WO2014130746A1 - Use of an omega-3 lipid-based emulsion for protecting human organs from ischemic injury - Google Patents

Abstract

The present invention provides methods of limiting cell death or damage or reperfusion damage resulting from hypoxic-ischemia, comprising administering an omega-3 lipid-based emulsion after a hypoxic-ischemia insult. The omega-3 lipid-based emulsion preferably comprises from about 10% to 35% omega-6 oil by weight in grams per 100 ml of emulsion; the omega-3 oil comprises about 20% to 100% triglyceride by weight per total weight of the omega-3 oil and about 20% wt.-% to 100% of the acyl-groups of the omega-3 triglycerides consist of DHA; the omega-3 oil comprises less than 10% omega-6 fatty acids; and the mean diameter of lipid droplets in the emulsion is less than about 5 microns.

Description

USE OF AN OMEGA-3 LIPID-BASED EMULSION FOR PROTECTING HUMAN

ORGANS FROM ISCHEMIC INJURY

[0001] This application claims priority to U.S. provisional application Serial No. 61/767,248, titled, "Omega-3 Triglyceride-DHA Emulsions for the Treatment of Hypoxic-ischemic Injuries" filed on February 20, 2013, which is herein incorporated in its entirety.

BACKGROUND

[0002] Cerebral hypoxia-ischemia (stroke) is a major cause of morbidity and mortality through all stages of the life cycle, including for infants born prematurely, for children in intensive care units, and for elderly with cerebral vascular accidents. Infants and children who survive hypoxic- ischemic encephalopathy demonstrate lifelong neurologic handicaps, including cerebral palsy, mental retardation, epilepsy, and learning disabilities. Vannucci, R. C. (2000) "Hypoxic- ischemic encephalopathy," American Journal of Perinatology 17(3): 113-120.

[0003] Cerebral hypoxia-ischemia commonly occurs in critically ill children, most notably in association with cardiopulmonary arrest. Lipid emulsions are commonly used in pediatric intensive care and are an important source of calories in these critically-ill children. Most commercially available emulsions are formed from soybean oil, which have high concentrations of omega-6 (n-6) fatty acids. Lipid emulsions rich in omega-3 (n-3) fatty acids such as cc- linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are derived from fish oils, and are not yet widely available for clinical use. However, omega-3 oils have been shown to have beneficial effects in neurologic diseases such as epilepsy, depression, and behavioral disorders. Most studies support a neuroprotective effect due to dietary administration leading to altered membrane lipid composition. In one study, intravenous cc-linolenic acid given before and/or after neurologic insult was protective in two animal models, kainate-induced seizures and global ischemia via four vessel occlusion in adult Sprague-Dawley rats. Lauritzen I, et al., "Polyunsaturated fatty acids are potent neuroprotectors," The EMBO Journal, 2000 Apr 17; 19(8): 1784-93. However, there remains a need for methods to protect the brain and other organs and tissues against damage after an initial ischemic insult. The present invention fulfills this need. Stroke is the 3^Γ(1-4^ώ most common cause of death in adults and carries huge costs in term of not just mortality but care for the consequences of stroke in survivors. SUMMARY OF THE INVENTION

[0004] The present invention provides a method of limiting neurological damage resulting from hypoxic-ischemia comprising, administering an omega-3 lipid-based emulsion after a cerebral hypoxic-ischemia insult wherein the omega-3 lipid-based emulsion comprises omega-3 oil effective to confer protection against neurological damage.

[0005] The present invention also provides a method of limiting cell death resulting from hypoxic-ischemia comprising, administering an omega-3 lipid-based emulsion after a cerebral hypoxic-ischemia insult wherein the omega-3 lipid-based emulsion comprises omega-3 oil effective to confer protection to limit cell death. Administration of the omega-3 lipid-based emulsion may be either enteral or parenteral.

[0006] Methods of the present invention also provide further comprise administering a conventional stroke treatment or preventative medication.

