246623) utilized a 76% DHA concentrate derived from fish oil. The authors found that supplementation with this DHA product induced an increase in omega-3 fatty acids and DHA concentrations in seminal plasma, associated with an increase in total antioxidant capacity and a decrease in sperm DNA. After 10 weeks of supplementation, the percentage of sperm containing DNA damage decreased from 22.0% to 9.3%. In contrast, placebo supplementation with sunflower oil did not induce changes in semen parameters. Marinez et al. appears to have utilized a DHA concentrate similar in fatty acid composition to the commercially available DHA concentrate in Study No. 15 from the Giannaccare et al. publication, where chemical analysis in Applicant's laboratory demonstrated the presence of small amounts of VLCPUFA. See the discussion above related to Giannaccare et al.'s analysis of meta-studies for dry eye disease.

[0098] Gonzalez-Ravina C, Aguirre-Lipperheide M, Pinto F, et al. (Effect of dietary supplementation with a highly pure and concentrated docosahexaenoic acid (DHA) supplement on human sperm function. Reprod Biol. 2018;18(3): 282-288. doi:10.1016 / j.repbio.2018.06.002) similarly utilized a high DHA concentrate (NuaDHA) containing 85% DHA according to the manufacturer (https: / / nuabiological.com / nua-DHA / nua-DHA-composicion-e-ingredientes / ), which, based on the disclosure of this application, exhibits the presence of VLCPUFAs. In their summary, the authors conclude, "Our study supports previous findings highlighting the importance of DHA supplementation as a means of improving sperm quality in asthenozoospermic men." The results of the study provide a clear indication for DHA supplementation in asthenozoospermic patients and seem to suggest that nutritional DHA supplementation at 1 g / day may be particularly beneficial in this infertile population (Discussion section, last paragraph). ii) Study of using DHA-enriched algae oil

[0099] A publication by Conquer et al. (Conquer JA, Martin JB, Tummon I, Watson L, Tekpetey F. Effect of DHA supplementation on DHA status and sperm motility in asthenozoospermic males. Lipids. 2000;35(2):149-154. doi:10.1007 / BF02664764) utilized microalgae oil containing 38.6% DHA.

[0100] The authors note that seminal plasma phospholipid DHA concentrations are lower in asthenozoospermic men than in normozoospermic men. Their study demonstrated that DHA supplementation increased the concentration of this fatty acid in seminal plasma to levels comparable to those previously reported in normozoospermic men. However, although DHA supplementation altered serum and seminal plasma concentrations of this fatty acid, it did not affect sperm DHA concentrations, nor did DHA supplementation affect sperm motility in asthenozoospermic men. According to the authors, the lack of effect on sperm DHA concentrations is likely related to the inability of sperm to incorporate preformed DHA. In this regard, the authors refer to one study in normozoospermic men that "suggested that fish oil (a source of EPA + DHA) supplementation increased DHA concentrations."

[0101] None of the three publications referenced above mentions VLCPUFA. However, based on the disclosures herein, the inventors have determined that, in addition to DHA, supplementation with VLCn3 is crucial for healthy sperm. The first two studies, which yielded positive results in improving sperm quality, utilized DHA concentrates from fish oil, which likely also contain small amounts of VLCn3. The third study, which reported no impact on sperm quality, utilized algal oil, which is not known to contain VLCn3 with the structure useful as a "building block" disclosed herein. The inventors have discovered that, even though the fatty acid DHA may play a role in sperm quality, there is also a need for VLCFA, and such VLCFA can be provided by the compositions of the present invention. As demonstrated by the examples of the present invention, it was surprisingly found that dietary VLCFA compositions can be absorbed and transported to testicular tissue (Example 1A). Thus, supplemented VLCFAs beneficial to male fertility can reach the testes and exert their functions there. This means that supplementation with the VLCFA composition according to the present invention can be used to treat male fertility conditions such as reduced sperm function and / or viability, or reduced quantity of mature sperm cells, such as in individuals who have developed a reduced ability to synthesize VLCFAs in the body. Cognitive Health:

[0102] In yet another embodiment, the present invention provides a composition for use in treating a subject's disease associated with brain and nervous tissue by administering a lipid composition comprising VLCFAs to the subject. For example, the subject may have a deficiency in one or more endogenous elongation enzyme systems important for healthy brain and nervous tissue. This can result in direct positive health benefits, or VLCFAs function as "building blocks" for longer fatty acids that have direct positive benefits for healthy brain and nervous tissue. Thus, the present invention provides a composition comprising VLCFAs for use in treating a disease associated with brain and nervous tissue, resulting in increased VLCFA concentrations in specific tissues.

[0103] Low intakes of the omega-3 fatty acids EPA and DHA have been associated with delayed brain development and increased risk of cognitive decline, including Alzheimer's disease (AD), later in life. However, published studies in this area appear to show conflicting results. The benefits of fish consumption for healthy cognition appear to be well established. Albanese et al. (Dietary fish and meat intake and dementia in Latin America, China, and India: a 10 / 66 Dementia Research Group population-based study, Am J Clin Nutr 2009;90:392-400) found a significant association between a lower prevalence of dementia and higher dietary fish intake in a study of 14,960 individuals aged 65 years or older in China, India, Cuba, the Dominican Republic, Venezuela, Mexico, and Peru, and a meta-analysis combining data from all countries.

[0104] Freund-Levi et al. (ω-3 Fatty Acid Treatment in 174 Patients With Mild to Moderate Alzheimer Disease: OmegAD Study, A Randomized Double-blind Trial, Arch Neurol. 2006;63:1402-1408) found that administration of ω-3 fatty acids had positive results in a subset of patients with very mild AD. Combined with data from epidemiological studies suggesting that the risk of developing AD is reduced by fish intake, Freund-Levi et al. concluded that their study supports the idea that ω-3 fatty acids play a role in the primary prevention of AD but not in the treatment of overt disease. Freund-Levi et al. utilized a supplement containing 2.8 times more DHA than EPA, randomizing patients to receive 1 g capsules four times daily containing 430 mg DHA and 150 mg EPA (applicant's EPAX1050TG) or an isocaloric placebo containing corn oil. EPAX1050TG is a concentrate of DHA derived from fish oil. As explained below, this concentrate also contains some amounts of VLCFAs.

[0105] Kongai et al. (Effects of krill oil containing n-3 polyunsaturated fatty acids in phospholipid form on human brain function: a randomized controlled trial in healthy elderly volunteers, Clinical Interventions in Aging 2013:8 1247-1257) conducted a study in which men aged 61 to 72 years were treated for 12 weeks with medium-chain triglycerides as a placebo, krill oil enriched with n-3 PUFAs incorporated into phosphatidylcholine, or sardine oil enriched with n-3 PUFAs incorporated into triglycerides. Measuring changes in oxyhemoglobin (oxy-Hb) concentrations in the cerebral cortex during memory and calculation tasks, the authors found that the krill oil and sardine oil groups had significantly greater changes in oxy-Hb concentrations than the placebo group during the working memory task at 12 weeks. Krill oil produced the best results, leading the authors to conclude: "This study provides evidence that n-3 PUFAs stimulate cognitive function in older adults. This is particularly true for krill oil, in which the majority of n-3 PUFAs are incorporated into phosphatidylcholine, making it more effective than sardine oil, in which n-3 PUFAs exist as triglycerides."

[0106] Participants in the Kongai et al. study were administered 2 g (eight 0.25 g capsules) of oil per day, which corresponded to 193 mg EPA and 92 mg DHA per day for krill oil (i.e., 96.5 mg EPA and 46 mg DHA per g of krill oil) and 491 mg EPA and 251 mg DHA per day for "sardine oil" (i.e., 245.5 mg EPA and 125.5 mg DHA per g of "sardine oil"). Those skilled in the art will recognize that 245.5 mg / g EPA and 125.5 mg / g DHA (a total of 371 mg / g EPA+DHA) and a total of 460 mg / g of omega-3 acids are significantly higher than those found in natural fish oil, and thus the so-called sardine oil "SO" is a product containing a moderately high concentration of C20-C22 omega-3 fatty acids derived from fish oil. As discussed below, the overlooked minority VLCFAs that are components of krill oil and the moderately concentrated omega-3 fatty acids utilized by Kongai et al. represent fatty acids that may be surprisingly important for maintaining healthy cognitive performance. As mentioned above, Kongai et al. utilized increases in oxy-Hb concentrations in the cerebral cortex as a measure of increases in cerebral blood flow during memory and calculation tasks. Such a procedure has been previously utilized by Jackson et al. ("DHA-rich oil modulates the cerebral hemodynamic response to cognitive tasks in healthy young adults: a near IR spectroscopy pilot study," British Journal of Nutrition (2012), 107, 1093-1098), who found that supplementation with "DHA-rich FO" compared to placebo resulted in significant increases in oxy-Hb and total hemoglobin (Hb) concentrations, indicative of increased cerebral blood flow (CBF) during cognitive tasks. In comparison, no effect on CBF was observed after supplementation with "EPA-rich FO."Because "EPA-enriched FO" contains significant amounts of DHA (see details below), the authors concluded that CBF responses "are modulated only after supplementation with DHA at doses exceeding 200 mg / day." The acronym "FO" is used as an abbreviation for "fish oil," which in this case refers to EPA and DHA derived from fish oil. The therapeutic oil (see page 1094) was purchased from EPAX AS (Aalesund, Norway, i.e., the applicant of the present application) and encapsulated in 500 mg capsules. Based on information from the authors, two 500 mg capsules per day of DHA and EPA-enriched oil contained the following contents of EPA and DHA (concentrated contents relative to natural fish oil): "DHA-enriched FO": 450 mg DHA and 90 mg EPA (i.e., quite close to the 430 mg DHA and 150 mg EPA "EPAX1050TG" utilized by Freund-Levi et al. in their paper mentioned above) and "EPA-enriched FO": 300 mg EPA and 200 mg DHA.

[0107] The scientific publications cited above provide the following important information: Published studies have focused on the effects of the marine omega-3 fatty acids EPA and DHA, but these studies appear to show conflicting results. Fish consumption appears to be beneficial for healthy cognition. Krill oil appears to be beneficial for healthy cognition. Omega-3 concentrates of fish oil with a high DHA content relative to EPA appear to provide positive results. Omega-3 concentrates of fish oils with a higher EPA content relative to DHA appear to produce less positive results than omega-3 concentrates of fish oils with a higher DHA content relative to EPA.

[0108] This latter statement seems somewhat contradictory to the fact that most natural fish oils and krill oils contain more EPA than DHA. Furthermore, the amount of DHA utilized to obtain positive results varies greatly, as follows: Freund-Levi et al. utilized a daily dose of 1.7 g of DHA. Jackson et al. concluded that increases in cerebral blood flow during cognitive tasks were regulated only after DHA supplementation at doses exceeding 200 mg / day. Kongai et al., using an evaluation procedure very similar to that of Jackson et al., obtained significant positive results with only 92 mg / day of DHA supplementation.

[0109] However, in addition to DHA and its possible dominant role in phosphatidylcholine, the present inventors have recognized that a group of fatty acids completely overlooked in these studies, VLCFAs, may have an unexpectedly important role, which may explain the conflicting results in the scientific literature.

[0110] Natural fish oil and krill oil contain small but valuable amounts of VLCFAs. Marine omega-3 fatty acid concentrates, as shown in the scientific publications cited above, focus on enriching the fatty acids EPA (C20:5n3) and DHA (C22:6n3). To obtain these concentrates, components with molecular weights smaller than that of EPA and larger than that of DHA have typically been removed, for example, by molecular / short-path distillation or extraction procedures. Removal of components with molecular weights larger than DHA has been particularly desirable to remove high-molecular-weight impurities, such as oligomers produced by the oxidation / decomposition of marine LCPUFAs, which are easily oxidized and heat-labile. Because unsaturated fatty acids are susceptible to oxidation, components with chain lengths longer than DHA have typically been removed, for example, by distillation, extraction, and similar procedures, in order to comply with pharmacopoeias and voluntary standards imposing upper limits on oligomeric / polymeric oxidation products. Furthermore, these high molecular weight components of marine oils are typically associated with the oil's undesirable unsaponifiable components, including organic contaminants such as cholesterol and brominated diphenyl ethers. Unfortunately, removing the unwanted heavy components also means that a large portion of the valuable VLCFAs derived from the starting natural oil are also removed.

