Krill oil composition rich in LPC-DHA and LPC-EPA

Abstract

The present invention provides compositions containing phosphatidylcholine-derived compounds bearing omega-3 fatty acids for use in prophylaxis or treatment, particularly when administered systemically. The present invention further relates to improved krill oil compositions enriched with LPC-DHA and LPC-EPA, methods of making, and methods of use for treating neurological and ocular disorders.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 002,425, filed March 31, 2020, and U.S. Provisional Patent Application No. 63 / 113,908, filed November 15, 2020, both of which are incorporated by reference in their entireties herein.

[0002] [Technical field] The present invention provides compositions comprising phosphatidylcholine-derived compounds bearing omega-3 fatty acids for use in prophylaxis or treatment, particularly when administered systemically.The present invention further relates to improved krill oil compositions enriched in LPC-DHA and LPC-EPA, methods of manufacture, and methods of use for treating neurological and ocular disorders.

[0003] BACKGROUND OF THEINVENTION The brain has very high concentrations of the omega-3 fatty acid DHA, which plays an important role in brain development and function. Deficiency of brain DHA has been associated with several neurological disorders, including Alzheimer's disease, Parkinson's disease, schizophrenia, and depression (1-3). DHA has also been shown to be protective against complications of stroke (4) and traumatic brain injury (5). Although beneficial effects of dietary DHA have been reported in various animal models of neurodegenerative diseases (6-9), clinical trials with currently available supplements (fish oil, algal oil, krill oil, ethyl esters) have provided disappointing results in patients suffering from Alzheimer's disease (10-12), Huntington's disease (13), and schizophrenia (14). The failure of these trials has been proposed to be due to the inability of the supplements to enrich the brain for DHA, since DHA from these supplements is absorbed primarily in the form of triacylglycerol (TAG), while transporters at the BBB require the lysophosphatidylcholine form of DHA (LPC-DHA) (15). Indeed, it has recently been demonstrated that feeding LPC-form DHA increased brain DHA by 100% in normal adult mice, while the same dose of free DHA had no effect (16). It has also been shown that LPC-DHA is superior to either PC-DHA or TAG-DHA in enriching DHA in rat brains (17). In contrast to DHA, the brain has very little EPA, and EPA-rich supplements do not significantly increase brain EPA content (18-21), although dietary EPA has been shown to be more beneficial than DHA for the prevention and treatment of depression (22-24). The lack of brain EPA abundance has been proposed to be due to its rapid oxidation in the brain (25). However, it has been demonstrated that brain EPA can actually be increased up to 100-fold by providing dietary EPA in the LPC form (26), indicating that the low levels of EPA are primarily due to low levels of LPC-EPA in plasma.

[0004] Additionally, the retina has the highest concentration of DHA in the body. It also has a majority of very long-chain fatty acids derived from EPA and DHA (32–36 n-3), and phospholipids that contain omega-3 fatty acids at both the sn-1 and sn-2 positions. These fatty acids and unique phospholipids serve important functions in maintaining the integrity of photoreceptor cells (41).

[0005] DHA has anti-inflammatory, anti-angiogenic, and pro-homeostatic roles in the retina, and DHA deficiency is associated with macular degeneration and other retinopathies (42). Several epidemiological studies have shown the beneficial effects of omega-3 fatty acid intake in the prevention of various retinopathies (42)(43). Furthermore, Tikhonenkho et al. (44) reported that diabetic retinopathy could be prevented in experimental animals by providing DHA in the diet. DHA has also been shown to be protective against dry eye disease (45) and age-related macular degeneration (46) in animal models. However, despite the positive results of epidemiological and experimental studies, intervention studies in patients using dietary supplements have not shown clear benefits (47)(48), possibly due to the inefficient absorption of conventional DHA supplements by the retina. It should be noted that Nishizawa et al.(49) reported that although they could increase retinal DHA in developing rats, they could not increase retinal DHA content in adults, even with a high DHA dose (8.5% calorie), which may explain the failure of previous clinical trials.

[0006] Ocular infections and inflammation include a wide range of pathological conditions that can be primary or secondary to a primary disease.

[0007] One example is dry eye disease (DED), a highly prevalent, multifactorial disorder of the tear film and ocular surface that results in ocular pain and vision loss. The focus of treatment for DED is supportive rather than curative, including the instillation of lubricating eye drops to provide temporary symptomatic relief. Inflammation is a central mechanism in the pathogenesis of DED, with the hyperosmolarity and instability of tears contributing to the inflammatory response. This is responsible for the chronic irritation and pain experienced by those suffering from DED. Topical anti-inflammatory agents such as corticosteroids are currently used to address DED, but potential long-term side effects limit their use.

[0008] The omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are well accepted as essential components of a healthy, balanced diet, as having beneficial effects on development, and as ameliorating various pathological conditions, including ocular conditions (e.g., retinal diseases and age-related macular degeneration (AMD)).

[0009] As reviewed by Querques et al., the retina has the highest concentration of DHA in the body, and several studies suggest that omega-3 polyunsaturated fatty acids may have a protective role in reducing the development and progression of retinal diseases (Querques et al., J Nutr Metab. 2011; 2011: 748361). High concentrations of DHA have been suggested to optimize photoreceptor membrane fluidity, retinal integrity, and visual function. Furthermore, many studies have demonstrated that DHA has a protective, e.g., anti-apoptotic, role in the retina. In contrast to DHA, the brain, nervous tissue and retina contain very little eicosapentaenoic acid (EPA), but it has been shown that EPA may have a protective role in preventing neurodegeneration (Yalagala et al J Lipid Res. 2019 Mar;60(3):566-578. doi: 10.1194 / jlr.M090464. Epub 2018 Dec 10). From a nutritional point of view, it is known that Western populations, especially older individuals, have a higher than optimal omega-6 / omega-3 ratio and would be better off enriching their diet with more omega-3 polyunsaturated fatty acids to improve inflammation-related pathogenesis.

[0010] Dry eye is a common eye condition. It affects 5-34% of people to some degree, depending on the population studied. In older adults, it affects up to 70%. Dietary omega-3 supplements have been proposed for the prevention and treatment of some eye diseases, but one clinical trial to treat DED with omega-3 derived from fish oil and phospholipid-rich krill oil did not demonstrate a significant change in the frequency of use of lubricating eye drops (Deinema et al. Ophthalmology. 2017 Jan;124(1):43-52).

[0011] Despite efforts in this field, the problem of dry eye remains highly prevalent and unsolved, and represents an unmet medical need. It would therefore be desirable to provide new and improved treatments for dry eye.

[0012] It would be preferable if such treatment could provide both symptomatic and therapeutic relief.

[0013] In particular, it would be desirable if such improved treatments could prevent ocular pathologies such as glaucoma, AMD, diabetic retinopathy and neuropathy by preventing degeneration of ocular tissues and the retina.

[0014] Summary of the Invention Although brain DHA deficiency is associated with several neurodegenerative diseases, current supplements do not enrich the brain for DHA because of the need for transporters at the blood-brain barrier for lysophosphatidylcholine (LPC-DHA). Krill oil phospholipids contain DHA and EPA at the sn-2 position, but do not generate LPC-DHA and LPC-EPA because pancreatic phospholipase A2 releases them as free acids.

[0015] However, we found that if dietary krill oil is pretreated with sn-1 specific lipase, LPC-DHA and LPC-EPA should be generated, thus enriching brain DHA and EPA. We tested this hypothesis by feeding untreated krill oil (UTKO) and lipase-treated krill oil (LTKO) to normal mice and measuring the fatty acid composition of brain and other tissues. UTKO increased brain DHA and EPA slightly, whereas LTKO was 5-fold and 70-fold more effective, respectively, in enriching brain DHA and EPA, and increased brain BDNF. In contrast, fish oil (without phospholipids) had no effect on either brain DHA or EPA, with or without lipase treatment. LTKO was also more effective than fish oil or UTKO in enriching liver DHA and EPA, but less effective than either fish oil or UTKO in enriching adipose tissue and heart. These results provide a novel strategy to target dietary DHA and EPA to the brain to prevent and treat neurological disorders such as Alzheimer's disease.

[0016] Accordingly, in a first aspect, the present invention relates to a method for treating, preventing and / or alleviating one or more symptoms and / or signs of an ocular disease or condition, comprising the step of administering to a subject in need thereof an effective amount of a formulation, said formulation comprising a lysophosphatidylcholine (LPC) composition comprising an LPC compound selected from the group consisting of any one of formulas 1-8, and any combination thereof, for improving, controlling, reducing or alleviating the symptoms of the disease or condition:

[0017] [ka]

[0018] During the ceremony, R1 is OH or O-CO-(CH2) n -CH3; R2 is OH or O-CO-(CH2) n -CH3; and n is 0, 1 or 2.

[0019] In one embodiment of the first aspect, R1 is OH and R2 is OH.

[0020] In one embodiment of the first aspect, when the LPC composition comprises i) a compound according to formula 1 in which R2 is OH; and / or ii) a compound according to formula 3 in which R1 is OH, the LPC composition has the proviso that it further comprises at least one other LPC compound.

[0021] In one embodiment of the first aspect, the one or more LPC compounds are: A compound according to formula 1; and / or a compound according to formula 3; And a compound according to formula 2; and / or a compound according to formula 4.

[0022] In one embodiment of the first aspect, R1 and R2 are OH; and the molar ratio of lyso-PC-DHA:lyso-PC-EPA is in the range of 1:1 to 3:1; or the molar ratio of lyso-PC-EPA:lyso-PC-DHA is in the range of 1:1 to 5:1; provided that i) the number of moles of lyso-PC-EPA is the number of moles of 1-lyso-PC-EPA plus the number of moles of 2-lyso-PC-EPA; and ii) the number of moles of lyso-PC-DHA is the number of moles of 1-lyso-PC-DHA plus the number of moles of 2-lyso-PC-DHA.

[0023] In one embodiment of the first aspect, the disease or condition is dry eye, such as a dry eye disease selected from the group consisting of ocular inflammation, corneal nerve disorders, and abrasions on the surface of the eye.

[0024] In one embodiment of the first aspect, the disease or condition is a neurodegenerative disease of the eye.

[0025] In one embodiment of the first aspect, the ocular neurodegenerative disease is selected from the group consisting of age-related macular degeneration, diabetic retinopathy, non-proliferative retinopathy, proliferative retinopathy, diabetic macular edema, retinitis pigmentosa, central vein occlusion and glaucoma.

[0026] In one embodiment of the first aspect, administration is by a modality selected from the group consisting of oral administration, and intravascular or intravenous administration.

[0027] In one embodiment of the first aspect, the mode of administration is oral administration.

[0028] In one embodiment of the first aspect, the mode of administration is intravascular or intravenous administration.

[0029] In one embodiment of the first aspect, the LPC composition in the formulation comprises an LPC compound selected from the group consisting of any one of formulas 1 to 8, and said LPC composition comprises total LPC in an amount corresponding to 10% to 100% by weight of the LPC composition.

[0030] In one embodiment of the first aspect, the LPC composition comprises an additional lipid distinct from the LPC.

[0031] In one embodiment of the first aspect, the LPC composition comprises between 5% and 12% by weight DHA, which is a free fatty acid or an ethyl ester, or is a lipid in the LPC composition.

[0032] In one embodiment of the first aspect, the LPC composition comprises 10% to 24% by weight EPA, which is either a free fatty acid or an ethyl ester, or bound to any lipid in the LPC composition.

[0033] In one embodiment of the first aspect, the LPC composition further comprises palmitoleic acid and / or palmitic acid.

[0034] In one embodiment of the first aspect, the LPC composition comprises 2% to 5% by weight palmitoleic acid, which is bound to any lipid in the LPC composition.

[0035] In one embodiment of the first aspect, the LPC composition contains 10% to 15% by weight palmitic acid, and the palmitic acid is bound to any lipid in the LPC composition.

[0036] In one embodiment of the first aspect, the additional lipid different to LPC is selected from the group consisting of triglycerides, ethyl esters, free fatty acids, and phospholipids such as phosphatidylethanolamines and phosphatidylcholines.

[0037] In one embodiment of the first aspect, the LPC composition comprises total phospholipids in an amount representing at least 35% by weight of the LPC composition.

[0038] In one embodiment of the first aspect, the LPC composition comprises total LPC in an amount representing at least 23% by weight of the LPC composition.

[0039] In one embodiment of the first aspect, the LPC composition comprises total LPC in an amount representing at least 40% by weight of the LPC composition.

[0040] In one embodiment of the first aspect, the LPC composition comprises total LPC in an amount representing at least 60% by weight of the LPC composition.

[0041] In one embodiment of the first aspect, the LPC composition comprises total LPC in an amount representing at least 90% by weight of the LPC composition.

[0042] In one embodiment of the first aspect, the LPC composition comprises total LPC in an amount representing at least 90%-98% by weight of the LPC composition.

[0043] In one embodiment of the first aspect, the LPC composition comprises total LPC in an amount representing at least 95% by weight of the LPC composition.

[0044] In one embodiment of the first aspect, the formulation comprises between 1% and 35% by weight of the LPC composition.

[0045] In one embodiment of the first aspect, the formulation comprises 25% by weight of the LPC composition.

[0046] In one embodiment of the first aspect, the formulation comprises 40% by weight of the LPC composition.

[0047] In one embodiment of the first aspect, the LPC composition comprises an amount of the LPC compound that is greater than the amount of phosphatidylcholine.

[0048] In one embodiment of the first aspect, the LPC compound is selected from LPC-EPA, LPC-DHA, and any combination thereof.

[0049] In one embodiment of the first aspect, the LPC composition comprises an amount of the LPC compound that is greater than the amount of phosphatidylcholine.

[0050] In one embodiment of the first aspect, the formulation further comprises an ingredient selected from the group consisting of lutein, astaxanthin, zeaxanthin, and any combination thereof.

[0051] In one embodiment of the first aspect, the subject is a mammalian subject.

[0052] In one embodiment of the first aspect, the subject is a human subject.

[0053] In a second aspect, the present invention relates to a method for preparing a krill oil composition enriched in LPC-DHA and LPC-EPA, comprising treating krill oil with a lipase specific for the sn-1 position of DHA- and EPA-containing phospholipids in the krill oil.

[0054] In one embodiment of the second aspect, the invention is a krill oil composition enriched in LPC-DHA and LPC-EPA, prepared by treating krill oil with a lipase specific for the sn-1 position of DHA- and EPA-containing phospholipids in said krill oil.

[0055] In one embodiment of the second aspect, the lipase comprises a lipase from Mucor meihei. In another embodiment, the lipase comprises an immobilized lipase from Mucor meihei. In another embodiment, the krill oil composition comprises a lysophosphatidylcholine comprising greater than about 80 weight percent LPC-DHA and LPC-EPA.

[0056] In one embodiment of the second aspect, the krill oil composition with lysophosphatidylcholine comprises greater than about 80 weight percent LPC-DHA and LPC-EPA.

[0057] In one embodiment of the second aspect, the krill oil composition with lysophosphatidylcholine comprises greater than about 70 weight percent LPC-DHA and LPC-EPA.

[0058] In one embodiment of the second aspect, the krill oil composition with lysophosphatidylcholine comprises greater than about 60 weight percent LPC-DHA and LPC-EPA.

[0059] In one embodiment of the second aspect, the krill oil composition with lysophosphatidylcholine comprises greater than about 50 weight percent LPC-DHA and LPC-EPA.

[0060] In one embodiment of the second aspect, the krill oil composition with lysophosphatidylcholine comprises greater than about 40 weight percent LPC-DHA and LPC-EPA.

[0061] In one embodiment of the second aspect, the krill oil composition with lysophosphatidylcholine comprises greater than about 30 weight percent LPC-DHA and LPC-EPA.

[0062] In one embodiment of the second aspect, the krill oil composition with lysophosphatidylcholine comprises greater than about 20 weight percent LPC-DHA and LPC-EPA.

[0063] In one embodiment of the second aspect, the krill oil composition with lysophosphatidylcholine comprises greater than about 10 weight percent LPC-DHA and LPC-EPA.

[0064] In one embodiment of the second aspect, the present invention provides a functional food composition comprising the krill oil composition described above.

[0065] In one embodiment of the second aspect, the present invention provides a pharmaceutical composition comprising the krill oil composition described above.

[0066] In a third aspect, the present invention relates to a method for treating or preventing a neurological disease or disorder in a subject, comprising administering to the subject an effective amount of a krill oil composition rich in LPC-DHA and LPC-EPA. In some embodiments, the neurological disease or disorder is selected from Parkinson's disease, schizophrenia, traumatic brain injury, stroke and Alzheimer's disease. In certain embodiments, the neurological disease or disorder is Alzheimer's disease.

[0067] The inventors further discovered that feeding normal mice LPC-DHA and LPC-EPA-enriched krill oil compositions according to the present invention significantly increased retinal EPA and DHA, whereas similar treatment with fish oil containing EPA and DHA only in the triglyceride form had no effect on either retinal EPA or DHA.

[0068] Thus, in another aspect, the present invention relates to a method of treating or preventing a retinal disease or disorder in a subject, comprising administering to the subject an effective amount of a krill oil composition enriched in LPC-DHA and LPC-EPA, hi one embodiment, the retinal disease or disorder is selected from age-related macular degeneration, diabetic retinopathy and dry eye disease.

[0069] Additionally, because lipase-treated krill oil is superior to untreated krill oil with fish oil in enriching the liver with omega-3 fatty acids, the compositions of the present invention are useful in treating liver diseases such as non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), which are known to be ameliorated by enriching the liver with DHA.