[0007] Omega-3 lipid-based emulsions of the present invention comprise at least 10%, preferably at least 20%, omega-3 oil, by weight. In certain embodiments, the omega-3 oil comprises at least 10%, preferably at least 20%, omega-3 triglyceride and/or omega-3 diglyceride and the fatty acids of the omega 3-triglyceride and/or omega-3 diglyceride comprise at least 40% EPA and/or DHA.

[0008] Omega-3 lipid-based emulsions may be administered at any effective dose, such as a dose of 0.05 g/kg to 4 g/kg, and may be administered any time after a hypoxic-ischemic insult, such as 20 minutes to six hours after the ischemic insult or 0-12 hours after the ischemic insult. Additional later administrations are also contemplated, for example an additional later administration is provided 1-24 hours after the insult. The omega-3 lipid based emulsions should be administered as soon as possible after the hypoxic-ischemic insult, preferably within the first one to two hours after the insult.

[0009] The methods of the present invention are useful when ischemia has occurred in the organs selected from the group consisting of brain, heart, kidney, spinal cord, large or small intestine, pancreas and lung [0010] The present invention also provides an omega-3 lipid-based emulsion suitable for enteral or parenteral administration, wherein said emulsion confers a protective benefit on cells against cell death following a hypoxic-ischemic insult, said emulsion comprising at least 20% omega-3 oil, by weight, and wherein the omega-3 oil comprises at least 20% omega-3 triglycerides and/or diglycerides, and wherein fatty acids of the omega-3 triglyceride and/or diglycerides comprise at least 40% EPA and/or DHA.

[0011] The present invention also provides the use of an omega-3 lipid-based emulsion as described herein to make a medicament to limit neurological damage and/or cell death resulting from hypoxic-ischemia.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention is illustrated by way of example, and not by way of limitation, in the figures.

[0013] FIG. 1A-1B are graphs that illustrate blood TG and glucose levels after TG emulsion injections. FIG. 1A is a graph that illustrates total plasma TG concentrations (mg/dL) in non- fasting neonatal mice (plO), acutely injected intraperitoneally ("i.p.") with saline or n-3 TG emulsion (0.75 g n-3 TG/kg body weight). *p < 0.05 (n = 3-8 in each group). Each data point represents the mean + SEM of 3 separate experiments. FIG. IB is a bar graph that illustrates plasma glucose concentrations (mg/dL) in non-fasting mice (plO) in post- H/I treatment of n-3 TG or n-6 TG or vehicle (saline) comparing to the time between before H/I and after H/I. **p < 0.001 (n = 5-9 in each group).

[0014] FIG. 2 is a graph that illustrates n-3 TG injection and cerebral blood flow after H/I. Cerebral blood flow (CBF) was measured by laser Doppler flowmetry (LDF) in neonatal mice after carotid artery ligation. Relative CBF was measured every two minutes during hypoxia in ipsilateral (right) hemispheres using a laser Doppler flowmeter. Changes in CBF in response to hypoxia were recorded for 20 min and expressed as percentage of the pre-hypoxia level for n-3 TG treated (n = 3) and saline treated (n = 5) neonatal mice.

[0015] FIG. 3A-3C are graphs that illustrate TTC stained coronal sections of mouse brain and quantification of injury after H/I. FIG. 3A is a micrograph that illustrates TTC- stained coronal sections of representative mouse brains from saline treated, n-3 TG treated and n-6 TG treated. The top panel shows images of coronal mouse brain that are sliced and then stained with TTC (grey for living tissue and white for the infarcted tissue), and the lower panel shows the infarcted areas that are traced in black for quantification. FIG. 3B is a bar graph that illustrates percent of cerebral infarct volume from pre-H/I mice treated with n-3 TG emulsion (n = 28) or n-6 TG emulsion (n = 10) or saline control (n = 27). FIG. 3C is a bar graph that illustrates percent of cerebral infarct volume after H/I in the post-H/I treatment protocol in mice treated with n-3 TG emulsion (n = 18) or saline control (n = 18). Each bar represents the mean + SEM of 5-7 independent experiments.