[0111] The removal of VLCFAs is particularly true during the production of concentrates highly enriched in EPA, which also removes a portion of the C22 fraction containing DHA. On the other hand, when producing a DHA concentrate, the inventors of the present invention have found that a significant amount of VLCFAs can remain in the product. For example, when analyzing an existing concentrate containing only 50% DHA, 6% DPA, and 8.5% EPA, the applicants found that the product contained 1.4% C24-C30 VLCn3s [Giannaccare et al., Study No. 15]. When analyzing a sample from a batch of DHA-enriched EPAX1050TG, the applicants found that it contained 0.2% VLCPUFAs and 0.6% VLCMUFAs. The authors of various publications on studies using concentrates from natural oils have not mentioned the presence or effects of VLCPUFAs / VLCn3s from natural oils, and it is clear that the positive effects of VLCPUFAs from natural oils have not been evident to the scientific community.

[0112] Moderately high concentrations of EPA+DHA from natural oils may also contain small amounts of VLCFAs, since these concentrates are typically produced by removing only a more limited portion of the fatty acids than those of DHA.

[0113] When analyzing commercially encapsulated krill oil, applicants found that it contained 0.2% C24-C30 VLCPUFAs and 0.2% VLCMUFAs, although the exact concentration of VLCFAs in commercially available krill oil may vary slightly from these figures, as commercial krill oil production methods appear to be based on several distinct manufacturing processes.

[0114] Thus, in Jackson et al.'s paper, "DHA-enriched FO" contains significantly higher relative concentrations of VLCFAs than "EPA-enriched FO," which appears to have a positive effect on cerebral blood flow during cognitive tasks. Similarly, in Kongai et al.'s paper, although krill oil contains much less EPA and DHA than SO oil, SO's omega-3 fish oil concentrate contains fewer VLCFAs than krill oil, which may contribute to the positive results of krill oil.

[0115] The brains of higher animals, especially myelin, contain VLCFAs. The concentration of VLCFAs in the brain increases with development. The brain and myelin contain saturated, monounsaturated, and polyunsaturated VLCFAs. The normal young human brain contains at least polyunsaturated VLCFAs with up to 38 carbon atoms. α-Hydroxy VLCFAs also occur in the brain. (A. Poulos (1995) "Very long chain fatty acids in higher animals - a review," Lipids, 30: 1-14.)

[0116] According to Steinberg et al., in humans, one specific very-long-chain acyl-CoA synthetases (VLCSs) are pre-expressed in the brain (Steinberg SJ, PA (2000) "Very Long-chain Acyl-CoA Synthetases. Human "Bubblegum" represents a new family of proteins capable of activating very long chain fatty acids," Journal of Biological Chemistry, 275, No. 45, pp. 35162-35169). VLCFA concentrations increase during development, and these VLCFAs are components of complex lipids such as gangliosides, cerebrosides, sulfatides, sphingomyelin, and other phospholipids. Incorporation of VLCFAs into these complex lipids requires activation by VLCSs. Many of these VLCFA-containing lipids are components of myelin membranes in the brain.

[0117] The inventors of the present invention have found that when an individual's capacity for in vivo synthesis of valuable brain VLCFAs or for incorporation of VLCFAs into complex lipids is reduced, for example due to age-related reasons, supplementation with a composition according to the present invention can ameliorate the subsequent adverse effects on the individual's cognitive health.

[0118] This surprising disclosure stands in stark contrast to the state of the art. For example, in a very recent review article on algae for the production of omega-3 acids, the authors make no mention of VLCPUFAs as defined by the present invention (Harwood JL, "Review: Algae: Critical Sources of Very Long-Chain Polyunsaturated Fatty Acids," Biomolecules 2019, 9, 708; doi:10.3390 / biom9110708). Harwood states that "there is abundant evidence that dietary EPA and DHA have beneficial health effects," and that these benefits include improved brain function (Introduction, last paragraph). As shown in the text and tables, there is no mention of the production or use of fatty acids with chain lengths greater than C22.

[0119] In contrast to this view, the inventors of the present invention have found that although the fatty acids DHA and EPA are very important for brain function, there is also a need for VLCFA, and such VLCFA can be provided by the composition of the present invention.As shown by the examples of the present invention, it was very unexpectedly discovered that VLCFA compositions added to feed can be absorbed and transported to the brain (Examples 1A, 2B, and 3).Therefore, supplemented VLCFAs that are beneficial to cognitive health can reach the brain, particularly to be incorporated into myelin, where they can exert their functions.This means that supplementation with the VLCFA composition of the present invention can be used as a treatment to improve the adverse effects on cognitive health, such as in individuals who have developed a reduced ability to synthesize VLCFAs in vivo.

[0120] In a further embodiment, the present invention discloses a composition for use in treating a disease associated with skin and / or endothelial and mucosal tissues / mucosa in a subject by introducing a lipid composition containing VLCFAs into the subject, the subject having a defect in one or more endogenous elongation enzyme systems important for healthy skin and / or endothelial and mucosal tissues. This may result in a direct positive health effect, or the VLCFAs may function as fatty acid "building blocks" that provide a direct positive health effect on healthy skin and / or endothelial and mucosal tissues / mucosa. Thus, the present invention provides a composition containing VLCFAs for use in treating a disease of skin and / or endothelial and mucosal tissues / mucosa, thereby increasing the concentration of VLCFAs in such specific tissues. In one embodiment, the composition used includes the treatment of one or more diseases of skin and / or endothelial and mucosal tissues / mucosa, such as dry skin, eczema, and allergies. See the Examples. Examples 1A and 2B demonstrate the uptake of VLCFAs in skin tissue. Examples 5 and 6 demonstrate the positive effect of VLCFAs on wound healing and in promoting a thicker epidermis and improving scale development. In another embodiment, the compositions used include treatments for lung and airway conditions such as asthma.

[0121] In summary, the compositions used can be used to treat one or more of the following diseases: i) Eye diseases such as macular degeneration (AMD), eye diseases caused by diabetic inflammation, and dominant Stargardt macular degeneration (STGD3). ii) Male fertility disorders such as reduced sperm function and / or viability or reduced quantity of mature sperm cells. iii) Skin and endothelial diseases such as eczema, psoriasis, acne, and rosacea, which involve either dry and wrinkled skin, rough, uncomfortable or sensitive skin, and wound healing capabilities that protect against the adverse effects of the sun's ultraviolet radiation on the skin, adverse effects on hair follicles, and a decline in hair health, including the risk of hair loss. iv) Diseases of mucosal tissue / mucosa such as lung diseases, respiratory diseases including asthma, liver diseases, allergies, diseases of the urinary and digestive systems. v) Mental health decline and diseases of the brain and nervous tissue, including the central nervous system, such as multiple sclerosis, Parkinson's disease, schizophrenia, dementia, Alzheimer's disease, cognitive impairment, migraine, seizures, and demyelinating diseases such as epilepsy. vi) Inflammation-related diseases such as cardiovascular diseases, for example atherosclerosis and rheumatoid arthritis.

[0122] The present invention further provides methods for increasing blood levels of VLCFAs in subjects with reduced endogenous VLCFA synthesis, particularly in subjects with inefficient elongation enzyme systems. The increase or correction of VLCFAs achieved using the methods or compositions of the present invention can be quantified as VLCFA concentrations in blood, such as red blood cells or plasma. The present invention also provides methods for increasing or normalizing VLCFA concentrations in specific tissues, including those affected by the disease being treated. As specifically shown in the Examples, applicants have investigated the uptake of VLCFAs in specific tissues of animals (mice, salmon, and rats) fed diets containing VLCFAs and found that VLCFAs can be quantified as VLCFA concentrations in specific tissues. In one study, salmon and rats were fed marine oil, and applicants analyzed eye, brain, testes, liver, heart, and skin tissues and identified that VLCFAs were incorporated into these tissues. Analysis and quantification of fatty acids present in tissues can be performed according to the art, for example, by in vivo chromatography, often coupled with mass spectrometry, after extraction of the relevant tissues with an appropriate solvent. The examples included in this application provide data demonstrating that the VLCFA content of several tissue types and animal / fish forms can be directly affected by VLCFA supplementation. For example, VLCFAs can be directly incorporated into the diet from administered VLCFA-containing compositions. Previous studies have shown that elongases and desaturases are responsible for the production of VLCPUFAs. However, Applicant has now discovered an alternative method for incorporating these "essential" fatty acids into various tissues. The examples in this application clearly demonstrate that VLCFAs are taken up from the gastrointestinal tract and transported to various tissues, such as the liver, skin, brain, retina, and eye, as well as into plasma. Compared to a control diet containing a similar fatty acid composition without VLCFAs, it clearly demonstrates that the observed increase in VLCFAs in tissues is not solely the result of in vivo synthesis from fatty acids containing shorter fatty acids, such as LCPUFAs. In the studies in the examples, VLCFAs are administered orally by including them in the feed. Alternative routes of administration are described below.

[0123] In one embodiment, the present invention provides a method for increasing VLCFA concentrations or correcting a VLCFA deficiency in a subject's blood, particularly in subjects with a reduced capacity for endogenous VLCFA synthesis. The compositions used achieve a substantial increase in the amount of VLCFA in plasma. The present invention also provides the disclosed methods for correcting an imbalance in the ratio of LCPUFA to VLCPUFA in blood. In one embodiment, using the methods of the present invention, the change in VLCFA in red blood cells, e.g., as a percentage of total fatty acids, is at least 10%, e.g., at least 20%, e.g., a 30-60% increase. Alternatively, quantitative measurements of actual red blood cell VLCFA can be performed. The compositions used achieve a substantial increase in the amount of VLCFA in blood. In one embodiment, the present invention provides a method for increasing VLCFA concentrations or correcting a VLCFA deficiency in a subject's blood, particularly in subjects with a reduced capacity for endogenous VLCFA synthesis. The present invention also provides the disclosed methods for correcting an imbalance in the ratio of LCPUFA to VLCPUFA in blood. The compositions used achieve a substantial increase in the amount of erythrocyte VLCFAs. In one embodiment, the present invention provides a method for increasing VLCFA concentrations or correcting VLCFA deficiencies in a subject's tissue, particularly in subjects with reduced endogenous VLCFA synthesis. The present invention also provides the disclosed method for correcting an imbalance in the ratio of LCFAs to VLCFAs in a tissue, the tissue being selected from the group consisting of epidermal tissues and mucosal / mucosal tissues, including, for example, the eye, retina or meibum, sperm and testes, brain and nervous system, and tissues of the lungs and airways, tissues of the cardiovascular system, and tissues of the bladder, urinary system, and digestive system. Composition:

[0124] The VLCFAs of the lipid composition belong to one or more groups of the fatty acid group VLCPUFA, i.e., belong to any of the VLCMUFAs, including but not limited to VLCn3 and VLCn6, or VLCMUFAn7, VLCMUFAn9, VLCMUFAn11, VLCMUFAn13, and VLCSA. In one embodiment, the lipid composition used in the treatment of the present invention contains at least 5% by weight of VLCFAs. In some (preferred) embodiments, the main component of the VLCFAs is omega-3 fatty acids and / or monounsaturated fatty acids. The fatty acids are obtained or isolated from natural sources, such as marine oils, as described in more detail below.

[0125] The present invention therefore provides a composition comprising at least 5% by weight of VLCFAs for use in treating a disease in a subject, particularly a disease associated with a deficiency in one or more endogenous elongation enzyme systems and / or a reduced capacity for endogenous synthesis of VLCFAs.

[0126] In one embodiment, the lipid composition comprises at least 4.0% by weight very long chain monounsaturated fatty acids and at least 1.0% by weight very long chain polyunsaturated fatty acids, hi another embodiment, the lipid composition comprises at least 1.0% by weight very long chain monounsaturated fatty acids and at least 4.0% by weight very long chain polyunsaturated fatty acids.

[0127] In a further embodiment, the lipid composition comprises at least 8% by weight of VLCMUFAs, such as at least 15% by weight of VLCMUFAs.

[0128] In one embodiment, the lipid composition comprises at least 2% by weight of VLCPUFA, for example at least 5% by weight of VLCPUFA. The VLCPUFA is preferably an omega-3 fatty acid or an omega-6 fatty acid. For some specific applications, such as treating male fertility, the composition comprises an omega-6 VLCPUFA.

[0129] In one embodiment, the lipid composition comprises a total of at least 8%, 10%, 12%, 15%, such as at least 20%, at least 25%, more preferably at least 30% by weight of very long chain fatty acids.

