WO2025146681A1 - Pro-nano-emulsions comprising omega 3 and use thereof - Google Patents

Abstract

The present invention relates to pro-nano-emulsion compositions comprising at least one type of Omega 3 fatty acid capable of converting to nano-emulsion composition having average droplet size of below Ip, typically below 0.5p, and use thereof, particularly as dietary supplement, for preferential delivery of the Omega 3 fatty acid to heart and/or liver tissue of a subject consuming the composition, thereby maintaining normal cardiovascular/hepatic function and/or improving cardiovascular/hepatic disease or disorders.

Description

PRO-NANO-EMULSIONS COMPRISING OMEGA 3 AND USE THEREOF

FIELD OF THE INVENTION

The present invention relates to pro-nano-emulsion compositions comprising at least one type of Omega 3 fatty acid capable of converting to nano-emulsion compositions having an average droplet size of below Ip, typically below 0.5 , and use thereof, particularly as dietary composition, for preferential delivery of the Omega 3 fatty acid to heart and/or liver tissue of a subject consuming the composition.

BACKGROUND OF THE INVENTION

The two major classes of polyunsaturated fatty acids (PUFAs) are the omega-3 and omega-6 fatty acids. PUFAs are distinguished from saturated and monounsaturated fatty acids by the presence of two or more double bonds between carbons within the fatty acid chain.

Omega 3 fatty acids (referred to herein as “Omega 3 s”) have a carbon-carbon double bond located three carbons from the methyl end of the chain. Omega 3 s are important constituents of animal lipid metabolism, and play an important role in human diet and physiology. Several different Omega 3s exist, but the majority of scientific research focuses on three: alpha-linolenic acid (ALA), found in plants; and eicosapentaenoic acid (EP A) and docosahexaenoic acid (DHA) found in algae and fish. ALA contains 18 carbon atoms, whereas EPA and DHA are considered “long-chain” (LC) Omega 3s because EPA contains 20 carbons and DHA contains 22.

The human body can only form carbon-carbon double bonds after the 9th carbon from the methyl end of a fatty acid. Therefore, ALA and linoleic acid are considered essential fatty acids, which must be obtained through dietary intake. ALA can be converted into EPA and then to DHA, but the conversion (which occurs primarily in the liver) is very limited, with reported rates of less than 15%. Therefore, consuming EPA and DHA directly from foods and/or dietary supplements is the only practical way to increase levels of these fatty acids in the body.

Omega 3 fatty acids are essential dietary components since their absence is dangerous to human health. Awareness of the health benefits of essential fatty acids has dramatically increased over the years. According to the World Health Organization (WHO), cardiovascular diseases (CVDs) are the leading cause of death globally, taking an estimated 17.9 million lives each year. CVDs are a group of disorders of the heart and blood vessels and include coronary heart disease, cerebrovascular disease, rheumatic heart disease and other conditions. In 2004, the FDA gave "qualified health claim" status to EPA and DHA Omega 3s, stating that their consumption may reduce the risk of coronary heart disease.

In addition, evidence suggests that Omega-3 s reduce blood triglyceride levels and are also anti-inflammatory agents. Brain function and vision also rely on dietary intake of DHA, which is a major structural component of the mammalian brain.

In liver diseases, Omega-3 s may reduce hepatic lipogenesis, inflammation, and hepatic fibrosis (e.g., Zhang K et al. 2016. Sci Rep. 6:30029). A recent meta-analysis conducted by Yan et al. suggests that Omega-3 s supplementation may decrease liver fat and hepatic enzyme parameters in patients with non-alcoholic fatty liver disease (NAFLD, Yan et al. 2018. Medicine 97:37). Supplementation with fish Omega 3s is also considered as an option to lower liver fat in obese adults and children with NAFLD.

Dietary supplements, nutraceuticals, and prescribed drugs containing Omega 3 s and/or Omega 3 s fatty acid esters, are thus used for the treatment of CVDs and liver diseases (e.g., Moertl D et al., 2011. American Heart Journal 161(5): 915. el-915. e9. Doi: 10.1016/j.ahj.2011.02.011; Shorn onov-Wagner L and Leikin-Frenkel L. 2015. Lipids in Health and Disease 14: 14. Doi:10.1186/sl2944-015-0012-7; Harari A et al., 2020. Nutrition, Metabolism & Cardiovascular Diseases 30:709e716; Lechner K et al., 2022. Clinical Research in Cardiology 111:308-321, Doi:10.1007/s00392-021-01925-9; Rodriguez D et al., 2022. Nutrients, 14:5146. Doi:.3390/nul4235146).

International (PCT) Patent Application Publication No. WO 2016/057915 describes compositions comprising at least one omega-3 fatty acid (either in the triglyceride, ester, or free fatty acid ester form) and at least one surface active agent, which forms micelles when in contact with an aqueous medium. The compositions are useful for treating certain disease states which may include (1) malabsorption syndromes, (2) primary sclerosing cholangitis (PSC), (3) non-alcoholic fatty liver disease (NAFLD), (4) sickle cell disease (SCD), (5) age-related macular degeneration (AMD), and (6) neurodegenerative disease, including, Parkinson's Disease (PD), Alzheimer's Disease (AD), Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease), Epilepsy, Bi-polar Syndrome, traumatic brain injury, peripheral neuropathy, and Multiple Sclerosis (MS).

U.S. Patent No. 9,302,017 describes use of the omega-3 fatty acid self-forming micelles for treating cardiovascular conditions or disorders.

However, the absorption of lipids by the digestive system is poor when taken while fasting or with a low-fat meal. When taken with fatty foods, the absorption of Omega 3 fatty acids may improve, but the ability to properly dose patients is often a significant challenge. Further complicating matters, for example, the over-introduction of certain lipids, such as the fat-soluble vitamins, can be harmful.

The emerging evidence on Omega 3 s supplementation for cardiovascular health suggests that high doses may be more effective, especially in populations with lower baseline Omega 3s levels (Elagizi et al., 2021. Nutrients 13:204). Clinical studies have demonstrated notable cardiovascular benefits with 4 g/day of Omega 3 s (EPA + DHA), surpassing standard care in reducing adverse left ventricular remodeling, inflammation, and triglyceride levels. Notably, the concept of a "threshold effect" proposes that certain populations may require higher doses to reach a therapeutic window, particularly in regions with lower baseline Omega 3s levels, such as the United States. The delivery method of the Omega-3 fatty acids may also have an impact on its advantage (Lanea K E and Derbyshireb E J. 2018. Critical Reviews in Food Science and Nutrition 58(l):62-69).

The European Food Safety Authority underscores the safety of consuming omega- 3 fatty acid supplements at doses up to 5 grams daily (Scientific Opinion on the Tolerable Upper Intake Level of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DP A), 2012, EFSA Journal, Volume 10, Issue 7 2815).

However, it is essential to recognize that there are potential side effects associated with excessive intake of fish oil or omega-3 fatty acids. Taking too high a dose may lead to side effects such as diarrhea and flatulence in some individuals (Bradberry J C and Hilleman D E. 2013P T. 2013 Nov; 38(11): 681-691. PMID: 24391388; PMCID: PMC3 875260).

Another review highlights the dual nature of EPA/DHA, which can ameliorate inflammation associated with chronic cardiovascular or autoimmune conditions but may hinder pathogen clearance during acute infections. (Fenton J I et al., 2013. Prostaglandins Leukot Essent Fatty Acids. 89(6):379-90). Additionally, the association of high serum Omega 3 fatty acids levels with an increased risk of prostate cancer and atrial fibrillation raises concerns about potential adverse health outcomes.

Therefore, a need exists to provide compositions comprising Omega 3s which are more effectively absorbed when administered orally and reach specific target organs, thereby potentially enabling administration of the Omega 3s at a lower dose while preserving the Omega 3 s beneficial effects.

SUMMARY OF THE INVENTION

The present invention addresses the above-described needs, providing nanoemulsion-generating composition comprising at least one type of Omega 3 fatty acid (Omega 3s), for oral delivery of the Omega 3 fatty acid to a subject, wherein the composition provides for preferential delivery and improved accumulation of the Omega 3s within tissue of the subject. The compositions of the present invention can be used as dietary compositions for maintaining a normal cardiovascular function, particularly normal heart function, and/or normal hepatic function in a subject consuming the dietary composition. The dietary compositions can be also used for reducing the risks to develop cardiovascular and/or hepatic diseases in a subject consuming the dietary composition. The compositions of the present invention may also be used as pharmaceuticals for treating and/or regulating and/or improving liver and/or heart diseases or disorders.

The present invention is based in part on the unexpected finding that oral consumption of the Omega 3s-comprising pro-nano-emulsion composition of the invention resulted in a significant accumulation of the Omega 3 s in the heart and/or the liver of the subject consuming the composition. Without wishing to be bound by any specific theory or mechanism of action, the Omega 3s accumulation may be attributed to the formation of nano-emulsion having an average particle size of below 1 micron (Ip), typically below 0.5 , upon contact of the pro-nano-emulsion composition with the aqueous content of the subject stomach and potentially further exposure to the digestive motility of the intestines. The contact of the pro-nano-emulsion with the aqueous solution of the stomach and the gastro-intestinal tract in general, together with the gastro-intestinal tract motility, causes immediate conversion of the pro-nano-emulsion and the said aqueous solution into the desired nano-emulsion. The nanometric particle size contributes to the absorbance of the Omega 3s comprising nano-emulsion from the gastrointestinal tract and into the subject cells, while accumulating in specific organs. However, as exemplified hereinbelow, while somewhat elevated levels of Docosahexaenoic acid (DHA) were observed in brain tissue of mice receiving the pro-nano-emulsion of the invention compared to control mice not receiving any form of Omega 3s, these levels were not significantly different from the DHA level in brain tissue of mice receiving fish oil comprising Omega 3s, and no accumulation of Eicosapentaenoic acid (EP A) was observed. Thus, without wishing to be bound by any specific theory or mechanism of action, the phenomenon of Omega 3 s accumulation in heart and/or liver tissue cannot be solely attributed to the nano-size of the emulsion particles, as oral administration of equivalent sized nano-emulsion comprising pomegranate seed oil, specifically punicic acid, previously developed by inventors of the present invention, resulted in significant amounts of the pomegranate seed oil/punicic acid in the brain of model mice and shown to be effective in treating neurodegenerative diseases (U.S. Patent Nos. 10,154,961 and 11,801,220).

Unexpectedly, the present invention shows that the absorbed Omega 3s accumulate in the liver and/or heart at quantities exceeding the quantities of non-emulsified Omega 3 s administered as a fish oil. Accordingly, the compositions and methods of the present invention may enable providing lower total doses of Omega 3 s, providing for better compliance by the subjects and optionally for avoiding adverse effects associated with high doses of Omega 3 s.