[0070] Thus, in another aspect, the present invention relates to a method for treating or preventing a liver disease that is ameliorated by enriching the liver with DHA, comprising administering to a subject in need of treatment an effective amount of a krill oil composition enriched in LPC-DHA and LPC-EPA, hi one embodiment, the liver disease is selected from non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

[0071] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the retinal EPA content (AUC / mg dry tissue) at the end of the study.

[0072] FIG. 2 shows the retinal EPA / ETA ratio at the end of the study.

[0073] FIG. 3 shows plasma LPC-EPA (ng / ml) collected after 0 weeks (T0, baseline), 2 weeks (T1) and 3 weeks (T2) of oral gavage.

[0074] FIG. 4 shows the retinal DHA / ETA ratio at the end of the study.

[0075] FIG. 5 shows plasma LPC-DHA (ng / ml) collected after 0 weeks (T0, baseline), 2 weeks (T1) and 3 weeks (T2) of oral gavage.

[0076] FIG. 6 shows retinal EPA concentrations as a % of total fatty acids for the six experimental groups.

[0077] FIG. 7 shows the relationship between retinal ARA (20:4 n-6) concentration and DHA dose for each of the six experimental groups.

[0078] FIG. 8 shows the area under the concentration time curve in ocular tissues following oral and iv administration.

[0079] FIG. 9 shows the area under the concentration-time curve for ocular tissues (whole eye) following oral and iv administration of LPC-EPA and LPC-DHA for the first 24 hours.

[0080] FIG. 10 shows the area under the time concentration curve for ocular tissues (whole eye) following oral and iv administration of LPC-EPA and LPC-DHA for the first 72 hours.

[0081] FIG. 11 shows that krill oil contains EPA and DHA at the sn-2 position of PC, and thus EPA and DHA are released as free fatty acids during degradation by pancreatic phospholipase A2. The released EPA and DHA are then absorbed as TAG, which does not generate either LPC-EPA or LPC-DHA required for transport to the brain. Thus, feeding krill oil does not result in significant enrichment of EPA and DHA in the brain. However, when krill oil is pretreated with lipase specific to sn-1 ester bonds, LPC-EPA and LPC-DHA are generated. Feeding this improved krill oil should result in the absorption of phospholipid forms of EPA and DHA, leading to their enrichment in the brain. On the other hand, lipase treatment of fish oil does not generate either LPC-EPA or LPC-DHA, and therefore does not result in enrichment of EPA and DHA in the brain (not shown).

[0082] Figure 12 shows the effect of dietary treatment on plasma levels of long-chain PUFAs. Total lipids of fasting plasma were extracted, transmethylated, and analyzed for fatty acid composition by GC / MS as described in the text. Values ​​shown are the mean ± standard deviation of five animals in each group. Statistical significance was determined by one-way ANOVA with post-hoc Tukey multiple comparison test (GraphPad Prism 8.0 software). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to the control group.

[0083] Figure 13 shows the effect of feeding native krill oil and fish oil, and lipase-treated krill oil and fish oil on plasma LPC species. Plasma samples were extracted with acidified methanol as described herein and analyzed by LC / MS using a HILIC column to analyze the concentration and isomeric composition of omega-3 LPC species. LPC concentrations were determined using 17:0 LPC as an internal standard. Values ​​were calculated applying a correction factor (x 0.233) since the intensity values ​​of the 17:0 LPC and 22:6 LPC standards differ by this amount under the conditions used. Bars with non-identical superscripts are significantly different from each other (total LPC) as determined by one-way ANOVA with post-hoc Tukey multiple comparison test.

[0084] Figure 14 shows the PUFA composition of the prefrontal cortex. Total fatty acid composition was analyzed by GC / MS, and the percentage composition of only 20:4 (ARA), 20:5 (EPA), 22:5 (DPA), and 22:6 (DHA) is shown. Values ​​shown are the mean ± standard deviation of five animals in each group. Statistical significance was determined by one-way ANOVA, and symbols are the same as under Figure 12 above.

[0085] Figure 15 shows the PUFA composition of hippocampus. Fatty acid composition was analyzed as described in the text. Values ​​shown are the mean ± standard deviation of 5 animals in each group. Symbols for statistical significance are the same as those under Figure 12 above.

[0086] Figure 16 shows the PUFA composition of the striatum. The PUFA composition was analyzed as described in the text. Values ​​shown are the mean ± standard deviation of five mice in each group. Statistical significance between control and experimental diets was determined by one-way ANOVA with post-hoc Tukey multiple comparison test. Significance symbols are the same as those under Figure 12 above.

[0087] Figure 17 shows the PUA composition in the cerebellum. The fatty acid composition was analyzed by GC / MS as described in the text. Values ​​shown are the mean ± standard deviation of six mice in each group. Statistical significance between control and experimental diets was determined by one-way ANOVA with post-hoc Tukey multiple comparison test. Significance symbols are the same as under Figure 12 above.

[0088] FIG. 18 shows the effect of dietary treatment on BDNF levels in the prefrontal cortex and hippocampus. Immunoassay of BDNF levels was performed as described in the text, using β-actin as an internal standard. BDNF / β-actin ratios shown are the mean ± standard deviation of three mice in each group. Inset shows a representative immunoblot. Statistical significance between control and experimental diets was determined by one-way ANOVA with post-hoc Tukey multiple comparison test. ***p<0.001 vs control; ****p<0.0001 vs control.

[0089] Figure 19 shows the PUFA composition of perigonadal adipose tissue. The percentage values ​​shown are the mean ± standard deviation of 5 mice for each group. The statistical significance values ​​(one-way ANOVA) are between control and experimental diets, and the symbols are the same as those under Figure 12 above.

[0090] Figure 20 shows the PUFA composition of inguinal adipose tissue. The percentage values ​​shown are the mean values ​​± standard deviation of 5 mice for each group. The statistical significance values ​​(one-way ANOVA) are between control and experimental diets, and the symbols are the same as those under Figure 12 above.

[0091] Figure 21 shows the effect of dietary fat on liver fatty acid composition. Fatty acid analysis of total lipids by GC / MS was as described in the text. Values ​​shown are the mean ± standard deviation of five mice in each group. Statistical significance between control and experimental diets was determined by one-way ANOVA with post-hoc Tukey multiple comparison test. Significance symbols are the same as under Figure 12 above.

[0092] Figure 22 shows the effect of dietary fat on cardiac fatty acid composition. Total cardiac lipids were extracted and analyzed for fatty acid composition by GC / MS as described in the text. Values ​​shown are the mean ± standard deviation of five mice in each group. Statistical significance between control and experimental diets was determined by one-way ANOVA with post-hoc Tukey multiple comparison test. Significance symbols are the same as under Figure 12 above.

[0093] Figure 23 shows that krill oil containing EPA and DHA at the sn-2 position of PC treated with lipase that specifically hydrolyzes sn-1 fatty acids produces LPC-EPA and LPC-DHA. Feeding this preparation to normal mice significantly increased retinal EPA and DHA. Similar treatment with fish oil containing EPA and DHA only in triglyceride form did not affect either retinal EPA or DHA.

[0094] Figure 24 shows the incorporation of dietary DHA into rat retinal lipids, given in PC, TAG, or LPC form. Normal male Sprague-Dawley rats (8 weeks old; n=5 per group) were gavaged daily for 30 days with 10 mg DHA in 250 μL corn oil in the form of TAG-DHA, di-DHA PC, or LPC-DHA. In addition, a half dose of LPC-DHA (5 mg DHA) was used to match the amount of LPC-DHA expected to be generated by di-DHA PC during digestion. All animals were fed a normal rodent chow diet that contained no DHA but contained 17.4 mg α-linolenic acid (18:3, n-3) per gram of chow. Retinal FA was analyzed by GC / MS as described in the text. The percentage compositions of 20:4(n-6), 20:5(n-3), 22:5(n-3), and 22:6(n-3) are shown here. Total FA composition is shown in Table 1. The significance of differences between treatment groups was determined by one-way ANOVA with post-hoc Tukey multiple comparison test (GraphPad Prism 8.0 software). Bars without a common letter above are significantly different from each other (p<0.05). TG: triacylglycerol; PC: phosphatidylcholine; LPC: lysophosphatidylcholine; FA: fatty acid; GC / MS: gas chromatography / mass spectrometry.

[0095] Figure 25 shows the effect of dietary free (unesterified) DHA, sn-1 DHA-LPC, and sn-2 DHA LPC on mouse retinal FA. Normal male mice (C57 BL / J6, 16 weeks old) were gavaged daily with 1 mg DHA in the form of free DHA, sn-1 DHA LPC, or sn-2 DHA LPC in 80 μL of corn oil for 30 days. Retinal FA composition was analyzed by GC / MS. Only values ​​for arachidonic acid and omega-3 FA are shown here (mean ± standard deviation, 8 animals / group). Total FA composition is shown in Table 2. Bars not topped with a common letter are significantly different from each other (p<0.05) by one-way ANOVA with post-hoc Tukey correction. SD: standard deviation; sn-1 and sn-2: stereospecific numbering 1 and 2, respectively; LPC: lysophosphatidylcholine; DHA: docosahexaenoic acid.

[0096] Figure 26 shows the molecular species of mouse retinal phospholipids containing DHA. Animals were gavaged with free (unesterified) DHA or the two isomers of LPC-DHA as described in Figure 2. The molecular species of PC (A) and PE (B) containing DHA were analyzed using multiple reaction monitoring by LC / MS / MS as described in the text, with 17:0 LPC, 17:0-17:0 PC, and 17:0-17:0 PE as internal standards. Bars without a common letter above them are significantly different from each other (mean ± standard deviation, 6 animals / group) by one-way ANOVA and post hoc Tukey multiple comparison test. PE: phosphatidylethanolamine; PC: phosphatidylcholine; sn-1 and sn-2: stereospecific numbering 1 and 2, respectively; DHA: docosahexaenoic acid; LC / MS / MS: liquid chromatography / tandem mass spectrometry; SD: standard deviation; LPC: lysophosphatidylcholine; DHA: docosahexaenoic acid.

[0097] Figure 27 shows the effect of feeding untreated or lipase-treated fish oil and krill oil on retinal FA in mice. Normal male mice (8 weeks old) were fed a diet (AIN93G) containing 7% total fat and supplemented with 0.264% EPA+DHA in the form of fish oil or krill oil treated with Mucor lipase (or untreated). Mice were fed the diet ad libitum for 30 days and retinal FA were analyzed by GC / MS. The percentage composition of 20:4(n-6), 20:5(n-3), 22:5(n-3) and 22:6(n-3) is shown here (mean ± standard deviation, 5 mice / group). Total FA composition is shown in Table 3. Bars not topped with a common letter are significantly different from each other by one-way ANOVA and post hoc Tukey multiple comparison test. KO: krill oil; FO: fish oil; FA: fatty acids; EPA: eicosapentaenoic acid; GC / MS: gas chromatography / mass spectrometry; DHA: docosahexaenoic acid.

[0098] [Definition] Throughout this disclosure, relevant terms are to be understood consistently with their typical meanings established in the relevant art, ie, the fields of medicinal chemistry, medicine, biology, biochemistry and physiology.

[0099] However, further explanation and clarification is provided for certain terms set forth below.

[0100] The following abbreviations indicate: ARA: arachidonic acid (20:4, n-6); BBB: blood-brain barrier; BDNF: brain-derived neurotrophic factor; DHA: docosahexaenoic acid (22:6, n-3); DPA: docosapentaenoic acid (22:5, n-3); EPA: eicosapentaenoic acid (20:5, n-3); FA: fatty acid(s); FO: fish oil; GC / MS: gas chromatography / mass spectrometry; KO: krill oil; LC / MS / MS: liquid chromatography / tandem mass spectrometry; LPC: lysophosphatidylcholine; PC: phosphatidylcholine, TAG: triacylglycerol.

[0101] "Krill oil" refers to an extract prepared from the seeds of Antarctic krill (Euphausia superba). Two of the most important components in krill oil are omega-3 fatty acids in the form of DHA and EPA, and phospholipid-derived fatty acids (PLFA), primarily phosphatidylcholine (alternatively called marine lecithin). Krill oil is available commercially.

[0102] [ka]

[0103] The terms "2-lyso-PC-DHA" and "2-LPC-DHA" are used interchangeably herein and refer to a compound according to Formula 1 where R2 is OH.

[0104] The terms "2-lyso-PC-EPA" and "2-LPC-EPA" are used interchangeably herein and refer to a compound according to formula 2 where R2 is OH.

[0105] The terms "2-lyso-PC-DPA" and "2-LPC-DPA" are used interchangeably herein and refer to a compound according to formula 5 where R2 is OH. The terms "2-lyso-PC-SDA" and "2-LPC-SDA" are used interchangeably herein and refer to a compound according to formula 6 where R2 is OH.

[0106] The terms "1-lyso-PC-DHA" and "1-LPC-DHA" are used interchangeably herein and refer to a compound according to formula 3 where R1 is OH.

[0107] The terms "1-lyso-PC-EPA" and "1-LPC-EPA" are used interchangeably herein and refer to a compound according to formula 4 where R1 is OH.

[0108] The terms "1-lyso-PC-DPA" and "1-LPC-DPA" are used interchangeably herein and refer to a compound according to formula 7 where R1 is OH.

[0109] The terms "1-lyso-PC-SDA" and "1-LPC-SDA" are used interchangeably herein and refer to a compound according to formula 8 where R1 is OH.

[0110] The terms "lyso-PC-DHA" and "LPC-DHA" are used interchangeably herein and include both 1-lyso-PC-DHA and 2-lyso-PC-DHA.

[0111] The terms "lyso-PC-EPA" and "LPC-EPA" are used interchangeably herein and include both 1-lyso-PC-EPA and 2-lyso-PC-EPA.

[0112] The terms "lysoPC-DPA" and "LPC-DPA" are used interchangeably herein and include both 1-lysoPC-DPA and 2-lysoPC-DPA.

[0113] The terms "lyso-PC-SDA" and "LPC-SDA" are used interchangeably herein and include both 1-lyso-PC-SDA and 2-lyso-PC-SDA.

[0114] The term "EPA" refers to eicosapentaenoic acid.

[0115] The term "DHA" refers to docosahexaenoic acid.

[0116] The term "DPA" refers to n3-docosapentaenoic acid. The term "n3" specifies that the compound is an omega-3 fatty acid.

[0117] The term "SDA" refers to stearidonic acid.

[0118] EPA, DHA, DPA, SDA, as used herein in connection with the compositions of the present invention, refer to fatty acid chains that may be attached to a lipid backbone, such as a phospholipid, lysophospholipid, triacylglyceride, diacylglyceride, monoacylglyceride or any other lipid backbone, or which may be present in the composition as free fatty acids or ethyl esters.

[0119] The term "total LPC" is used herein to describe the total lysophosphatidylcholine content in a composition.

[0120] The term "total phospholipids" is used herein to describe the total phospholipid content, including lysophospholipids, in a composition.

[0121] The term "intravenous administration" as used herein refers to a mode of administration in which a liquid substance is delivered directly into a vein. The intravenous route of administration can be used for injection (using a higher pressure syringe) or infusion (typically using only pressure provided by gravity).

[0122] The term "pharmaceutical acceptable excipient" refers to a substance other than the components of the LPC compositions referred to in the claims, which is commonly used with oil-based pharmaceuticals. Such excipients include, but are not limited to, triolein, soybean oil, safflower oil, sesame oil, castor oil, coconut oil, triglycerides, tributyrin, tricaproin, tricaprylin, vitamin E, antioxidants, alpha-tocopherol, ascorbic acid, deferoxamine mesylate, thioglycolic acid, emulsifiers, lecithin, polysorbate 80, methylcellulose, gelatin, serum albumin, sorbitan laurate, sorbitan oleate, sorbitan trioleate, polyethylene glycol (PEG), PEG 400, polyethylene glycol modified phosphatidylethanolamine (PEG-PE), poloxamer, glycerin, sorbitol, xylitol, pH adjusting agents; sodium hydroxide, antimicrobial agents EDTA, sodium benzoate, benzyl alcohol, and proteins such as albumin. Pharmaceutically acceptable excipients should be acceptable in the sense of being compatible with the other components of the composition and not deleterious to the recipient thereof.

[0123] The term "pharmaceutical acceptable salt" as used herein refers to pharmaceutical acceptable salt derived from various organic and inorganic counterions well known in the art, including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, and, where the molecule contains a basic functional group, includes the salt of organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate.Suitable salts include those described in P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of pharmaceutical salts properties, Selection, and Use; 2002.

[0124] The term "prophylaxis" refers to measures taken to prevent but not treat a disease or condition.

[0125] The term "effective amount" refers to an amount of a disclosed compound or composition provided herein that is sufficient to affect a beneficial or desired biological, emotional, medical, or clinical response in a cell, tissue, system, animal, or human. An effective amount may be administered in one or more administrations, applications, or dosages. The term may also include within its scope an amount effective to enhance or restore substantially normal physiological function.

[0126] The term "therapeutically effective amount" is an art-recognized term. In an embodiment, the term refers to an amount of a composition disclosed herein that produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. In an embodiment, the term refers to an amount necessary or sufficient to eliminate, reduce or alleviate a medical symptom for a period of time. The effective amount may vary depending on factors such as the disease or condition being treated, the particular composition being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular composition without necessitating undue experimentation.