[0130] In one embodiment, the composition comprises a mixture of several different fatty acids of varying lengths and degrees of unsaturation. The composition may comprise at least two different VLCFAs, such as at least three different VLCFAs. In one embodiment, the composition comprises LCPUFAs in addition to VLCFAs, as further disclosed below. For example, the composition comprises at least two LCPUFAs and at least two VLCFAs. Furthermore, the composition may comprise both omega-3 and / or omega-6 VLCPUFAs and VLCMUFAs. In one embodiment, the composition comprises either omega-3 or omega-6 VLCPUFAs with more than six double bonds.

[0131] VLCFAs that may be present in the composition are selected from, but not limited to, any one of the following groups of fatty acids: C24:1n9 (nervonic acid) and other isomers of tetracosenoic acid; C26:1n9 and other isomers of hexacosenoic acid; C28:1n9 and isomers of octacosenoic acid; C30:1, C32:1, C32:1, and longer chain monounsaturated fatty acids; C24:4n3, C24:5n3, C24:6n3, especially C24:5n3; C26:3n3, C26:4n3, C26:5n3, C26:6n3, C26:7n3, especially C26:6n3; C28:3n3, C28:4n3, C28:5n3, C28:6n3, C28:7n3, C28:8n3, especially C28:7n3, C28:8n3; C30:3n3, C30:4n3, C30:5n3, C30:6n3, C30:7n3, C30:8n3; C32:3n3, C32:4n3, C32:5n3, C32:6n3, C32:7n3, C32:8n3, C32:9n3, especially C32:7n3 and C32:8n3; C34:4n3, C34:5n3, C34:6n3, C34:7n3, C34:8n3, C34:9n3, especially C34:7n3 and C34:8n3; C36:4n3, C36:5n3, C36:6n3, C36:7n3, C36:8n3, C36:9n3, especially C36:7n3, C36:8n3, or even longer chain omega-3 fatty acids; C24:2n6, C24:4n6, C24:5n6; C26:4n6, C26:5n6, C26:6n6; C28:4n6, C28:5n6, C28:6n6, C28:7n6; C30:4n6, C30:5n6, C30:6n6, C30:7n6; C32:4n6, C32:5n6, C32:6n6, C32:7n6, C32:8n6; C34:4n6, C34:5n6, C34:6n6, C34:8n6; C36:4n6, C36:5n6, C36:6n6, C34:8n6, or longer chain omega-6 fatty acids; It may also contain VLCSAs C24:0, C26:0, C28:0, C30:0, C32:0 or longer chain saturated fatty acids.

[0132] In certain embodiments, compositions for use in accordance with the present invention may contain any amount of fatty acids having chain lengths longer than C32, including, but not limited to, fatty acids having chain lengths of C34, C36, C38, and C40. Additionally, other positional isomers of the fatty acids listed above, as well as fatty acids having a different number of fatty acids and / or a different number of double bonds than the fatty acids listed above, may be present in the composition.

[0133] The examples show that VLCFAs from the administered composition are incorporated into various tissues and plasma. In one embodiment, the composition used contains any of the incorporated fatty acids shown in the examples. The predominant fatty acids present in the feed are those that increase most significantly in particular tissues. In one particular embodiment, the composition used contains at least one fatty acid selected from the group consisting of C24:5n3, C26:6n3, and C28:8n3.

[0134] In diseases where a particular VLCFA is known to accumulate, that fatty acid should not be included in the composition used for treatment.

[0135] In one embodiment, the composition comprises at least 4% by weight of VLCMUFAs having a chain length of C24 to C32, and in one embodiment, the composition comprises the VLCMUFA C24:1. High concentrations of this fatty acid may be beneficial for the treatment of certain conditions, particularly those related to the brain and nervous tissue. However, for conditions known to result in the accumulation of VLCMUFAs, this fatty acid should not be included in the treatment. In one embodiment, the method comprises administering a lipid composition comprising C24:1 fatty acids in an amount of 4.0-50.0%, e.g., 7.0-40.0%, 8.0-20.0%, e.g., 13.0-20.0%, e.g., about 40%. Furthermore, for the treatment of conditions of the brain and nervous tissue, or for eye health and prenatal and postnatal health, the composition preferably comprises a high concentration of DHA. As shown in Example 2B, with regard to uptake into brain tissue, the fatty acid C28:8 is absorbed into brain tissue to a greater extent than other fatty acids, supporting its potential inclusion in brain health compositions.

[0136] According to the above-described embodiments, the fatty acids of the administered lipid composition may be present in the form of free fatty acids, free fatty acid salts, monoglycerides, diglycerides, triglycerides, ethyl esters, wax esters, (O)-acetylated ω-hydroxy fatty acids (OAHFAs), cholesteryl esters, ceramides, phospholipids, or sphingomyelins, alone or in combination. Alternatively, the fatty acids may be in any form that can be absorbed in the digestive tract or by specific tissues upon topical administration. Preferably, the fatty acids are in the form of free fatty acids, fatty acid salts, ethyl esters, glycerides, or wax esters. In the case of topical administration to deliver a preparation containing a VLCFA composition, the fatty acids are preferably in the form of free fatty acids, fatty acid salts such as glycerides (monoglycerides, diglycerides, or triglycerides, alone or in combination), OAHFAs, cholesteryl esters, ceramides, phospholipids, sphingomyelins, or wax esters; in even more preferred embodiments, the VLCFAs are in the form of wax esters. In one embodiment, for topical administration of the composition, the composition comprises a salt, and therefore at least some of the fatty acids of the composition, such as at least some of the VLCPUFAs, may be in the form of a fatty acid salt.

[0137] In addition to VLCFAs, the lipid composition used may further comprise other fatty acids, such as long-chain polyunsaturated fatty acids. In one embodiment, the composition used comprises at least 5% by weight of one or more LCPUFAs, such as one or more C20-C22 PUFAs. In certain embodiments, the compositions of the present invention comprise at least 10%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% by weight of at least one LCPUFA, such as one or more C20-C22 long-chain PUFAs. In one embodiment, the LCPUFA comprises at least one of EPA, DHA, and omega-3 DPA (n3DPA, all-cis-7,10,13,16,19-docosapentaenoic acid). In further embodiments, the compositions of the present invention comprise at least 5%, at least 10%, or at least 20%, at least 30%, or at least 40% by weight of DHA. In yet another embodiment, the compositions of the present invention comprise at least 5% by weight, at least 8% by weight, or at least 10% by weight of DPA(22:5n3). In some embodiments of the present invention, the EPA:DHA weight ratio of the compositions ranges from about 1:15 to about 10:1, about 1:10 to about 8:1, about 1:8 to about 6:1, about 1:5 to about 5:1, about 1:4 to about 4:1, about 1:3 to about 3:1, or about 1:2 to about 2:1. In one embodiment, the compositions used comprise 5-30% by weight of VLCFAs and 50-90% by weight of LCPUFAs, based on the weight of the composition. In one embodiment, the lipid composition used primarily comprises fatty acids and / or fatty acid derivatives, preferably at least 90.0% by weight, e.g., at least 95.0% by weight, of the lipid composition is fatty acid.

[0138] Furthermore, in some embodiments, the VLCFA-enriched lipid composition further comprises a significant amount of monounsaturated fatty acids. In one embodiment, the composition used comprises at least 5% by weight of one or more LCMUFAs, such as one or more C20-C22 MUFAs. In certain embodiments, the compositions of the present invention comprise at least 10%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% by weight of at least one LCMUFA, such as one or more C20-C22 long-chain MUFAs. In some embodiments, the VLCFA-enriched composition also comprises a quantity of C18 MUFAs, such as C18:1n9 and / or C18:1n7.

[0139] Furthermore, in some embodiments, the VLCFA-enriched lipid composition contains small amounts of saturated fatty acids of all lengths. Overall, the composition contains less than 1.0% saturated fatty acids, more preferably less than 0.5% saturated fatty acids. In particular, the amounts of C16:0 (palmitic acid), C18:0 (stearic acid), and C20:0 (arachidic acid) are low, preferably less than 1.0% in total. Stearic acid is particularly low, preferably less than 1.0%, more preferably less than 0.5%. Furthermore, the amount of very long chain saturated fatty acids (VLCSFA) is low, with the amounts of fatty acids C24:0, C26:0, C28:0, and C30:0 preferably less than 2.0%, more preferably 1.0% by weight, and most preferably less than 0.5% by weight of the fatty acid mixture. However, in other embodiments, for example, when the composition is used to treat skin or mucous membranes, the composition may contain very long chain saturated fatty acids (VLCSAs). For example, the composition comprises more than 1.0%, e.g., more than 2.0%, of a VLCSA, and suitable VLCSAs for inclusion in the composition are, for example, lignoceric acid (C24:0) and cerotic acid (C26:0). In one example, the composition comprises C24:0 and is for the treatment of a skin disorder, particularly papulopustular rosacea.

[0140] In their book chapters on current progress in understanding the importance of VLCPUFA in the retina, Bennett and Anderson (2016) ("Current Progress in Deciphering Importance of VLC-PUFA in the retina," In: C. Bowes Rickman et al. (eds.) and "Retinal Degenerative Diseases," Advances in Experimental Medicine and Biology 854, Springer, Switzerland) state that while the reconstitution of VLCPUFA in the deficient retina would confirm the importance of these fatty acids, "it is not possible to chemically synthesize VLCPUFA in sufficient quantities to allow feeding studies in mice." This is despite the fact that much research has focused on the synthetic production of VLCPUFA using recombinant techniques. For example, Anderson et al. (U.S. Patent Nos. 2009 / 0203787A1, 2012 / 0071558A1, and 2014 / 0100280A1) disclose a recombinant process for producing C28-C38 VLCPUFAs using the ELOVL4 gene. Anderson et al. state (in paragraph 13 of U.S. Patent No. 2009 / 0203787A1) that such a recombinant process is necessary because VLCPUFAs are naturally found only in small amounts in a few organs or in specific animal species, stating, "To obtain even microgram amounts of these VLCPUFAs, they must be extracted from natural sources, such as bovine retina. As a result, research into C28-C38 VLCPUFAs is limited, and no means exist for their commercial production." Furthermore, Raman et al. (U.S. Patent No. 2013 / 0190399) disclose the chemical synthesis of VLCPUFAs. According to Raman, in

[0009] , "Due to the limited enzymatic production rates and limited amounts of VLCPUFAs found in some known biological sources, research into the compounds and their therapeutic utility has been severely limited. Therefore, there is a need for reliable and efficient chemical methods for producing VLCPUFAs..."In

[0010] , Raman states that "traditional sources of VLCPUFAs, such as the retina, brain, and sperm, contain only small amounts of these long-chain fatty acids." Raman et al. began their synthesis with C20-C22 LCPUFAs, such as DHA and DPA. Through chemical synthesis using "saturated zinc weighting agents" or aldehydes, selected LCPUFAs are chemically linked to additional carbon atom chains not present in oils to provide synthetic VLCPUFAs. However, the disclosed chemical reactions between LCPUFAs, such as EPA, DHA, and DPA, which have distinct, unnatural carbon atom chains, via synthetic "weighting agents" result in synthetic VLCPUFAs with the same number of double bonds as the original PUFAs, i.e., five double bonds when starting with EPA and DPA, and six double bonds when starting with DHA. While Raman discloses the synthesis of VLCPUFAs with four, five, and six double bonds, he does not teach how to synthesize all biologically important VLCPUFAs with various numbers of double bonds.

[0141] In nature, the double bonds of fatty acids are all cis-form. In polyunsaturated omega-3 and omega-6 fatty acids, each double bond is separated from the next by a single methylene (-CH2-) group. The all-cis form and the exact location of the double bond in the fatty acid molecule are crucial for the biological transformation and function of the fatty acid. The polyunsaturated fatty acids of the composition used are substantially all cis-form. Due to the function of natural fatty acids in the body, they can be distinguished from chemically synthesized fatty acids, which always contain a certain amount of trans isomers, and from fatty acids in which the double bond position deviates from that of beneficial natural fatty acids, including fatty acid isomers with conjugated double bonds. In complex biological reactions involving VLCPUFAs, including VLCn3 and VLCn6, trans and conjugated isomers are converted along with the natural all-cis isomers to produce molecules that compete with the natural fatty acid isomers and modify their biological effects.