Accordingly, the present invention discloses use of the Omega 3s-comprising pro- and nano-emulsions of the invention as dietary compositions and optionally pharmaceutical compositions for preferentially delivering at least one type of Omega 3 s to heart and/or liver tissue, which may be effective in maintaining the heart and/or liver function as well as in reducing the risks for, preventing, regulating, and/or treating cardiovascular and hepatic diseases and/or disorders in a subject consuming the Omega 3 s compositions of the invention as is described in details hereinbelow.

According to certain aspects, the present invention provides a method for preferentially delivering at least one type of Omega 3 fatty acid (Omega 3 s) to heart and/or liver tissue of a subject, the method comprising orally administering to the subject a pro-nano-emulsion composition comprising: an oil comprising at least one type of Omega 3 s; a combination of at least two liquid surface-active agents; and at least one water-soluble organic solvent, thereby preferentially delivering the at least one type of Omega 3s to said subject heart and/or liver tissue.

According to certain embodiments, the subject is a human.

According to certain embodiments, the preferential delivery of the Omega 3 s to the heart and/or liver tissue of a human subject results in maintaining the cardiovascular and/or hepatic function of the subject. According to certain embodiments, the preferential delivery of the Omega 3 s to the heart tissue of a human subject results in maintaining the heart function of the subject. According to certain embodiments, the preferential delivery of the Omega 3s to the liver tissue of a human subject results in maintaining the hepatic function of the subject. According to certain embodiments, the preferential delivery of the Omega 3s to the heart and/or liver tissue of a human subject results in reducing the risk of the subject to develop a cardiovascular and/or hepatic disease or disorder.

According to certain embodiments, the human subject is a patient having a cardiovascular and/or hepatic disease or disorder. According to certain embodiments, the preferential delivery of the Omega 3 s to the heart and/or liver tissue of a human patient having a cardiovascular and/or hepatic disease or disorder results in improving and/or treating and/or regulating the cardiovascular and/or hepatic disease or disorder.

According to certain embodiments, the pro-nano-emulsion composition comprises at least 25% w/w oil out of the total weight of the composition. According to certain exemplary embodiments, the composition comprises from about 25% to about 35% w/w oil out of the total weight of the composition.

According to certain embodiments, the oil comprises at least 50% w/w of at least one type of Omega 3 s. According to certain embodiments, the oil comprises at least 60%, at least 70%, at least 80% or more of the at least one type of Omega 3 s.

According to certain embodiments, the oil of the pro-nano-emulsion composition consists of the at least one Omega 3 s. According to certain embodiments, the composition comprises at least 50% w/w of the combination of liquid surface active agents out of the total weight of the composition. According to certain exemplary embodiments, the composition comprises from about 50% to about 75% w/w of the combination of liquid surface active agents out of the total weight of the composition.

According to certain embodiments, the composition comprises at least 5% w/w of the at least one water-soluble organic solvent out of the total weight of the composition. According to certain embodiments, the composition comprises from about 5% to about 10% w/w of the at least one water-soluble organic solvent out of the total weight of the composition. According to certain exemplary embodiments, the composition comprises from about 5% to about 7.5% w/w of the at least one water-soluble organic solvent out of the total weight of the composition.

According to certain embodiments, the combination of liquid surface-active agents comprises at least two polysorbates. According to certain exemplary embodiments, the combination of liquid surface-active agents consists of at least two polysorbates.

According to certain embodiments, the polysorbates are selected from polyoxyethylene sorbitan monooleate (also known as Tween™) and sorbitan fatty acid ester (also known as Span™). Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the combination of polysorbates comprises at least one polyoxyethylene sorbitan ester selected from polyoxyethylene sorbitan monolaurate (Tween™ 20), polyoxyethylene sorbitan monopalmitate (Tween™ 40), polyoxyethylene sorbitan monostearate (Tween™ 60) and polyoxyethylene sorbitan monooleate (Tween™ 80); and at least one sorbitan fatty acid ester selected from the group consisting of sorbitan monolaurate (Span™ 20), sorbitan monopalmitate (Span™ 40) and sorbitan monooleate (Span™ 80). Each possibility represents a separate embodiment of the present invention.

According to certain exemplary embodiments, the combination of surface active agents consists of polyoxyethylene sorbitan monooleate (Tween™ 80) and sorbitan monooleate (Span™ 80).

According to certain embodiments, the composition comprises from about 30% to about 60% w/w of Tween™ 80 out of the total weight of the composition. According to certain embodiments, the composition comprises from about 34% to about 52% w/w Tween™ 80 out of the total weight of the composition. According to certain embodiments, the composition comprises from about 15% to about 30% w/w Span™ 80 out of the total weight of the composition. According to certain embodiments, the composition comprises from about 15% to about 23% w/w Span™ 80 out of the total weight of the composition.

According to certain embodiments, the combination of surface-active agents is devoid of poloxamer.

According to certain embodiments, the water-soluble organic solvent is selected from the group consisting of edible liquids, including but not limited to, ethanol, propylene glycol and liquid polyethylene glycol. Each possibility represents a separate embodiment of the present invention.

According to certain exemplary embodiments, the water-soluble organic solvent is ethanol.

According to yet further exemplary embodiments, the pro-nano-emulsion composition of the present invention comprises from about 25% to about 35% w/w Omega 3s; from about 34% to about 52% w/w Tween™ 80, from about 15% to about 23%w/w Span™ 80; and from about 1% to about 7.5% w/w ethanol, based on the total weight of the composition.

According to certain embodiments, the at least one type of omega-3 fatty acid is in a form selected from the group consisting of triglyceride, an ester, and a free fatty acid. Each possibility represents a separate embodiment of the present invention.

According to certain exemplary embodiments, the at least one type of Omega 3s is selected from the group consisting of docosahexaenoic acid (DHA); docosapentaenoic acid (DPA); and eicosapentaenoic acid (EP A). Each possibility represents a separate embodiment of the present invention. It is to be explicitly understood that the Omega 3 s can be in a form of triglycerides, free fatty acid acids or ethyl esters.

According to certain embodiments, the oil of the pro-nano-emulsion comprises

DHA and EP A. According to certain embodiments, the pro-nano-emulsion composition comprises from about 40 mg to about 200 mg DHA. According to certain embodiments, the DHA concentration within the pro-nano-emulsion composition is below 100 mg. According to certain embodiments, the pro-nano-emulsion composition comprises from about 100 mg to about 500 mg EPA. According to certain embodiments, the EPA concentration within the pro-nano-emulsion composition is below 200 mg.

According to certain exemplary embodiments, the Omega 3s is fish-oil Omega 3s. According to certain embodiments, the oil of the pro-nano-emulsion consists of fish oil.

The pro-nano-emulsion or nano-emulsion composition of the invention may be administered per se or within a composition including, but not limited to, a dietary supplement, a nutraceutical or a functional food as described hereinbelow. The pro-nano- emulsion or nano-emulsion composition of the invention may also be administered within a pharmaceutical composition.

According to certain embodiments, the pro-nano-emulsion composition of the present invention or a formulation comprising the same comprises at least one additional active agent other than the Omega 3s selected from the group consisting of a nutritional supplement; an antioxidant; a cannabinoid; and any combination thereof. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the nutritional supplement is selected from the group consisting of folic acid, vitamin C, vitamin E, beta-carotene, lycopene, vitamin A, vitamin D, vitamin KI, vitaminK2, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the antioxidant is selected from the group consisting of tocopherols, Co-Q 10, ascorbic acid and derivatives thereof, carotenoids, astaxanthin, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the at least one additional active agent is other than statin.

According to certain embodiments, the cardiovascular disorder is selected from the group consisting of, but not limited to, congestive heart failure, hypertension, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, transient ischemic attack, systolic dysfunction, diastolic dysfunction, aneurysm, aortic dissection, myocardial ischemia, acute myocardial infarction (AMI), acute ST-segment elevation myocardial infarction (STEMI), acute non-ST-segment elevation myocardial infarction (NSTEMI), angina pectoris, congenital heart disease, unstable angina (UA), stable angina (SA), myocardial infarction, dilated congestive cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, Cor-pulmonale, arrhythmia, valvular heart disease, endocarditis, pulmonary embolism, venous thrombosis, peripheral vascular disease, peripheral artery disease, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the congestive heart failure is heart failure with preserved ejection fraction (HFpEF).

According to certain embodiments, the preferential delivering the Omega 3 s to the heart tissue improves the diastolic function of the heart.

According to certain embodiments, the hepatic disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD) and/or a symptom associated with NAFLD and parenteral nutrition (PN)- associated liver disease (PNALD).

According to certain embodiments, the NAFLD is selected from the group consisting of non-alcoholic steatosis and non-alcoholic steatohepatitis (NASH).

According to certain embodiments, the symptom associated with NAFLD is selected from the group consisting of fat droplet accumulation, inflammation, fibrosis, hepatocyte cell death and any combination thereof. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, improving the cardiovascular function results in reducing the risks associated with cardiovascular disease or disorder including, but not limited to, high low-density lipoprotein cholesterol, high triglyceride levels, and high level of factors that contribute to coagulation of blood.

According to certain embodiments, improving the hepatic function results in a reduced risk to develop NAFLD.

The therapeutic amount required and the form and regime of administration of the omega 3s-comprising pro-nano-emulsion of the present invention depend on the type of the disease, disorder, or risk factor to be treated and parameters related thereto, including severity and stage, and on parameters related to the subject to be treated, including age, gender, and weight. A significant advantage of using Omega 3 s within the composition of the present invention is the improved delivery to, and accumulation of the Omega 3 s within, the target organs, the heart, and the liver. This advantage enables use of lower amounts of Omega 3 s to achieve at least the same beneficial effect as hitherto known doses, preferably while avoiding adverse effects of high Omega 3s consumption. It is also to be understood that the subject can be of any age and health condition. Accordingly, the methods of the present invention can be used for treating vulnerable populations including elderly, obese, diabetic, sick, or very young subjects, as well as subjects not yet showing significant signs of a disease.

According to certain embodiments, the pro-nano-emulsion composition of the present invention is a liquid composition.

The liquid pro-nano-emulsion can be orally administered as is, or while present within a capsule or any other oral delivery formulation.

According to certain aspects, the present invention provides a pro-nano-emulsion composition comprising an oil comprising at least one type of Omega 3 fatty acid; a combination of at least two liquid surface-active agents; and at least one water-soluble organic solvent.

According to certain embodiments, the pro-nano-emulsion of the present invention is formulated in a form of a pharmaceutical composition.

According to certain embodiments, the pro-nano-emulsion of the present invention is formulated in a form selected from the group consisting of dietary supplement, nutraceutical composition and functional food. Each possibility represents a separate embodiment of the present invention.

According to certain currently exemplary embodiments, the pro-nano-emulsion composition is formulated as a dietary supplement.

According to certain embodiments, the pharmaceutical composition comprising the pro-nano-emulsion of the present invention further comprises a pharmaceutically acceptable diluent or carrier.

According to certain embodiments, the nutraceutical composition comprising the pro-nano-emulsion of the present invention further comprises a nutritionally acceptable diluent or carrier.