[0016] FIG. 4A-4B are graphs that illustrate the effect of Tri-DHA versus Tri-EPA on cerebral infarct volume after H/I. FIG. 4A is a bar graph that illustrates mice subjected to 15 min ischemia followed by 24-hr reperfusion and received 2 i.p. administrations (immediately after ischemia and 1 hr of reperfusion) at 2 doses (0.1 g n-3 TG/kg and 0.375 g n-3 TG/kg). Each bar represents the mean + SEM of 5-7 independent experiments performed using the same H/I model. FIG. 4B are micrographs that illustrate TTC-stained coronal sections of representative mouse brains from saline treated, 0.1 g Tri-DHA, 0.375 g Tri-DHA, 0.1 g Tri-EPA and 0.375 g Tri-EPA. * p < 0.05 compared to other groups except 0.1 g Tri-DHA/kg . ** /? < 0.05 compared to other groups except 0.375 g Tri-DHA/kg and 0.375 g Tri-EPA/kg.

[0017] FIG. 5 is a bar graph that illustrates the effects of delayed treatment with Tri-DHA on cerebral infarct volume after H/I. Mice were subjected to 15-min ischemia followed by 24-hr reperfusion and received 2 i.p. administrations at four-time points (immediate [0,1 hr], delayed 1-hr [1,2 hr], or 2-hr [2,3 hr] or 4-hr [4,5 hr] treatments). Each bar represents the mean + SEM of 5-7 independent experiments. * p < 0.05; ** p < 0.001 vs. saline control (n = 10-20 in each group).

[0018] FIG. 6 is a bar graph that illustrates the long-term effect of Tri-DHA on cerebral tissue death at 8 wk after H/I. Mice were subjected to 15-min H/I and received 2 i.p. administrations of 0.375 g Tri-DHA/kg (n = 6) vs. saline (n = 5). At 8 wk after H/I mice were sacrificed and brains were fixed with 4% paraformaldehyde and 10 μιη- thick slices were cut and preserved. Nissl staining was used for identifying neuronal and brain structure. As described in Methods of Example 6 right brain tissue loss in relation to the contralateral hemisphere was calculated and expressed as a percentage. Each bar represents the mean + SEM. * p < 0.05.

[0019] FIG. 7A-7C are bar graphs that illustrate the reduction of acute MI in vivo after administration of n-3 TG. FIG. 7A is a bar graph that illustrates the reduction of infarct size area (%) for (left) control and (right) after administration of n-3 TG in the mouse heart after H/I injury. FIG. 7B is a bar graph that illustrates a decrease in LDH release which is a marker for heart cell damage for (left) control and (right) after administration of n-3 TG. FIG. 7C is a bar graph that illustrates n-3 TG maintenance of heart function via fractional shortening (%) for (left) control and (right) after administration of n-3 TG. Each bar represents the mean + SEM. * p < 0.01.

[0020] FIG. 8 is a bar graph that illustrates increase in gene expression and mouse heart protein expression of BCL-2 which is an anti-apoptotic marker for (left) normoxia, (middle) control, and (right) after administration of n-3 TG post ischemic event. Each bar represents the mean + SEM. * /? < 0.05.

[0021] FIG. 9 are bar graphs that illustrate acute n-3 TG neuroprotection in hypoxia/ischemia as quantified for the (left) juvenile rat, (middle) adult mouse, and (right) neonatal mouse. % infarct volume was measured and it was determined that the n3-TG treated group on right in each bar graph had significantly less damage as compared to the saline treated controls on the left of each bar graph.

[0022] FIG. 10 is a graph that illustrates attenuation of brain injury by n-3 FA after H/I. After injection of n-3 FA, different regions of the brain are markedly protected from stroke injury after H/I.

[0023] FIG. 11A-11B are graphs that illustrate acute n-3 TG injection decrease of brain Ca²⁺ induced opening of mitrochondrial permeability transition pores (mPTP) after H/I. After H/I and after n-3 TG injection, mitochondrial function is maintained.

[0024] FIG. 12 are graphs that illustrate navigational memory assessment in vivo. Eight weeks after stroke, neuronal function is maintained and is much better in mice that had been treated initially with pure omega-3 triDHA after stroke. Results were much better with triDHA has compared to triEPA. Mice were given 275 mg triDHA/kg immediately after H/I injury and 1 hour later.