[0142] In some embodiments, the fatty acids of the lipid composition are derived from, i.e., isolated from, natural sources, such as oils from aquatic animals or aquatic plants, natural non-aquatic plant oils, or a combination of these oils. Preferably, the fatty acids are derived from oils or combinations of oils from aquatic animals or aquatic plants, such as marine or freshwater organisms. More preferably, the fatty acids are derived from marine oils, i.e., oils derived from marine animals or marine plants. Marine oils may be selected from the group including, but not limited to, fish oil, mollusk oil, crustacean oil, marine mammal oil, plankton oil, algae oil, and microalgae oil. The fatty acids of the lipid composition may also be derived from a combination of two or more natural sources, as described above. The term "fish oil" encompasses all lipid fractions present in any fish species. "Fish" is a term that includes bony fishes, cartilaginous fishes (such as sharks, rays, and chimaeras), cyclostomes, and jawless fishes. While the choice of raw material is not limited, preferred species of bony fish can be found in the families Engraulidae, Carangidae, Clupeidae, Smeltidae, Salmonidae, and Scombridae. Specific fish species from which such oils may be derived include herring, capelin, anchovy, mackerel, whiting, sand lance, cod, and Alaska pollock. The oil may be derived from the whole fish or from parts of the fish, such as the liver or the remains after removing fillets. In cartilaginous fish species such as sharks, the oil may preferably be obtained from the liver. The term "mollusk oil" includes all lipid fractions present in all species of the phylum Mollusca, including cephalopods such as squid and octopus. As used herein, the term "planktonic oil" refers to all lipid fractions that can be obtained from a diverse population of organisms that inhabit a wide range of waters and are unable to swim against ocean currents, excluding large organisms such as jellyfish. The term "natural vegetable oil" is meant to include oils from algae and microalgae, and also oils from single-cell organisms. Thus, natural vegetable oils may be selected from all oils derived from non-transgenic plants, vegetables, seeds, algae, microalgae, and single-cell organisms.As used herein, the terms "natural oil" and "oil from a natural source" refer to any fatty acid-containing lipid, including, but not limited to, one or more of a glyceride, a phospholipid, a diacylglyceryl ether, a wax ester, a sterol, a sterol ester, a ceramide, or a sphingomyelin, obtained from a natural organism that is not genetically modified (non-GMO).

[0143] The VLCPUFAs in the lipid compositions of the present invention are substantially all-cis. Therefore, the VLCFA compositions used in accordance with the present invention are substantially free of trans fatty acids. The amount of trans isomers is less than 2% by weight, less than 1% by weight, e.g., less than 0.9% by weight, preferably less than 0.5% by weight, and more preferably less than 0.3% by weight of the total fatty acids. In one embodiment, the amount of trans isomers is in the range of 0.1-0.3% by weight of the oil, and in another embodiment, the amount of VLCFA trans isomers is in the range of 0.2-0.5% by weight of the oil. Therefore, as an optimal composition, VLCFAs concentrated from natural oils are more preferable from a biological standpoint. The amount of trans fatty acids in the composition may be measured, particularly by GC-FID, where trans fatty acids appear just before or just after the main peak and are assumed to have the same response factor as all-cis fatty acids.

[0144] The fatty acid composition according to the present invention may typically be obtained and isolated by appropriate procedures for transesterification or hydrolysis of fatty acids from natural oils, followed by a physicochemical refining process. The composition according to the present invention can be produced based on the natural oils and methods disclosed in International Patent Application WO 2016 / 182452, in particular, but is not limited to the starting oils and methods disclosed therein. The fatty acids of the composition used are not chemically synthesized. The fatty acids of the lipid composition are isolated and concentrated from natural sources to obtain concentrated amounts of fatty acids. In one embodiment, the VLCFAs of the composition are unmodified compared to oils isolated from natural sources. Thus, in one embodiment, the chain length of the VLCPUFAs is not altered, and the composition preferably contains natural VLCPUFAs without any elongation step prior to administration. Furthermore, the composition does not contain any lipid-producing cells that secrete or produce VLCFAs. Instead, the composition contains a certain amount of VLCFAs, isolated and concentrated from natural sources using methods suitable for scale-up and production for commercial use. Fatty acids are generally unstable, and the fatty acids used should be prepared by methods using mild conditions (e.g., low temperature and low pressure) to avoid degradation and isomerization, e.g., to avoid converting naturally occurring all-cis fatty acids to trans or conjugated fatty acids.

[0145] The compositions for use may be included in various products and should be formulated according to the intended use. The compositions may be administered by any route, including, but not limited to, oral, intravenous, intramuscular, sublingual, subcutaneous, intrathecal, buccal, rectal, vaginal, ocular, intranasal, inhalation, transdermal, and intradermal. For oral use, the compositions of the present disclosure may be formulated in various forms, such as oral dosage forms, such as tablets, soft or hard capsules, chewable capsules or beads, or fluid compositions. By ingesting a concentrate of the VLCFA fraction of natural oils, subjects will benefit from greater positive effects and much smaller amounts of medications / supplements than by ingesting natural oils such as fish oil, krill oil, algae oil, or calanus oil. At the same time, subjects will benefit from the elimination of calories and potential negative effects from fatty acid and lipid components that do not promote relief and / or healing as disclosed herein.

[0146] In one embodiment of the invention, the administration of the lipid composition is performed by the oral route. In another embodiment of the invention, the administration of the lipid composition is performed by the parenteral route.

[0147] In a preferred embodiment, the lipid composition for parenteral administration is administered with a diluent suitable for parenteral use, which may be a lipid composition utilized for use as parenteral nutrition, i.e., a lipid composition incorporated into a commercially available lipid emulsion formulation, such as an intravenous lipid emulsion used as a source of calories and essential fatty acids, e.g., Intralipid.

[0148] In one embodiment, treatment of diseases associated with lung tissue and airways is carried out by an inhalation device according to the art.

[0149] In one embodiment, treatment of skin and mucosal-associated diseases is carried out, for example, by direct application to the skin and mucosal membranes, by lotions or creams, or through transdermal delivery by patches or suppositories (and similar treatment devices) according to the art. In another more general embodiment, a patch can be used to introduce lipid compositions into the body, and fatty acids can be delivered transdermally through the skin and into the bloodstream. Cosmetics containing compositions for use according to the present invention include lotions and creams, skin hydrating formulations, and sunscreen formulations, which are typically applied directly to the skin. In one embodiment, the composition is applied topically to the eye or eyelids or in or around the eye. For topical application, such preparations may be in the form of, for example, eye drops, ointments, topical skin preparations, lotions, gels, eye mini-tablets, etc.

[0150] In some embodiments of the present disclosure, the composition acts as an active pharmaceutical ingredient (API), and the composition is for use as a medicament. In some embodiments, the fatty acid of the composition is present in a pharmaceutically acceptable amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat, e.g., reduce and / or alleviate, the effects and symptoms of at least one health problem in a subject in need thereof. In some embodiments of the present invention, the composition does not contain an additional active agent. In this embodiment, the composition can be used for the pharmaceutical treatment of a subject, for example, a subject diagnosed with a reduced capacity for endogenous synthesis of VLCFAs. Related diseases are also disclosed above. In another embodiment, the composition according to the present invention is a food supplement, nutritional supplement, or dietary supplement containing VLCFAs. In a related embodiment, the present invention provides a composition selected from the group consisting of Enteral Formulas for Special Medical Use, Foods for Specified Health Uses, Foods for Special Medical Purposes (FSMP), Foods for Special Dietary Use (FSDU), Medical Nutrition, and Medical Food. Such compositions, such as VLCFAs, are particularly suitable for subjects with specific nutrient deficiencies. The compositions are suitable for nutritional management of subjects with specific nutrient needs. Such compositions are typically administered to subjects under medical supervision. The compositions contain appropriate VLCFAs for increasing or correcting VLCFA concentrations in blood or specific tissues, such as subjects diagnosed with reduced endogenous VLCFA synthesis. Thus, the VLCFA compositions are particularly suitable for treating subjects with reduced endogenous VLCFA synthesis. The compositions and methods of the present invention have the ability to correct nutritional deficiencies in such target populations.

[0151] Nutritional supplements according to the present invention may be delivered in any suitable manner, including, but not limited to, oral delivery, dermal delivery, or mucosal delivery such as eye drops. The components of the nutritional supplement may include excipients and / or carriers acceptable for oral ingestion, particularly in the form of an oral delivery vehicle such as a capsule, preferably a gelatin capsule, a liquid, an emulsion, a tablet, or a powder. dose:

[0152] The total daily dose depends on several factors, including the disease the subject has, the severity of the disease, the subject, the composition, the formulation, the form of use, and the mode of administration. In one embodiment, the dose of the lipid composition ranges from about 0.600 g to about 6.0 g. For example, in some embodiments, the total dose of the composition ranges from about 0.8 g to about 4.0 g, about 1.0 g to about 4.0 g, e.g., about 3.0 g, or about 1.0 g to about 2.0 g. When using high-concentration VLCFA compositions, including those containing concentrations significantly greater than 5%, the dose may be much lower, e.g., about 0.06 g to 0.6 g. The composition may be administered in 1 to 10 doses, e.g., 1 to 4 times daily, e.g., once, twice, three times, or four times daily, e.g., once, twice, or three times daily. In one embodiment, the dose is adjusted according to the VLCFA concentration measured in the subject. The composition is preferably administered over an extended period of time, such as 12 to 52 weeks, for example, 24 to 46 weeks. It is expected that adequate VLCFA levels will be reached after 12 to 16 weeks, and the subject will need to continue treatment to maintain these levels. In one embodiment, the subject will need to continue taking the composition for the rest of their life. [Example]

[0153] Example 1: VLCPUFA supplementation in mice - effects on ocular (pupil) and plasma fatty acid composition

[0154] Lipid composition: Lipid mixtures 1 and 2 were prepared from standard anchovy fish oil. The crude fish oil was purified and ethylated, and the ethylated oil was fractionated and concentrated by distillation, urea precipitation, and, in the case of lipid mixture 1, lithium precipitation to obtain the desired composition. This fraction was finally re-esterified to triglycerides by enzymatic reaction with glycerol.

[0155] The fatty acid composition of lipid mixtures 1 and 2 was analyzed on a Scion 436-GC equipped with a split / non-split injection system (1 min for split), using a Restek Rxi-5ms capillary column (30 m length, 0.25 mm internal diameter, 0.25 μM film thickness), a flame ionization detector, and TotalChrom software. Hydrogen was used as the carrier gas. Fatty acid amounts were calculated using C23:0, EPA, and DHA standards. When standards were unavailable, the same response factors were assumed for VLCPUFA as for DHA. The fatty acid compositions of lipid mixtures 1 and 2 are shown in Table 1. [Table 1] Test meals were prepared with the following compositions:

[0156] Test diet 1: 10% fat (5% soybean oil, 5% lard), 17% protein, 5% fiber, 62% carbohydrates, minerals, and vitamins (i.e., standard mouse chow). Test diet 2: 10% fat (5% lipid mix 1 (containing VLCPUFA), 5% lard), 17% protein, 5% fiber, 62% carbohydrates, minerals, and vitamins (i.e., containing VLCPUFA). Test diet 3: 10% fat (5% lipid mix 2, 5% lard), 17% protein, 5% fiber, 62% carbohydrates, minerals, and vitamins (i.e., no VLCPUFA). All test meals were stored at -20°C.

[0157] animal: Mice from the C57 / bl6 strain from Charles River were used in the feeding study. They weighed approximately 25 g. The animals were housed at room temperature in cages with free access to food and water.

[0158] Eye tissue Eight animals from test diet group 1 and nine from test diet groups 2 and 3 were sacrificed 29–33 days after the start of the feeding study. Whole eye pupils, including retinal tissue, were carefully dissected from the animals by trained personnel. Samples were immediately frozen on dry ice and sent to Nofima Laboratories in Norway for phospholipid extraction and isolation. Fatty acid analysis of the prepared samples was performed by EPAx Norway. Total lipids were analyzed using the method described by Folch et al. 1 Lipids were extracted from mouse eye tissues by the method described in. Lipid species were separated using thin-layer chromatography (TLC). The phospholipid fraction was used for fatty acid analysis.