According to certain embodiments, the carrier further assists in targeting the Omega 3 s to its place of function. According to certain embodiments, the carrier further enhances the bioavailability of the Omega 3s within the subject body.

The pro-nano-emulsion of the present invention converts to a nano-emulsion upon contact with an aqueous solution, optionally further upon exposure to at least one mixing force.

According to certain exemplary embodiments, the pro-nano-emulsion composition is converted to a nano-emulsion upon entering the gastrointestinal tract of a subject orally consuming the pro-nano-emulsion.

According to certain embodiments, the pro-nano-emulsion or the nano-emulsion is encapsulated within a soft or hard capsule commonly used in pharmaceutical and foodsupplement products, to be administered orally.

According to certain embodiments, the pro-nano-emulsion is used as a liquid to be administered orally.

According to certain embodiments, before use, the pro-nano-emulsion is mixed with water or any other edible aqueous liquid to form the nano-emulsion to be administered orally.

It is to be explicitly understood that the present invention encompasses a nanoemulsion comprising Omega 3s produced from the pro-nano-emulsion of the present invention.

According to certain embodiments, the average droplet size of the nano-emulsion converted from the pro-nano-emulsion is below Ip. According to some embodiments, the average droplet size of the nano-emulsion is below 0.5p. According to certain exemplary embodiments, the Z Average peak of the nano-emulsion measured by dynamic light scattering (DLS) is from about 150 nm to about 300 nm. According to certain embodiments, the Z Average peak of the nano-emulsion measured by dynamic light scattering (DLS) is from about 200 nm to about 250 nm.

It is to be understood that any combination of each of the aspects and the embodiments disclosed herein is explicitly encompassed within the disclosure of the present invention.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show the level of DHA accumulation in the liver (Fig. 1A) and heart (Fig. IB) in samples taken from mice that received either fish oil (Omega 3s), the pro-nano- emulsion omega-3 composition (Assay), or were untreated (Control). Shown in the fish oil (Omega 3s) group is accumulation of DHA after two weeks of supplementation with fish oil (dotted pattern column) and one week after cessation of supplementation (white column); Shown in the pro-nano-emulsion omega-3 composition (Assay) group is accumulation of DHA after two weeks of supplementation with the pro-nano-emulsion omega-3 composition (shown in diagonal stripes) and one week after treatment cessation (shown in black). DHA levels for non-treated mice are shown in vertical stripes.

FIGS. 2A-2C show the level of EPA in the plasma (Fig. 2A), the liver (Fig. 2B) and the heart (Fig. 2C) in WT C57BL mice that were treated with fish oil (co 3 oil) or treated with the pro-nano-emulsion omega-3 composition (nano-m3).

FIGS. 2D-2F show the level of DHA in the plasma (Fig. 2D), the liver (Fig. 2E) and the heart (Fig. 2F) in WT C57BL mice that were treated with fish oil (m3 oil) or treated with the pro-nano-emulsion omega-3 composition (nano-m3).

FIGS. 3A-3D show the level of EPA in the plasma (Fig. 3 A), the liver (Fig. 3B), the brain (Fig. 3C), and the heart (Fig. 3D) in WT C57BL mice that were untreated, treated with fish oil (m3 oil), or treated with the pro-nano-emulsion omega-3 composition (nano-m3). Mean of results is indicated by the bar, and error bars indicate the standard error of the mean (SEM). Comparisons were performed by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, *** p < 0.001, *** p < 0.0001, ns (not significant) p > 0.05.

FIGS. 3E-3H show the level of DHA in the plasma (Fig. 3E), the liver (Fig. 3F), the brain (Fig. 3G), and the heart (Fig. 3H) in WT C57BL mice that were untreated, treated with fish oil (o3 oil), or treated with the pro-nano-emulsion omega-3 composition (nano-o3). Mean of results is indicated by the bar, and error bars indicate the SEM. Comparisons were performed by one-way ANOVA with Tukey’ s multiple comparisons test. *p < 0.05, **p < 0.01, *** p < 0.001, *** p < 0.0001, ns (not significant) p > 0.05.

FIGS. 4A-4D show the results of various parameters that were assessed in a mouse model for heart failure with preserved ejection fraction (HFpEF). HFpEF mice were either untreated, treated with fish oil (co 3 oil), or treated with the pro-nano-emulsion omega-3 composition (nano-c 3). Fig. 4A shows early diastolic flow peak velocity of the mitral valve (E) values; Fig. 4B shows early diastolic peak velocity of mitral valve annulus (E’) values; Fig. 4C shows mitral valve EZE’ (MV E/E’) ratio values; Fig. 4D shows global longitudinal strain (GLS) values. Mean of results is indicated by the bar, and error bars indicate the SEM. Comparisons were performed by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, *** p < 0.001, *** p < 0.0001, ns (not significant) p > 0.05.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions comprising at least one type of Omega 3 fatty acid (Omega 3 s), unexpectedly providing for preferential delivery and accumulation of the Omega 3s in the heart and/or liver of a subject orally consuming the composition. The composition of the invention, defined herein as “pro-nano-emulsion” comprises an oil component comprising the at least one type of Omega 3s, at least one liquid surface active agent, and at least one water soluble organic solvent, and is characterized by: a) conversion to a nano-emulsion upon contact with the aqueous environment and optionally the motility of the gastrointestinal tract of a subject consuming the pro-nano-emulsion, and b) by the capability of the nano-emulsion to deliver Omega 3s to, and accumulate in, the liver and/or heart of a mammal. The nanoemulsion is characterized by nano-sized droplets, typically having a Z average peak of less than 300 nm.

Definitions

Polyunsaturated fatty acids (PUFAs) are frequently designated by their number of carbon atoms and double bonds. Alpha-linolenic acid (ALA), for example, is known as Cl 8:3 n-3 because it has 18 carbons and 3 double bonds, the first double bond located at the carbon numbered 3 (n-3), starting from the methyl end of the fatty acid chain. Similarly, Eicosapentaenoic acid (EP A) is known as C20:5 (n-3), Docosahexaenoic acid (DHA) as C22:6 (n-3), and Docosapentaenoic acid (DPA) as 22:5 (n-3). DHA, EPA and DPA are the most common Omega 3 fatty acids in food. The terms “omega 3”, omega 3 fatty acids” and “omega 3 s” are used herein interchangeably and refer to omega 3 fatty acid described above including natural and synthetic omega-3 fatty acids, as well as pharmaceutically-acceptable esters, free acids, triglycerides, derivatives, conjugates, precursors, salts, and mixtures thereof. Esters of omega-3 fatty acids with glycerol such as mono-, di- and triglycerides; and esters of the omega-3 fatty acids and a primary, secondary, and/or tertiary alcohol, such as, for example, fatty acid methyl esters and fatty acid ethyl esters are also encompassed in the teachings of the present invention. The omega-3 fatty acids, esters, triglycerides, derivatives, conjugates, precursors, salts, and/or mixtures thereof according to the present invention can be used in their pure form and/or as a component of an oil, for example, as marine oil (e.g., fish oil and purified fish oil concentrates), algae oils, microbial oils, and plant-based oils.

The term “composition” as used herein includes dietary compositions including, but not limited to, dietary supplements, nutraceutical formulations, and functional food, or therapeutic compositions including pharmaceutical formulations.

As used herein, the term “dietary supplement” refers to a product intended to supplement the diet and provide nutrients that may be missing or insufficient in a subject regular diet. Dietary supplements may be available in various forms, including, but not limited to, tablets, capsules, softgels, powders, liquids, and gummies.

As used herein, the term "nutraceutical" refers to a product derived from food source containing exogenous ingredients and/or endogenous ingredients, conferring to the product a health benefit beyond basic nutritional value. Nutraceuticals may include dietary supplements, functional foods, medicinal foods, and herbal products.

As used herein, the term "functional food" refers to a food containing exogenous ingredients and/or endogenous ingredients at a higher concentration conferring to the food additional function besides providing nutrients and energy. Functional foods include conventional foods - naturally nutrient-rich foods, for example, fruits, vegetables, nuts, whole grains, and fish; and modified foods - foods that have been fortified, enriched, or enhanced with additional nutrients or bioactive compounds.

According to certain embodiments, the dietary compositions comprising Omega 3s according to the teachings of the invention provide for preferential delivery of the Omega 3s to the heart and/or liver tissue of a subject consuming the same, enabling the Omega 3s to exert their activity in maintaining the heart/liver function and/or reducing the risk of developing a cardiovascular and/or hepatic disorder or disease.

According to certain embodiments, the pharmaceutical compositions comprising Omega 3 s according to the teachings of the invention provide for preferential delivery of the Omega 3s to the heart and/or liver tissue of a subject consuming the same, enabling the Omega 3 s to exert their activity in preventing, treating, and/or improving a cardiovascular and/or hepatic disease or disorder affecting the subject.

As used herein, the term “about” is to be understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within ± 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. All values provided herein are understood to be modified by the term “about”. According to certain embodiments, the term “about” refers to ± 10%.

According to certain aspects, the present invention provides a pro-nano-emulsion composition comprising: an oil comprising at least one type of Omega 3 fatty acid (Omega 3 s); a combination of at least two liquid surface active agents; and at least one water-soluble organic solvent.

According to certain embodiments, the pro-nano-emulsion composition is devoid of an aqueous solvent, particularly, devoid of water.

According to certain embodiments, the oil of the pro-nano-emulsion composition consists of Omega 3s.

According to certain embodiments, the pro-nano-emulsion composition comprises at least 25%, at least 30% at least 35% at least 40% at least 45% at least 50%, at least 55% or at least 60% oil w/w out of the total weight of the composition.

According to certain embodiments, the composition comprises at least 25%, at least 26% at least 27% at least 28% at least 29% at least 30%, at least 31% at least 32%, at least 33%, at least 34%, or at least 35% w/w oil out of the total weight of the composition. According to certain exemplary embodiments, the composition comprises from about 25% to about 35% w/w oil out of the total weight of the composition. According to further certain exemplary embodiments, the oil concentration within the pro-nano-emulsion is about 31% w/w out of the total weight of the composition.

According to certain embodiments, the oil component of the pro-nano-emulsion composition comprises EPA and DHA.

According to certain embodiments, the oil further comprises at least one additional fatty acid other than EPA and DHA. Examples of such fatty acids include, but are not limited to, Omega-3 fatty acids other than EPA and DHA, including, but not limited to, a-linolenic acid (ALA), heneicosapentaenoic acid (HP A), docosapentaenoic acid (DPA), eicosatetraenoic acid (ETA), eicosatrienoic acid (ETE), and stearidonic acid (STA), and Omega-6 fatty acids. According to certain embodiments, the at least one fatty acid other than EPA and DHA is in a form chosen from ethyl ester and triglyceride.