[0127] For example, it is well within the skill of the art to begin administering the compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for administration purposes. Thus, a single dosage composition can contain such amounts, or sub-fractions thereof, to constitute a daily dose. The dose can be adjusted by the individual physician in the event of any contraindications. It is generally preferred to use the maximum dose of the pharmacological agent of the present invention (alone or in combination with other therapeutic agents), i.e., the highest safe dose according to sound medical judgment. However, it will be understood by those skilled in the art that a patient may require a lower dose or a tolerable dose for medical reasons, psychological reasons, or virtually any other reason.

[0128] For example, the response to a therapeutically effective amount of the disclosed compounds and / or pharmaceutical compositions can be measured by determining the physiological effect of the treatment or drug (such as a reduction or absence of symptoms of a disease following administration of a treatment or pharmacological agent). Other assays are known to those of skill in the art and can be employed to measure the level of response. The amount of treatment can be varied, for example, by increasing or decreasing the amount of the disclosed compounds and / or pharmaceutical compositions, by changing the disclosed compounds and / or pharmaceutical compositions administered, by changing the route of administration, by changing the timing of dosing, etc. Doses can vary and can be administered in one or more dose administrations per day for one or several days. Guidance can be found in the literature for appropriate doses for a given class of pharmaceuticals.

[0129] The term "treating" is art-recognized and includes preventing a disease, disorder, or condition from occurring in a subject who may be susceptible to, but has not yet been diagnosed as having, the disease, disorder, or condition; inhibiting (e.g., preventing the progression of) a disease, disorder, or condition; and alleviating (e.g., causing regression of the disease, disorder, and / or condition) a disease, disorder, or condition. Treating a disease or condition (such as treating dry eye disease selected from ocular inflammation, corneal neuropathy and abrasions on the surface of the eye, or ocular neurodegenerative diseases selected from age-related macular degeneration, diabetic retinopathy, non-proliferative retinopathy, proliferative retinopathy, diabetic macular edema, retinitis pigmentosa, central vein occlusion and glaucoma, as well as other related diseases or any other medical condition) is well understood in the art to include ameliorating at least one symptom of a particular disease or condition, even if the underlying pathology is not affected, and includes administration of a composition that reduces the frequency or delays the onset of symptoms of the medical condition in a subject compared to subjects not receiving the composition.

[0130] Detailed Description of the Invention Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art of medicine, pharmacology, pharmaceutical chemistry, biology, biochemistry, and physiology.

[0131] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and suitable methods and materials are described herein.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, shall prevail.

[0132] Where a numerical limit or range is stated herein, the endpoints are included, and all values ​​and subranges within that numerical limit or range are specifically included, as if expressly written.

[0133] As mentioned above, there are many medical conditions, including dry eye disease (DED) (Deinema et al., American Academy of Ophthalmology 2016, ISSN 0161-6420), that are associated with low retinal omega-3 levels or that would benefit from increased levels of retinal long-chain omega-3 levels. DHA, EPA, DPA and SDA are omega-3 fatty acids of particular interest in this regard.

[0134] Thus, there is a need for means to increase the levels of omega-3 fatty acids in the retina, and in particular to increase the levels of DHA, EPA, DPA and / or SDA in the retina.

[0135] Unlike other tissues, omega-3 uptake does not occur via lipoprotein receptors in the retina. Previous studies in animal models have reported that the LPC form of DHA crosses the blood-retinal barrier via Mfsd2a, a sodium-dependent lysophosphatidylcholine (LPC) cotransporter that transports LPC containing DHA and other long-chain fatty acids.

[0136] The photoreceptor membrane discs of the eye in the outer segments of rod photoreceptors are highly enriched in the visual pigment rhodopsin and the omega-3 fatty acid docosahexaenoic acid (DHA). The eye obtains DHA from the blood, and Mfsd2a-mediated LPC transport has been demonstrated as a critical pathway for DHA uptake in the eye and photoreceptor membrane disc development.

[0137] Furthermore, it has been demonstrated that Mfsd2a is highly expressed in the retinal pigment epithelium of the embryonic eye prior to photoreceptor development, and that the retinal pigment epithelium of the embryonic eye is the major site of Mfsd2a expression in the eye.

[0138] As reviewed by Querques et al., the retina has the highest concentration of DHA in the body, and several studies suggest that omega-3 polyunsaturated fatty acids may have a protective role in reducing the onset and progression of retinal disease (Querques et al., J Nutr Metab. 2011; 2011: 748361). High concentrations of DHA have been suggested to optimize photoreceptor membrane fluidity, retinal integrity, and visual function. Furthermore, many studies have demonstrated that DHA has a protective (e.g., anti-apoptotic) role in the retina. The review discloses that dietary intake of omega-3 affects the incidence of AMD.

[0139] Additionally, a study using krill oil supplementation demonstrated that moderate daily dosing of omega-3 EFAs, primarily in phospholipid form (such as krill oil), for three months resulted in reduced tear osmolality and increased tear stability in people with DED. It was also demonstrated that phospholipids derived from krill oil may confer additional therapeutic benefit, with improved DED symptoms and reduced basal tear levels of interleukin-17A compared to placebo (Deinema et al., 2017).

[0140] Dietary supplements containing omega-3 EFAs in general may have potential to influence the prevalence of eye diseases and in the treatment of such diseases, but the form in which the omega-3 is delivered may affect uptake and efficacy.

[0141] This is demonstrated in the examples. For example, Example 4 explains that the content of both retinal EPA and DHA is richer from lipase-treated krill oil than from normal krill oil and fish oil. From the viewpoint of the results, one of the objectives of the present invention is to increase the uptake of EPA and DHA in ocular tissue and retina, and optimize the omega-6 / omega-3 ratio in ocular tissue. Without being bound by theory, it is believed that the increase in the uptake of omega-3 essential fatty acids changes the inflammatory state of the eye by regulating cytokine production, thus helping to treat and prevent inflammation. Since most eicosanoids derived from the omega-6 fatty acid pathway are proinflammatory, omega-3 EFAs bias prostaglandin metabolism toward the production of anti-inflammatory eicosanoids, limiting and resolving inflammation.

[0142] It has been shown in the prior art that a length of 14 or more carbon atoms is essential for transport across the blood-brain barrier (BBB) ​​or blood-retinal barrier (BRB) by the Mfsd2A transporter. DHA, EPA, SDA and DPA are considered to be very important for positive health effects in humans, and all of them have more than 14 carbon atoms. Therefore, based on the information we currently have, each and every one of these fatty acids should be efficiently transported across the blood-retinal barrier when bound to LPC. Therefore, although LPC-DHA and LPC-EPA were selected as model molecules in this study (Examples 2-4), all data provided herein on ocular uptake is also believed to show the expected uptake profile of the other two omega-3 fatty acids mentioned above, namely SDA and DPA.

[0143] LPC-DHA and LPC-EPA were to be administered both orally and intravenously in the study. For intravenous administration, it was decided to mix LPC-DHA and LPC-EPA with one or more pharma- ceutically acceptable excipients. Intralipid (IV) provided by Sigma Aldrich is compatible with oily substances and was therefore selected as one or more pharma-ceutically acceptable excipients. For further details on the pharmaceutical compositions used in Examples 2-4, see Example 1.

[0144] Thirty-two male Sprague Dawley rats were administered either LPC-DHA or LPC-EPA orally or intravenously as a single dose. The intravenous dose was administered directly into the tail vein as a slow bolus over 30 seconds. Each rat was euthanized by carbon dioxide gas overdose at each of the following times: 0.5, 3, 8, 24, 72, 96, 168, and 336 hours after dosing. Each carcass was flash frozen in a hexane / solid carbon dioxide mixture immediately after collection and then stored at approximately -20°C until further analysis.

[0145] As detailed in Example 2, frozen carcasses were subjected to quantitative ocular autoradiography to study DHA and EPA ocular tissue uptake at 0.5, 3, 8, 24, 72, 96, 168 and 336 hours post-dosing.

[0146] The final results for LPC-DHA are shown in Example 2, Tables 1a and 1b. The final results for LPC-EPA are shown in Example 3, Tables 2a and 2b. The data are also shown in Figures 8-10.

[0147] When administered intravascularly to avoid initial exposure to the intestine, DHA and surprisingly also its LPC form EPA (measured by their respective 14C radiolabeled carboxylic acid residues) show very rapid and sustained uptake and large accumulation in ocular tissues. For LPC-EPA given as a short intravascular (iv) bolus, the area under the concentration-time curve (AUC) for the first 24 hours was demonstrated to be more than 5 times greater than the equivalent measurement for orally administered LPC. For LPC-DHA given as a short intravascular (iv) bolus, the area under the concentration-time curve (AUC) for the first 24 hours was demonstrated to be more than 40 times greater than the equivalent measurement for orally administered LPC-DHA. This demonstrates that a route of administration that can avoid initial exposure to the intestine results in very rapid and sustained uptake of LPC-EPA and LPC-DHA into ocular tissues.

[0148] Example 3 provides data on daily dosing of various krill oil lysophospholipids, including LPC-EPA and LPC-DHA, for three weeks. Twenty-four male rats were divided into groups and given daily oral gavage doses containing olive oil, different doses of LPC composition, and Superba Boost krill oil for three weeks. Retinal FA was extracted and analyzed.

[0149] The EPA results are shown in Figures 1 and 2. There is a strong dose-response relationship, with higher doses of EPA shown to be associated with higher retinal EPA concentrations (Figure 1) and increased EPA / ETA (20:4) ratios (Figure 2). One isomer of eicosatetraenoic acid (ETA) is arachidonic acid, an omega-6 fatty acid. The EPA / ETA ratio is an indicator of the omega-3 / omega-6 shift that occurs with enhanced uptake of omega-3 PUFAs. As explained, it is believed to have a positive impact on ocular inflammatory conditions. As described in the introduction, omega-6 fatty acids are associated with inflammation, while omega-3 fatty acids such as EPA and DHA are associated with anti-inflammatory signals. As can be seen in Figure 15, rats fed the crude lysophospholipid composition show a higher retinal EPA / ETA ratio than rats fed Superba Boost krill oil at a similar dose. This means that rats fed the LPC composition exhibited a more favorable EPA profile in the retina compared to rats fed Superba Boost krill oil and olive oil, and furthermore, that higher doses of the lysophospholipid composition are associated with a more favorable EPA profile in the retina.

[0150] Inspection of Figure 3 suggests that the improved retinal EPA profile is likely related to the greater amount of LPC-EPA present in the plasma and the subsequent increased EPA uptake into the retina via mfsd2a. The example crude provided at the high dose (Group 4) showed both the highest levels of LPC-EPA in plasma, as well as the highest retinal EPA content and improved EPA to ETA ratio.

[0151] Figure 4 and Figure 5 are related to the results of DHA.For DHA, it also shows a strong dose-response relationship, and higher doses of lysophospholipid composition are associated with higher DHA / ETA ratio, and the increase in DHA / ETA ratio is particularly significant in the crude high dose group.This suggests that DHA is dose-dependently taken up into retina from lysophospholipid composition, regardless of the difference in purity of lysophospholipid product.

[0152] FIG. 5 further shows that the crude high dose group (Group 4) exhibited the highest levels of LPC-DHA at the end of the study period (T2), supporting the notion that DHA uptake across the blood-retinal barrier occurs via Mfsd2a.

[0153] Figure 6 shows ocular EPA concentrations relative to total fatty acids. As can be seen from Figure 6, higher doses of the "crude" lysophospholipid composition are associated with higher retinal EPA concentrations relative to total fatty acids.

[0154] Figure 7 shows the relationship between arachidonic acid (ARA; 20:4 n-6), DHA dose and test product. Figure 7 shows that higher doses of "crude" DHA / EPA lysophospholipid composition are associated with lower levels of retinal ARA concentration compared to total fatty acids, and crude lysophospholipid composition shows more significant ARA reduction compared to Superba Boost at equivalent doses. This again suggests that increasing doses of lysophospholipid composition are associated with more beneficial fatty acid profile in retina.

[0155] Therefore, Example 3 demonstrates that oral administration route can bring about favorable fatty acid content of retina, which has a positive effect on retinal inflammatory state.Based on these results, the present inventors propose an oral dietary supplement comprising the LPC-composition described herein for use in the treatment, such as preventive therapy, of eye diseases disclosed herein.

[0156] Accordingly, in a first aspect, the present invention relates to a method for treating, preventing and / or alleviating one or more symptoms and / or signs of an ocular disease or condition, comprising the step of administering to a subject in need thereof an effective amount of a formulation, wherein the formulation comprises a lysophosphatidylcholine (LPC) composition comprising an LPC compound selected from the group consisting of any one of formulas 1-8, and any combination thereof, thereby improving, controlling, reducing or alleviating the symptoms of the disease or condition:

[0157] [ka]

[0158] During the ceremony, R1 is OH or O-CO-(CH2) n -CH3; R2 is OH or O-CO-(CH2) n -CH3; and n is 0, 1 or 2.

[0159] In one embodiment of the present invention, R1 is OH and R2 is OH.

[0160] An alternative embodiment of the present invention relates to the first embodiment of the present invention, where R1 is OH or a protecting group and R2 is OH or a protecting group. An example of a protecting group is O-CO-(CH2) where n is 0, 1 or 2. n -CH3.

[0161] The protecting group is preferably a group that does not interfere with binding to Mfsd2a transporter and at the same time inhibits the transfer of omega-3 (i.e., DHA, EPA, SDA and DPA) acyl groups.When the omega-3 fatty acid moiety (e.g., DHA, EPA, SDA and DPA moiety) is located at the sn-1 position of the glycerol backbone, the protecting group typically inhibits the transfer of the omega-3 fatty acid moiety from sn-1 position to sn-2 position.When the omega-3 fatty acid moiety (e.g., DHA moiety) is located at the sn-2 position of the glycerol backbone, the protecting group typically inhibits the transfer of the omega-3 fatty acid moiety from sn-2 position to sn-1 position.

[0162] Formulas 1 and 3 refer to compounds with DHA moieties attached. Formulas 2 and 4 refer to compounds with EPA moieties attached. Formulas 5 and 7 refer to compounds with n-3DPA moieties attached. Formulas 6 and 8 refer to compounds with SDA moieties attached. In fact, the DHA, EPA, DPA and SDA moieties can in principle be replaced by any omega-3 fatty acid, as long as the omega-3 fatty acid has 14 or more C atoms. However, DHA, EPA, DPA and SDA are considered to be the most relevant in terms of human eye health.

[0163] An alternative embodiment of the present invention relates to the first embodiment of the present invention, in which the DHA, EPA, DPA and SDA moieties are replaced by any omega-3 moiety; at least i) any omega-3 moiety having 14 or more C atoms in the chain, or ii) any omega-3 moiety having a length corresponding to a chain length of 14 or more C atoms.

[0164] An alternative embodiment according to the present invention relates to the first embodiment of the invention, wherein the DHA, EPA, DPA and SDA moieties are replaced by DHA, EPA, DPA, ALA and SDA moieties.

[0165] Throughout this application, the terms LPC compound and "active ingredient" / "active component" all refer to compounds of formulas 1-8.

[0166] The one or more active ingredients in which R1 is OH and R2 is OH referred to in the first aspect of the present invention are all LPC molecules having any of DHA, EPA, DPA or SDA molecules attached to the triacylglycerol portion of the LPC. Technical effects have been demonstrated for LPC-DHA and LPC-EPA. Based on the data presented in WO2018162617 and WO2008068413, the one or more active ingredients in which R1 is O-CO-(CH2) n -CH3 and R2 is O-CO-(CH2) nIt is believed that similar benefits will be obtained with one or more active ingredients in which: -CH3; and n is 0, 1 or 2, particularly where n=0.

[0167] Although the results presented herein are impressive for intravenous administration of formulations containing LPC compositions, the effect can be further improved, for example, by including a pharma- ceutically acceptable carrier.For example, liposomes can be a suitable carrier for the oily constituents of the present invention by providing a hydrophobic interior for the oily substance and a hydrophilic exterior facing a hydrophilic environment.In addition, it is also known that LPC typically associates with blood proteins such as albumin, reducing the effective concentration of LPC.

[0168] The formulations containing the LPC compositions of the present invention may or may not contain one or more solvents, such as ethanol and / or water.When the composition contains one or more solvents, the amount of one or more active ingredients in the composition may be referred to as the dry weight % of the composition.However, when the composition does not contain one or more solvents, the amount of one or more active ingredients in the composition may be referred to as the weight % of the composition.

[0169] In one embodiment of the invention, a formulation containing an LPC composition may include a combination of two or more of one or more active ingredients, one of which may have a DHA moiety attached to a glycerol backbone and another of which may have an EPA moiety attached to a glycerol backbone.

[0170] Thus, in one embodiment of the present invention, the formulation comprising the LPC composition comprises a combination of two or more of one or more active ingredients. One of the active ingredients has a DHA moiety attached to the glycerol backbone, and the other active ingredient has an EPA moiety attached to the glycerol backbone. In a preferred embodiment, there is a specific molar ratio of the active ingredient having a DHA moiety attached to the glycerol backbone to the active ingredient having an EPA moiety attached to the glycerol backbone. The molar ratio of the active ingredient having a DHA moiety attached to the glycerol backbone: the active ingredient having an EPA moiety attached to the glycerol backbone is preferably in the range of 1:1 to 10:1, such as in the range of 1:1 to 7:1, or in the range of 1:1 to 5:1, or in the range of 1:1 to 3:1. In another embodiment of the present invention, the molar ratio of the active ingredient having an EPA moiety attached to the glycerol backbone: the active ingredient having a DHA moiety attached to the glycerol backbone is preferably in the range of 1:1 to 10:1, such as in the range of 1:1 to 7:1, or in the range of 1:1 to 5:1, or in the range of 1:1 to 3:1.