[0159] plasma Blood samples were collected from two mice from each test diet group that were sacrificed 33 days after the start of the feeding study. Samples were taken from the aorta immediately after death. Samples were immediately frozen on dry ice and sent to EPAx Norway for analysis. One mL of a solution containing 0.05157 mg / mL of C23:0 internal standard was added to the test tube, and the solvent was evaporated under a stream of nitrogen. Plasma was then added to the same tube, and the tissue weight was recorded. 3.5 mL of 0.5 M sodium methoxide in methanol was added, and the test tube was then heated in a boiling water bath for 1 hour. After cooling, 5 mL of BCL3 was added, and the test tube was heated in a boiling water bath for 5 minutes. After heating, 0.6 mL of isooctane was added to the test tube, which was then washed with 5 mL of saturated aqueous sodium chloride. The isooctane phase was transferred to a microvial and injected directly into a GC column. Fatty acid analysis of ocular tissue samples:

[0160] Fatty acid analysis was performed on a Perkin Elmer Clarius 680 / 600T GC-MS using an Agilent CP Wax 52 B (CP7713) column. Peak areas from chromatograms obtained from simultaneous single-ion scans of 67, 79, and 91 m / z were used to quantify LC and VLCPUFA fatty acids. The response factor for DHA (relative to C23:0) using this instrument was calculated using standard solutions of known concentrations of DHA and C23:0. Since no standards were available for VLCPUFA, the same response factor as DHA was assumed and used to calculate mg fatty acids / g tissue for VLCPUFA.

[0161] Ocular Results The results of the analysis of PUFAs with 22 or more carbons are shown in Table 2 below, and the results for each fatty acid are shown in Figures 1-8. Figure 1: EPA content (mg / g tissue) in the eyes (pupils) of mice fed test diets 1, 2, and 3. Figure 2: DHA content (mg / g tissue) in the eyes (pupils) of mice fed test diets 1, 2, and 3. Figure 3: DPAn3 content (mg / g tissue) in the eyes (pupils) of mice fed test diets 1, 2, and 3. Figure 4: C24:5n3 content (μg / g tissue) in the eyes (pupils) of mice fed test diets 1, 2, and 3. Figure 5: C24:6n3 content (μg / g tissue) in the eyes (pupils) of mice fed test diets 1, 2, and 3. Figure 6: C26:5n3 content (μg / g tissue) in the eyes (pupils) of mice fed test diets 1, 2, and 3. Figure 7: C26:6n3 content (μg / g tissue) in the eyes (pupils) of mice fed test diets 1, 2, and 3. Figure 8: C28:8n3 content (μg / g tissue) in the eyes (pupils) of mice fed test diets 1, 2, and 3.

[0162] [Table 2] The results of tissue analysis show that the concentrations of EPA, DPA, and DHA in the eye tissue of mice fed test diets 2 and 3 were slightly higher compared to the control (test diet 1). There appeared to be no difference between test diets 2 and 3. These diets contained similar amounts of EPA, DPA, and DHA.

[0163] PL extracts from mice fed test diet 2 (which contains VLCPUFA) show higher concentrations of VLCPUFA than mice fed test diets 1 and 3. This is particularly evident for the C26:6 and C28:8 VLCPUFAs. Plasma results

[0164] The results of the analysis of PUFA fatty acids with 20 or more carbons found in plasma are shown in Table 3. The results for each fatty acid are shown in Figures 9 to 16. Figure 9: EPA content (μg / g tissue) in plasma from mice fed test diets 1, 2, and 3. Figure 10: DHA content (μg / g tissue) in plasma of mice fed test diets 1, 2, and 3 Figure 11: DPAn3 content (μg / g tissue) in plasma of mice fed test diets 1, 2, and 3 Figure 12: C24:5n3 content (μg / g tissue) in plasma of mice fed test diets 1, 2, and 3 Figure 13: C24:6n3 content (μg / g tissue) in plasma of mice fed test diets 1, 2, and 3 Figure 14: C26:5n3 content (μg / g tissue) in plasma of mice fed test diets 1, 2, and 3 Figure 15: C26:6n3 content (μg / g tissue) in plasma of mice fed test diets 1, 2, and 3 Figure 16: C28:8n3 content (μg / g tissue) in plasma of mice fed test diets 1, 2, and 3

[0165] [Table 3] The results show that EPA, DHA, and DPA concentrations tended to be higher in groups fed test diets 2 and 3 than in controls fed standard mouse chow (test diet 1), and this was similar across all samples. This result is expected, as test diets 2 and 3 contain EPA, DHA, and DPA, whereas standard mouse chow does not contain these fatty acids.

[0166] Significantly higher concentrations of VLC fatty acids were found in the plasma of the group fed test diet 2, which contained VLC fatty acids in its diet. This was particularly evident for the fatty acids C26:5, C26:6, and C28:8, which were found in significant concentrations in the group fed test diet 2, but not in detectable amounts in the other two groups. Conclusion:

[0167] Feeding studies in mice have shown that orally administered VLC fatty acids are taken up by ocular tissue: ocular tissues of mice that received VLCPUFAs in their diet contained higher concentrations of VLCPUFAs than controls.

[0168] Very long chain lipid components in ocular tissues are known to play an important role in the retina and retinal function. This example supports the present invention that compositions of VLCFAs can be taken up into tissues and used to treat ocular diseases and generally maintain good ocular health.

[0169] Feeding studies in mice also demonstrated that orally administered VLC fatty acids were incorporated into plasma: plasma from mice fed a diet containing VLCPUFA contained measurable and significantly higher concentrations of VLC fatty acids than controls.

[0170] This example supports the present invention, which demonstrates that VLCFA compositions can be transported into plasma for further distribution in other tissues. Absorption and transport in living organisms are important steps for the role of active compounds in various diseases and for maintaining health in general. Example 1A: Supplementation of VLCPUFA in Mice - Effects on Fatty Acid Composition of Skin, Brain, Testes, Liver, and Heart Lipid composition and test meals

[0171] The same lipid composition, test diets, and animals were used as described in Example 1. As provided in Example 1, Test Diet 2 contains VLCPUFA. Tissue preparation

[0172] Eight animals from test diet group 1 and nine animals from test diet groups 2 and 3 were sacrificed 29–33 days after the start of the feeding study. Tissue samples of skin, brain, testes, liver, and heart were carefully dissected by trained personnel from five animals in each diet group. Samples were immediately frozen on dry ice and sent to Nofima, Norway, for phospholipid extraction and isolation. Fatty acid analysis of the prepared samples was performed by EPAx Norway.

[0173] Total lipids were calculated using the method of Folch et al. 1 Lipids were extracted from tissues by the method described in (1). Lipid species were separated using thin-layer chromatography (TLC). The phospholipid (PL) fraction was used for fatty acid analysis of all tissue samples, and the triglyceride (TAG) fraction was also analyzed for liver and heart samples. Fatty acid analysis of skin, brain, testis, liver, and heart tissue samples:

[0174] Fatty acid analysis was performed on a Perkin Elmer Clarius 680 / 600T GC-MS using an Agilent CP Wax 52 B (CP7713) column. Peak areas from chromatograms obtained from simultaneous single-ion scans of 67, 79, and 91 m / z were used to quantify LC and VLCPUFA fatty acids. The response factor for DHA (relative to C23:0) on this instrument was calculated using standard solutions of known concentrations of DHA and C23:0. Since no standards were available for VLCPUFA, the same response factor as DHA was assumed and used to calculate mg fatty acids / g tissue for VLCPUFA. Skin Results

[0175] The results of the analysis of PUFAs with 22 or more carbon atoms in skin tissue are shown in Table A1 below, and the results for each fatty acid are shown in Figures 31 to 33. Figure 31: C24:5n3 content (mg / g tissue) in skin from mice fed test diets 1, 2, and 3 Figure 32: C26:6n3 content (mg / g tissue) in skin from mice fed test diets 1, 2, and 3 Figure 33: C28:8n3 content (mg / g tissue) in skin from mice fed test diets 1, 2, and 3

[0176] [Table A1] Figures 31 to 33 show the contents of several major VLC fatty acids in the skin tissue of mice fed various diets. Each square in the figure represents the mean ± standard deviation, and the brackets indicate the highest and lowest values ​​in each group. Brain results

[0177] The results of the analysis of PUFAs with 22 or more carbons in brain tissue are shown in Table A2 below, and the results for each fatty acid are shown in Figures 34-37. Figure 34: EPA content (mg / g tissue) in the brain from mice fed test diets 1, 2, and 3 Figure 35: DHA content (mg / g tissue) in the brain from mice fed test diets 1, 2, and 3 Figure 36: C24:5n3 content (mg / g tissue) in brains from mice fed test diets 1, 2, and 3 Figure 37: C28:8n3 content (mg / g tissue) in the brain from mice fed test diets 1, 2, and 3

[0178] [Table A2] Figures 34 to 37 show the contents of major VLC fatty acids in the brain tissue of mice fed various diets. Each square in the figure represents the mean value ± one standard deviation, and the bracketed boxes represent the highest and lowest values ​​in each group. Testicular results

[0179] The results of the analysis of PUFAs with 22 or more carbons in testis tissue are shown in Table A3 below, and the results for each fatty acid are shown in Figures 38-41. Figure 38: C24:5n3 content (mg / g tissue) in testes from mice fed test diets 1, 2, and 3 Figure 39: C24:6n3 content (mg / g tissue) in testes from mice fed test diets 1, 2, and 3 Figure 40: C26:6n3 content (mg / g tissue) in testes from mice fed test diets 1, 2, and 3 Figure 41: C28:8n3 content (mg / g tissue) in testes from mice fed test diets 1, 2, and 3

[0180] [Table A3] Figures 38 to 41 show the contents of major VLC fatty acids in the testis tissue of mice fed various diets. Each square in the figure represents the mean value ± one standard deviation, and the brackets indicate the highest and lowest values ​​in each group. Liver results PL fraction - liver

[0181] The results of the analysis of PUFAs with 22 or more carbons in the PL fraction of liver tissue are shown in Table A4 below, and the results for each fatty acid are shown in Figures 42-43. Figure 42: C24:6n3 content (mg / g tissue) of PL in liver from mice fed test diets 1, 2, and 3 Figure 43: C26:6n3 content (mg / g tissue) of PL in liver from mice fed test diets 1, 2, and 3

[0182] [Table A4] Figures 42 and 43 show the contents of several major VLC fatty acids in the PL fraction of liver tissue from mice fed various diets. Each square in the figure represents the mean ± one standard deviation, and the bracketed boxes represent the highest and lowest values ​​in each group. TAG fraction-liver

[0183] The results of the analysis of PUFAs with 22 or more carbons in the TAG fraction of liver tissue are shown in Table A5 below, and the results for each fatty acid are shown in Figures 44-46. Figure 44: C24:5n3 content (mg / g tissue) in the TAG fraction of liver from mice fed test diets 1, 2, and 3 Figure 45: C26:6n3 content (mg / g tissue) in the TAG fraction of liver from mice fed test diets 1, 2, and 3 Figure 46: C28:8n3 content (mg / g tissue) in the TAG fraction of liver from mice fed test diets 1, 2, and 3

[0184] [Table A5] Figures 44-46 show the contents of several major VLC fatty acids in the TAG fraction of liver tissue from mice fed various diets. Each square in the figure represents the mean ± one standard deviation, and the bracketed boxes represent the highest and lowest values ​​in each group. Cardiac Results PL fraction - heart

[0185] The results of the analysis of PUFAs with 22 or more carbons from the cardiac PL fraction are shown in Table A6 below, and the results for each fatty acid are shown in Figures 47-48. Figure 47: C24:5n3 content (μg / g tissue) in the PL fraction of hearts from mice fed test diets 1, 2, and 3. Figure 48: C26:6n3 content (μg / g tissue) in the PL fraction of hearts from mice fed test diets 1, 2, and 3.

[0186] [Table A6] Figures 47-48 show the contents of several major VLC fatty acids in the PL fraction of cardiac tissue from mice fed various diets. Each square in the figure represents the mean ± one standard deviation, and the bracketed boxes indicate the highest and lowest values ​​in each group. TAG fraction - heart

[0187] The results of the analysis of PUFAs with 22 or more carbons in the TAG fraction of heart tissue are shown in Table A7 below, and the results for each fatty acid are shown in Figures 49-51. Figure 49: C24:5n3 content (mg / g tissue) in the TAG fraction of hearts from mice fed test diets 1, 2, and 3 Figure 50: C26:6n3 content (mg / g tissue) in the TAG fraction of hearts from mice fed test diets 1, 2, and 3 Figure 51: C28:8n3 content (mg / g tissue) in the TAG fraction of hearts from mice fed test diets 1, 2, and 3

[0188] [Table A7] Figures 49-51 show the contents of several major VLC fatty acids in the TAG fraction of cardiac tissue from mice fed various diets. Each square in the figure represents the mean value ± one standard deviation, and the bracketed boxes indicate the highest and lowest values ​​in each group. Conclusion:

[0189] Feeding studies in mice have shown that orally administered VLCPUFAs are taken up by tissues in the skin, brain, testes, liver, and heart. Tissues from mice with VLCPUFAs in their diets had higher VLCPUFA concentrations than controls. Fatty acids were generally incorporated into both polar lipid fractions, including phospholipids, and neutral triglyceride lipid fractions, such as triglycerides, in tissues.