According to certain embodiments, the pro-nano-emulsion of the present invention comprises fish oil as the oil component of the pro-nano-emulsion. According to certain embodiments, the fish oil comprises at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more Omega 3s. According to certain embodiments, the fish oil comprises from about 50% to about 60% EPA and from about 20% to about 30% DHA.

According to certain embodiments, the pro-nano-emulsion composition comprises from about 40 mg to about 200 mg DHA, or from about 50 mg to about 190 mg, or from about 60 mg to about 180 mg, or from about 70 mg to about 170 mg, or from about 80 mg to about 160 mg, or from about 90 mg to about 150 mg DHA. According to certain embodiments, the DHA concentration within the pro-nano-emulsion composition is below 100 mg. According to certain embodiments, the pro-nano-emulsion composition comprises from about 100 mg to about 500 mg EP A, or from about 150 mg to about 450 ng, or from about 200 mg to about 400 mg EP A. According to certain embodiments, the EPA concentration within the pro-nano-emulsion composition is below 200 mg.

According to certain embodiments, the composition comprises at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, or at least 63%, 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, or at least 75%, w/w of the combination of surface-active agents out of the total weight of the composition. According to certain exemplary embodiments, the composition comprises from about 50% to about 75% w/w or from about 60% to about 70% w/w of the combination of surface-active agents out of the total weight of the composition. According to further certain currently exemplary embodiments, the concentration of the combination of surface-active agents within the pro-nano-emulsion composition is from about 63% to about 64% w/w out of the total weight of the composition.

According to certain embodiments, the combination of surface-active agents comprises at least two polysorbates. According to certain exemplary embodiments, the combination of surface-active agents consists of at least two polysorbates.

According to certain embodiments, the polysorbates are selected from polyoxyethylene sorbitan ester (also known as Tween™) and sorbitan fatty acid ester (also known as Span™). Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the combination of polysorbates comprises at least one polyoxyethylene sorbitan ester selected from polyoxyethylene sorbitan monolaurate (Tween™ 20), polyoxyethylene sorbitan monopalmitate (Tween™ 40), polyoxyethylene sorbitan monostearate (Tween™ 60) and polyoxyethylene sorbitan monooleate (Tween™ 80); and at least one sorbitan fatty acid ester selected from the group consisting of sorbitan monolaurate (Span™ 20), sorbitan monopalmitate (Span™ 40) and sorbitan monooleate (Span™ 80). Each possibility represents a separate embodiment of the present invention. According to certain embodiments, the composition comprises from about 30% to about 60%, from about 35% to about 55%, or from about 40% to about 50% w/w Tween™ 80 out of the total weight of the composition. According to certain currently exemplary embodiments, the concentration of the Tween™ 80 within the pro-nano- emulsion composition is about 43%, or about 43.5% or about 44% w/w out of the total weight of the composition.

According to certain embodiments, the composition comprises from about 15% to about 30%, from about 15.5% to about 23%, or from about 17.5% to about 21.5% w/w Span™ 80 out of the total weight of the composition. According to certain exemplary embodiments, the concentration of the Span™ 80 within the pro-nano-emulsion composition is about 19.5% w/w out of the total weight of the composition.

It is to be explicitly understood that the surface active agent poloxamer is not included in the surface active agent combination according to the teachings of the present invention.

According to certain embodiments, the water soluble organic solvent is selected from the group consisting of ethanol, propylene glycol, and liquid polyethylene glycol, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

According to certain exemplary embodiments, the water soluble organic solvent is ethanol.

According to certain embodiments, the composition comprises at least 5%, at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, at least 5.6%, at least 5.7%, at least 5.8%, at least 5.9%, or at least 6.0% w/w of the at least one water-soluble organic solvent out of the total weight of the composition. According to certain embodiments, the composition comprises from about 5% to about 10% w/w of the at least one water-soluble organic solvent out of the total weight of the composition. According to certain exemplary embodiments, the composition comprises from about 5% to about 7.5% w/w of the at least one water-soluble organic solvent out of the total weight of the composition. According to further exemplary embodiments, the concentration of the at least one water- soluble organic solvent within the pro-nano-emulsion composition is about 6.1%.

According to some embodiments, the pro-nano-emulsion is stable at a temperature of up to 45°C. According to some embodiments, the pro-nano-emulsion is stable at a temperature range of between about 18°C to about 45°C, including each value within the specified range. As used herein, the term “stable” means that the pro-nano-emulsion preserves its ability to self-emulsify to a nano-emulsion with the physicochemical properties and health benefits described herein.

According to some embodiments, the pro-nano-emulsion is stable at room temperature for at least 1, 2, 3, 6, 9, 12, 15, 18, or 22 months. Each possibility represents a separate embodiment of the invention. According to certain exemplary embodiments, the pro-nano-emulsion is stable at room temperature for at least 12 months.

The pro-nano-emulsion of the present invention converts to a nano-emulsion upon contact with an aqueous solution, optionally further upon exposure to at least one mixing force. According to certain exemplary embodiments, the pro-nano-emulsion composition is converted to the nano-emulsion upon entering the gastrointestinal tract of a subject orally consuming the pro-nano-emulsion.

According to certain embodiments, the present invention provides a nano-emulsion comprising the pro-nano-emulsion of the present invention and an aqueous solution. According to certain embodiments, the aqueous solution is selected from the group consisting of water, simulated intestinal fluid solution, and simulated gastric juice solution.

According to some embodiments, the nano-emulsion converted from the pro-nano- emulsion is an oil-in-water (O/W) nano-emulsion.

According to certain embodiments, the average droplet size of the nano-emulsion converted from the pro-nano-emulsion is below 1,000 nm, below 950 nm, below 900 nm, below 850 nm, below 800 nm, below 750 nm, below 700 nm, below 650 nm, below 600 nm, below 550 nm, or below 500 nm. Each possibility represents a separate embodiment of the invention. According to certain exemplary embodiments, the Z-Average of the nano-emulsion measured by dynamic light scattering (DLS) is from about 150 nm to about 300 nm, from about 170 nm to about 280 nm, from about 190 nm to about 260 nm, or from about 210 nm to about 240 nm. According to certain exemplary embodiments, the Z-Average of the nano-emulsion measured by dynamic light scattering (DLS) about According to some embodiments, the polydispersity index (PDI) of the nanoemulsion converted from the pro-nano-emulsion is below 0.6, below 0.5, or below 0.4 (per volume). Each possibility represents a separate embodiment of the invention. According to some embodiments, the PDI is from about 0.2 to about 0.5 (per volume), including each value within the specified range. According to some embodiments, the PDI is from about 0.25 to about 0.4 (per volume). According to some embodiments, the PDI is from about 0.25 to about 0.375 (per volume).

Omega-3 fatty acids may regulate plasma lipid levels, cardiovascular and immune functions, insulin action, neuronal development, and visual function. Marine oils, also commonly referred to as fish oils, are a source of omega-3 fatty acids, including eicosapentaenoic acid (EP A) and docosahexaenoic acid (DHA), have been found to regulate lipid metabolism. Plant-based oils and microbial oils are also sources of omega- 3 fatty acids. Plant-based oils comprise alpha-linolenic acid (ALA) that may be converted within a human body to EPA and then to DHA. However, the efficacy of ALA conversion with the human body deteriorates with age, such that for elderly people (typically over 60 years of age), plant-based oils are not sufficient as EPA and DHA supplement.

Omega-3 fatty acids may have beneficial effects on the risk factors for cardiovascular diseases, for example hypertension and hypertriglyceridemia, and on the coagulation factor VII phospholipid complex activity. Omega-3 fatty acids may also lower serum triglycerides, increase serum HDL cholesterol, lower systolic and diastolic blood pressure and/or pulse rate, and may lower the activity of the blood coagulation factor VH-phospholipid complex. Further, omega-3 fatty acids are generally well- tolerated, without giving rise to severe side effects.

According to certain aspects, the present invention provides a method for preferentially delivering at least one type of Omega 3 fatty acid (Omega 3 s) to heart and/or liver tissue of a subject, the method comprising orally administering to the subject a pro-nano-emulsion composition comprising an oil component comprising at least one type of Omega 3 s; a combination of at least two liquid surface-active agents; and at least one water-soluble organic solvent, thereby preferentially delivering the at least one type of Omega 3s to said subject heart and/or liver tissue.

According some embodiments, the present invention provides a method for preferentially delivering at least one type of Omega 3 fatty acid (Omega 3 s) to heart tissue of a subject, the method comprising orally administering to the subject a pro-nano- emulsion composition comprising an oil component comprising at least one type of Omega 3 s; a combination of at least two liquid surface-active agents; and at least one water-soluble organic solvent, thereby preferentially delivering the at least one type of Omega 3s to the heart tissue of said subject.

According to certain embodiments, the preferential delivery of the Omega 3 s results in accumulation of said Omega 3s in the heart tissue of the subject.

According to certain embodiments, the DHA and EPA content in the heart tissue is at least 4 times higher compared to the content of said DHA and EPA when orally administered to the subject in an oil composition.

According to certain aspects, the present invention provides a method for preferentially delivering at least one type of Omega 3 fatty acid (Omega 3 s) to liver tissue of a subject, the method comprising orally administering to the subject a pro-nano- emulsion composition comprising an oil component comprising at least one type of Omega 3 s; a combination of at least two liquid surface-active agents; and at least one water-soluble organic solvent, thereby preferentially delivering the at least one type of Omega 3s to the liver tissue of said subject.

According to certain embodiments, the preferential delivery of the Omega 3 s results in accumulation of said Omega 3a in the liver tissue of the subject.

According to certain embodiments, the DHA and EPA content in the liver tissue is at least 1.5 times higher compared to the content of said DHA and EPA when orally administered to the subject in an oil composition.

According to certain aspects, the pro-nano-emulsion of the present invention or a composition comprising the same is for use in preferentially delivering at least one type of Omega 3 fatty acid (Omega 3s) to heart and/or liver tissue of a subject.

According to some embodiments, the pro-nano-emulsion of the present invention or a composition comprising the same is for use in preferentially delivering at least one type of Omega 3 fatty acid (Omega 3s) to heart tissue of a subject.

According to other embodiments, the pro-nano-emulsion of the present invention or a composition comprising the same is for use in preferentially delivering at least one type of Omega 3 fatty acid (Omega 3s) to liver tissue of a subject.

According to certain embodiments, the subject is a human.

According to certain embodiments, the preferential delivery of the Omega 3 s to the heart and/or liver tissue of a human subject results in maintaining the cardiovascular and/or hepatic function of the subject. According to certain embodiments, the preferential delivery of the Omega 3 s to the heart tissue of a human subject results in maintaining the heart function of the subject. According to certain embodiments, the preferential delivery of the Omega 3s to the liver tissue of a human subject results in maintaining the hepatic function of the subject. According to certain embodiments, the preferential delivery of the Omega 3s to the heart and/or liver tissue of a human subject results in the risk of the subject to develop a cardiovascular and/or hepatic disease or disorder.