[0171] The following example describes how to calculate the molar ratio.If a composition contains 10 moles of LPC-DHA and 2 moles of LPC-EPA, the molar ratio of the active ingredient with DHA moiety bound to glycerol backbone and the active ingredient with EPA moiety bound to glycerol backbone is 10:2, i.e. 5:1.Unless otherwise stated, the moles of LPC-EPA are the moles of 1-LPC-EPA+the moles of 2-LPC-EPA, and the moles of LPC-DHA are the moles of 1-LPC-DHA+the moles of 2-LPC-DHA.

[0172] It has been previously discussed that the position of the omega-3 fatty acid moiety on the glycerol backbone may affect the ocular uptake of the fatty acid. Thus, in one embodiment of the present invention, the enumerated omega-3 fatty acid moiety is attached to the sn-1 position of the glycerol backbone. In another embodiment of the present invention, the enumerated omega-3 fatty acid moiety is attached to the sn-2 position of the glycerol backbone. In an alternative embodiment of the invention, there is a specific molar ratio of active ingredient having an omega-3 fatty acid moiety attached to the sn-1 position of the glycerol backbone to active ingredient having an omega-3 fatty acid moiety attached to the sn-1 position of the glycerol backbone. The molar ratio of active ingredient having an omega-3 fatty acid moiety attached to the sn-2 position of the glycerol backbone:active ingredient having an omega-3 fatty acid moiety attached to the sn-1 position of the glycerol backbone is preferably in the range of 1:8 to 18:1, such as in the range of 1:8 to 15:1, or in the range of 1:8 to 10:1.

[0173] Reference is made to the following example which illustrates how to calculate the molar ratio: If a composition comprises 5 moles of 2-LPC-DHA, 5 moles of 2-LPC-EPA and 2 moles of 1-LPC-DHA, the molar ratio of active ingredient having an omega-3 fatty acid moiety attached to the sn-1 position of the glycerol backbone to active ingredient having an omega-3 fatty acid moiety attached to the sn-2 position of the glycerol backbone is 10:2, i.e. 5:1.

[0174] The second aspect of the present invention relates to a krill oil composition enriched in LPC-DHA and LPC-EPA. Krill oil contains significant amounts of EPA and DHA in the form of phospholipids, but these fatty acids are at the sn-2 position of the phospholipids, are released by pancreatic PLA2 as free acids during digestion, are then absorbed as TAG, and are therefore not efficiently transported into the brain (16). However, when krill oil is pretreated with lipase specific for the sn-1 position, EPA and DHA are released as LPC, which makes them more likely to be absorbed as phospholipids and taken up into the brain (Figure 11).

[0175] On the other hand, since fish oil does not contain phospholipids, treatment of fish oil with lipase does not generate either LPC-EPA or LPC-DHA, but releases omega-3 fatty acids as either free acids or monoacylglycerols, which are absorbed as TAGs and therefore do not enrich the brain for omega-3 fatty acids. In this study, we compared the ability of fish oil and krill oil to enrich the brain for omega-3 fatty acids in normal mice before and after treatment with lipase. The results clearly show that lipase treatment of krill oil enriches the brain for EPA and DHA, but lipase treatment of fish oil showed no effect on its ability to increase either EPA or DHA.

[0176] The krill oil composition of some further embodiments of the present invention is prepared by hydrolyzing krill oil in the presence of an excess of a lipase specific for the sn-1 position of krill oil phospholipids at a temperature of about 40° C. to about 50° C. under an inert atmosphere for a time sufficient to ensure substantially complete hydrolysis of the sn-1 ester bond. Suitable lipases include lipase from Mucor meihei, Rhizopus oryzae lipase, Novozyme 435 (from Candida sp.) and Lipozyme TLIM (from Thermomyces sp.). In some embodiments, the lipase comprises lipase from Mucor meihei. In some embodiments, up to greater than 50 weight percent of the lipase is used to hydrolyze the krill oil. In some embodiments, the lipase is immobilized.

[0177] In a particular embodiment, a solution of krill oil in 95% ethanol is treated with 1.33 times its weight of lipase from Mucor meihei by stirring at 40° C. for 72 hours, after which the ethanol is evaporated to provide a lipase-treated krill oil composition.

[0178] Lipase-treated krill oil prepared as described herein may be further purified for use as a food additive or for incorporation into functional food or pharmaceutical compositions. Methods for purifying krill oil compositions are known in the art and may include solvent extraction, chromatography, and the like. Chromatographic purification typically involves loading the lipid extract onto a silica gel column and eluting with solvents of increasing polarity. After removing neutral lipids and untreated phospholipids, lyso-PC was eluted with methanol to obtain a pure compound containing about 85% EPA and DHA.

[0179] Also disclosed herein is a composition comprising the lipase-treated krill oil of the present invention.The formulation, composition, or substance of the present invention may be prepared in many ways, such as as a dry powder, as a capsule, as a ready-to-drink juice, or as a food additive.Alternatively, the formulation may also be brewed, fermented, boiled to make a compote prepared as an alcoholic or water extract.In general, any formulation using the extract described herein in any manner is within the contemplated scope of the present invention.

[0180] In one embodiment, the lipase-treated krill oil composition may be packaged and sold as a powder. The powder may then be reconstituted with water or blended with food and ingested for medicinal, prophylactic, nutritional, or other health-related purposes. Alternatively, the powder may be encapsulated. The capsule may then be swallowed or broken open and used as the powder described above. Finally, the powder may be formed into a solid pill. The solid pill may be dissolved in water for ingestion, crushed into a powder as described above, or swallowed.

[0181] The lipase treated krill oil can be incorporated into solid compositions which include conventional non-toxic solid carriers including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.

[0182] For example, liquid compositions can be prepared by dissolving, dispersing, etc., the lipase-treated krill oil and any adjuvants described herein in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, etc., to form a solution or suspension. If desired, the liquid compositions can also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, etc. (e.g., sodium acetate, sorbitan monolaurate, sodium triethanolamine acetate, triethanolamine oleate, etc.). Actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington's Pharmaceutical Sciences, referenced above.

[0183] Other embodiments include the use of permeation enhancer excipients including polymers such as polycations (chitosan and its quaternary ammonium derivatives, poly-L-arginine, aminated gelatin); polyanions (N-carboxymethylchitosan, poly-acrylic acid); and thiolated polymers (carboxymethylcellulose-cysteine, polycarbophil-cysteine, chitosan-thiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugates).

[0184] For oral administration, the composition generally takes the form of a tablet, capsule, soft gel capsule, or may be an aqueous or non-aqueous solution, suspension, or syrup. Tablets and capsules for oral use may contain one or more commonly used carriers, such as lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. Typically, the composition of the present disclosure can be combined with an oral non-toxic pharma- ceutically acceptable inert carrier, such as lactose, starch, sucrose, glucose, methylcellulose, hypromellose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, and the like. In addition, suitable binders, lubricants, disintegrants, and coloring agents can be incorporated into the mixture, as desired or necessary. Suitable binders include starch, gelatin, natural sugars (e.g., glucose or beta lactose), corn-derived sweeteners, natural and synthetic gums (e.g., acacia, tragacanth, or sodium alginate), carboxymethylcellulose, polyethylene glycol, wax, and the like. Lubricants used in these dosages include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc.

[0185] Formulations for oral administration include tablets, lozenges, gels, and the like. Alternatively, oral administration can be performed using transmucosal delivery systems known to those skilled in the art. The extracts of the present disclosure can be delivered through the skin or mucosal tissue using conventional transmucosal delivery systems, i.e., transdermal "patches," in which the drug is typically contained within a laminated structure that serves as a drug delivery device that is secured to the body surface. In such structures, the drug composition is typically contained in a layer, or layers, "reservoirs" that are underneath an upper backing layer. The laminated device can contain a single reservoir, or can contain multiple reservoirs. In one aspect, the reservoir comprises a polymeric matrix of a pharma- ceutically acceptable contact adhesive material that serves to secure the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylene, polysiloxane, polyisobutylene, polyacrylate, polyurethane, and the like. Alternatively, the drug-containing reservoir and the skin contact adhesive are present as separate, distinct layers (adhesive underneath the reservoir), in which case the reservoir may be a polymer matrix as described above, or a liquid or gel reservoir, or may take some other form. The backing layer in these laminates serves as the upper surface of the device, and acts as the main structural element of the laminate structure, providing the device with high flexibility. The material selected for the backing layer should be substantially impermeable to the active agent and any other materials present.

[0186] When liquid suspension is used, the active agent can be combined with any oral non-toxic pharma- ceutically acceptable inert carrier (such as ethanol, glycerol, water, etc.), as well as emulsifiers and suspending agents.If desired, flavoring agents, coloring agents, and / or sweetening agents can also be added.Other optional components for incorporation into the oral formulations herein include, but are not limited to, preservatives, suspending agents, thickening agents, etc.

[0187] Lipase-treated krill oil can be formulated as an ointment or cream. Ointments are semi-solid preparations typically based on petrolatum or other petrolatum derivatives. Creams containing the selected active agent are viscous liquids or semi-solid emulsions, either oil-in-water or water-in-oil, as known in the art. Cream bases are water-washable and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also called the "internal" phase, is generally composed of petrolatum and a fatty alcohol, such as cetyl or stearyl alcohol; the aqueous phase usually, but not necessarily, exceeds the oil phase in volume and generally contains a humectant. Emulsifiers in cream formulations are generally nonionic, anionic, cationic, or amphoteric surfactants. The specific ointment or cream base used is one that provides optimal drug delivery, as will be understood by those skilled in the art. As with other carriers or vehicles, ointment bases should be inert, stable, non-irritating, and non-sensitizing.

[0188] Another aspect of the present invention provides a food composition comprising the lipase-treated krill oil of the present invention. In a particular embodiment, the lipase-treated krill oil according to the present invention can be used as an active ingredient in health functional foods or general foods. The food composition of the present invention may be used to prevent or treat neurological diseases such as Alzheimer's disease. In this case, the lipase-treated krill oil of the present invention may be added without further processing or may be used together with other foods or food ingredients according to methods known in the art. The amount of the active ingredient may be appropriately determined depending on its intended purpose, such as prevention, health management or treatment purposes. The food composition of the present invention may further contain one or more additives selected from nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonation additives for carbonated drinks. The food composition of the present invention may further contain fruit pulp or plant pulp for producing natural fruit juices, fruit juice drinks, and vegetable drinks. Such materials can be used independently or as mixtures thereof. The proportion of such additives is not limited, but is typically selected from the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

[0189] When intended to produce food or beverage, the lipase-treated krill oil of the present invention is typically added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of the raw material of the food or beverage. When the food or beverage is consumed for a long period of time for health purposes and hygiene or health management, the amount of broccoli or extract may be adjusted to be below the lower limit defined above. The food composition of the present invention uses a broccoli plant extract consisting only of natural products or naturally occurring chemicals, so there are no safety-related issues. Therefore, broccoli or extract may also be used in an amount exceeding the upper limit defined above.

[0190] There is no particular limitation on the type of food. Examples of foods to which lipase treated krill oil can be added include common foods such as meat, sausages, bread, chocolate, candy, snacks, cookies, pizza, instant noodles, other noodles, chewing gum, dairy products including ice cream, soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes.

[0191] The beverage may contain one or more additional ingredients, such as flavorings or natural carbohydrates, as in a typical beverage. The natural carbohydrates may be monosaccharides, such as glucose and fructose, disaccharides, such as maltose and sucrose, polysaccharides, such as dextrin and cyclodextrin, and sugar alcohols, such as xylitol, sorbitol, and erythritol. The beverage may contain one or more sweeteners. For example, natural sweeteners, such as thaumatin and stevia extract, and synthetic sweeteners, such as saccharin and aspartame, may be used as sweeteners.

[0192] A third aspect of the invention relates to a formulation comprising an LPC composition according to the first or second aspect of the invention for use in prophylaxis and / or treatment. In some preferred embodiments, the formulation is to be administered orally or intravascularly.

[0193] The ocular surface can be subject to certain disorders such as dry eye (1) and corneal injury (2).

[0194] Proper hygiene of the periocular area (3) is essential for the health of the ocular surface and the entire eye.

[0195] In a preferred embodiment according to the fourth aspect of the invention, the condition that would benefit from increased levels of retinal DHA and / or EPA levels is an ocular surface disorder.

[0196] Dry eye disease (DED) is one of the most common eye conditions worldwide and a leading reason for visits to an ophthalmologist.

[0197] DED is a multifactorial disease affecting the tear film and ocular surface, causing symptoms of tear film discomfort, visual disturbance and tear film instability, with potential damage to the ocular surface, combined with increased tear film osmolality and ocular surface inflammation (DEWS II-2017 definition).

[0198] In a preferred embodiment according to the fourth aspect of the invention, the condition that would benefit from increased levels of retinal DHA and / or EPA levels is dry eye disease.

[0199] corneal damage The cornea is a transparent membrane located in front of the iris and functions as the human eye's strongest converging lens.

[0200] Lesions affecting the cornea are very frequent and are usually associated with subsequent development of inflammation.

[0201] Corneal injuries include abrasions (such as scratches or scrapes limited to the ocular surface), chemical injuries (caused by fluids entering the eye), and injuries from contact lenses or foreign bodies.

[0202] There are also corneal injuries due to surgical treatments involving the anterior segment of the eye.

[0203] Glaucoma is a chronic degenerative disease affecting the optic nerve and characterized by damage to the nerve fibers that compose it and the resulting loss of visual field. If left untreated, the gradual loss of visual field can lead to blindness.

[0204] Glaucoma is a major social problem: it is the second leading cause of blindness worldwide, affecting approximately 60 million people and causing more than 8 million blindness. In addition, approximately 50% of subjects with glaucoma are unaware that they have the condition.

[0205] It is an insidious disease because the disease is often discovered before symptoms are noticed, although vision changes are already very advanced. However, if diagnosed early and treated appropriately, it can be effectively controlled, allowing good vision for the rest of the patient's life.

[0206] There are many risk factors associated with the development of the disease. The main ones are: -High intraocular pressure (IOP) -Older age -Familial predisposition It is.

[0207] The IOP value is determined by the fluid that circulates inside the eye, namely the aqueous humor. In a healthy eye, the ratio between the aqueous humor produced and the aqueous humor drained is such that it maintains a constant intraocular pressure, typically between 11 and 20 mmHg. In the presence of glaucoma, this ratio is altered due to a reduction in the drainage outflow of aqueous humor that occurs at the level of the trabecular meshwork (the structure that allows the outflow of this fluid from the eye).

[0208] Glaucoma can be classified in several ways: - According to etiology, glaucoma can be primary, when it occurs in the absence of other ocular or systemic diseases, or secondary, when combined with a pre-existing condition; - distinguish between open-angle glaucoma, caused by increased outflow resistance at the level of the trabecular meshwork and closed-angle glaucoma, in which there is an anatomical problem preventing the reach of aqueous humor to the trabecular meshwork, depending on the alteration of the aqueous humor outflow; - Differentiate between high-pressure and normal-tension glaucoma based on the primary risk factor, IOP.

[0209] If the IOP becomes higher than normal after birth, it is considered to be congenital or acquired glaucoma, whereas if it develops during the first few years of life, it is considered to be infantile glaucoma.

[0210] In a preferred embodiment of the invention, a formulation comprising an LPC composition according to the first or second aspect is provided for use to inhibit, prevent or treat glaucoma.

[0211] Retinal disorders A healthy retina is essential for vision because the retina's specialized photoreceptors, cones and rods, convert light stimuli into electrical impulses that are transmitted via the optic nerve to the brain, where the processing and perception of visual images occurs.

[0212] Many serious medical conditions affect the retina, including: -Age-related macular degeneration (AMD) causes progressive damage to the macula, the central and most critical area of ​​the retina, resulting in gradual loss of central vision.

[0213] -Diabetic Retinopathy (DRP): The major and most common eye complication of diabetes. DRP is characterized by damage affecting retinal capillaries in patients with both type 1 or type 2 diabetes mellitus (DM). If left undiagnosed and untreated, it can cause blindness.

[0214] -Diabetic macular edema (DME) is one of the major complications of diabetes along with DRP and, if untreated, can significantly impair central vision. The likelihood of developing DME increases with the length and severity of diabetes: approximately 30% of patients who have had DM for more than 20 years will develop DME.

[0215] In a preferred embodiment according to any of the above aspects, the condition that would benefit from increased levels of retinal DHA and / or EPA levels is dry eye disease, such as ocular inflammation, corneal neuropathy, and abrasions on the ocular surface.

[0216] In some embodiments, ocular inflammatory diseases include corneal dystrophies, trachoma, onchocerciasis, uveitis, sympathetic ophthalmia, and endophthalmitis.

[0217] In an alternative embodiment according to any of the above aspects, the condition that would benefit from increased levels of retinal DHA and / or EPA levels is an ocular neurodegenerative disease selected from the group consisting of age-related macular degeneration, diabetic retinopathy, non-proliferative retinopathy, proliferative retinopathy, diabetic macular edema, retinitis pigmentosa, central vein occlusion and glaucoma.

[0218] LPC composition In some preferred embodiments, the LPC composition is analyzed according to the methods described in WO2019 / 123015, which is incorporated by reference in its entirety.

[0219] In some embodiments according to any of the above aspects, the LPC composition comprises at least one of the LPC compounds of formulas 1-8, and any combination thereof:

[0220] [ka]

[0221] In some embodiments, the LPC composition further comprises a lipid distinct from the LPC selected from the group consisting of triglycerides, ethyl esters, free fatty acids, and phospholipids such as phosphatidylethanolamine and phosphatidylcholine. In some embodiments, the lipid distinct from the LPC is derived from a different source, such as a non-cryl source.