[0025] FIG. 13A-13B are graphs that illustrate n-3 DG decreases brain injury and infarct volume in H/I neonatal mice.

[0026] FIG. 14 is a bar graph that illustrates DHA content in brain mitochondria. At 4 hours after injection of the n-3 TG emulsion after stroke, DHA content in brain mitochondria is increased and that this increase likely contributes to the beneficial effects of DHA. Note that EPA content was not increased in brain mitochondria (data not shown).

[0027] FIG. 15 is a bar graph that illustrates infarct volume at 24 hours post H/I in mice post- treated with NS (vehicle) or NPD1 (20 ng). NPD1 is a catabolic product of DHA.

[0028] FIG. 16 is a diagram that illustrates an in vivo left anterior descending coronary artery (LAD) occlusion model.

[0029] FIG. 17 is a graph showing that n-3 TG decreases mitochondrial production of reactive oxidation species [ROS].

[0030] Before the present embodiments of the invention are described, it is to be understood that the inventions are not limited to the particular processes, compositions, or methodologies described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein, are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. DETAILED DESCRIPTION

[0031] In the following description, for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without these specific details.

Definitions

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0033] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein, and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et ah, Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lan, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Principles of Neural Science, 4th ed., Eric R. Kandel, James H. Schwart, Thomas M. Jessell editors. McGraw-Hill/ Appleton & Lange: New York, N. (2000).

[0034] As used herein "omega-3 lipid-based emulsion" is an oil-in-water emulsion comprising at least 10% omega-3 oil (and up to 100% omega-3 oil). Preferably the omega-3 lipid-based emulsion comprises at least 20% - 35% omega-3 oil. [0035] As used herein, "omega-3 oils" means any omega-3 fatty acid, including free omega-3 fatty acids and omega-3 triglycerides, diglycerides and monoglycerides.

[0036] The term "omega-3 fatty acid" means a polyunsaturated fatty acid wherein one of the carbon-carbon double bonds is between the third and fourth carbon atoms from the distal end of the hydrocarbon side chain of the fatty acid. Examples of "omega-3 fatty acid" include a- linolenic acid (18:3n-3; a-ALA; A³'⁶'⁹), eicosapentaenoic acid (20:5n-3; EPA; A⁵'⁸'¹¹'¹⁴'¹⁷), docosahexaenoic acid (22:6n-3; DHA) and docosapentaenoic acid (22:5n-3; DPA; A⁷'¹⁰'¹³'¹⁶'¹⁹), wherein EPA and DHA are most preferred. Omega-3 fatty acids having at least 20 carbon atoms are herein called "long chain omega-3 fatty acids."

[0037] The term "omega-3 triglyceride" or "omega-3 diglyceride" or "omega-3 monoglyceride" refers to a triglyceride or a diglyceride or monoglyceride, respectively, comprising at least one omega-3 fatty acid esterified with a glycerol moiety. As used herein, the term "omega-3 tri/diglyceride" means that omega-3 fatty acid comprises an omega-3 triglyceride and/or a diglyceride or any combination thereof.

[0038] The amount of omega-3 oil or omega-6 oil in the lipid-based oil-in-water emulsion is expressed by weight in grams of omega-3 or omega-6 oil per 100 mL emulsion.

[0039] The amount of glyceride (mono-, di-, or triglyceride) in the omega-3 oil or omega-6 oil is expressed as the percentage of the glyceride by weight per total weight of the omega-3 or omega- 6 oil.

[0040] The amount of fatty acid such as EPA or DHA in a glyceride (mono-, di-, or triglyceride) is expressed as the wt. of the acyl groups of the respective glyceride.