According to certain aspects, the present invention provides a method for maintaining the heart function in a subject, comprising orally administering to the subject an effective amount of the pro-nano-emulsion of the present invention or a composition comprising the same.

According to certain aspects, the present invention provides a method for improving cardiovascular function in a subject in need thereof, comprising orally administering to the subject an effective amount of the pro-nano-emulsion of the present invention or a composition comprising the same.

According to certain aspects, the pro-nano-emulsion of the present invention or a composition comprising the same is for use in maintaining the heart function in a subject consuming the same.

According to certain aspects, the pro-nano-emulsion of the present invention or a composition comprising the same is for use in improving the hepatic function in a subject consuming the same.

According to certain aspects, the present invention provides a method for treating and/or regulating a cardiovascular disease and/or disorder, comprising orally administering to a subject in need thereof a therapeutically effective amount of the pro- nano-emulsion of the present invention or a composition comprising same. As used herein, the terms “cardiovascular disease”, “cardiovascular condition” and "cardiovascular disorder" include disorders of the heart and vasculature, including, for examples, congestive heart failure, hypertension, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, transient ischemic attack, systolic dysfunction, diastolic dysfunction, aneurysm, aortic dissection, myocardial ischemia, acute myocardial infarction (AMI), acute ST-segment elevation myocardial infarction (STEMI), acute non- ST-segment elevation myocardial infarction (NSTEMI), angina pectoris, unstable angina (UA), stable angina (SA), myocardial infarction, dilated congestive cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, Cor-pulmonale, arrhythmia, valvular heart disease, endocarditis, pulmonary embolism, venous thrombosis, peripheral vascular disease, and peripheral artery disease. Each possibility represents a separate embodiment of the present invention.

In humans, cholesterol and triglycerides are part of lipoprotein complexes in the bloodstream and can be separated via ultracentrifugation into high-density lipoprotein (HDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and very- low-density lipoprotein (VLDL) fractions. Cholesterol and triglycerides are synthesized in the liver, incorporated into VLDL, and released into the plasma. High levels of total cholesterol (total-C), LDL-C, and apolipoprotein B (a membrane complex for LDL-C and VLDL-C) promote human atherosclerosis and decreased levels of HDL-C and its transport complex; apolipoprotein A is also associated with the development of atherosclerosis. Furthermore, cardiovascular morbidity and mortality in humans can vary directly with the level of total-C and LDL-C and inversely with the level of HDL-C. In addition, research suggests that non-HDL cholesterol is an indicator of hypertriglyceridemia, vascular disease, atherosclerotic disease, and related conditions. In fact, the US National Cholesterol Education Program Adult Treatment Panel II (NCEP ATP III) specifies non-HDL cholesterol reduction as a treatment objective.

Hypertriglyceridemia, for example, is a condition related to cardiovascular disease in which fasting blood serum concentrations of triglycerides are ^150 mg/dL. Blood concentrations can rise from moderately high levels of 200 mg/dL to 500 mg/dL, or in severe cases, above 500 mg/dL. The American Heart Association has categorized triglyceride concentrations as “normal” (below 150 mg/dL), “elevated” (150 to 199 mg/dL), “high” (200 to 499 mg/dL), and “very high” (above 500 mg/dL). It will be evident to the skilled practitioner that the categorization of hypertriglyceridemia can vary from country to country. For example, Canadian and European guidelines recommend fasting blood serum triglyceride levels of less than 1.7 mmol/L as “desirable”, from 1.7 to 2.2 mmol/L as “borderline high” and 2.3 to 5.6 mmol/L as “high” and above 5.6 mmol/L as “very high”. The skilled practitioner will also appreciate that what constitutes elevated blood serum triglyceride levels may vary based on age and gender.

Heart Failure with Preserved Ejection Fraction (HFpEF) is a type of heart failure where the heart maintains its ability to pump blood (preserved ejection fraction, typically >50%) but has difficulty relaxing and filling with blood during the diastolic phase. This results in increased pressure in the heart and lungs, leading to symptoms of heart failure despite a relatively normal ejection fraction. The prevalence of heart failure (HF) with HFpEF relative to heart failure with reduced ejection fraction (HFrEF) continues to increase, while established pharmacological treatment options are limited. HFpEF clinically presents as a heterogeneous condition with numerous underlying aetiologies but conceptually relates to a distinct metabolic phenotype characterized by extracardiac comorbidities such as obesity, arterial hypertension (HT), and type 2 diabetes mellitus (T2D). A unifying hallmark of these metabolic features is the accumulation of ectopic adipose tissue in the epicardium, driving left ventricular remodeling and dysfunction through altered paracrine signaling to cardiomyocytes, through macrophage infiltration and interstitial fibrosis, and through systemic inflammation and coronary microvascular endothelial dysfunction.

Unexpectedly, the present invention shows that accumulation of Omega 3 s in the heart of HFpEF model mice, obtained after oral administration of the pro-nano-emulsion of the present invention, resulted in a significant improvement in the diastolic function (relaxation) of the heart muscles.

Omega 3s has been also reported as a potential treatment and/or prevention of liver diseases and inflammation associated with liver disease.

According to certain aspects, the present invention provides a method for maintaining the hepatic function of a subject, comprising orally administering to the subject an effective amount of the pro-nano-emulsion of the present invention or a composition comprising the same. According to certain aspects, the pro-nano-emulsion of the present invention or a composition comprising the same is for use in maintaining the hepatic function in a subject consuming the same. According to certain aspects, the present invention provides a method for treating and/or regulating a hepatic disease and/or disorder, comprising orally administering to a subject in need thereof a therapeutically effective amount of the pro-nano-emulsion of the present invention or a composition comprising the same.

According to certain aspects, the pro-nano-emulsion of the present invention or a composition comprising the same is for use in treating and/or regulating a hepatic disease and/or disorder.

According to certain aspects, the present invention provides a method for improving hepatic function in a subject in need thereof, the method comprising orally administering to the subject an effective amount of the pro-nano-emulsion of the present invention or a composition comprising the same.

According to certain aspects, the pro-nano-emulsion of the present invention or a composition comprising the same is for use in improving hepatic function of a subject in need thereof.

As used herein the terms “liver diseases” or "hepatic diseases" refer to a group of medical conditions that affects the liver’s structure or function. The liver is a vital organ that performs over 500 vital functions in the body, including blood filtration, metabolism, bile production, and storage of nutrients. Liver diseases can range from mild and temporary conditions to severe and chronic illness. Some common liver diseases include: hepatitis, cirrhosis, fatty liver disease, including non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), liver cancer, liver failure, autoimmune liver diseases, and genetic liver diseases.

Liver diseases may also result from long term total parenteral nutrition (TPN) or parenteral nutrition (PN). TPN or PN may be required for treating malabsorption syndromes, such as short bowel syndrome (SBS) in infant as well as in adults, when nutrients, including vitamins, minerals, carbohydrates, proteins, and fats, as well as drugs need to be administered through the veins. Patients on long-term PN frequently experience serious metabolic complications. The most common complications are hepatic and biliary disorders manifested by steatosis, fibrosis, and cholestasis. These disorders can progress to fulminant liver failure.

The effective amount of the Omega 3s pro-nano-emulsion of the present invention to be orally administered to a subject depends on the pro-nano-emulsion composition, the amount of Omega 3s within the composition, the purpose of administration and characteristics of the individual, including general health, age, gender, body weight, etc.

According to certain embodiments, maintaining the heart function of a subject comprises orally administrating to the subject at least 250 mg/day of a combination of EPA and DHA within the pro-nano-emulsion of the present invention. According to certain embodiments, the EPA:DHA ratio in the combination is about 2.5: 1.

The pro-nano-emulsion compositions of the invention may be administered per se or within a composition.

It is to be explicitly understood that the present invention also encompasses administering a nano-emulsion converted from the pro-nano-emulsion of the present invention before administration, in all methods disclosed herein.

According to certain exemplary embodiments of the invention, the pro-nano- emulsion of the present invention is administered within a dietary composition formulation. The dietary composition may further comprise any one of food-grade fillers, binders, preservatives, colorant, sweeteners and other flavoring agents, and the like.

According to certain currently exemplary embodiments, the pro-nano-emulsion of the present invention is administered within a dietary supplement.

Dietary supplements are available in various forms, including pills, capsules, tablets, powders, liquids, gels, and chewable forms. According to some embodiments, the dietary supplement is selected from the group consisting of pills, capsules, tablets, powders, liquids, gels, and chewable forms. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the dietary supplement comprising the pro- nano-emulsion of the present invention is in a form of a capsule. According to certain embodiments, the dietary supplement comprising the nano-emulsion of the present invention is in a form of a capsule. As used herein, the term “capsule” refers to an oral dosage form that is a safe, readily dissolvable enclosure for carrying an active agent. Capsules include a shell surrounding and containing the active agent or formulation comprising the same. The capsule prevents degradation of the active agent or formulation comprising the same and allows for storage for a period of at least several months at room temperature. The capsule is adapted for self-administration by an individual. The capsule shell dissolves or disintegrates in the digestive tract after the capsule is ingested, thereby releasing the active ingredient to be absorbed by the body of the individual ingesting the capsule.

According to some embodiments, the capsule is a soft or hard capsule. According to some embodiments, the soft or hard capsule shell is based on gelatin or on a non-gelatin alternative, such as cellulose, hydroxypropyl methylcellulose, vegetable starch, tapioca starch, carrageenan, potato starch, cassava starch, cornstarch, arrowroot or combinations thereof. According to specific embodiments, the soft or hard capsule shell is based on gelatin. According to some embodiments, the soft or hard capsule shell is based on fish- derived, plant-derived, or mammal-derived gelatin. According to some embodiments, the plant-based gelatin is vegetable, tapioca, or grass-derived gelatin. According to some embodiments, the mammal-derived gelatin is bovine-derived gelatin. According to some embodiments, the gelatin is derived from fish or plant-based sources. According to some embodiments, the soft or hard gelatin capsule shell is based on fish-derived gelatin. According to some embodiments, the soft or hard gelatin capsule shell is based on fish- derived gelatin. According to some embodiments, the soft capsule shell is based on fish- derived gelatin. According to some embodiments, the hard gelatin capsule shell is based on fish-derived gelatin.

As used herein, the term "soft capsule" may be used interchangeably with "soft gel capsule" and “softgel”. Soft capsules are particularly suitable for containing liquid-based ingredients, such as those of the present invention.

According to some embodiments, the capsule is a soft capsule. According to some embodiments, the capsule shell is a soft capsule shell. The shell of a soft capsule is typically made of animal-based components such as gelatin combined with a plasticizer such as glycerin and a solvent such as water. Other suitable synthetic, animal-based or plant-based components with properties similar to gelatin or starch may also be used as described hereinabove. Soft capsule shells are typically made and filled with active agents or formulations thereof in continuous processes that are known in the art.