[0222] In some embodiments, the LPC composition comprises an amount of total LPC equivalent to 10-100% by weight of the LPC composition (e.g., 10-100% by weight of the LPC composition, preferably 15-100% by weight of the LPC composition, more preferably 20-100% by weight of the LPC composition, even more preferably 30-100% by weight of the LPC composition, and most preferably 50-100% by weight of the LPC composition).

[0223] In some embodiments, the LPC composition comprises a fatty acid profile in which the amount of DHA corresponds to 5% to 12% by weight of the composition, where DHA is present as a free fatty acid, or as an ethyl ester, or bound to any lipid in the LPC composition.

[0224] In one embodiment, the LPC composition comprises a fatty acid profile in which the amount of EPA corresponds to 10% to 24% by weight of the composition, where EPA is present as a free fatty acid, or as an ethyl ester, or bound to any lipid in the LPC composition.

[0225] In one embodiment, the LPC composition further comprises palmitoleic acid and / or palmitic acid.

[0226] In one embodiment, the LPC composition comprises a fatty acid profile in which the amount of palmitoleic acid corresponds to 2% to 5% by weight of the composition, where the palmitoleic acid is present as a free fatty acid, or as an ethyl ester, or bound to any lipid in the LPC composition.

[0227] In one embodiment, the LPC composition comprises a fatty acid profile in an amount of 10% to 15% by weight of the composition, wherein palmitic acid is present as a free fatty acid, or as an ethyl ester, or bound to any lipid in the LPC composition.

[0228] In one embodiment, the LPC composition comprises total phospholipids in an amount representing at least 35% by weight of the LPC composition.

[0229] In a further embodiment, the LPC composition comprises a major or majority amount of at least one LPC compound according to the invention compared to the amount of phosphatidylcholine.

[0230] In one embodiment, the LPC composition comprises total LPC in an amount representing at least 23, 24, 25, 26, 27 or 28% by weight of the LPC composition.

[0231] In one embodiment, the LPC composition comprises an amount of total LPC representing at least 60% by weight of the LPC composition.

[0232] In one embodiment of the first aspect, the LPC composition comprises total LPC in an amount representing at least 90% by weight of the LPC composition.

[0233] In one embodiment of the first aspect, the LPC composition comprises total LPC in an amount corresponding to 90% to 98% by weight of the LPC composition.

[0234] In one embodiment of the first aspect, the LPC composition comprises total LPC in an amount equivalent to about 95% by weight of the LPC composition.

[0235] For example, in one embodiment, the LPC composition comprises 60% to 100% total LPC by weight (such as about 60% to 95% total LPC by weight).

[0236] In some embodiments of the invention, the LPC compound is any krill-derived processed phospholipid product that contains significantly higher amounts of LPC compared to the PC produced by Aker Biomarine.

[0237] In a further embodiment of any of the above aspects, the LPC compound is selected from LPC-EPA, LPC-DHA, and any combination thereof.

[0238] In further embodiments of any of the above aspects, the LPC composition is a krill oil LPC composition. In some preferred embodiments, the krill oil LPC composition is enriched in LPC-EPA and LPC-DHA. In some preferred embodiments, the krill oil composition is lipase-treated krill oil. In some embodiments, the lipase-treated krill oil is made as described in Example 4. In some preferred embodiments, the lipase-treated krill oil comprises at least 80% by weight LPC-DHA and LPC-EPA. In some preferred embodiments, the lipase is a lipase from Mucor meihei. In some preferred embodiments, the lipase is immobilized.

[0239] Other Therapeutic Uses In a sixth aspect, the present invention relates to the use of the compositions and preparations described in the first and second aspects above for treating neurological disorders.In some embodiments, the neurological disorder is selected from Parkinson's disease, schizophrenia, traumatic brain injury, stroke and Alzheimer's disease.In some particularly preferred embodiments, the neurological disorder is Alzheimer's disease.

[0240] In a seventh aspect, the present invention relates to the use of the compositions and preparations described in the first and second aspects above for treating liver diseases that are alleviated by enriching the liver with DHA. In some preferred embodiments, the liver disease is selected from non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

[0241] Having generally described the invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only and are not intended to be limiting, unless otherwise specified.

[0242] 〔experiment〕 Example 1: Preparation of oral and intravenous formulations used in Examples 2-4 (material)

[0243] [Table 1]

[0244] [Table 2]

[0245] [Table 3]

[0246] [Table 4]

[0247] ([ 14 C]-LPC-DHA formulation, herein referred to as formulation A) The formulations subsequently administered intravenously were prepared according to the following target specifications:

[0248] [Table 5]

[0249] [ 14 C]-LPC-DHA was mixed with the Intralipid formulation to give a final concentration of 190 mg / kg phospholipid and a concentration of approximately 1.5 mg / kg (155 μCi / kg) of [ 14 A dosage formulation containing [C]-LPC-DHA was obtained: 0.394mL of ethanol [ 14 C-LPC-DHA (2361 μCi / mL) was dispensed into a 20 mL glass vial and reduced to a final volume of approximately 0.30 mL under a nitrogen stream at ambient temperature. 5.70 mL of 20% Intralipid was diluted with concentrated ethanolic [ 14 C]-LPC-DHA solution and gently vortexed to ensure homogeneity.

[0250] ([ 14C]-LPC-EPA formulation, herein referred to as formulation B) The formulations subsequently administered intravenously were prepared according to the following target specifications:

[0251] [Table 6]

[0252] [ 14 C]-LPC-EPA was mixed with the Intralipid formulation to give a final concentration of 190 mg / kg phospholipid and a concentration of approximately 1.5 mg / kg (155 μCi / kg) of [ 14 A dosing formulation containing [C]-LPC-EPA was obtained as follows: 0.394mL of ethanol [ 14 C]-LPC-EPA (2361 μCi / mL) was dispensed into a 20 mL glass vial and reduced to a final volume of approximately 0.30 mL under a nitrogen stream at ambient temperature. 5.70 mL of 20% Intralipid was diluted with concentrated ethanolic [ 14 C]-LPC-EPA solution and gently vortexed to ensure homogeneity.

[0253] ([ 14 C]-LPC-EPA and [ 14 Oral Dosage Formulation of [C]-LPC-DHA [ 14 C]-LPC-EPA and [ 14 Oral dosing formulations of [C]-LPC-DHA were prepared by adding an ethanolic solution of each radiolabeled compound to the modified krill oil LPC formulation at a concentration ratio of 10:90 v / v radiolabeled ethanol:modified krill oil.

[0254] Each formulation subsequently administered orally was prepared according to the following target specifications:

[0255] [Table 7]

[0256] [ 14C]-LPC-EPA was mixed with the phospholipid formulation to a final concentration of 855 mg / mL phospholipid and approximately 1.5 mg / kg (155 μCi / kg) of [ 14 A dosing formulation containing [C]-LPC-EPA was obtained as follows: The final total concentration of ethanol in the formulation was approximately 10%.

[0257] 0.328mL of ethanol 14 C]-LPC-EPA (2361 μCi / mL) was dispensed into a 20 mL glass vial. 0.172 mL of ethanol was added (to 10% by volume). 4.5 mL of the modified krill oil LPC formulation (37 °C) was diluted with concentrated ethanolic [ 14 C]-LPC-EPA solution and mixed by vortexing and vigorous pipetting to ensure homogeneity.

[0258] the above[ 14 As described for [C]-LPC-EPA, 14 C]-LPC-DHA formulation was added to the phospholipid formulation.

[0259] Example 2: Uptake of LPC in Ocular Tissues - Oral and Intravenous Administration Thirty-two male Sprague Dawley rats, approximately 7-8 weeks of age and weighing 213-289 g at the time of dose administration, were housed in polypropylene cages and left undisturbed except for a short period during dosing. The rooms in which the animals were located were thermostatically monitored and data were recorded continuously (typically temperature range 21 ± 2°C and humidity range 55 ± 10%) and exposed to 12 hours of fluorescent light and 12 hours of darkness per day. The animals were equilibrated under standard animal housing conditions for a minimum of 3 days prior to use. The health of the animals was monitored throughout this period and the suitability of each animal for experimental use was confirmed prior to use.

[0260] Pelleted food (RM1 (E) SQC, Special Diets Services, Witham, Essex, UK) and water (from a domestic water supply) were available ad libitum throughout the holding, acclimation and post-dosing periods.

[0261] Sixteen rats were administered a single intravenous dose of either Formulation A or Formulation B (8 rats per formulation) according to the dosing specifications specified in Example 1. Each rat was weighed prior to administration of the dose, and the individual dose administered was calculated based on body weight and the specific dosing volume.

[0262] Sixteen rats selected for oral dosing were fasted overnight and the dose was administered 1 hour after reintroduction of food. The dosing equipment for oral administration consisted of a syringe and a gavage tube. During dose administration, the tube was fed into the esophagus so that the formulation could be administered directly into the stomach.

[0263] The dosing equipment for intravenous administration consisted of a hypodermic syringe and needle. The dose was administered directly into the tail vein as a slow bolus over 30 seconds.

[0264] After administration of the formulation to male rats, one rat was euthanized by carbon dioxide gas overdose at each of the following times: 0.5, 3, 8, 24, 72, 96, 168, and 336 hours post-dosing.

[0265] Each carcass was flash frozen in a hexane / solid carbon dioxide mixture immediately after collection and then stored at approximately −20° C. until analysis by QWBA (quantitative whole body autoradiography).

[0266] Frozen carcasses were subjected to QWBA using a procedure based on the work of Ullberg (Acta. Radiol. Suppl 118, 22 31, 1954). Sections were presented at up to five different levels of the rat's body to include 30-40 tissues (exposed in the presence of sufficient radioactivity) whose uptake in the brain, blood, kidneys and spleen is disclosed herein.

[0267] Lyophilized whole-body autoradiography sections were exposed to phosphor-storage imaging plates and incubated at ambient temperature in the dark for a minimum of 5 days.

[0268] A series of calibrated autoradiographs containing known amounts of radioactivity [ 14 C] Microscale (nCi / g, Perkin Elmer) was exposed alongside the animal sections on each plate.

[0269] The distribution of radioactivity was measured in the ocular samples and microscale and quantified using a Fuji FLA-5100 fluorescence image analysis system and associated Tina (version 2.09) and SeeScan (version 2.0) software.

[0270] A representative background radioactivity measurement was performed for each exposure plate used. The limit of accurate quantification was considered to be the lowest visible [14C] microscale. A standard curve was generated from the microscale using Seescan, from which tissue concentrations of radioactivity were determined (nCi / g). For calculation of weight equivalent / g data, the nCi / g data were divided by the associated specific activity (nCi / μg).

[0271] Table 1a shows that male albino rats were given an average of 1.5510 mg / kg 14 1 shows the total amount of radioactivity in ocular tissues following a single intravenous administration of [C]-LPC-DHA.

[0272] Table 1b presents the same data as 1a in molar concentrations, normalized to a dose of 2.80 micromoles / kg (1.5949 mg / kg).

[0273] Table 2a shows the total amount of radioactivity in the eye following a single intravenous dose of [14C]-LPC-EPA at an average dose of 1.4968 mg / kg in male albino rats.

[0274] Table 2b presents the same data as 2a in molar concentrations, normalized to a dose of 2.80 micromoles / kg (1.5218 mg / kg).

[0275] Table 3a shows that male albino rats received an average of 1.6914 mg / kg 14 1 shows the total amount of radioactivity in the eye following a single oral dose of [C]-LPC-DHA.

[0276] Table 3b presents the same data as 3a in molar concentrations, normalized to a dose of 2.80 micromoles / kg (1.5949 mg / kg).

[0277] Table 4a shows that male albino rats were given an average of 1.6759 mg / kg 14 3 shows the total amount of radioactivity in the eye following a single oral dose of [C]-LPC-EPA.

[0278] Table 4b presents the same data as 4a in molar concentrations, normalized to a dose of 2.80 micromoles / kg (1.5218 mg / kg).

[0279] The results are also presented in Figures 8-10.

[0280] [Table 8]

[0281] [Table 9]

[0282] [Table 10]

[0283] [Table 11]

[0284] When administered orally to avoid initial exposure and degradation in the mouth and intestines, DHA and surprisingly, its LPC form, EPA (their respective 14 The uptake and accumulation of 1,2-dichlorophenyl phosphate (as measured by C radiolabeled carboxylic acid residues) in ocular tissues is also very rapid and sustained, as shown in Tables 1-4 and Figures 8-10.

[0285] It was demonstrated that for LPC-EPA given as a brief intravenous (iv) bolus, the area under the concentration-time curve (AUC) for the first 24 hours was more than 5-fold greater than the equivalent measurement for orally dosed LPC (Figure 9). It was demonstrated that for LPC-DHA given as a brief intravenous (iv) bolus, the area under the concentration-time curve (AUC) for the first 24 hours was more than 40-fold higher than the equivalent measurement for orally dosed LPC-DHA (Figure 9).

[0286] Example 3: Effect of a composition containing a combination of LPC EPA and LPC DHA.

[0287] This example provides data on daily dosing of various krill oil lysophospholipids containing LPC-EPA and LPC-DHA for three weeks.It was of interest to determine whether krill oil lysophospholipid compositions cause an increase in retinal EPA and DHA content.The EPA, DHA and total omega-3 content of these oils are shown in Table 5 below.The krill oil lysophospholipid compositions of various purity and their production have been described in detail previously (WO2019 / 123015).

[0288] [Table 12]

[0289] Twenty-four male rats were divided into six groups and gavaged daily for three weeks with: group 1) olive oil (0 mg / kg / day EPA and 0 mg / kg / day DHA); group 2) crude (27% LPC), low dose (185 mg / kg / day EPA and 108 mg / kg / day DHA); 3) crude (27% LPC), medium dose (370 mg / kg / day EPA and 217 mg / kg / day DHA); 4) crude (27% LPC), high dose (926 mg / kg / day EPA and 543 mg / kg / day DHA); 5) pure (89% LPC), medium dose (324 mg / kg / day EPA and 160 mg / kg / day DHA); 6) Superba Boost krill oil, medium dose (379 mg / kg / day EPA and 219 mg / kg / day DHA).

[0290] After tissue homogenization, retinal FAs were extracted and analyzed by HPLC. As seen in Figures 1 and 2, there is a strong dose-response relationship, with higher doses of EPA being associated with higher retinal EPA concentrations (Figure 1) and increased EPA / ETA (20:4) ratios (Figure 2). Surprisingly, Figure 1 shows no difference in retinal EPA concentrations between the "crude" krill oil lysophospholipid composition and the "pure" krill oil lysophospholipid composition. However, rats fed the crude lysophospholipid composition show a higher retinal EPA / ETA ratio than rats fed Superba Boost krill oil at a similar dose. This means that rats fed the LPC composition show a more favorable EPA profile in the retina compared to rats fed Superba Boost krill oil and olive oil, and furthermore, higher doses of the lysophospholipid composition are associated with a more favorable EPA profile in the retina. Inspection of Figure 3 suggests that the improved retinal EPA profile is likely related to the greater amount of LPC-EPA present in the plasma (and the subsequent increased EPA uptake via mfsd2a), with the high crude dose showing both the highest levels of LPC-EPA in plasma as well as the highest retinal EPA content and EPA / ETA ratio.

[0291] Figure 4 shows a strong dose-response relationship, with higher doses of the lysophospholipid composition associated with higher DHA / ETA ratios, and a particularly pronounced increase in the DHA / ETA ratio in the crude high-dose group. This suggests that DHA is dose-dependently taken up into the retina from the lysophospholipid composition, regardless of the difference in purity of the lysophospholipid product. Figure 5 further shows that the crude high-dose group shows the highest level of LPC-DHA at the end of the study period (T2), which supports the notion that DHA uptake across the blood-retinal barrier occurs via Mfsd2a.

[0292] Analysis of fatty acid methyl esters (FAMEs) by GC-FID was used to further evaluate brain EPA concentrations relative to total fatty acids (Figure 6), as well as the relationship between arachidonic acid (ARA; 20:4 n-6), DHA dose and test product (Figure 7). GC-fid further supported the HPLC data, with higher doses of the "crude" lysophospholipid composition being associated with higher retinal EPA concentrations compared to total fatty acids (Figure 6). Furthermore, Figure 7 shows that higher doses of the "crude" DHA / EPA lysophospholipid composition were associated with lower levels of retinal ARA concentrations compared to total fatty acids, with the crude lysophospholipid composition showing a more pronounced ARA reduction compared to Superba Boost at comparable doses. This again suggests that increased doses of the lysophospholipid composition are associated with a more beneficial fatty acid profile in the retina.

[0293] Example 4: Use of chrylysophospholipids for delivery of EPA and DHA to the brain, retina and liver.

[0294] 〔material and method〕 Krill oil and fish oil were generous gifts from Bioriginal Food & Science Corporation, Saskatoon, Canada. Antibody against BDNF was obtained from Abcam, Cambridge, MA. Standards of 17:0 LPC, 22:6 LPC were purchased from Avanti Polar Lipids (Alabaster, AL). Immobilized lipase from Mucor meihei was purchased from Creative enzymes (Shirley, NY). Krill oil and fish oil were treated with lipase as follows: oil (15 g) was dissolved in 300 mL of 95% aqueous ethanol (v / v) and 20 g of immobilized lipase was added. The reaction mixture was shaken under nitrogen in an orbital incubator at 175 rpm at 40 °C in the dark for 72 h. Ethanol was evaporated under vacuum and samples were analyzed for lipid and fatty acid composition by TLC and GC / MS. Control and lipase-treated krill oil samples were analyzed by TLC using a solvent system of chloroform:methanol:water (65:25:4, by volume). Spots corresponding to LPC, PC, and PE were scraped and analyzed for fatty acid composition by GC / MS. Control and lipase-treated fish oil samples were separated on TLC plates using a solvent system of hexane:diethyl ether:acetic acid (90:10:1, by volume), and spots corresponding to MAG, DAG, free fatty acids, and TAG were analyzed by GC / MS.