[0190] This example supports the invention that compositions of VLCFAs are taken up by tissues and can be used to treat diseases caused by a lack of VLCFAs and generally to maintain good function of these organs. Example 2: Supplementation of Atlantic salmon diet with VLCPUFA - Effect on ocular fatty acid composition Lipid composition:

[0191] Lipid mixture A was prepared from standard anchovy fish oil. The crude fish oil was purified and ethylated, and the ethylated oil was fractionated and concentrated by distillation, urea precipitation, and lithium precipitation to obtain the desired composition. The VLCPUFA fraction was finally re-esterified to triglycerides by enzymatic reaction with glycerol. Lipid mixture A was in triglyceride form, containing small amounts of monoglycerides and diglycerides. Fatty acid analysis of lipid mixture A was performed on a Perkin-Elmer Clarius 500 equipped with a split / non-split injection system (1 min for non-split), using an Agilent CP Wax 52 B (CP7713) column, a flame ionization detector, and TotalChrom software. Hydrogen was used as the carrier gas. The amount of fatty acids was calculated using an internal standard of 23:0. The response factor for DHA (relative to C23:0) was calculated using standard solutions of known concentrations of EPA, DHA, and C23:0. Since no standards are available for VLCPUFA, the same response factor as for DHA was assumed and used to calculate VLCPUFA in mg / g. The results of the analysis of PUFA fatty acids with 20 or more carbons in lipid mixture A are shown in Table 4.

[0192] [Table 4] Lipid blend A contained 175 mg / g VLCPUFA from fish oil and was used to prepare test meals with various VLCPUFA contents. Test meal:

[0193] Five different test meals were prepared (a, b, c, d, and e). The amounts of ingredients were adjusted to have the same concentration in all test meals. The EPA and DHA contents were also adjusted to the same concentrations. The only difference was the VLCPUFA content in the test meals. The VLCPUFA concentration in the test meals was adjusted by adding various amounts of lipid mixture A to the test meals. The composition of the various test meals is shown in Table 5. [Table 5] Feeding Experiments:

[0194] Farmed Atlantic salmon (Salmo salar) juveniles weighing approximately 5 g were used in the experiment. Five different test diets (a-e) containing 0.00-1.41 wt% VLCPUFA in the diet were prepared. Three aquaria (n = 3) were set up for each test diet. 100 individual fish were placed in each aquaria with recirculated freshwater. The feeding period was 4 weeks. At the end of the feeding experiment, 10 individual fish from each aquaria were terminated as a group, frozen on dry ice, and stored at -40°C before organ dissection. Individual body weights had increased to approximately 11 g. Sample preparation:

[0195] Whole eye pupils were dissected from 10 individuals from each aquarium, balanced to create group samples of 10 fish, frozen in liquid nitrogen, and stored at -40°C for subsequent analysis of lipids. Three samples from each test diet (group samples from three aquaria) were included.

[0196] Total lipids were extracted from salmon eye tissue by the method of Folch et al. Lipid species were separated using thin-layer chromatography (TLC). The phospholipid fraction was used for fatty acid analysis. Fatty acid analysis of ocular tissue:

[0197] Fatty acid analysis was performed on a Perkin Elmer Clarius 680 / 600T GC-MS using an Agilent CP Wax 52 B (CP7713) column. Peak areas from chromatograms obtained from simultaneous single-ion scans of 67, 79, and 91 m / z were used to quantify LC and VLCPUFA fatty acids. The response factor for DHA (relative to C23:0) using this instrument was calculated using standard solutions of known concentrations of DHA and C23:0. Since no standards were available for VLCPUFA, the same response factor as DHA was assumed and used to calculate mg fatty acids / g tissue for VLCPUFA. result:

[0198] The analysis results of PUFAs with 20 or more carbon atoms in salmon eye tissue are shown in Table 6. The results for each fatty acid are shown in Figures 17 to 24. FIG. 17 shows the EPA content (mg / g tissue) in pupil tissue from Atlantic salmon fed test diets a, b, c, d, and e. FIG. 18 shows the DHA content (mg / g tissue) in pupil tissue from Atlantic salmon fed test diets a, b, c, d, and e. FIG. 19 shows DPAn3 content (mg / g tissue) in pupil tissue from Atlantic salmon fed test diets a, b, c, d, and e. FIG. 20 shows the C24:5n3 content (mg / g tissue) in pupil tissue from Atlantic salmon fed test diets a, b, c, d, and e. FIG. 21 shows the C24:6n3 content (mg / g tissue) in pupil tissue from Atlantic salmon fed test diets a, b, c, d, and e. FIG. 22 shows the C26:5n3 content (mg / g tissue) in pupil tissue from Atlantic salmon fed test diets a, b, c, d, and e. FIG. 23 shows the C26:6n3 content (mg / g tissue) in pupil tissue from Atlantic salmon fed test diets a, b, c, d, and e. FIG. 24 shows the C28:8n3 content (mg / g tissue) in pupil tissue from Atlantic salmon fed test diets a, b, c, d, and e.

[0199] [Table 6] These data indicate that VLCPUFA content in the eye tissue (pupil) of Atlantic salmon tends to increase as the concentration of VLCPUFA in the test diet increases. This effect is most pronounced for C26:5, C26:6, and C28:8 in test diets d and e, which have the highest VLCPUFA content compared to test diets that did not contain any VLCPUFA. Conclusion:

[0200] A feeding study in salmon demonstrated that orally administered VLCPUFAs increased the levels of some VLCPUFAs in ocular tissues (pupils). VLCPUFAs are known to play important roles in the human eye, and the present inventors now demonstrate that VLCPUFAs are also involved in the salmon eye. The retina of the eye is known to have high expression of the ELOVL4 protein and a relatively high VLCPUFA content. Previous studies have shown that VLCPUFA concentrations in the eye are determined solely by endogenous elongation and desaturation reactions. This study is the first to demonstrate that VLCPUFAs can be obtained from dietary sources.

[0201] This example supports the invention that compositions of VLCFAs can be used for supplementation and possible treatment and relief of eye-related diseases or general eye health. Example 2B: Supplementation of Atlantic salmon diets with VLCPUFA - Effects on fatty acid composition of skin, brain, heart, and liver

[0202] The same lipid composition and test diet as in Example 2 were used, and the details of the feeding experiment and sample preparation are as shown in Example 2. In all tissues, the PL fraction was analyzed, and in heart and liver tissues, the TAG fraction was also analyzed. Fatty acid analysis of skin, brain, heart, and liver tissues:

[0203] Fatty acid analysis of tissue extracts was performed on a Perkin Elmer Clarius 680 / 600T GC-MS using an Agilent CP Wax 52 B (CP7713) column. Peak areas from chromatograms obtained from simultaneous single-ion scans of 67, 79, and 91 m / z were used to quantify LC and VLCPUFA fatty acids. The response factor for DHA (relative to C23:0) using this instrument was calculated using standard solutions of known concentrations of DHA and C23:0. Because no standards were available for VLCPUFA, the same response factor as for DHA was assumed and used to calculate mg fatty acids / g tissue for VLCPUFA. Skin tissue results:

[0204] The analysis results for PUFAs with 20 or more carbon atoms in salmon skin tissue are shown in Table B1. The results for each fatty acid are shown in Figures 52 to 54. Figure 52 shows the C24:5n3 content (μg / g tissue) in skin tissue from Atlantic salmon fed test diets a, b, c, d, and e. Figure 53 shows the C26:6n3 content (μg / g tissue) in skin tissue from Atlantic salmon fed test diets a, b, c, d, and e. Figure 54 shows the C28:8n3 content (μg / g tissue) in skin tissue from Atlantic salmon fed test diets a, b, c, d, and e.

[0205] [Table B1] Figures 52-54 show the contents of several major VLC fatty acids in the skin tissue of salmon fed various diets. Each square in the figure represents the mean ± standard deviation, and the brackets indicate the highest and lowest values ​​in each group. Results in brain tissue:

[0206] The analysis results for PUFAs with 20 or more carbon atoms in salmon brain tissue are shown in Table B2. The results for each fatty acid are shown in Figures 55-56. Figure 55 shows the C26:6n3 content (μg / g tissue) in brain tissue from Atlantic salmon fed test diets a, b, c, d, and e. Figure 56 shows the C28:8n3 content (μg / g tissue) in brain tissue from Atlantic salmon fed test diets a, b, c, d, and e.

[0207] [Table B2] C28:8n3 fatty acids are observed to be incorporated in significantly greater amounts than other fatty acids. Figures 55-56 show the contents of several major VLC fatty acids in the brain tissue of salmon fed various diets. Each square in the figure represents the mean ± standard deviation, and the brackets indicate the highest and lowest values ​​in each group. Liver tissue results: PL fraction - liver

[0208] The results of the analysis of PUFAs with 20 or more carbons in salmon liver PL tissue are shown in Table B3. The results for selected fatty acids are shown in Figures 57-59. Figure 57 shows the C24:5n3 content (μg / g tissue) in the PL fraction of liver tissue from Atlantic salmon fed test diets a, b, c, d, and e. Figure 58 shows the C26:6n3 content (μg / g tissue) in the PL fraction of liver tissue from Atlantic salmon fed test diets a, b, c, d, and e. Figure 59 shows the C28:8n3 content (μg / g tissue) in the PL fraction of liver tissue from Atlantic salmon fed test diets a, b, c, d, and e.

[0209] [Table B3] In test diet e, it was observed that the fatty acids C24:5 and C26:6 were very clearly incorporated into polar phospholipid liver tissue, which was more highly incorporated than into the TAG fraction of liver tissue as shown in the results in Table B4 below.

[0210] Figures 57-59 show the contents of several major VLC fatty acids in the PL fraction of liver tissue from salmon fed various diets. Each square in the figure represents the mean ± standard deviation, and the brackets indicate the highest and lowest values ​​in each group. TAG fraction-liver

[0211] The results of the analysis of PUFAs with 20 or more carbons in the TAG fraction of salmon liver tissue are shown in Table B4. The results for selected fatty acids are shown in Figures 60-62. Figure 60 shows the C24:5n3 content (μg / g tissue) in the TAG fraction of liver tissue from Atlantic salmon fed test diets a, b, c, d, and e. Figure 61 shows the C26:6n3 content (μg / g tissue) in the TAG fraction of liver tissue from Atlantic salmon fed test diets a, b, c, d, and e. Figure 62 shows the C28:8n3 content (μg / g tissue) in the TAG fraction of liver tissue from Atlantic salmon fed test diets a, b, c, d, and e.

[0212] [Table B4] Figures 60-62 show the contents of several major VLC fatty acids in the TAG fraction of liver tissue from salmon fed various diets. Each square in the figure represents the mean ± standard deviation, and the bracketed boxes represent the highest and lowest values ​​in each group. Heart tissue results: PL fraction - cardiac tissue

[0213] The results of the analysis of PUFAs with 20 or more carbon atoms in the PL tissue of salmon heart tissue are shown in Table B5. The results for each fatty acid are shown in Figures 63 to 65. Figure 63 shows the C24:5n3 content (μg / g tissue) in the PL fraction of heart tissue from Atlantic salmon fed test diets a, b, c, d, and e. Figure 64 shows the C26:6n3 content (μg / g tissue) in the PL fraction of heart tissue from Atlantic salmon fed test diets a, b, c, d, and e. Figure 65 shows the C28:8n3 content (μg / g tissue) in the PL fraction of heart tissue from Atlantic salmon fed test diets a, b, c, d, and e.

[0214] [Table B5] Figures 63-65 show the contents of several major VLC fatty acids in the PL fraction of cardiac tissue from salmon fed various diets. Each square in the figure represents the mean ± standard deviation, and the brackets indicate the highest and lowest values ​​in each group. TAG fraction-heart tissue

[0215] The results of the analysis of PUFAs with 20 or more carbons in the TAG fraction of salmon heart tissue are shown in Table B6. The results for each fatty acid are shown in Figures 66-68. Figure 66 shows the C24:5n3 content (μg / g tissue) in the TAG fraction of heart tissue from Atlantic salmon fed test diets a, b, c, d, and e. Figure 67 shows the C26:6n3 content (μg / g tissue) in the TAG fraction of heart tissue from Atlantic salmon fed test diets a, b, c, d, and e. Figure 68 shows the C28:8n3 content (μg / g tissue) in the TAG fraction of heart tissue from Atlantic salmon fed test diets a, b, c, d, and e.