[0041] As used herein, hypoxia refers to a shortage of oxygen in the body or in a specific organ or tissue

[0042] As used herein ischemia refers to insufficient blood flow to provide adequate oxygenation. The most common causes of ischemia are acute arterial thrombus formation, chronic narrowing (stenosis) of a supply artery that is often caused by atherosclerotic disease, and arterial vasospasm. As blood flow is reduced to an organ, oxygen extraction increases. When the tissue is unable to extract adequate oxygen, the partial pressure of oxygen within the +tissue fails (hypoxia) leading to a reduction in mitochondrial respiration and oxidative metabolism. Further, in many acute situations of organ ischemia-hypoxia (e.g., stroke, myocardial infarction, intestinal volvulus, etc.) the patient is far too ill to have oral or enteral administration of therapeutic agents and thus needs parenteral injections, such as from lipid emulsions for immediate action.

[0043] As used herein hypoxia-ischemia refers to the occurrence of both hypoxia and ischemia in a tissue or organ.

[0044] As used herein reperfusion damage or reperfusion injury refer to damage caused with restoration of blood supply to hypoxic-ischemic (H/I) tissues. Reperfusion injury can be more damaging than the initial ischemia. Reintroduction of blood flow brings oxygen back to the tissues, causing a greater production of free radicals and reactive oxygen species that damage cells. It also brings more calcium ions to the tissues causing further calcium overloading and can result in potentially fatal myocardial infarction or heart attack and also accelerates cellular self- destruction. The restored blood flow also exaggerates the inflammation response of damaged tissues, causing white blood cells to destroy damaged cells that may otherwise still be viable. See Sims, N.R.; Muyderman, H., Mitochondria, oxidative metabolism and cell death in stroke, Biochimica et Biophysica Acta, January 2010, 1802(1):80-91. Epub September 12, 2009. PubMed PMID 19751827.

[0045] While not wishing it to be bound by theory, it is believed that organ death or injury is frequently precipitated by hypoxia-ischemia, or associated with reperfusion damage, cardiac infarct, organ transplantation, endothelial dysfunction, impaired organ micro perfusion, increased risk of thrombus formation, or ectopic fat deposition, etc. Ectopic fat depositions usually occur in organs not specialized in fat deposition, such as liver, pancreas, or heart.

[0046] Typically, post-operative and post-traumatic conditions as well as severe septic episodes are characterized by a substantial stimulation of the immune system ischemia reperfusion syndrome, and tendency for thrombosis formation. The immune response is activated by the release of pro-inflammatory cytokines (e.g., tumor necrosis factor and interleukins) which at high levels may cause severe tissue damage. [0047] In such clinical conditions, it is of particular importance to provide exogenous lipids that are hydrolyzed and eliminated faster than endogenous lipids (to avoid excessive increases of plasma triglyceride concentration). These lipids supply omega-3 fatty acids capable of reducing cytokine production as well as cytokine toxicity on tissues. Thus, fatty acids are optimally administered via lipid glycerides such as triglycerides. The fatty acids are released and used after the lipids are catabolized in the body via lipolysis. This effect is obtained when fatty acids are cleaved from the lipid molecules and incorporated (in free form or as components of phospholipids) in cell membranes where they influence membrane structure and cell function, serve as secondary messengers (thus affecting regulation of cell metabolism), influence the regulation of nuclear transcription factors, and are precursors of eicosanoids. Thus, it is desirable that this process takes place as quickly as possible.

[0048] The human body is capable of synthesizing certain types of fatty acids. However, long chain omega-3 and omega-6 are designated as "essential" fatty acids because they cannot be produced by the human body and must be obtained through other sources. For example, fish oils from cold-water fish have high omega-3 polyunsaturated fatty acids content with lower omega-6 fatty acid content. Table 1 was supplied by the manufacturer of the n-3 Tri-DHA oil Fresenius Kabi, and it describes the make-up of the n-3 Tri-DHA used in some of the experiments described herein Examples 6 and 7. Table 1 estimates (in column 2) that the n-3 fish oil comprises a range of 1-7% in gm/100 ml Linoleic acid (C18:2n-6), and 1-4% Arachidonic acid (C20: 4n-6), which together have a theoretical upper limit of 11%. However, to date no fish oil has been reported that has over 10% omega-6 fatty acids. The di- and tri-glyceride omega-3 emulsions of the present invention when made from fish oil, use fish oil with 10% or less omega- 6 fatty acid. Most vegetable oils (i.e., soybean and safflower) have high omega-6 polyunsaturated fatty acids (most in the form of 18:2 (Δ 9 ' 12 ) -linoleic acid) content but low omega-3 (predominantly 18:3(Δ⁹' ¹²' ¹⁵)-a-linolenic acid) content.