[0295] Diets and Treatments. Treated and untreated krill oil and fish oil containing equivalent amounts of total EPA and DHA (approximately 1.2 μmol / g diet) were mixed with corn oil to provide 7% total fat in the diet, blended with AIN93G rodent diet, pelleted and vacuum sealed by Diets Inc. The control diet contained only corn oil, and diets were stored at -20°C and thawed weekly before use. The fatty acid composition of the edible oils (including the mixed corn oil) is shown in Table 6. As expected, lipase treatment resulted in the production of LPCs rich in EPA and DHA. The percentage of EPA+DHA in the LPC of untreated krill oil was 18%, whereas it was >80% in lipase-treated krill oil. Table 7 shows the distribution of omega-3 fatty acids (EPA+DHA) among the major lipid fractions of the edible oils. PC contained the greatest percentage of total EPA+DHA in untreated krill oil, while LPC contained the greatest percentage in lipase-treated krill oil. Lipase treatment of fish oil resulted in a shift of EPA and DHA from TAGs to free fatty acids and MAGs.

[0296] All experiments were performed under ethical approval from the UIC Institutional Animal Care and Use Committee protocol. Male C57BL / 6 mice (age, 2 months) were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were randomly divided into 5 groups of 5 mice each and fed ad libitum for 30 days. Body weights were measured weekly. After the 30-day period, mice were anesthetized with ketamine (90 mg / ml) and xylazine (10 mg / ml). Blood was collected by cardiac puncture using a heparinized syringe, and plasma was prepared by centrifugation at 1500 × g for 15 min. Mice were then perfused transcardially with ice-cold 100 mM PBS (pH 7.4), and liver, heart, brain, and adipose tissue were harvested. Brains were further dissected to isolate the prefrontal cortex, hippocampus, striatum, and cerebellum. All samples were snap frozen in liquid nitrogen and stored at 80 °C until analysis.

[0297] Statistics Statistical analysis was performed using GraphPad Prism 8.0 software (La Jolla, Calif.). Significance between treatments was determined by one-way ANOVA with post-hoc Tukey multiple comparison tests.

[0298] Lipid extraction and fatty acid analysis Plasma samples for LC / MS analysis of LPC species and isomers were extracted essentially as described by Ivanova et al. (27). Plasma samples (100 μl) were deproteinized with 900 μl of acidified methanol (0.01 N HCl, pH 4.0) after the addition of an internal standard of 17:0 LPC (10 μg). Samples were sonicated for 3 min in a Branson sonicator and centrifuged at 21,500 × g for 10 min at 4 °C. The supernatant was collected and 10 μl was injected into the HPLC system. Analysis of LPC isomers was performed using an Atlantis (Waters Corp., Milford, MA) HILIC column and by multiple reaction monitoring in positive mode electrospray mass spectrometry as previously described (28). Quantification of LPC species was performed with the internal standard 17:0, and a correction factor (× 0.233) was applied to account for differences in ionic intensities of 17:0 LPC and 22:6 LPC species determined separately with known standards. Our previous studies did not include this correction factor, and therefore previously reported plasma values ​​of omega-3 LPC species (16, 17, 29) were approximately 4-fold higher than those reported here.

[0299] For fatty acid analysis, lipids were extracted by the procedure of Bligh and Dyer ( 30 ), and fatty acid methyl esters were prepared using methanolic HCl and analyzed by GC / MS as previously described ( 16 ).

[0300] Western blot analysis BDNF levels in brain fractions were determined by Western blot analysis. Briefly, tissues were homogenized in lysis buffer (50 mM Tris, pH 8.0, 25 mM KCl, 0.5 mM EDTA, Nonidet P-40, 0.1 mM EGTA, 10 μL / mL aprotinin, 1 mM MgCl2, 1 mM CaCl2, 1 mM Na4P2O7, 1 mM Na4MO4, 1% protease inhibitor cocktail, 1 mM phenylsulfonyl fluoride, 10 mM NaF, 1 mM NaV), the suspension was left on ice for 30 min, and centrifuged at 13000×g for 10 min at 4°C. The supernatant (20 μg protein) was subjected to electrophoresis on a 10% SDS gel at 80 V for 2 h. The separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA) rinsed with methanol, and the membrane was blocked with 5% (w / v) nonfat dry milk in Tris-buffered saline (TBST) containing 0.1% Tween 20 for 1 h at 4 °C. The membrane was probed with the primary antibody (BDNF, EPR1292, Abcam, Cambridge, MA) in TBST overnight at 4 °C. The membrane was washed three times with TBST and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technologies, Danvers, MA) for 1 h at room temperature. Finally, the membrane was washed three times with TBST and developed with an enhanced chemiluminescence (ECL) detection kit (Bio-Rad, Hercules, CA) for 1 min, and the respective proteins were quantified by a Bio-Rad ChemiDoc MP Imaging System, and the results were expressed as the ratio of BDNF / β-actin.

[0301] 〔result〕 There were no significant differences in food intake or body weight of mice treated with the different diets (results not shown).

[0302] The percentage composition of plasma ARA (20:4), EPA (20:5), DPA (22:5) and DHA (22:6) is shown in Figure 12. Lipase treatment of fish oil and krill oil resulted in higher accumulation of EPA and DHA in plasma, whereas untreated fish oil and krill oil only modestly increased EPA and DHA. Lipase treatment increased the potency of krill oil over fish oil in increasing plasma EPA and DHA (9-fold increase in EPA and 3-fold increase in DHA for krill oil, compared with 2.2-fold increase in EPA and 70% increase in DHA for fish oil). These results suggest that lipase treatment increases the overall absorption of krill oil over fish oil. Arachidonate content was significantly decreased only in mice fed untreated krill oil or lipase-treated fish oil.

[0303] The concentrations and isomeric composition of LPC species, including EPA, DPA, and DHA, were measured by LC / MS (Figure 13). Although the three LPCs were increased by all diets, the increase after feeding lipase-treated krill oil was 4-5 times greater than the other diets. Furthermore, most of the increase was due to sn-2 acyl isomers in all cases. Although lipase-treated krill oil contained sn-2 acyl LPC isomers, the LPC found in plasma may not all be due to recently absorbed LPC, since plasma sn-2 acyl LPC also increased slightly after feeding fish oil without LPC. Furthermore, the LPC-DHA / LPC-EPA ratio was much higher in plasma compared to the ratio in the diet, suggesting that some conversion of EPA to DHA probably occurred in the liver. Lipase treatment of krill oil resulted in a 4-fold increase in plasma levels of omega-3 LPC compared to untreated krill oil, whereas there was no increase with lipase treatment of fish oil.

[0304] PUFA in Brain Regions Four separate brain regions were analyzed for fatty acid composition. In the prefrontal cortex (Figure 14), EPA content was significantly increased by untreated krill oil (from 0.23% to 1.66% of total FA). However, lipase treatment of krill oil resulted in a further 3-fold increase in cortical EPA compared to untreated krill oil. In contrast, fish oil had no effect on EPA content, with or without treatment with lipase. Cortical DHA content increased by 25% after feeding untreated krill oil, whereas lipase treatment of krill oil resulted in a 118% increase, indicating a 4.7-fold stimulation by lipase treatment. Fish oil had no effect on cortical DHA content before or after lipase treatment. DPA(22:5) was increased 4.5-fold by lipase-treated krill oil, but not by the other preparations. There was a significant decrease in the ARA(20:4) content of the cortex with both the krill oil preparation and the lipase-treated fish oil, but not with the untreated fish oil. The decrease in ARA was apparently due to its replacement with EPA or DHA, but in the case of the lipase-treated fish oil, there was no net increase in omega-3 fatty acids.

[0305] In the hippocampus (Figure 15), EPA content was significantly increased only by lipase-treated krill oil (12.5-fold increase compared to control). DHA content was increased by 15% by untreated krill oil, while lipase treatment produced a 46% increase (3-fold stimulation by lipase treatment). ARA content was significantly decreased by all preparations, although untreated fish oil reduced it less than the other preparations. None of the diets changed DPA.

[0306] In the striatum (Figure 16), EPA content was increased by lipase-treated krill oil (70-fold), but not by untreated krill oil. Interestingly, lipase-treated fish oil also increased EPA content (12-fold), but untreated fish oil had no effect. DHA content was increased by 23% by untreated krill oil and 107% by lipase-treated krill oil (4.7-fold stimulation by lipase treatment). DHA content in the striatum was not affected by untreated or treated fish oil. The percentage of DPA was significantly increased only by lipase-treated krill oil (from 0.14% to 1.45%). ARA content was significantly decreased only by untreated krill oil (-51%) and untreated fish oil (-27%).

[0307] In the cerebellum, only lipase-treated krill oil significantly increased EPA content (11-fold) (Figure 17). DHA content was increased by both untreated and lipase-treated krill oil, but the increase was greater after lipase treatment (12% stimulation by lipase treatment). Fish oil, with or without lipase treatment, showed no significant effect. A significant decrease in ARA content occurred with lipase-treated krill oil. ARA was also decreased by lipase-treated fish oil, although there was no significant increase in omega-3 fatty acids.

[0308] These results indicate that EPA and DHA accumulation in all brain regions was increased several-fold by partial lipolysis of krill oil, whereas the effect of fish oil was minimal even after lipase treatment. These results support the conclusion that the production of LPC-EPA and LPC-DHA is essential for brain accumulation of omega-3 fatty acids. Although LPC-EPA levels were higher than those in plasma, the increase in brain DHA was greater than that of EPA, suggesting that EPA is either converted to DHA in the brain or converted to LPC-DHA before entering the brain.

[0309] BDNF in the brain: Our previous studies have shown that increasing brain DHA or EPA with LPC-DHA and LPC-EPA results in an increase in brain BDNF, a neurotrophin essential for neuronal survival and cognitive function (16, 26). To determine whether similar changes occurred after feeding krill oil and fish oil, we determined the levels of BDNF in the prefrontal cortex and hippocampus by Western blot. As shown in Figure 18, BDNF levels in both brain regions were significantly increased by both untreated and treated krill oil, but the increase with lipase-treated krill oil was three-fold greater than with untreated krill oil. There was a small but significant increase in BDNF in the hippocampus with the fish oil diet, but not in the cortex, and lipase treatment did not show any further increase.

[0310] Adipose tissue In contrast to the brain region, lipase-treated krill oil had no effect on the EPA or DHA content of perigonadal (Figure 19) or inguinal adipose tissue (Figure 20). However, untreated and treated fish oil, as well as untreated krill oil, all increased the EPA and DHA content of both types of adipose tissue. None of the preparations had an effect on the ARA concentration in inguinal adipose tissue. Here, the increase in omega-3 fatty acids did not replace ARA, as occurred in the brain region, but at the expense of 18:1 and 18:2 (not shown). However, in perigonadal adipose tissue, all preparations except lipase-treated krill oil decreased the ARA content in response to the increase in omega-3 fatty acids.

[0311] Liver In the liver, EPA content was significantly increased by both untreated and lipase-treated krill oil (Figure 21). However, the increase after lipase treatment was six-fold greater. DHA levels were significantly increased only by lipase-treated krill oil. The increase in EPA and DHA occurred at the expense of 16:0, which decreased from 28.8% in the control to 25.5% in the lipase-treated krill oil group. Fish oil, with or without lipase treatment, had no effect on either EPA or DHA. However, a decrease in 18:0 was observed in the lipase-treated fish oil group (8.3% vs. 10.6% in the control group). None of the preparations produced significant changes in the hepatic ARA or DPA content.

[0312] Heart In the heart, all preparations increased both EPA and DHA (Figure 22). Interestingly, however, lipase treatment of fish oil or krill oil resulted in a decrease in the heart's ability to enrich with omega-3 fatty acids. No changes were observed in ARA or DPA levels for any of the supplements. No significant changes occurred in other major fatty acids, including 16:0, 18:0, 18:1, or 18:2 (not shown).

[0313] [Consideration] Feed EPA and DHA most commonly occur in the form of TAGs (fish oil, algae oil) or phospholipids (krill oil). Currently, supplements are also available as ethyl esters (e.g., Lovaza, Vascepa) and free fatty acids (e.g., Epanova) of EPA and DHA. However, none of these preparations significantly enrich brain EPA or DHA at recommended doses, although they enrich most other tissues for EPA or DHA. This is evident because these supplements are absorbed primarily as TAGs (16, 31), whereas transporters at the BBB are specific for the LPC forms of EPA or DHA (15). Krill oil has about 40% phospholipids by weight, but the EPA and DHA in the phospholipids are present in the sn-2 position and are released as free fatty acids during digestion by pancreatic PLA2. The released EPA and DHA are then absorbed as TAGs and therefore do not enrich brain omega-3 fatty acids. The aim of this study is to demonstrate that the potency of krill oil to enrich brain EPA and DHA can be increased by predigesting the PC of krill oil with a lipase specific for the sn-1 position, thereby generating LPC-EPA and LPC-DHA, which are then absorbed as phospholipids and preferentially enrich brain omega-3 fatty acids. For comparison, we also treated fish oil, which does not contain phospholipids, with lipase, thus not generating LPC, but generating MAG and free fatty acids. The results presented here show that lipase treatment of krill oil enhances its ability to enrich brain EPA by 10-70 fold and brain DHA by 15-83% compared to untreated krill oil, whereas fish oil, with or without lipase treatment, showed no effect on brain omega-3 fatty acids. It should be noted that there was also a small but significant increase in brain DHA with unprocessed krill oil, apparently due to the small amounts of LPC-EPA and LPC-DHA present in it, and therefore stimulation by lipase treatment is likely to be less pronounced.Furthermore, the potential for increasing brain omega-3 fatty acids by lipase treatment of krill oil is even greater than that shown here, since lipase treatment did not convert all PC to LPC under the conditions used.

[0314] Lipase-treated krill oil was highly efficient in increasing brain EPA and DHA but showed no effect on omega-3 fatty acids in adipose tissue. Similarly, in the heart, all other preparations increased EPA and DHA more efficiently than lipase-treated krill oil, but in the liver, lipase-treated krill oil was more efficient than all other preparations. These results indicate different metabolic fates of omega-3 fatty acids absorbed as either TAG or phospholipids, as previously proposed (17). Because chylomicrons first pass through adipose tissue, muscle, and heart before entering the liver as remnants, the majority of omega-3 fatty acids derived from chylomicron TAG are taken up by lipoprotein lipase-mediated uptake in adipose tissue, muscle, and heart. On the other hand, phospholipid-derived omega-3 fatty acids are taken up by the liver mainly in the form of chylomicron remnants. EPA and DHA are hydrolyzed, incorporated into membranes or lipoproteins, and partially secreted by the liver in the form of LPC (32-34), and then taken up by the brain via the Mfsd2a pathway (15). It is possible that some of the LPC-EPA and LPC-DHA in lipase-treated krill oil is absorbed into the circulation as LPC, bypassing the liver and transporting directly to the brain, although there is no direct evidence for this.

[0315] There is some debate regarding the relative bioavailability of omega-3 fatty acids from fish oil and krill oil. Although many studies claim a higher bioavailability of krill oil fatty acids (35, 36), others have questioned this conclusion due to the methodology used for the comparison (37). Based on the fasting plasma values ​​of EPA and DHA in this study, unprocessed fish oil and krill oil showed similar bioavailability. However, although pretreatment of the oils with lipase increased plasma EPA and DHA in both cases, krill oil showed a greater increase after lipase treatment compared to fish oil. These results suggest that partial hydrolysis with lipase not only increases brain omega-3 fatty acids but may be beneficial for the overall bioavailability of omega-3 fatty acids from fish oil and krill oil to all tissues, which may be particularly important in patients with impaired pancreatic function.

[0316] Our study also showed that lipase-treated krill oil significantly increased BDNF levels in the brain, indicating a functional effect of increasing omega-3 fatty acids in the brain. The increase in BDNF by krill oil was consistent with an increase in brain DHA and EPA, indicating a direct effect of these fatty acids on the expression of BDNF, as reported by other studies (38, 39). Interestingly, fish oil also showed some increase in hippocampal BDNF, although there was no increase in DHA. BDNF plays an important role in neurogenesis, neuroplasticity, and neuroprotection. BDNF is known to decrease in aging, and low levels of BDNF are associated with various psychiatric and neurodegenerative disorders (40). Therefore, increasing brain BDNF by lipase-treated krill oil may potentially provide a safe and effective functional food approach for the prevention and treatment of these diseases.

[0317] Fatty acid analysis of the oils used was performed by GC / MS. The values ​​shown (% of total fatty acids) are for the final oil mixture (corn oil and either fish oil or krill oil). Analysis of fatty acids of the LPC was performed after TLC separation of chloroform extracts of the krill oil mixtures.

[0318] [Table 13]

[0319] Oils used in feed preparations (including corn oil filler) were extracted and separated by TLC using a phospholipid solvent system for krill oil (chloroform:methanol:water, 65:25:4, by volume) or a neutral lipid solvent system for fish oil (hexane:diethyl ether:acetic acid, 70:30:1, by volume). Spots of the lipids shown below were scraped and their fatty acid composition determined by GC / MS as described in the text. Values ​​shown are percentages of total EPA+DPA recovered in a given lipid and are from one representative preparation. Small amounts of DAG present in the fish oil samples, and PE present in the krill samples, are not shown here.