[0216] [Table B6] Figures 66-68 show the contents of several major VLC fatty acids in the TAG fraction of heart tissue from salmon fed various diets. Each square in the figure represents the mean ± standard deviation, and the brackets indicate the highest and lowest values ​​in each group. Conclusion:

[0217] Salmon feeding studies have shown that orally administered VLCPUFAs result in increased amounts of some VLCPUFAs in the skin, brain, heart, and liver, in addition to ocular uptake as shown in Example 2.

[0218] This study demonstrates that VLCPUFA can be ingested from dietary sources and contribute to their abundance in various tissues, and further demonstrates that differences exist in the extent of incorporation into the neutral and polar lipid fractions of tissues. Example 3: VLCPUFA content in rat brain, eye, and skin tissues - effect of dietary fish oil amount

[0219] Sixteen male Zucker fa / fa rats (Crl:ZUC(Orl)-Lepr fa, from Charles River Laboratories, Italy) were assigned to three experimental groups of six rats with comparable mean body weights for each dietary group. During the 4-week feeding intervention period, rats were fed diets containing either vegetable oil, fish oil, or a 1:1 vegetable oil / fish oil mixture (PO, FO, or a 1:1 PO / FO mixture). Skin, eye, and brain organs were dissected and stored at -80°C for subsequent VLCPUFA analysis. Fish oil contained 0.3–0.5% VLCPUFA. Organ material was available (from Nofima) under the MarOmega3 project managed by Pelagia / EPAx. VLCPUFA in rat tissues

[0220] Total lipids were calculated using the method of Folch et al. 1 Lipids were extracted from rat tissues (brain, eye, and skin) according to the method described by

[14] . Six individual organ samples were analyzed per dietary group. The major lipid classes were separated using thin-layer chromatography (TLC). The phospholipid fraction was used to measure VLCPUFA concentrations in the organs.

[0221] The VLCPUFA concentrations identified in the brain, eye, and skin tissues of rats in the various dietary groups are shown in Figure 25, where VLCPUFA is the percentage of total fatty acids in the PL of the brain, eye, and skin of rats fed three different diets (PO, FO, or a 1:1 PO / FO mixture). Results are expressed as means with standard deviations (SEM), and each value is from three to four rats. Data were analyzed by one-way ANOVA. There were no significant differences between dietary groups within tissues (P < 0.05), but there was a trend toward increased concentrations in the eye with increasing dietary fish oil concentration, consistent with the results found in salmon tissues. Conclusion:

[0222] VLCPUFAs were detected in all tissue samples. In rat eyes, VLCPUFA concentrations tended to increase with increasing dietary fish oil concentrations. Fish oil contained small amounts of VLCPUFAs, ranging from 0.3 to 0.5%. This example demonstrates that VLCPUFA content in important tissues can be influenced by dietary intake. Therefore, the VLCFA composition (concentrate) may be used as a novel VLCFA supplement to treat or alleviate disease or help maintain good health. Example 4: VLCPUFA content in brain, eye, and skin tissues of Atlantic salmon - effect of dietary fish oil level 1. Salmon feeding test

[0223] Experimental fish were fed three dietary concentrations of two different fish oils (fish oil 1 and fish oil 2, both containing approximately 0.3-0.5% VLCPUFA) until they reached approximately double their starting weight of 100 g. Three tanks were prepared for each dietary group. When fish reached 200 g on the various diets, brain, eye, and skin samples were collected, frozen in liquid nitrogen, and stored at -40°C for subsequent analysis of VLCPUFA content in organs. The objective of this study was to examine how increasing dietary concentrations of fish oil affected the VLCPUFA content of the eyes, brain, and skin of Atlantic salmon. VLCPUFA in salmon tissue:

[0224] Total lipids were determined according to Folch et al. 1 The VLCPUFAs were extracted from salmon tissues by the method described above. Five organ samples per tissue were used per tank. The major lipid classes were separated using thin-layer chromatography (TLC). Phospholipid (PL) fractions from three organs were used to measure VLCPUFA concentrations.

[0225] VLCPUFA methyl esters were analyzed on a Scion 436-GC equipped with a split / split injection system (split injection time 1 min) using a Restek Rxi-5ms capillary column (30 m length, 0.25 mm internal diameter, 0.25 μM film thickness), a flame ionization detector, and TotalChrom software. Hydrogen was used as the carrier gas. As shown in Figure 26, the detected concentrations of VLCPUFA (as a percentage of total FAs) differed significantly in the PL of eye tissues from fish fed increasing dietary concentrations of fish oil 1. Figure 26 shows the VLCPUFAs identified in the PL of brain, eye, and skin from Atlantic salmon fed three different concentrations of two fish oils (fish oil 1 and fish oil 2). Results are expressed as means with standard deviations. Data were analyzed by one-way analysis of variance. An asterisk (*) indicates a significant difference (P < 0.05). All organs showed a trend toward increased VLCPUFA concentrations with increasing dietary fish oil dose, although not significant. The VLCPUFA content of the fish oil was low, and presumably higher concentrations of VLCPUFA in the diet would be needed to demonstrate significant effects. Conclusion:

[0226] VLCPUFAs showed a tendency to increase in all salmon tissues tested as fish oil concentrations increased in the diet. Significant differences were present in fish eye tissue. This example demonstrates that VLCPUFA content in important tissues can be influenced by dietary intake. Thus, the VLCFA composition (concentrate) may be used as a novel VLCFA supplement to help treat or alleviate disease or help maintain good health. Example 5: Effects of VLCPUFA on Skin Cells - Effects on Wound Healing and Skin Health in General

[0227] The role of VLCPUFAs was investigated in in vitro wound healing models. Human (1) and salmon skin cell (2) models were used to test synthetic C26:6n3 and VLCPUFA concentrates from fish oil. Lipid composition:

[0228] Lipid composition A: a VLCPUFA concentrate from fish oil, and lipid composition B: C26:6n3, a pure synthetic fatty acid purchased from BOC Sciences (NY, USA) were tested.

[0229] Lipid composition A was prepared from standard anchovy fish oil. The crude fish oil was purified and ethylated, and the ethylated oil was fractionated and concentrated by distillation, urea precipitation, and lithium precipitation to obtain the desired composition. The fractions were finally re-esterified to triglycerides by enzymatic reaction with glycerin.

[0230] VLCPUFA methyl esters were analyzed on a Scion 436-GC equipped with a split / split injection system (split injection time 1 min) using a Restek Rxi-5ms capillary column (30 m length, 0.25 mm internal diameter, 0.25 μM film thickness), flame ionization detection, and TotalChrom software. Hydrogen was used as the carrier gas. The analysis of PUFAs with 20 or more carbons is shown in Table 7. [Table 7] 1. In vitro study of human dermal fibroblasts in cell culture medium

[0231] A commercially available human dermal fibroblast cell line (ATCC PCS-201-012) was prepared as described by Vuong et al. 2 The cells were cultured in Dulbecco's modified Eagle's medium according to the method described by. 1A. Cell culture study using lipid composition A

[0232] ATCC cells were seeded into wells containing 2 mL of culture medium supplemented with 1, 2, and 4 μM lipid composition A. Albumin in PBS served as a control. Scratch wounds were created at 90–100% confluence, and the wells were photographed at several time points up to 24 hours. Cell migration into the scratch / wound closure was examined over time by light microscopy and images were taken. The rate of scratch / wound closure was measured by the % confluence of the scratch opening (higher values ​​indicate better wound closure). 2 μM lipid composition A resulted in significantly higher wound closure rates in terms of % confluence after 24 hours compared to the control (Figure 27). This data demonstrates significantly better "wound healing" with 2 μM lipid composition A compared to the control.

[0233] Figure 27 shows fluorescence images of ATCC human fibroblasts whose culture medium was supplemented with 4 μM lipid composition A. The right panel shows the percentage of confluence in the scratch wound for the various concentration groups. 1B. Cell culture study using lipid composition B

[0234] ATCC cells were cultured for 4 days in medium supplemented with 10 μM and 20 μM lipid composition B before being harvested to measure fatty acid composition. Folch et al. 1 Three replicate samples were generated per group prior to lipid extraction by the method of.

[0235] The content of C26:6n3 in ATCC human skin cells was affected by adding lipid composition B to the culture medium. The results showed that C26:6n3 significantly increased from 0.7% to 5.5% of total FAs (p=0.001 (ANOVA)). Proliferation measurements:

[0236] Proliferation assays measure the density / number of cells in culture by fluorescent staining of nucleic acids. The results show that ATCC cells cultured in medium supplemented with lipid composition B resulted in significantly higher cell numbers compared to the control (Figure 28).

[0237] Figure 28 shows measurements of cell proliferation after culturing to approximately 50% confluence with lipid composition B. Results are presented as mean ± standard deviation (n=4). Asterisks (*) indicate significant differences between groups. Controls 1 and 2 show albumin concentrations equivalent to the albumin concentration in the 20 μM lipid composition B matrix. Scratch wound assay (in vitro wound healing model):

[0238] Cells were seeded (6 replicates) into wells containing 2 mL of culture medium supplemented with 10 μM lipid composition B. Albumin in PBS served as a control. Scratch wounds were created at 90-100% confluence, and the wells were photographed at several time points up to 24 hours. Cell migration into the scratch wound / wound closure was examined and images were taken over time by light microscopy (Figure 29). Figure 29 shows the effect of lipid composition B on the rate of scratch wound closure. Human ATCC dermal fibroblasts were cultured with lipid composition B (10 μM) or control (albumin in PBS) for 24 hours until the monolayer was confluent. Cells were then scratched, and cell migration into the wound was tracked at various time points (0 h to 24 h, as indicated) using Fiji / ImageJ software, as shown in Panel A. Scratch wound size was analyzed as the average percent reduction (calculated from the change in each well's original size at 0 h to the measured size at 24 h) (n=6, results shown in Panel B).

[0239] In the 10 μM lipid composition B group, the diameter size of the wounds was reduced after 24 hours compared to the control, indicating a tendency for an increased wound closure rate. 2. In vitro studies using primary cell cultures of skin cells from Atlantic salmon

[0240] Primary cell cultures of salmon skin cells (keratocytes) were isolated from freshwater Atlantic salmon. The skins were carefully placed in plate wells and cultured at 13°C in growth medium (L-15) supplemented with 10 μM or 20 μM lipid composition B (26:6n3) or 25 ng / mL fibroblast growth factor (FGF) as a positive control. Albumin in PBS was used as a control.

[0241] Supplementation of the culture medium with lipid composition B resulted in an increase in the cellular content of C26:6n3 fatty acids from 0% in the control group to 1.4% in the 20 μM lipid composition B group. Analysis of cell migration from salmon skin

[0242] Two days after isolation of the exine shells, all wells of the different treatments were examined microscopically and pictures were taken.

[0243] Figure 30 shows the results of cell migration from salmon rinds. Salmon rinds were collected from freshwater salmon, placed in wells containing culture medium, and incubated at 13°C without CO2. Cell migration was examined the following day. Treatments included 25 ng / mL FGF (n=7), 10 μM lipid composition B (n=5), and 20 μM lipid composition B (n=5). Albumin in PBS served as a control (n=6). The y-axis scale ranges from no cell migration (0%) to cell migration from all rinds (100%). Various letters indicate significant differences (p≦0.05).

[0244] At the first time point, there was a significant difference between the groups, with 1 μM lipid composition B (vs. albumin control) showing an immediate cell migration effect similar to FGF. At the second time point, the difference was not significant (P=0.061), but the control still showed significantly less cell migration compared to the other groups. At the final time point, there was a significant difference between the groups, with the low dose (10 μM) of lipid composition B having the greatest effect on cell migration. Conclusion:

[0245] Lipid composition A (VLCPUFA concentrate from fish oil) significantly increased cell migration in human fibroblasts at 2 μM compared to control. Lipid composition B (synthetic C26:6n3) significantly increased cell migration in salmon skin cell cultures at 10 μM compared to controls. The same trend was observed with lipid composition B in human fibroblasts.

[0246] This example demonstrates novel effects of VLCPUFAs on two different skin cell models and demonstrates the direct effects of VLCPUFA supplementation on both human and fish skin cells, demonstrating the beneficial health effects of these fatty acids on wound healing.