The term “plasticizer” refers to a substance that is added to the gelatin or starch to form the soft or hard capsule. Plasticizers may include glycerin, sorbitol, propylene glycol, other suitable polyols, or combinations thereof. The amount of plasticizer can be adjusted to arrive at soft capsule or hard capsule shells with the desired level of softness and flexibility. According to some specific embodiments, the plasticizer is glycerin.

According to other embodiments, the capsule is a hard capsule wherein the shell is based on gelatin. According to other embodiments, the capsule is a hard capsule wherein the shell is based on fish-derived gelatin. According to some embodiments, the capsule is a hard capsule wherein the shell is based on gelatin and the plasticizer is glycerin.

According to some embodiments, the capsule is a soft capsule wherein the shell is based on gelatin. According to some specific embodiments, the capsule is a soft capsule wherein the shell is based on fish-derived gelatin. According to some embodiments, the capsule is a soft capsule wherein the shell is based on gelatin and wherein the plasticizer is glycerin. According to some specific embodiments, the capsule is a soft capsule wherein the shell is based on fish-derived gelatin and wherein the plasticizer is glycerin.

According to some embodiments, the dietary composition is a liquid. According to some embodiments, the liquid is provided as drops.

According to some embodiments, the dietary composition is provided as a stick or sachet.

According to some embodiments, the chewable form is selected from the group consisting of gummies, chewing gums, lozenges, and chewable tablets.

As used herein, a “therapeutically effective amount” of a pro-nano-emulsion composition as described in some embodiments herein can be a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, for example, an amount which results in the prevention of, or a decrease in the symptoms associated with, a disease that is being treated. The amount of composition administered to the subject, particularly one in need of the composition, can depend on the type and severity of the disease and on the characteristics of the individual, as described above further taking into account tolerance to drugs. According to certain embodiments, the pre-nano-emulsion formulation of the present invention enable use of lower Omega 3 s doses compared to hitherto known doses while achieving at least an equal therapeutic or prophylactic effect. According to certain embodiments, the doses enabled by the formulations of the present invention are not associated with high-doses adverse effects of Omega 3 s.

According to certain embodiments, the pro-nano-emulsion of the invention is in a form of a pharmaceutical composition to be administered orally.

The pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, but are not limited to, carriers, preservatives, and/or coloring agents. General considerations in the composition and/or manufacture of pharmaceutical compositions may be found, for example, in Remington The Science and Practice of Pharmacy 21^st ed., Lippincott Williams & Wilkins, 2005.

The dietary or pharmaceutical compositions of the invention may further comprise at least one additional active agent other than the Omega 3 s including, but not limited to, a nutritional supplement; an antioxidant; a cannabinoid and any combination thereof. Each possibility represents a separate embodiment of the present invention.

According to certain embodiments, the nutritional supplement is selected from the group consisting of folic acid, vitamin C, vitamin E, beta-carotene, lycopene, vitamin A, vitamin D, vitamin KI, vitamin K2, astaxanthins and any combination thereof. Each possibility represents a separate embodiment of the present invention. According to certain embodiments, the dietary or pharmaceutical compositions of the invention is devoid of statin(s).

According to certain embodiments, the antioxidant is selected from the group consisting of tocopherols, Co-Q 10, ascorbic acid, and derivatives thereof, and carotenoids.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention. EXAMPLES

Example 1: Preparation of Omega-3 nano-emulsion composition

Materials and Methods

245 g of Tween™ 80, 109 g of Span™ 80 and 175 g of Omega-3 oil, were mixed by a mechanical stirrer, (40 power) for 5 min. 34.6 g of ethanol were then added to the mixture and mixed for an additional 2.5 hours to form the pro-nano-emulsion. The entire experiment was conducted under nitrogen atmosphere, (99.999). Converting the pro- nano-emulsion to nano-emulsion was performed by adding 1-2 droplets of the mixture to 3 ml of deionized water or artificial gastric liquids. After vortexing for 30-60 seconds an emulsion with nano-sized droplets was obtained.

Component concentrations at the pro-nano-emulsion:

31.0503% Omega-3 oil (Fish oil, Supherb, Israel; minimum EPA:500 mg/g; minimum DHA, 200 mg/g)

43.4705% Tween 80

19.3401% Span 80

6.1391% Ethanol

The droplets size of the oil-in-water (O/W) nano-emulsion which result upon contact with water was measured by Dynamic Light Scattering (DLS).

The measured droplets size was 220.6 nm; Z-Average, peak 1 : 47.59 nm, 5.4%; peak 2: 260.3 nm, 89%; peak 3: 4778 nm, 5.5%. Poly dispersity Index (PDI) was 0.313 (by volume). Peak 1 : 45.19 nm, 28.7%; peak 2: 117.1 nm, 71.3%, (by number).

Volume distribution shows the total volume of particles in the different size bins; Number distribution shows the number of particles in the different size bins.

Example 2: Droplets size stability

DLS measurements were conducted at the same way described above, for:

1. Omega 3 pro-nano-emulsion that was prepared as described hereinabove on December 14, 2021 (22 month old formulation) and stored at room temperature (RT). At the end of the storage, the pre-nano-emulsion was converted to nano-emulsion. The droplets size of the nano-emulsion was 202 nm; Z-Average, peak 1 : 220.5nm, 95.4%; peak 2: 4887 nm, 4.6%; PDI: 0.346. (by volume). Peak 1: 108.6nm, 100%, (by number).

2. Omega3 formulation that was prepared on December 14, 2021 (22 month old formulation) and stored at 45°C. At the end of the storage, the pre-nano- emulsion was converted to nano-emulsion.

The droplets size of the nano-emulsion was 169.3 nm; Z-Average, peak 1 : 23.76nm, 17.9%; peak 2: 97.9 nm, 44.2%; peak 3: 306 nm, 35.4% PDI: 0.280. (by volume) peak 1 : 20.98nm, 32.3%; peak 2: 74.94nm, 67.7%, (by number).

The above results demonstrate good stability of the pro-nano-emulsion at temperature as high as 45°C.

Example 3: Droplets size measurements of nano-emulsions prepared with simulated gastric juice or simulated intestinal fluid solutions

22-month old Omega 3 pro-nano-emulsion stored at RT or at 45°C and freshly prepared pro-nano-emulsion were added (1-2 droplets), to gastric juice, pH 1.2 or to intestinal fluid pH 6.8, simulated solutions at 37°C and vortexed for 30 sec. to form nanoemulsions.

Nano-emulsion formed with simulated gastric juice solution. pH=l,2

The droplets size for the 22 month formulation, stored at RT, was 158.2 nm; Z- Average, peak 1 : 189.7nm, 89.1%; peak 2: 4702 nm, 1.1%; peak 3: 28.98 nm, 9.8%. Pdi: 0.260 (by volume). Peak 1 : 27.91 nm, 32%; peak 2: 67.63 nm, 68% (by number).

The droplets size for the 22 month formulation, stored at 45°C, was 153.1 nm; Z- Average, peak 1 : 143.5 nm, 97.4%; peak 2: 4853 nm, 2.6%. PDI: 0.262 (by volume). Peak 1 : 35.04 nm, 56.6%; peak 2: 79.86nm, 43.4% (by number).

Nano-emulsion formed with simulated intestinal fluid solution. pH=6,83

The droplets size for the 22 month formulation, stored at RT, was 162 nm; Z- Average, peak 1 : 73.67 nm, 65.8%; peak 2: 304.4 nm, 32.3%; peak 3: 4951 nm, 1.9%. Pdi: 0.284 (by volume). Peak 1 : 39.66 nm, 100%, (by number).

The droplets size for the 22 month formulation, stored at 45°C, was 143.6 nm; Z- Average, peak 1 : 137.4nm, 99.5%; peak 2: 4789 nm, 0.5%; PDI: 0.262. (by volume).

Peakl : 24.5nm, 24.5%; peak 2: 51.2nm, 75.5%, (by number). with

9.8 g of Tween™ 80, 4.36 g of Span™ 80 and 7 g of Omega-3 oil were mixed by a magnetic stirrer, for 10 min. 1.384 g of propylene glycol were then added to the mixture and mixed for an additional 4 hours to form the pro-nano-emulsion. Converting the pro- nano-emulsion to nano-emulsion was performed by adding 1-2 droplets of the mixture, to 3 ml of deionized water. After vortexing for 30-60 seconds an emulsion with nanosized droplets was obtained.

i-nano-emulsion:

31.0503% Omega-3 oil (Fish oil, Supherb, Israel, as above)

43.4705% Tween 80

19.3401% Span 80

6.1391% propylene glycol, (cas: 57-55-6)

The droplets size of the nano-emulsion was 175.4 nm; Z-Average. peak 1 : 47.41nm, 12.9%; peak 2: 176.2 nm, 80.6%; peak 3: 141 Inm, 0.3% PDI: 0.345. (by volume). Peak 1 : 42.69nm, 27.6%; peak 2: 88.67nm, 72.4%, (by number).

The above results show no significant difference in terms of droplet size when using ethanol or propylene glycol for freshly prepared emulsions.

Example 5: Preparation of Omega-3 nano-emulsion composition with polyethylene glycol 400:

Materials and Methods

9.8 g of Tween™ 80, 4.36 g of Span™ 80 and 7 g of Omega-3 oil, were mixed by a magnetic stirrer, for 10 min. 1.384 g of polyethylene glycol 400 were then added to the mixture and mixed for an additional 4 hours to form the pro-nano-emulsion. Converting the pro-nano-emulsion to nano-emulsion was performed by adding 1-2 droplets of the mixture to 3 ml of deionized water. After vortexing for 30-60 seconds an emulsion with nano-sized droplets was obtained. Components concentrations at the pro-nano-emulsion:

31.0503% Omega-3 oil (Fish oil, Supherb, Israel, as above)

43.4705% Tween 80

19.3401% Span 80

6.1391% polyethylene glycol 400 MW, (cas: 23522-68-3)

The droplets size of the nano-emulsion was 216.5 nm; Z-Average, peak 1 : 44.97 nm, 13.7%; peak 2: 185.1 nm, 65.4%; peak 3: 712.3 nm, 8.8%. Pdi: 0.554 (by volume). Peak 1 : 39.51 nm, 29.9%; peak 2: 91.57 nm, 70.1%, (by number).

In this experiment, the high PDI values indicate the formation of non-homogenous emulsion.

Example 6: Large-scale preparation of pro-nano-emulsion and droplet size of the resulting nano-emulsion

Omega 3 pro-nano-emulsion was prepared at Supherb (Israel), on August 9, 2023, as described in Example 1 hereinabove (Zero batch) and was stored at 4°C, for two months as scale up preparation of 150 L.

Omega 3 source: Fish oil (Supherb, Israel, as above). The fish species used in this oil is the anchovies which is harvested from the world cleanest ocean area at the Peruvian and Chilean coasts.