[0320] [Table 14]

[0321] Example 5: Use of Krill Oil LPC Composition to Increase DHA Content in the Retina 〔material and method〕 Animals and dietary treatments: All studies in animals described herein were approved by the Institutional Animal Care and Use Committee at the University of Illinois at Chicago. Retinal samples were obtained from previous studies of brain deposition of DHA in mice and rats published previously [16, 17, 67]. Male Sprague-Dawley rats (8 weeks old) were purchased from Harlan Laboratories (Indianapolis, IN, USA). Male C57 BL / J6 mice (2–4 months old) were purchased from Jackson Laboratories (Bar Harbor, Maine).

[0322] In the first study, male Sprague-Dawley rats (n = 5 per group, 8 weeks old) were gavaged daily with 10 mg of DHA (40 mg DHA / kg body weight) for 30 days in the form of TAG-DHA (DHASCO algal oil, DSM Nutritional Products, Columbia, MD, USA), synthetic di-DHA PC (phosphatidylcholine), or synthetic LPC-DHA (sn-1 acyl)

[17] . DHA was distributed equally among the three positions of TAG-DHA

[17] . The study also included another group of rats gavaged with a half-dose of LPC-DHA (20 mg DHA / kg), comparable to the expected amount of LPC-DHA generated from di-DHA PC during digestion. In the second study, 4-month-old male C57 BL / J6 mice (n = 8 per group) were gavaged daily with 40 mg DHA / kg body weight in the form of free (unesterified) DHA, sn-1 acyl LPC-DHA, or sn-2 acyl LPC-DHA for 30 days as previously described

[16] . In the third study, male C57 BL / J6 mice (2 months old, n = 5 per group) were fed diets enriched with either native or lipase-treated fish oil or krill oil for 30 days. The total amount of omega-3 FAs (eicosapentaenoic acid (EPA) + DHA) was 2.64 g / kg diet in all diets. Untreated krill oil contained 18% of the total omega-3 FAs as LPC, whereas lipase-treated krill oil contained >80% of the total omega-3 FAs as LPC. The fish oil diet did not contain LPC-EPA or LPC-DHA.Animals were perfused transcardially with ice-cold phosphate-buffered saline under anesthesia, and retinas were harvested and frozen at -80°C until analysis.

[0323] Analytical procedure: Total retinal lipids were extracted by the procedure of Bligh and Dyer

[30] and fatty acids were methylated using methanolic HCl. Fatty acid analysis was performed by GC / MS (gas chromatography / mass spectrometry) using a Shimadzu QP2010SE equipped with a Supelco Omegawax column as previously described

[16] . A total ion current in the range of 50–400 m / z was used for quantification of methyl esters. For LC / MS / MS (liquid chromatography / tandem mass spectrometry) analysis, lipids were extracted by the procedure of Ivanova et al.

[27] . Analysis of phospholipid molecular species was performed by multiple reaction monitoring

[31] on an ABSciex QTRAP mass spectrometer (Redwood City, CA, USA) coupled to an Agilent 2600 UPLC system (Santa Clara, CA, USA). The internal standards 17:0 LPC, di17:0 PC, and di17:0 PE (phosphatidylethanolamine) were used for quantification of the corresponding molecular species, but no correction factors for differences in ion intensities of the different molecular species were applied.

[0324] Statistics and correlations: The significance of differences between treatment groups was determined by one-way ANOVA with Tukey's post hoc multiple comparisons correction or unpaired t-tests between control and treated samples adjusted by the Holm-Sidak method (Graphpad Prism 8.0, San Diego, CA, USA).

[0325] 〔result〕 Comparative Effects of Dietary DHA in the Forms of PC, TAG, and LPC in Rats We have previously demonstrated that rat brain DHA is efficiently enriched by dietary LPC-DHA and di-DHA PC, but not by TAG-DHA

[17] . Because the mechanism of DHA uptake appears to be similar for brain and retina

[66] , we determined the abundance of retinal DHA in the same group of animals. Normal rats were gavaged with 10 mg DHA / day (40 mg DHA / kg) in the form of TAG-DHA, di-DHA PC, or LPC-DHA (sn-1 acyl) for 30 days, and the FA composition of the retina was determined by GC / MS. Furthermore, a half dose of LPC-DHA (20 mg / kg) was used to equate to the expected generation of LPC-DHA by digestion of di-DHA PC in the intestinal tract by pancreatic phospholipase A2 (PLA2). The concentrations of omega-3 FAs and arachidonic acid are shown in Figure 23, while the total FA composition is shown in Table 8. As shown in Figure 24, the percentage of DHA in the retina was significantly increased by di-DHA PC (+45%) and LPC-DHA (+101%), but not by TAG-DHA (+13%, not significant). Half the dose of LPC-DHA (5 mg / rat) was more efficient (+75%) than the total dose of di-DHA PC (+45%), indicating that hydrolysis of di-DHA PC by pancreatic PLA2 (phospholipase A2) may not be efficient. Of note, retinal DHA content in normal rats was very high (16.88% of total FAs), but was doubled (to 33.98% of total FAs) by feeding a total dose of LPC-DHA, indicating a wide range of retinal DHA content that can be achieved by diet. The increase in DHA occurred mainly at the expense of arachidonic acid, as we found in the brain

[17] . However, DHA also replaced saturated FAs (16:0 and 18:0) in the retina (Table 8). None of the treatments altered either retinal EPA (20:5(n-3)) or DPA (22:5(n-3)).

[0326] Comparative effects of free DHA and LPC-DHA isomers in mice Previous studies suggested that for DHA uptake by the brain, DHA must be in the sn-2 position of LPC, as this is the natural position of phospholipids in vivo [68, 69]. However, our recent study showed that sn-1 DHA LPC and sn-2 DHA LPC are equally effective in enriching brain DHA and improving brain function in mice

[16] . We analyzed the FA composition of the retina in mice fed free (unesterified) DHA, sn-1 DHA LPC, and sn-2 DHA LPC to determine whether there is a preference for the sn-2 acyl isomer of LPC for transport across the blood-retinal barrier. As shown in Figure 25 and Table 9, free DHA had no effect on retinal DHA, similar to the effect of TAG-DHA in rats. However, both isomers of LPC-DHA increased retinal DHA by 80%, indicating that DHA uptake by the retina is similar to that of the brain and is associated with the Mfsd2a transporter as shown by others

[66] . Furthermore, transporters at the blood-retinal barrier did not distinguish between the two isomers of LPC-DHA, as we found for the brain

[16] . There was no effect on either the EPA (20:5, n-3) or DPA (22:5, n-3) content by either DHA treatment. There was a significant reduction in arachidonate by the two isomers of LPC-DHA, but not by free DHA. In addition, there was a significant reduction in saturated fatty acids (16:0 and 18:0) as well as 18:1 by LPC-DHA, but not by free DHA. These results, similar to those in rats, suggest that the increase in mouse retinal DHA occurred by replacement of not only arachidonate but also saturated FAs and 18:1.

[0327] To determine whether the metabolic fates of the two LPC isomers in the retina differed from each other, the molecular species of PC and PE containing DHA were analyzed by LC / MS / MS. As shown in Figure 26, both isomers of LPC-DHA increased most of the major PC and PE containing DHA, except for 20:4-22:6 PC and 20:4-22:6 PE, probably due to the decrease of 20:4 in the retina by LPC treatment. There was no significant difference between the effects of the two isomers of LPC on the molecular species composition of PC or PE. Although the total DHA content in the retina was not significantly increased by free DHA (Figure 27), a few individual species of PC and PE containing DHA were increased. However, this increase was at a much lower level compared to LPC-DHA. Retinal LPC-DHA was increased after treatment with dietary LPC-DHA, but not LPE-DHA. Unlike the brain, where DHA is more prevalent in the PE species

[16] , the retina contained more DHA in the PC species. PE: phosphatidylethanolamine.

[0328] Effects of fish oil and krill oil on retinal omega-3 FAs in mice Although previous studies with fish oil did not show significant enrichment of retinal DHA or EPA in adult animals

[49] , we recently demonstrated that pretreatment of krill oil with lipase to generate LPC-EPA and LPC-DHA allowed for significant enrichment of both DHA and EPA in the brain of adult mice

[67] . On the other hand, similar treatment of fish oil to generate free EPA and DHA or monoacylglycerol EPA and DHA had no effect on brain omega-3 FAs. To determine whether lipase-treated krill oil could also be used to enrich retinal DHA and EPA, we analyzed the FA composition of the retina in mice treated with fish oil and krill oil, with or without lipase treatment.

[0329] As shown in Figure 27 and Table 10, retinal DHA was increased by 33% over control values ​​after feeding untreated krill oil, likely due to the presence of small amounts of LPC-DHA in the krill oil preparation

[67] . However, feeding lipase-treated krill oil increased retinal DHA by 76%, indicating a 2.3-fold stimulation of enriching DHA by lipase treatment. Furthermore, there was a 100-fold increase in retinal EPA with lipase-treated krill oil, but not with untreated krill oil or fish oil. This supports our previous observation that feeding LPC-EPA increases EPA levels in the brain and retina, contrary to previous reports that EPA in these tissues is not enriched by diets rich in omega-3 FAs [18, 19, 20]. In contrast to the results with pure LPC-DHA, we found no significant displacement of retinal arachidonate by DHA after treatment with lipase-treated krill oil. Instead, DHA and EPA appeared to replace saturated FAs and oleic acid (18:1, n-9). There was also some reduction in saturated FAs and 18:1 in animals fed unprocessed krill oil, but these reductions did not reach statistical significance (Table 10).

[0330] [Consideration] Retinal DHA is known to decrease with age

[54] , as well as in diabetes [57, 58, 59]. Furthermore, reduced DHA levels are associated with several retinal diseases, most notably diabetic retinopathy (DR) [54, 56, 44]. DR affects nearly 100 million people worldwide and is the most common cause of blindness in the adult population

[70] . Hyperglycemia-induced oxidative stress and chronic inflammation are thought to be the primary underlying causes of DR

[71] . DHA, which is uniquely enriched in the retina, has been shown to have both antioxidant and anti-inflammatory properties [54, 72, 73]. In addition to DR, deficiencies in DHA have also been implicated in other diseases of the eye, including retinitis pigmentosa

[74] , glaucoma [75, 76], age-related macular degeneration

[55] , dry eye disease

[77] , and Alzheimer's disease-related blindness

[41] . The common element in all of these diseases is chronic inflammation. It is therefore important to investigate whether retinal DHA levels can be increased through diet in adult mammals, thereby preventing or treating these diseases. The results presented here show that, despite the very high initial levels of DHA in normal retinas, it can be further increased to 100% through dietary LPC-DHA, but not by dietary free or TAG-DHA. To our knowledge, this is the highest enrichment of retinal DHA achieved by dietary supplementation in normal adult animals. Previous studies have found no or only slight increases in retinal omega-3 levels, even after treatment with very high concentrations of dietary omega-3 FA. For example, Prokopiou et al.

[78] fed aged (2-year-old) mice 200 mg omega-3 FA / day in the form of fish oil for 60 days and indeed found a decrease in retinal DHA (-21%), whereas the minor component, retinal EPA, increased by 42%. Similarly, when ABCA4- / - mice (Stargardt disease) were gavaged with 206 mg / day of omega-3 FAs (172 mg EPA + 34 mg DHA) for 3 months, there was no change in retinal DHA but a 67% increase in retinal EPA (from 0.93% to 1.56% total)

[79] .A study by Schnebelen et al.

[80] showed that feeding 3-week-old rats a 5% fat diet containing 20% ​​omega-3 FA for 3 months only increased retinal DHA by 8%. In contrast to these studies, Suh et al.

[82] and Tanito et al.

[81] , using transgenic mice carrying the fat-1 gene that converts endogenous omega-6 FA to omega-3 FA, reported a near doubling of retinal DHA compared to wild-type controls. However, as reported by these researchers, increasing retinal DHA by genetic manipulation rather than diet seems to cause abnormal electroretinograms and susceptibility to oxidative stress. On the other hand, Connor et al.

[83] reported that dietary increase in retinal DHA content in neonatal mice prevents retinopathy of prematurity by inhibiting pathological neovascularization. Furthermore, Sapieha et al.

[56] showed that supplementation of a diet containing a high concentration of omega-3 FA (2% of the diet) preserved retinal function and enhanced glucose tolerance in a mouse model of type 2 diabetes, although no changes in retinal DHA levels were reported. Similar benefits against diabetic retinopathy were shown in rats by Tikhonenko et al.

[44] who fed 5% calories as herring oil. However, the dose of omega-3 FA required to achieve these beneficial effects is not practical in a clinical setting, since the equivalent dose in humans would be approximately 14 g omega-3 FA / day for a 70 kg human, based on the study of Sapieha et al., or approximately 16 mL fish oil / day according to the study of Tikhonenko et al., using allometric calculations

[84] . In contrast, the dose of LPC-DHA required to nearly double retinal DHA is approximately 50-fold lower than in the above studies and is therefore easily applicable to clinical conditions.

[0331] Unlike the brain, which acquires DHA mainly through the Mfsd2a pathway, the retina appears to acquire DHA through multiple pathways. This is because Mfs2a deficiency only results in a 45% decrease in retinal DHA and a 57% decrease in VLCFAs

[85] . Thus, although a study by Bazan

[41] showed the importance of adiponectin receptors for maintaining retinal DHA levels, roles of FA-binding proteins and lipoprotein receptors have been proposed by other studies [54, 65]. These pathways may explain the slight increase in retinal DHA with dietary free DHA and TAG. The retina also has an efficient recycling mechanism that retains DHA by phagocytosis of retinal pigment epithelial cells [52, 41], possibly explaining the milder effect of congenital Mfsd2a deficiency on retinal function [66, 85] compared to brain function

[15] . Another difference between the brain and retina was that in the brain, DHA was largely replaced by arachidonic acid [16, 17, 67], whereas in the retina, DHA replaced more saturated fatty acids and oleic acid than arachidonic acid. Although a reduction in arachidonic acid may be beneficial due to its role in the generation of proinflammatory eicosanoids, the physiological effects of reducing saturated fatty acids and oleic acid in the retina are unclear.

[0332] In contrast to DHA, retinal EPA content is very low and does not increase substantially even after feeding EPA-rich supplements, whereas DHA levels are increased by these treatments [80, 83]. It is therefore hypothesized that EPA does not enter the brain and retina and is not rapidly converted to DHA or oxidized without net accumulation [25, 86]. In the present study, retinal EPA levels did not increase after feeding pure LPC-DHA, but a marked increase occurred after feeding lipase-treated krill oil containing both LPC-EPA and LPC-DHA. Thus, we suggest that the failure of previous studies to show an increase in brain or retinal EPA is due to the inability of the supplement to generate LPC-EPA in vivo. We have previously shown that similar enrichment of brain EPA and retinal EPA occurred after feeding pure LPC-EPA

[26] . Increasing retinal EPA in addition to DHA may be more beneficial than increasing its DHA content alone, as EPA is the preferred substrate for the synthesis of VLCFAs

[89] that have unique functional importance in the retina [87, 88]. EPA is also known to compete more effectively than DHA for arachidonate, thereby inhibiting the synthesis of proinflammatory prostaglandins.

[0333] The majority of studies on omega-3 FAs have focused on increasing tissue EPA and DHA levels alone. However, an ancillary benefit of dietary LPC-EPA / DHA, compared to fish oil or ethyl esters, is that for each molecule of DHA or EPA taken up by the retina and brain via the Mfsd2a pathway, a molecule of choline is simultaneously taken up. Choline is an essential component of acetylcholine as well as membrane phospholipids, which play a key role in vision

[90] . In fact, citicoline (CDP-choline), a precursor of choline phospholipids, is used clinically to treat retinopathy and glaucoma

[90] . Thus, LPC-EPA / DHA could provide the combined benefits of omega-3 FAs and citicoline in a single effective preparation.