[0049] Essential fatty acids may be obtained through diet or other enteral or parenteral administration. However, the rate of EPA and DHA omega-3 fatty acid enrichment following oral supplementation varies substantially between different tissues and is particularly low in some regions of the brain and in the retina especially when given as the essential fatty acid precursor, a-linolenic acid. Further, human consumption of omega-3 fatty acids has decreased over the past thirty years, while consumption of omega-6 fatty acids has increased, especially in Western populations.

[0050] Cao et al., "Chronic administration of ethyl docosahexaenoate decreases mortality and cerebral edema in ischemic gerbils.", Life Sci. 2005 Nov 19;78(1):74-81 alleges that dietary docosahexaenoic acid (DHA) intake can decrease the level of membrane arachidonic acid (AA), which is liberated during cerebral ischemia and implicated in the pathogenesis of brain damage. Cao investigated the effects of chronic ethyl docosahexaenoate (E-DHA) administration on mortality and cerebral edema induced by transient forebrain ischemia in gerbils.

[0051] GB 2388026, incorporated herein by reference in its entirety, refers to use n-3 polyunsaturated fatty acids EPA and/or DHA in the preparation of an oral medicament for preventing cerebral damage in patients having symptoms of atherosclerosis of arteries supplying the brain.

[0052] Strokin M, Neuroscience. 2006 Jun 30;140(2):547-53, incorporated herein by reference in its entirety, investigated the role of docosahexaenoic acid (22:6n-3) in brain phospholipids for neuronal survival.

[0053] WO 2004/028470 (PCT/US2003/030484), incorporated herein by reference in its entirety, purports to disclose methods and compositions which impede the development and progression of diseases associated with subclinical inflammation.

[0054] The following U.S. Patent applications are hereby incorporated by reference as if fully set forth herein: Provisional Application No. 60/735,862, filed 11/14/2005; Provisional Application No. 60/799,677, filed 5/12/2006; Application No. 11/558,568, filed 11/10/2006; PCT/US06/60777, filed 11/10/2006; Application No. 13/336,290, filed 12/23/2011; Provisional Application No. 60/845,518, filed 9/19/2006; PCT/US07/20364, filed 9/19/2007; Application No. 12/441,795, filed 12/8/2009, now U.S. Patent No. 9,410,181; Application No. 13/783,779, filed 03/04/2013; Application 13/953,718, filed 07/29/2013 and Application No. 14/102,146, filed 12/10/2013.

[0055] See also Qi, K. (2002) "Omega-3 triglycerides modify blood clearance and tissue targeting pathways of lipid emulsions," Biochemistry 41: 3119-3127, incorporated herein by reference in its entirety, refers to omega-3 rich triglycerides which are recognized as having modulating roles in many physiological and pathological conditions.

[0056] It is now well-established that cerebral hypoxia-ischemia of sufficient duration to deplete high energy reserves in neural cells initiates a cascade of events over the hours to days of reperfusion that culminates in extensive death, both necrotic and apoptotic. These events include the generation of reactive oxygen species and oxidative damage to cells, release of inflammatory mediators and initiation of prolonged inflammatory reactions, and ongoing apoptosis that can continue for weeks to months. This applies to ischemic injury to organs in young, adult and elderly humans.