[0247] Ceramide is the main component of the stratum corneum of the epidermis of human skin.It is known that very long chain lipid components are bound to ceramide.This example supports the present invention in that VLCFA composition can be used for wound healing, inflammatory skin conditions, and other various skin-related diseases in both humans and animals / fish. Example 6: Supplementation of Atlantic salmon diets with VLCPUFA - Evaluation of skin from Atlantic salmon juveniles fed different concentrations of VLCPUFA

[0248] To assess how different dietary concentrations of VLCPUFAs affect skin and scale development in juvenile Atlantic salmon, fish fed no, medium, or high concentrations of a VLCPUFA concentrate diet were analyzed. A VLCPUFA concentrate, referred to as Lipid Mix A as described in Example 2, was included in the fish diet at three different concentrations and fed to three groups of fish. Test diet a: 0% VLCPUFA; Test diet c: 0.71% VLCPUFA, and Test meal e: 1.41% VLCPUFA.

[0249] To follow the changes involved in mesenchymal stem cell recruitment, scale mineralization, and skin maturation, juvenile fish were used when scales were just beginning to develop. Figure 69 shows the ultrastructure of Atlantic salmon skin, showing the various layers including the epidermis with mucus cells, scales, dermis, and underlying adipose tissue and muscle.

[0250] Skin from three different groups of fish fed various concentrations of VLCPUFA was embedded in paraffin, sectioned, and stained with histological stains to visualize cellular structures and mucosal cells (AB / PAS, Figure 70). These sections were analyzed microscopically at 40x magnification using a Leica scanner and ImageScope software. Fifteen fish from each group were used for these analyses. Figure 70 shows the measurements performed in this study, including mucous cell counts, epidermal and dermal thickness, and assessment of scale development.

[0251] Results showed that fish fed 0% VLCPUFA (test diet a) had fewer developed scales compared to fish fed medium (test diet c) and high (test diet e) VLCPUFA concentrations. Fish from the test diet a group also had thinner epidermis, indicating an immature skin structure. Assessments were performed at two different time points. Figure 71 illustrates the development over time in fish from the test diet a group, with more mature scales in the final sample. The first sample (left photo) when the fish was 9.5 g shows mineralized scales (black arrow), while the final sample (right photo) when the fish was 12 g shows developing scales (white arrow).

[0252] Skin thickness measurements indicated that fish fed higher doses of VLCPUFA had thicker skin compared to fish fed lower doses (Figure 72). This result, as seen in other salmon experiments, may be a sign of more mature skin and may be due to more pronounced scale development. As shown in Figure 72, the measured skin thickness significantly increased when juvenile salmon were fed diets supplemented with VLCPUFA. Solid vertical bars indicate skin thickness measured in μm for test diet groups a, c, and e. The diets for these dietary groups contained 0%, 0.71%, and 1.41% VCLPUFA by weight, respectively. Fifteen skin samples were analyzed for each group of salmon. Significant differences are marked with different letters (P < 0.05).

[0253] Further analysis of samples is required to assess the extent of mesenchymal stem cell mineralization and mobilization. Preliminary results indicate that salmon fed VLCPUFAs have better overall scale development and a more mature epidermis compared to fish without VLCPUFAs in their diet. Conclusion:

[0254] A salmon feeding study demonstrated the in vivo effects of maintaining a fish diet containing VLCPUFA on salmon skin. The results indicate that VLCPUFA in the fish diet promotes skin with a thicker epidermis, improved scale development, and a more mature skin structure, and that fish fed VLCPUFA have healthier skin. This study demonstrates that dietary VLCPUFA has a positive effect on skin development in salmon. This example supports the present invention, in that VLCPUFA compositions can be used for supplementation and the possible treatment and alleviation of skin diseases or general skin health. Example 7: Supplementation of mice with VLCFAs - Effects on skin and plasma fatty acid composition Lipid composition:

[0255] A VLCFA concentrate (see Table 8 below) was prepared from standard anchovy fish oil. The crude fish oil was refined and ethylated, and the ethylated oil was fractionated and concentrated by distillation to obtain the desired composition. The fractions were finally re-esterified to triglycerides by enzymatic reaction with glycerin.

[0256] [Table 8] [Table 9] Five different diets were prepared by mixing the above-mentioned VLCFA concentrates (test diets 4 and 5) or two different fish oils (EPAX 3000 TG and EPAX 0460 TGN) manufactured by Epax Norway AS with soybean oil. Mice were given 100 mg / day of the various fatty acid mixtures (by gavage). The dosage of the various fatty acids per mouse per day in the various dietary groups is shown in Table 10 below. [Table 10] All test meals were stored at 0°C. animal:

[0257] Mice from the C57 / BL6 strain from Charles River were used for feeding studies. The animals were housed at room temperature in cages with free access to regular mouse chow and water. Fatty acid analysis

[0258] The fatty acid composition of VLCFA concentrates, tissue extracts, and plasma was analyzed on a Scion 436-GC equipped with a pre-split / non-split injection system (1 min for non-split), a Restek Rxi-5ms capillary column (30 m length, 0.25 mm internal diameter, 0.25 μM film thickness), flame ionization detection, and Compass CDS software. Hydrogen was used as the carrier gas. Fatty acid abundances were calculated using standards for C23:0, EPA, and DHA. For VLCPUFA, the same response factor as DHA was assumed because no standard was available. VLCMUFA was assumed to have the same response factor as C23:0. Tissue preparation

[0259] Eight animals from each test diet were sacrificed 4 weeks after the start of the feeding study. Various tissues were carefully dissected from the animals by trained personnel. Samples were immediately frozen on dry ice and sent to Nofima, Norway, for extraction and separation of lipid classes. Fatty acid analysis of the prepared samples was performed by EPAx Norway. Total lipids were calculated using the method of Folch et al. 1 Lipids were extracted from mouse tissues by the method described in. Lipid species were separated using thin-layer chromatography (TLC). Total extracts and neutral lipid fractions were used for fatty acid analysis. Plasma samples were sent directly to EPAx Norway and prepared for analysis as described in Example 1. Total lipids - skin tissue results

[0260] The results of the analysis of PUFAs with 22 or more carbons are shown in Table 11 below, and the results for selected fatty acids are shown in Figures 73-74. Figure 73: C24:1 content (mg / g tissue) in skin from mice fed test diets 1, 2, and 3 Figure 74: C26:1 content (mg / g tissue) in skin from mice fed test diets 1, 2, and 3 [Table 11] VLCMUFA C24:1 was observed to be greatest in the group fed test meal number 5. Neutral Lipids - Skin Consequences

[0261] The results of the analysis of PUFAs with 22 or more carbon atoms in the skin's neutral lipids are shown in Table 12 below, and the results for each fatty acid are shown in Figures 75-76. Figure 75: C24:1 content (μg / g tissue) in skin from mice fed test diets 1, 2, 3, 4, and 5 Figure 76: C26:1 content (μg / g tissue) in skin from mice fed test diets 1, 2, 3, 4, and 5 [Table 12] Conclusion:

[0262] These results demonstrate that feeding mice a diet containing increased amounts of VLC fatty acids increases the concentrations of both VLCPUFA and VLCMUFA in skin tissue. plasma

[0263] The results of the analysis of PUFAs with 22 or more carbons in plasma are shown in Table 13 below, and the results for each fatty acid are shown in Figure 77. Figure 77: C24:1 content (μg / g plasma) from mice fed test meals 1, 2, 3, 4, and 5 [Table 13] Conclusion:

[0264] These results demonstrate that feeding mice a diet containing increased amounts of VLC fatty acids increases the concentration of VLCMUFA in their plasma. References

[0265] Folch, J., Lees, M., Sloane, Stanley, GH. "A simple method for the isolation and purification of total lipids from animal tissues." J Biol Chem. 1957;226(1):497-509. PMID: 13428781. 2) Vuong TT, Ronning SB, Ahmed TAE, Brathagen K, Host V, Hincke MT, et al. "Processed eggshell membrane powder regulates cellular functions and increases MMP activity important in early wound healing processes." PLoS One. 2018;13(8):e0201975. DOI: 10.1371 / journal.pone.0201975.

Claims

1. 1. A lipid composition comprising a mixture of several different fatty acids for use in treating a disease or condition in a subject, the lipid composition comprising at least 5% by weight of very long chain fatty acids (VLCFAs) having a chain length of at least 24 carbon atoms, the VLCFAs being isolated from oils derived from marine animals or marine plants, the composition comprising both omega-3 and / or omega-6 very long chain polyunsaturated fatty acids (VLCPUFAs) and very long chain monounsaturated fatty acids (VLCMUFAs), and the subject having a deficiency or abnormality in the concentration of VLCFAs present in tissues that play a role in the disease or condition; wherein the composition is administered to the subject for the treatment of a disease or condition caused by a deficiency in one or more elongation enzyme systems and / or desaturase systems and / or β-oxidation systems for the endogenous synthesis of VLCFAs; The lipid composition.

2. The lipid composition of claim 1, wherein the disease or condition is caused by a deficiency in one or more endogenous elongation enzyme systems for the endogenous synthesis of VLCFAs.

3. The lipid composition according to claim 1 or 2, wherein the disease or condition is associated with a deficiency in any one of the elongation enzyme systems ELOVL1 to ELOVL7.

4. 4. The lipid composition of claim 1, wherein the treatment is directed to a tissue selected from the group consisting of ocular tissue, meibomian glands, sperm and testes, brain and nervous system, skin tissue, epidermal tissue, and mucosal tissue including lung and airway tissue, and cardiovascular tissue.

5. The lipid composition according to any one of claims 1 to 4, wherein the use is intended for people suffering from an age-related decrease in the efficiency of one or more of the body's elongation enzyme systems or for people suffering from an inherited decrease in the efficiency of one or more of the body's elongation enzyme systems.

6. The lipid composition according to any one of claims 1 to 5, wherein the use is for preventive treatment such as maintaining normal tissue function or improving tissue function by supplying VLCFA to the tissue.

7. The lipid composition according to any one of claims 1 to 6, wherein the treatment is to increase or normalize VLCFA concentrations in specific tissues involved in the disease being treated.

8. The lipid composition of any one of claims 1 to 7, wherein the disease to be treated is any of: eye diseases; the ability to produce healthy sperm; diseases of skin tissue, endothelial tissue, and mucosal tissue / mucosa; diseases of the brain and nervous tissue; diseases of the cardiovascular system; and inflammatory diseases.

9. The lipid composition according to any one of claims 1 to 8, wherein the disease or condition is selected from the group consisting of: i) an eye disease selected from the group consisting of macular degeneration (AMD), eye diseases caused by diabetic inflammation, and dominant Stargardt macular degeneration (STGD3); ii) male fertility disorders selected from the group consisting of reduced sperm function and / or viability, and reduced mature sperm cell mass; iii) skin and endothelial diseases or conditions selected from the group consisting of eczema, psoriasis, acne, and rosacea, including any of dry and wrinkled skin, rough, uncomfortable or sensitive skin, reduced wound healing ability, inflammation of hair follicles, reduced hair health including risk of hair loss; iv) diseases of mucosal tissue / mucosa selected from the group consisting of respiratory diseases including lung diseases and asthma, liver diseases, allergies, and diseases of the urinary and digestive systems; v) diseases of the brain and nervous tissue, including the central nervous system, selected from the group consisting of poor mental health, demyelinating diseases including multiple sclerosis, Parkinson's disease, schizophrenia, dementia, Alzheimer's disease, cognitive impairment, migraine, seizures, and epilepsy; vi) Inflammation-related diseases as cardiovascular diseases selected from the group consisting of atherosclerosis and rheumatoid arthritis.

10. The lipid composition according to any one of claims 1 to 9, wherein the use is for wound healing and skin treatment.

11. The lipid composition according to any one of claims 1 to 10, wherein the composition comprises any of omega-3 VLCPUFAs and VLCMUFAs having more than six double bonds.

12. The lipid composition according to any one of claims 1 to 11, wherein the composition comprises at least one of C28:7n3 and C28:8n3.

13. The lipid composition according to any one of claims 1 to 12, wherein the composition further comprises very long chain saturated fatty acids.

14. The lipid composition of any one of claims 1 to 13, wherein the composition further comprises DHA (C22:6n3) or n3DPA (C22:5n3).

15. 15. The lipid composition of any one of claims 1 to 14, wherein the composition comprises a total of at least 10% by weight of very long chain monounsaturated fatty acids and very long chain polyunsaturated fatty acids.

16. 16. The lipid composition of any one of claims 1 to 15, wherein the VLCFAs of the composition are isolated from fish oil, mollusk oil, crustacean oil, marine mammal oil, plankton oil, algae oil, or microalgae oil.

17. The lipid composition according to any one of claims 1 to 16, wherein the VLCFAs of the composition are substantially all cis-isomers.

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