[0334]

Table 15

[0335] JPEG0007599077000020.jpg214169

[0336] JPEG0007599077000021.jpg214169

[0337] JPEG0007599077000022.jpg214169

[0338] JPEG0007599077000023.jpg215169

[0339] JPEG0007599077000024.jpg33169

[0340]

Table 16

[0341] JPEG0007599077000026.jpg222169

[0342] JPEG0007599077000027.jpg222169

[0343] JPEG0007599077000028.jpg210169

[0344] JPEG0007599077000029.jpg24169

[0345]

Table 17

[0346] JPEG0007599077000031.jpg212169

[0347] JPEG0007599077000032.jpg212169

[0348] JPEG0007599077000033.jpg212169

[0349] JPEG0007599077000034.jpg154169

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Schnebelen C., Viau S., Gregoire S., Joffre C., Creuzot-Garcher C.P., Bron A.M., Bretillon L., Acar N. Nutrition for the eye: Different susceptibility of the retina and the lacrimal gland to dietary omega-6 and omega-3 polyunsaturated fatty acid incorporation. Ophthalmic Res. 2009;41:216-224. doi: 10.1159 / 000217726. 81. Tanito M., Brush R.S., Elliott M.H., Wicker L.D., Henry K.R., Anderson R.E. High levels of retinal membrane docosahexaenoic acid increase susceptibility to stress-induced degeneration. J. Lipid Res. 2009;50:807-819. doi: 10.1194 / jlr.M800170-JLR200. 82. Suh M., Sauve Y., Merrells K.J., Kang J.X., Ma D.W.L. Supranormal electroretinogram in F at-1 mice with retinas enriched in docosahexaenoic acid and n-3 very long chain fatty acids (C24-C36) Investig. Ophtalmol. Vis. Sci. 2009;50:4394-4401. doi: 10.1167 / iovs.08-2565. 83. Connor K.M., SanGiovanni J.P., Lofqvist C., Aderman C.M., Chen J., Higuchi A., Hong S., Pravda E.A., Majchrzak S., Carper D., et al. Increased dietary intake of -e-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis. Nat. Med. 2007;13:868. doi: 10.1038 / nm1591. 84. Nair A.B., Jacob S. A simple practice guide for dose conversion between animals and human. J. Basic Clin. Pharm. 2016;7:27-31. doi: 10.4103 / 0976-0105.177703. 85. Lobanova E.S., Schuhmann K., Finkelstein S., Lewis T.R., Cady M.A., Hao Y., Keuthan C., Ash J.D., Burns M.E., Shevchenko A., et al. Disrupted blood-retina lysophosphatidylcholine transport impairs photoreceptor health but not visual signal transduction. J. Neurosci. 2019;39:9689-9701. doi: 10.1523 / JNEUROSCI.1142-19.2019. 86. Kaur G., Molero J.C., Weisinger H.S., Sinclair A.J. Orally administered [14C] DPA and [14C] DHA are metabolised differently to [14C] EPA in rats. Br. J. Nutr. 2013;109:441-448. doi: 10.1017 / S0007114512001419. 87. Suh M., Clandinin M.T. 20:5n-3 but not 22:6n-3 is a preferred substrate for synthesis of n-3 very-long-chain fatty acids (C24-C36) in retina. Curr. Eye Res. 2005;30:959-968. doi: 10.1080 / 02713680500246957. 88. Yu M., Benham A., Logan S., Brush R.S., Mandal M.N., Anderson R.E., Agbaga M.P. ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3. J. Lipid Res. 2012;53:494-504. doi: 10.1194 / jlr.M021386. 89. Jun B., Mukherjee P.K., Asatryan A., Kautzmann M.A., Heap J., Gordon W.C., Bhattacharjee S., Yang R., Petasis N.A., Bazan N.G. Elovanoids are novel cell-specific lipid mediators necessary for neuroprotective signaling for photoreceptor cell integrity. Sci. Rep. 2017;7:5279. doi: 10.1038 / s41598-017-05433-7. 90. Faiq M.A., Wollstein G., Schuman J.S., Chan K.C. Cholinergic nervous system and glaucoma: From basic science to clinical applications. Prog. Retin. Eye Res. 2019;72:100767. doi: 10.1016 / j.preteyeres.2019.06.003. All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems of the present invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims. [Brief description of the drawings]

[0351] [Figure 1] FIG. 1 shows the retinal EPA content (AUC / mg dry tissue) at the end of the study. [Diagram 2] FIG. 2 shows the retinal EPA / ETA ratio at the end of the study. [Diagram 3] FIG. 3 shows plasma LPC-EPA (ng / ml) collected after 0 weeks (T0, baseline), 2 weeks (T1) and 3 weeks (T2) of oral gavage. [Figure 4] FIG. 4 shows the retinal DHA / ETA ratio at the end of the study. [Diagram 5] FIG. 5 shows plasma LPC-DHA (ng / ml) collected after 0 weeks (T0, baseline), 2 weeks (T1) and 3 weeks (T2) of oral gavage. [Figure 6] FIG. 6 shows retinal EPA concentrations as a % of total fatty acids for the six experimental groups. [Figure 7] FIG. 7 shows the relationship between retinal ARA (20:4 n-6) concentration and DHA dose for each of the six experimental groups. [Figure 8] FIG. 8 shows the area under the concentration time curve in ocular tissues following oral and iv administration. [Figure 9] FIG. 9 shows the area under the concentration-time curve for ocular tissues (whole eye) following oral and iv administration of LPC-EPA and LPC-DHA for the first 24 hours. [Figure 10]FIG. 10 shows the area under the time concentration curve for ocular tissues (whole eye) following oral and iv administration of LPC-EPA and LPC-DHA for the first 72 hours. [Figure 11] FIG. 11 shows that krill oil contains EPA and DHA at the sn-2 position of PC, and thus EPA and DHA are released as free fatty acids during degradation by pancreatic phospholipase A2. The released EPA and DHA are then absorbed as TAG, which does not generate either LPC-EPA or LPC-DHA required for transport to the brain. Thus, feeding krill oil does not result in significant enrichment of EPA and DHA in the brain. However, when krill oil is pretreated with lipase specific to sn-1 ester bonds, LPC-EPA and LPC-DHA are generated. Feeding this improved krill oil should result in the absorption of phospholipid forms of EPA and DHA, leading to their enrichment in the brain. On the other hand, lipase treatment of fish oil does not generate either LPC-EPA or LPC-DHA, and therefore does not result in enrichment of EPA and DHA in the brain (not shown). [Figure 12] Figure 12 shows the effect of dietary treatment on plasma levels of long-chain PUFAs. Total lipids of fasting plasma were extracted, transmethylated, and analyzed for fatty acid composition by GC / MS as described in the text. Values ​​shown are the mean ± standard deviation of five animals in each group. Statistical significance was determined by one-way ANOVA with post-hoc Tukey multiple comparison test (GraphPad Prism 8.0 software). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared to the control group. [Figure 13]Figure 13 shows the effect of feeding native krill oil and fish oil, and lipase-treated krill oil and fish oil on plasma LPC species. Plasma samples were extracted with acidified methanol as described herein and analyzed by LC / MS using a HILIC column to analyze the concentration and isomeric composition of omega-3 LPC species. LPC concentrations were determined using 17:0 LPC as an internal standard. Values ​​were calculated applying a correction factor (x 0.233) since the intensity values ​​of the 17:0 LPC and 22:6 LPC standards differ by this amount under the conditions used. Bars with non-identical superscripts are significantly different from each other (total LPC) as determined by one-way ANOVA with post-hoc Tukey multiple comparison test. [Figure 14] Figure 14 shows the PUFA composition of the prefrontal cortex. Total fatty acid composition was analyzed by GC / MS, and the percentage composition of only 20:4 (ARA), 20:5 (EPA), 22:5 (DPA), and 22:6 (DHA) is shown. Values ​​shown are the mean ± standard deviation of five animals in each group. Statistical significance was determined by one-way ANOVA, and symbols are the same as under Figure 12 above. [Figure 15] Figure 15 shows the PUFA composition of hippocampus. Fatty acid composition was analyzed as described in the text. Values ​​shown are the mean ± standard deviation of 5 animals in each group. Symbols for statistical significance are the same as those under Figure 12 above. [Figure 16] Figure 16 shows the PUFA composition of the striatum. The PUFA composition was analyzed as described in the text. Values ​​shown are the mean ± standard deviation of five mice in each group. Statistical significance between control and experimental diets was determined by one-way ANOVA with post-hoc Tukey multiple comparison test. Significance symbols are the same as those under Figure 12 above. [Figure 17]Figure 17 shows the PUA composition in the cerebellum. The fatty acid composition was analyzed by GC / MS as described in the text. Values ​​shown are the mean ± standard deviation of six mice in each group. Statistical significance between control and experimental diets was determined by one-way ANOVA with post-hoc Tukey multiple comparison test. Significance symbols are the same as under Figure 12 above. [Figure 18] FIG. 18 shows the effect of dietary treatment on BDNF levels in the prefrontal cortex and hippocampus. Immunoassay of BDNF levels was performed as described in the text, using β-actin as an internal standard. BDNF / β-actin ratios shown are the mean ± standard deviation of three mice in each group. Inset shows a representative immunoblot. Statistical significance between control and experimental diets was determined by one-way ANOVA with post-hoc Tukey multiple comparison test. ***p<0.001 vs control; ****p<0.0001 vs control. [Figure 19] Figure 19 shows the PUFA composition of perigonadal adipose tissue. The percentage values ​​shown are the mean ± standard deviation of 5 mice for each group. The statistical significance values ​​(one-way ANOVA) are between control and experimental diets, and the symbols are the same as those under Figure 12 above. [Figure 20] Figure 20 shows the PUFA composition of inguinal adipose tissue. The percentage values ​​shown are the mean values ​​± standard deviation of 5 mice for each group. The statistical significance values ​​(one-way ANOVA) are between control and experimental diets, and the symbols are the same as those under Figure 12 above. [Figure 21] Figure 21 shows the effect of dietary fat on liver fatty acid composition. Fatty acid analysis of total lipids by GC / MS was as described in the text. Values ​​shown are the mean ± standard deviation of five mice in each group. Statistical significance between control and experimental diets was determined by one-way ANOVA with post-hoc Tukey multiple comparison test. Significance symbols are the same as under Figure 12 above. [Figure 22]Figure 22 shows the effect of dietary fat on cardiac fatty acid composition. Total cardiac lipids were extracted and analyzed for fatty acid composition by GC / MS as described in the text. Values ​​shown are the mean ± standard deviation of five mice in each group. Statistical significance between control and experimental diets was determined by one-way ANOVA with post-hoc Tukey multiple comparison test. Significance symbols are the same as under Figure 12 above. [Diagram 23] Figure 23 shows that krill oil containing EPA and DHA at the sn-2 position of PC treated with lipase that specifically hydrolyzes sn-1 fatty acids produces LPC-EPA and LPC-DHA. Feeding this preparation to normal mice significantly increased retinal EPA and DHA. Similar treatment with fish oil containing EPA and DHA only in triglyceride form did not affect either retinal EPA or DHA. [Figure 24]Figure 24 shows the incorporation of dietary DHA into rat retinal lipids, given in PC, TAG, or LPC form. Normal male Sprague-Dawley rats (8 weeks old; n=5 per group) were gavaged daily for 30 days with 10 mg DHA in 250 μL corn oil in the form of TAG-DHA, di-DHA PC, or LPC-DHA. In addition, a half dose of LPC-DHA (5 mg DHA) was used to match the amount of LPC-DHA expected to be generated by di-DHA PC during digestion. All animals were fed a normal rodent chow diet that contained no DHA but contained 17.4 mg α-linolenic acid (18:3, n-3) per gram of chow. Retinal FA was analyzed by GC / MS as described in the text. The percentage compositions of 20:4(n-6), 20:5(n-3), 22:5(n-3), and 22:6(n-3) are shown here. Total FA composition is shown in Table 1. The significance of differences between treatment groups was determined by one-way ANOVA with post-hoc Tukey multiple comparison test (GraphPad Prism 8.0 software). Bars without a common letter above are significantly different from each other (p<0.05). TG: triacylglycerol; PC: phosphatidylcholine; LPC: lysophosphatidylcholine; FA: fatty acid; GC / MS: gas chromatography / mass spectrometry. [Diagram 25]Figure 25 shows the effect of dietary free (unesterified) DHA, sn-1 DHA-LPC, and sn-2 DHA LPC on mouse retinal FA. Normal male mice (C57 BL / J6, 16 weeks old) were gavaged daily with 1 mg DHA in the form of free DHA, sn-1 DHA LPC, or sn-2 DHA LPC in 80 μL of corn oil for 30 days. Retinal FA composition was analyzed by GC / MS. Only values ​​for arachidonic acid and omega-3 FA are shown here (mean ± standard deviation, 8 animals / group). Total FA composition is shown in Table 2. Bars not topped with a common letter are significantly different from each other (p<0.05) by one-way ANOVA with post-hoc Tukey correction. SD: standard deviation; sn-1 and sn-2: stereospecific numbering 1 and 2, respectively; LPC: lysophosphatidylcholine; DHA: docosahexaenoic acid. [Figure 26A-B] Figure 26 shows the molecular species of mouse retinal phospholipids containing DHA. Animals were gavaged with free (unesterified) DHA or the two isomers of LPC-DHA as described in Figure 2. The molecular species of PC (A) and PE (B) containing DHA were analyzed using multiple reaction monitoring by LC / MS / MS as described in the text, with 17:0 LPC, 17:0-17:0 PC, and 17:0-17:0 PE as internal standards. Bars without a common letter above them are significantly different from each other (mean ± standard deviation, 6 animals / group) by one-way ANOVA and post hoc Tukey multiple comparison test. PE: phosphatidylethanolamine; PC: phosphatidylcholine; sn-1 and sn-2: stereospecific numbering 1 and 2, respectively; DHA: docosahexaenoic acid; LC / MS / MS: liquid chromatography / tandem mass spectrometry; SD: standard deviation; LPC: lysophosphatidylcholine; DHA: docosahexaenoic acid. [Figure 27]Figure 27 shows the effect of feeding untreated or lipase-treated fish oil and krill oil on retinal FA in mice. Normal male mice (8 weeks old) were fed a diet (AIN93G) containing 7% total fat and supplemented with 0.264% EPA+DHA in the form of fish oil or krill oil treated with Mucor lipase (or untreated). Mice were fed the diet ad libitum for 30 days and retinal FA were analyzed by GC / MS. The percentage composition of 20:4(n-6), 20:5(n-3), 22:5(n-3) and 22:6(n-3) is shown here (mean ± standard deviation, 5 mice / group). Total FA composition is shown in Table 3. Bars not topped with a common letter are significantly different from each other by one-way ANOVA and post hoc Tukey multiple comparison test. KO: krill oil; FO: fish oil; FA: fatty acids; EPA: eicosapentaenoic acid; GC / MS: gas chromatography / mass spectrometry; DHA: docosahexaenoic acid.

Claims

1. 1. A formulation for improving, controlling, reducing, or alleviating one or more symptoms and / or signs of an ocular disease or condition in a subject in need thereof, comprising: The formulation is administered to a subject in need thereof in an effective amount of a pharmaceutical or nutraceutical formulation; The preparation comprises a lysophosphatidylcholine (LPC) composition comprising at least an LPC compound represented by formula 1 or 3 and an LPC compound represented by formula 2 or 4, among the LPC compounds selected from the group consisting of any one of formulas 1 to 8, and any combination thereof; The formulation is an oral formulation, ameliorating, controlling, reducing or alleviating the symptoms of a disease or condition; formulation: 【Chemistry 1】 During the ceremony, R １ is OH or -OC(O)(CH ２ ) ｎ CH ３ and R ２ is OH or -OC(O)(CH ２ ) ｎ CH ３ and n is 0, 1 or 2 ； and the molar ratio of lyso-PC-EPA:lyso-PC-DHA is in the range of 1:1 to 5:1; moreover, i) the number of moles of lyso-PC-EPA is the number of moles of 1-lyso-PC-EPA plus the number of moles of 2-lyso-PC-EPA; and ii) The number of moles of lyso PC-DHA is the number of moles of 1-lyso PC-DHA plus the number of moles of 2-lyso PC-DHA.

2. R １ is OH and R ２ The formulation of claim 1 , wherein is OH.

3. The LPC composition i) R ２ A compound according to formula 1, wherein is OH; and / or ii) R １ Compounds according to formula 3, where is OH If it contains The formulation of claim 1, with the proviso that the LPC composition further comprises at least one of the other LPC compounds mentioned in claim 1.

4. One or more of the LPC compounds A compound according to formula 1; and / or a compound according to formula 3; And a compound according to formula 2; and / or a compound according to formula 4, The formulation of claim 1.

5. R １ and R ２ is OH; and the molar ratio of lyso-PC-DHA:lyso-PC-EPA is in the range of 1:1 to 3:1; 2. The formulation of claim 1, wherein i) the number of moles of lyso PC-EPA is the number of moles of 1-lyso PC-EPA plus the number of moles of 2-lyso PC-EPA; and ii) the number of moles of lyso PC-DHA is the number of moles of 1-lyso PC-DHA plus the number of moles of 2-lyso PC-DHA.

6. The formulation of claim 1 , wherein the disease or condition is dry eye.

7. The formulation of claim 1 , wherein the disease or condition is a neurodegenerative disease of the eye.

8. 8. The formulation of claim 7, wherein the ocular neurodegenerative disease is selected from the group consisting of age-related macular degeneration, diabetic retinopathy, non-proliferative retinopathy, proliferative retinopathy, diabetic macular edema, retinitis pigmentosa, central vein occlusion and glaucoma.

9. 2. The formulation of claim 1, wherein the LPC composition in the formulation comprises total LPC in an amount of 10% to 100% by weight of the LPC composition.

10. The formulation of claim 1 , wherein the LPC composition comprises an additional lipid different from the LPC.

11. 11. The formulation of claim 10, wherein the additional lipid is selected from the group consisting of triglycerides, free fatty acids, ethyl esters, and phospholipids selected from the group consisting of phosphatidylethanolamines and phosphatidylcholines.

12. The formulation of claim 11 , wherein the LPC composition comprises an amount of the LPC compound that is greater than the amount of phosphatidylcholine.

13. 10. The formulation of claim 1, wherein the formulation further comprises an ingredient selected from the group consisting of lutein, astaxanthin, zeaxanthin, and any combination thereof.

14. The formulation of claim 1 , wherein the subject is a mammalian subject.

15. The formulation of claim 14, wherein the subject is a human subject.

16. R １ and R ２ is OH; and the molar ratio of lyso-PC-EPA:lyso-PC-DHA is in the range of 1:1 to 5:1; 2. The formulation of claim 1, wherein i) the number of moles of lyso PC-EPA is the number of moles of 1-lyso PC-EPA plus the number of moles of 2-lyso PC-EPA; and ii) the number of moles of lyso PC-DHA is the number of moles of 1-lyso PC-DHA plus the number of moles of 2-lyso PC-DHA.

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