[0057] As an example, neuronal loss following hypoxia/ischemia is believed to result, at least in part, from elevated glutamate release and excitoxicity. Excess glutamate activation of N-methyl- D-aspartic acid (NMDA) receptors induces pro-apoptotic pathways and inhibits anti-apoptotic signaling pathways. Omega-3 fatty acids can modify a number of signaling pathways to effect transcriptional regulation. Not being bound by theory, since prior studies by the present inventors have shown that whole brain fatty acid profiles are not modified following acute administration of omega-3 triglyceride emulsions, it is believed that the omega-3 fatty acids protect neurons by modulating signaling pathways that counter the effects of hyper stimulated NMDA receptors, protection against free radical generation and consequent oxidative damage, maintaining mitochondrial function and thereby prevent/reduce post-ischemic inflammation and release of inflammatory mediators.

[0058] Recent evidence (see Qi K, Seo T, Al-Haideri M, Worgall TS, Vogel T, et al. (2002) Omega-3 triglycerides modify blood clearance and tissue targeting pathways of lipid emulsions. Biochemistry 41 : 3119-3127) also indicates that routes for blood clearance and tissue uptake of fish oil omega triglycerides are very different from those of omega-6 soy oil long chain triaglycerols (LCT). For example, removal of omega-3 very long chain triaglycerol (VLCT) emulsions from blood seems to depend far less on intravascular lipolysis than does LCT emulsions. While substantial amounts of both emulsions are delivered to tissues as intact triglyceride, this pathway is likely more important for omega-3 triglyceride particles. Omega-3 triglyceride particles, VLCT, are less dependent on "classical" lipoprotein receptor related clearance pathways, than are LCT. Fatty acid derived from omega-3 triglyceride appear to act as stronger inhibitors than LCT in sterol regulatory element (SRE) dependent gene expression- genes that are involved in both triglyceride and cholesterol synthesis.

[0059] The present invention provides methods of limiting or preventing cell death and cell/tissue damage resulting from hypoxic-ischemia. "Limiting" as used herein includes decreasing and/or preventing. Among the effects of treating the ischemia are a reduction in cell death, decreased inflammation, reduction in infarct size, reduction in production of inflammatory cytokines, reduction in production of reactive oxygen species, and maintenance of mitochondrial integrity. The methods of present invention comprise administering an omega-3 lipid-based emulsion of the present invention after an hypoxic-ischemia insult. The present invention also provides, in those cases where the hypoxic-ischemic insult can be predicted, methods of limiting or preventing cell death and cell/tissue damage comprising administering an omega-3 lipid-based emulsion of the present invention before the hypoxic-ischemia insult.

[0060] When the hypoxic-ischemic insult is cerebral, the present invention limits neural cell death and/or limits neurological damage. Since the basic mechanisms of cell death following ischemia after an hypoxic-ischemic insult are similar in most bodily organs, the present invention also provides limiting cell death in other organs such as the heart, large and small intestines, kidney and lung following an hypoxic-ischemia insult. For example, after a colonic ischemic event due to acute mesenteric artery ischemia, chronic mesenteric artery ischemia or ischemia due to mesenteric venous thrombosis, the present invention provides a method of limiting intestinal cell death. Similar prevention of cell death would apply to myocardial infarction. (See FIG. 7.)

[0061] Prior studies have shown that omega-6 fatty acids such as omega-6 linoleic acids are far less effective in neuroprotection and cardiac protection when provided before an ischemic event. The studies involved the administration of Intralipid®, a soy oil based emulsion containing 55% of its fatty acids as omega-6 linoleic acid, with a very low content of EPA and DHA (-2%). Further, direct injection of free fatty acids, as compared to triglycerides or diglycerides, can have serious side effects, such as encephalopathy.

[0062] Accordingly, some of the methods of the present invention comprise administering an omega-3 lipid-based emulsion comprising at least 10%, preferably at least 20% (up to 100%) by weight of omega-3 oil. Preferably the omega-3 oil comprises at least 10%, preferably at least 20% (up to 100%) omega-3 tri/diglycerides The fatty acids in the omega-3 tri/diglycerides preferably comprise at least 40% (up to 100%) EPA and/or DHA. The omega-3 oil in the present emulsions has less than 10%, preferably less than 5% omega-6 oil.