The pro-nano-emulsion was prepared and as described in Example 1 hereinabove and DLS measurements were conducted for the respective nano-emulsion formed as described below:

Droplets size of freshly prepared nano-emulsion formed with water:

The droplets size was 165.1 nm; Z-Average, peak 1 : 190.7 nm, 95.7%; peak 2: 4735 nm, 4.3%. Pdi: 0.376 (by volume). Peak 1 : 61.03 nm, 100% (by number).

Droplets size of nano-emulsion formed with simulated gastric juice solution at pH 1.2, 37°C was 174.1 nm; Z-Average, peak 1 : 93.66 nm, 55.2%; peak 2: 295.4 nm, 38%; peak 3: 425.9nm, 6.9%. PDI: 0.358 (by volume). Peak 1 : 71.42nm, 100%, (by number).

Droplets size of the nano-emulsion formed with simulated intestinal fluid solution at pH 6.8, 37°C was 179.9 nm; Z-Average, peak 1: 37. 3nm, 11.8%; peak 2: 106.4 nm, 41.6%; peak 3: 298.3 nm, 39.7%. PDI: 0.355 (by volume). Peak 1: 33.2 nm, 30.2%; peak 2: 85.74 nm, 69.8%, (by number).

7: Omega 3 accumulation in wild type C57BL mice tissue

Materials and Methods

C57BL mice were divided into three treatment groups, 6-10 mice per group:

“Control” - regular feed with no supplement of fish oil (Supherb, Israel; minimum EPA:500 mg/g; minimum DHA, 200 mg/g);

“Omega 3 s” - regular feed, supplemented with 50pl of the fish oil. The fish oil was administered once daily by gavage; and

“Assay” - regular feed, supplemented with 50pl the fish oil in a form of pro-nano- emulsion administered daily by gavage.

Treatments were applied for two weeks. After two weeks, one group of mice (3-5 mice) from each treatment group (including control) was sacrificed. Additional group of each treatment (including control) was sacrificed two weeks after the treatment had been stopped. Blood and designated organ samples (brain, liver, heart, and lungs) from all mice were collected and tested for EPA and DHA levels.

Assessment of Omega-3 s in organs

Organ samples were weighed and homogenized with saline at a ratio of 1 :5 (w:v) in plastic tubes on ice. Lipids were extracted from an aliquot of the homogenates according to previously known procedures (Folch et al. J biol Chem, 1957, 226(1):497- 509). Total Omega-3 lipid content was calculated based on the sample dry weight after the sampled aliquot was evaporated to achieve constant weight.

Fattv acid analysis

Fatty acids were analyzed as methyl ester derivatives (FAME) by gas chromatography (GC) in a Varian, 3800 Series (Walnut Creek, CA) chromatograph (FID) with a fused silica SGE capillary column 30 0.025 and Varian Star Workstation Advance Application software, version 6. Aliquots of the lipid extracted from the tissue homogenate as described hereinabove were processed as described before (Shomonov- Wagner 1 et al., 2015. Lipids in Health and Disease, 14: 14 Doi: 10.1186/sl2944-015- 0012-7). After lipid extraction and weight, aliquots representing 0.5 g tissue, kept frozen at -20°C before use, were taken into a screw-capped tube (teflon-lined) containing 5 pg heptadecanoic acid as Internal Standard. 1 ml 5% H2SO4 in methanol was added. The tubes were gassed with nitrogen, closed tightly, and heated at 85°C for 1.5 h with occasional shaking. After cooling, 1 ml of hexane was added, the tube content was mixed and, after a short centrifugation, the hexane layer was transferred into a new tube. Before GLC analysis, the hexane extracts were concentrated by evaporation under nitrogen. One- twentieth of the final re-suspension was applied in 1 pl hexane into the gas chromatograph. The fatty acid profiles were compared to that of a known mixture of fatty acids of animal source, PUFA2 (Supelco, USA) for identification.

Results

While the levels of DHA in the blood, brain, and lungs samples showed no difference between mice treated with Omega-3 (fish oil) and those treated with the pro- nano omega-3 composition (data not shown), the scenario differed for the heart and the liver tissue. The heart tissue exhibited a significant increase in DHA accumulation, while the liver tissue showed an extended accumulation, one week after the treatment stopped, as illustrated in Fig. 1A and Fig.lB.

Example 8: Omega 3 accumulation in wild type C57BL mice plasma, heart, and liver tissue

Accumulation of Omega 3 in mice plasma and heart and liver tissue was examined essentially as described in Example 7 hereinabove.

Two groups were examined:

“Omega 3s” (co-3) - regular feed supplemented with 50pl of fish oil (Supherb, Israel; minimum EPA:500 mg/g; minimum DHA, 200 mg/g). The fish oil was administered once daily by gavage to 6 mice; and

“Assay” - regular feed, supplemented with 160pl of pro-nano-emulsion comprising 50 pl of the fish oil (nano-co-3). The pro-nano-emulsion was administered daily by gavage, to 6 mice.

After 2 weeks of oil administration in both forms, mice plasma samples were taken and then the mice were sacrificed and the other tissue were collocated.

Fatty acid analyses were performed as described in Example 7 hereinabove.

Results

While the levels of EPA and DHA in the plasma showed relatively small difference between mice treated with Omega-3 and those treated with the pro-nano-emulsion omega-3 compound, the scenario differed for the heart and liver tissue, which exhibited a significant and unexpected increase in both EPA and DHA accumulation for mice treated with the pro-nano-emulsion omega-3 compound compared with mice treated with omega-3, as illustrated in Figs. 2A-2F. Specifically, Figs. 2A-2C show the level of EPA in the plasma (Fig. 2A), the liver (Fig. 2B) and the heart (Fig. 2C) in the mice that were treated with fish oil (co 3 oil) or treated with the pro-nano-emulsion omega-3 composition (nano-co3) and Figs. 2D-2F show the level of DHA in the plasma (Fig. 2D), the liver (Fig. 2E) and the heart (Fig. 2F) of the same mice.

Example 9: Omega 3 accumulation in wild type C57BL

In order to more fully characterize the unexpected increase of Omega 3 accumulation in mouse tissue of C57BL mice that received the pro-nano-emulsion comprising Omega 3 s of the invention compared with fish oil, the accumulation of Omega 3 in mice plasma, liver, brain, and heart tissue was examined using the methods described in Example 7 hereinabove.

Three groups were examined:

“Untreated” - regular feed with no supplement of fish oil administered at libido to 8 mice;

“co 3 oil” - regular feed administered at libido, supplemented with 50pl of fish oil (Supherb, Israel; minimum EPA: 500 mg/g; minimum DHA, 200 mg/g. The fish oil was administered once daily by gavage to 6 mice; and

“Nano-co3” - regular feed administered at libido supplemented with 160pl of pro- nano-emulsion comprising 50pl of the fish oil. The “nano-co3” was administered once daily by gavage to 6 mice.

After 14 days, mice plasma samples were taken. The mice were then sacrificed and the liver, brain, and heart were collected.

Fatty acid analyses were performed as described in Example 7 hereinabove.

Results

The results are presented in Figs 3A-3H. The data are presented as the mean ± standard error of the mean (SEM) and were considered significant when the p-value was <0.05. All experiments were independently repeated at least three times. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparisons.

The difference in the levels of EPA and DHA in the plasma between mice that received fish oil and those that received the pro-nano-emulsion Omega-3 compound was not statistically significant as shown in Fig. 3A and Fig. 3E, confirming the results presented in Figs. 2 A and 2D.

Advantageously, mice receiving fed supplemented with the pro-nano-emulsion omega-3 composition of the invention showed statistically significant accumulation of EPA and DHA in both heart and liver tissue compared with untreated mice, and unexpectedly, compared with mice that received fish oil, as shown in Fig. 3B and Fig. 3D and Fig. 3F and Fig. 3H, respectively.

In contrast, as shown in Fig. 3C, no accumulation of EPA was observed in the brain of mice in all groups. While a statistically significant accumulation of DHA was observed in mice that received the pro-nano-emulsion Omega-3 compound compared with the untreated mice, there was no statistically significant difference between the mice that received fish oil vs. the mice that received the Omega-3 pro-nano-emulsion composition.

Example 10: Effect of administering Omega3s in a form of pro-nano emulsion on heart disease or disorder

Heart failure with preserved ejection fraction (HFpEF) is a common, morbid, and potentially fatal syndrome for which there are no evidence-based therapies. Diastolic dysfunction, or an impairment of left ventricle relaxation, is an important cause of HFpEF and is predictive of developing overt heart failure. In order to assess a beneficial effect of the Omega-3 pro-nano-emulsion composition of the invention on HFpEF, a mouse model of HFpEF was induced in C57BL/6N mice by subjecting the mice to a combination of high fat diet (HFD) and constitutive nitric oxide (NO) synthase inhibition using N[w]- nitro-l-arginine methyl ester (L-NAME) as described in Schiattarella et al. (Schiattarella G et al., 2019. Nature 568(7752):351-356. doi: 10.1038/s41586-019-1100-z). This combination replicates the numerous systemic and cardiovascular features of human HFpEF.

Specifically, all mice were subjected to a high-fat diet (~35 g% fat, TD.06414, Envigo) along with drinking water containing 0.5g/L of L-NAME for a period of 10 weeks. During this period, the mice were allowed free access to food and water. After 10 weeks, all mice exhibited a HFpEF phenotype, including obesity as well as heart pathology (including reduction in the E/E’ ratio described below) as monitored by echocardiography using Doppler ultrasound (Schiattarella et al., 2019, ibid). The mice were then returned to regular feed and drinking water and the mice were split into three groups:

“Untreated” - no supplement of fish oil and no induction of HFpEF, 8 mice;

“co 3 oil” - 50pl of the fish oil (Supherb, Israel; minimum EPA:500 mg/g; minimum DHA, 200 mg/g). The fish oil was administered once daily by gavage to 6 mice; and

“Nano-o3” - 160pl of pro-nano-emulsion comprising 50pl of the fish oil was administered once daily by gavage, 6 mice.

The experiment was allowed to proceed for 65 days. At the end of the 65-day period, echocardiography using Doppler ultrasound was performed on the mice. The mice were sacrifice after additional 3 days (day 68).

Results

Early diastolic flow peak velocity of the mitral valve (E) and early diastolic peak velocity of mitral valve annulus (E’) as well as mitral valve E/E’ ratio (MV E/E’), are often used for evaluating left ventricular diastolic function. In particular, E’ reports on the diastolic function of the tissue by measuring the extent of tissue expansion during diastole. These values may be determined by echocardiography using Doppler ultrasound. A high ratio of E/E’ (in mice, over 30) or a low E’ (in mice, below 15mm/s) are indicators of left ventricular diastolic dysfunction.

As shown in Fig. 4A, E was not statistically different between the three groups. As shown in Fig. 4C, a statistically significant improvement in MV EZE’ was observed in the mice that received the Omega-3 pro-nano-emulsion composition compared with untreated mice. However, no statistically significant improvement was observed in the mice that received the Omega-3 pro-nano-emulsion composition compared with the mice that received Omega-3 oil.

However, as shown in Fig. 4B, a statistically significant improvement in E’ was observed in the mice that received the Omega-3 pro-nano-emulsion compared with the untreated mice. Importantly and unexpectedly, as can further be seen in Fig. 4B, a statistically significant improvement in E’ was also observed in the mice that received the Omega-3 pro-nano-emulsion compared with the mice that received Omega-3 oil. These findings indicate that diastolic function of HFpEF is improved specifically with the Omega-3 pro-nano-emulsion composition of the present invention.

In addition, echocardiographic global longitudinal strain (GLS) was assessed. GLS is a parameter that expresses longitudinal shortening as a percentage (change in length as a proportion to baseline length) that can be determined from a 2D-echocardiogram. This parameter reflects the function of sub -endocardial longitudinally oriented fibers, which are most prone to ischemic damage and wall stress. Such damage limits the ability of the fibers to shorten, and as such, a lower percentage is an indication of greater damage. Thus, GLS% is a sensitive marker for systolic dysfunction.

As shown in Fig. 4D, a statistically significant improvement in GLS% was observed in the mice that received the Omega-3 pro-nano-emulsion composition compared with the untreated mice. Furthermore, as can further be seen in Fig. 4D, a statistically significant improvement in GLS% was also observed in the mice that received the Omega-3 pro-nano-emulsion composition compared with the mice that received fish oil. Thus, remarkably, supplementation with the Omega-3 pro-nano- emulsion composition of the invention significantly improved systolic function.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A method for preferentially delivering at least one type of Omega 3 fatty acid (Omega 3s) to heart and/or liver tissue of a subject, the method comprising orally administering to the subject a pro-nano-emulsion composition comprising: an oil comprising at least one type of Omega 3s; a combination of at least two liquid surface-active agents; and at least one water-soluble organic solvent, thereby preferentially delivering the at least one type of Omega 3s to said subject heart and/or liver tissue.

2. The method of claim 1, wherein the subject is a human and wherein said method results in maintaining the cardiovascular and/or hepatic function of said subject.

3. The method of any one of claims 1-2, wherein the subject is a human and wherein said method results in maintaining the heart and/or the liver function of said subject.

4. The method of any one of claims 1-3, wherein the subject is a human and wherein said method results in reducing the risk of said subject to develop a cardiovascular and/or hepatic disease or disorder.

5. The method of claim 1, wherein the subject is a human patient having a cardiovascular and/or hepatic disease or disorder, and wherein said method results in improving and/or treating and/or regulating said cardiovascular and/or hepatic disease or disorder.

6. The method of any one of the preceding claims, wherein the pro-nano-emulsion composition comprises at least 25% w/w oil out of the total weight of the composition.

7. The method of any one of the preceding claims, wherein the oil comprises at least 50% w/w of the at least one type of Omega 3 s.

8. The method of claim 7, wherein the oil consists of the at least one type of Omega 3s.

9. The method of any one of the preceding claims, wherein the at least one type of Omega 3 fatty acid is selected from the group consisting of docosahexaenoic acid (DHA); eicosapentaenoic acid (EP A); and docosapentaenoic acid (DPA).

10. The method of any one of the preceding claims, wherein the pro-nano-emulsion composition comprises DHA and EP A.

11. The method of claim 10, wherein the pro-nano-emulsion composition comprises from about 40 mg to about 200 mg DHA and from about 100 mg to about 500 mg EPA.

12. The method of claim 11, wherein the DHA concentration within the pro-nano- emulsion composition is below 100 mg and the EPA concentration is below 200 mg.

13. The method of any one of the preceding claims, wherein the pro-nano-emulsion composition comprises at least 50% w/w of the combination of liquid surfaceactive agents out of the total weight of the composition.

14. The method of any one of the preceding claims, wherein the combination of liquid surface-active agents comprises at least two polysorbates.

15. The method of claim 14, wherein the combination of liquid surface-active agents consists of two polysorbates.

16. The method of any one of claims 14-15, wherein the polysorbates are selected from polyoxyethylene sorbitan ester (Tween™) and sorbitan fatty acid ester (Span™).

17. The method of claim 16, wherein the combination of polysorbates comprises at least one polyoxyethylene sorbitan ester selected from the group consisting of polyoxyethylene sorbitan monolaurate (Tween™ 20), polyoxyethylene sorbitan monopalmitate (Tween™ 40), polyoxyethylene sorbitan monostearate (Tween™ 60) and polyoxyethylene sorbitan monooleate (Tween™ 80); and at least one sorbitan fatty acid ester selected from the group consisting of sorbitan monolaurate (Span™ 20), sorbitan monopalmitate (Span™ 40) and sorbitan monooleate (Span™ 80).

18. The method of claim 17, wherein the combination of surface-active agents consists of polyoxyethylene sorbitan monooleate (Tween™ 80) and sorbitan monooleate (Span™ 80).

19. The method of any one of the preceding claims, wherein the water-soluble organic solvent is selected from the group consisting of ethanol, propylene glycol and liquid polyethylene glycol.

20. The method of any one of the preceding claims, wherein the pro-nano-emulsion composition comprises from about 25% to about 35% w/w oil comprising at least one type of Omega 3s; from about 34% to about 52% w/w Tween™ 80, from about 15% to about 23% w/w Span™ 80; and from about 5% to about 7.5% w/w ethanol, based on the total weight of the composition.

21. The method of any one of the preceding claims, wherein the pro-nano-emulsion composition further comprises at least one additional active agent other than the Omega 3s, wherein the at least one additional active agent is selected from the group consisting of a nutritional supplement; an antioxidant; a cannabinoid and any combination thereof.

22. The method of claim 21, wherein the nutritional supplement is selected from the group consisting of folic acid, vitamin C, vitamin E, beta-carotene, vitamin A, vitamin D, vitamin KI, vitamin K2, and any combination thereof.

23. The method of any one of claims 21-22, wherein the antioxidant is selected from the group consisting of tocopherols, Co-Q 10, ascorbic acid and derivatives thereof, carotenoids, astaxanthin, and any combination thereof.

24. The method of claim 5, wherein the cardiovascular disorder is selected from the group consisting of congestive heart failure, hypertension, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, transient ischemic attack, systolic dysfunction, diastolic dysfunction, aneurysm, aortic dissection, myocardial ischemia, acute myocardial infarction (AMI), acute ST-segment elevation myocardial infarction (STEMI), acute non-ST-segment elevation myocardial infarction (NSTEMI), angina pectoris, congenital heart disease, unstable angina (UA), stable angina (SA), myocardial infarction, rheumatic heart disease, dilated congestive cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, Cor-pulmonale, arrhythmia, valvular heart disease, endocarditis, pulmonary embolism, venous thrombosis, peripheral vascular disease, peripheral artery disease, and combinations thereof.

25. The method of claim 24, wherein the congestive heart failure is heart failure with preserved ejection fraction (HFpEF).

26. The method of claim 25, wherein said method improves the diastolic function of the heart.

27. The method of claim 5, wherein the hepatic disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD) and/or a symptom associated with NAFLD and parenteral nutrition (PN)-associated liver disease (PNALD) and/or a symptom associated with PNALD.

28. The method of any one of the preceding claims, wherein the pro-nano-emulsion composition is self-emulsified to a nano-emulsion upon contact with an aqueous solution, optionally further upon exposure to at least one mixing force, wherein the nano-emulsion has a Z-average droplet size of below Ip.

29. The method of claim 28, wherein the pro-nano-emulsion composition is selfemulsified to the nano-emulsion prior to the oral administration.

30. The method of any one of the preceding claims, wherein said method comprises administering the pro-nano-emulsion composition as a liquid composition.

31. The method of any one of claims 1-30, wherein said method comprises administering the pro-nano-emulsion composition in a dietary composition formulation.

32. The method of any one of claims 1-30, wherein said method comprises administering the pro-nano-emulsion composition in a pharmaceutical composition.

33. The method of any one of claims 31-32, wherein the dietary or pharmaceutical composition is in a form of a soft or hard capsule formulated for oral delivery.

34. A pro-nano-emulsion composition comprising: an oil comprising at least one type of Omega 3 fatty acid; a combination of at least two liquid surface-active agents; and at least one water-soluble organic solvent.

35. The pro-nano-emulsion composition of claim 34, wherein said composition comprises at least 25% w/w oil out of the total weight of the composition.

36. The pro-nano-emulsion composition of claim 35, wherein the oil comprises at least 50% w/w of the at least one type of Omega 3s.

37. The pro-nano-emulsion of any one of claims 35-36, wherein said pro-nano- emulsion composition comprises DHA and EPA.

38. The pro-nano-emulsion of claim 37, wherein said pro-nano-emulsion composition comprises from about 40 mg to about 200 mg DHA and from about 100 mg to about 500 mg EPA.

39. The pro-nano-emulsion of claim 38, wherein the DHA concentration within said pro-nano-emulsion composition is below 100 mg and the EPA concentration is below 200 mg.

40. The pro-nano-emulsion composition of any one of claims 34-39, wherein the combination of liquid surface-active agents comprises at least 50% w/w out of the total weight of the composition.

41. The pro-nano-emulsion composition of any one of claims 34-40, wherein the water-soluble organic solvent is selected from the group consisting of ethanol, propylene glycol and liquid polyethylene glycol.

42. The pro-nano-emulsion composition of any one of claims 34-41, wherein said composition is in a form of a liquid.

43. The pro-nano-emulsion composition of any one of claims 34-42, wherein said composition is self-emulsified to nano-emulsion upon contact with an aqueous solution, optionally further upon exposure to at least one mixing force, wherein the nano-emulsion has a Z-average droplet size of below Ip.

44. A self-emulsified nano-emulsion formed from the pro-nano-emulsion composition of any one of claims 34-42, wherein said nano-emulsion has a Z- average droplet size of below I .

45. The self-emulsified nano-emulsion of claim 44, wherein said nano-emulsion comprises an aqueous solvent.

46. The self-emulsified nano-emulsion of claim 45, wherein the aqueous solvent is selected from the group consisting of water, simulated intestinal fluid solution, and simulated gastric juice solution.

47. A pharmaceutical composition comprising the pro-nano-emulsion composition of any one of claims 34-43, or the self-emulsified nano-emulsion of any one of claims 44-46, further comprising a pharmaceutically acceptable diluent or carrier.

48. A dietary composition comprising the pro-nano-emulsion composition of any one of claims 34-43 or the self-emulsified nano-emulsion of any one of claims 44-46, further comprising at least one of a food-grade filer, carrier, colorant, and flavoring agent.

49. The dietary composition of claim 48, wherein said dietary composition is selected from the group consisting of a dietary supplement, a nutraceutical composition, and a functional food.

50. The pro-nano-emulsion composition of any one of claims 34-43, the selfemulsified nano-emulsion of any one of claims 44-46, the pharmaceutical composition of claim 47 or the dietary composition of any one of claims 48-49 encapsulated within a soft or hard capsule suitable for oral administration.