Triglyceride DHA omega-3 lipid-based oil-in-water emulsions

[0063] Other embodiments are directed to Triglyceride DHA omega-3 lipid-based oil-in-water emulsions (n-3 Tri-DHA emulsions) and to their therapeutic use in treating H/I and reperfusion injury, including in organ transplants. The results described in Example 7 show that administering n-3 Tri-DHA emulsions (Table 1) after H/I had a neuroprotective effect against cerebral infarction and reperfusion damage. These results are described in Williams (see Williams JJ, Mayurasakorn K, Vannucci SJ, Mastropietro C, Bazan NG, et al. (2013) N-3 Fatty Acid Rich Triglyceride Emulsions Are Neuroprotective after Cerebral Hypoxic-Ischemic Injury in Neonatal Mice. PLoS ONE 8(2): e56233. doi: 10.1371/journal.pone.0056233) and summarized here. Four different types of lipid emulsions were used including omega-3 (n-3) TG fish oil- based and omega-6 (n-6) TG.

Cerebral Hypoxia-Ischemia

[0064] The term "the omega-3 lipid-based emulsions of the present invention" generally includes the herein described n-3 Tri-DHA emulsions. The results described in Example 7 show that administering Triglyceride DHA omega-3 lipid-based oil-in-water emulsions (n-3 Tri-DHA emulsions) (Table 1) after H/I had a neuroprotective effect against cerebral infarction and reperfusion damage. These results are described in Williams JJ, Mayurasakorn K, Vannucci SJ, Mastropietro C, Bazan NG, et al. (2013) N-3 Fatty Acid Rich Triglyceride Emulsions Are Neuroprotective after Cerebral Hypoxic-Ischemic Injury in Neonatal Mice. PLoS ONE 8(2): e56233. doi: 10.1371/journal.pone.0056233) and summarized here. Four different types of lipid emulsions were used including omega-3 (n-3) TG fish oil-based and omega-6 (n-6) TG soy oil- based emulsions that were commercially prepared intravenous phospholipid- stabilized emulsions. In Example 6 and 7 and FIGS. 7-16, the omega-3 Tri-DHA emulsions are referred to as "n-3 TG" emulsions and they comprise 10% omega-3 fish oil (n-3) by weight in grams perl 00 ml of emulsion, wherein the omega-3 oil is >90 % triglyceride (TG) by weight per total weight of the omega-3 oil, and in which up to about 30% wt.% of the acyl groups of the TG are DHA and up to about 28% are EPA. These n-3 TG emulsions are also called n-3 TG90-DHA30 emulsions. Other emulsions having pure (99%) DHA or pure (99%) EPA were also tested as described.

[0065] n-6 TG emulsions having no DHA or EPA (Table 1) were also tested. These emulsions have 20% omega-6 oil (n-6) by weight in grams perlOO ml of emulsion, 0% DHA, 0% EPA and about 55% triglyceride by weight per total weight of the omega-3 oil from linoleic acid. The n-6 TG emulsions were produced from soy bean oil rich in n-6 FA with linoleic acid constituting about 55% of total FA.

[0066] Treatment with the n-3 TG90-DHA30 emulsions both before- and after- H/I significantly reduced total infarct volume when administered prior to H/I and when administered immediately after H/I.

[0067] Administration by i.p. injection of n-3 TG90-DHA30 raised blood TG levels up to threefold higher at 1.5 hours post-injection compared to baseline; this was followed by a decrease of levels to baseline at 3 and 5 hours (FIG. 1A) due to catabolism of n-3 TG90-DHA30 in the blood stream. After H/I it was determined that there was no difference in blood glucose levels among n-3 TG-treated vs. n-6 TG-treated vs. saline controls. FIG. IB. Further no difference was observed in capillary bleeding times in n-3 treated mice as compared to saline controls; and n-3 TG90-DHA30 did not change cerebral blood flow after H/I.

[0068] n-3 TG90-DHA30 but not n-6 TG emulsions protected the brain against H/I injury as evidenced by the subcortical region of the brain (FIG. 3A) where infarct volume was substantially decreased in n-3 TG treated mice, while a significant increase in infarct volume occurred with n-6 TG emulsion injection. Immediate post-H/I treatment with n-3 TG90-DHA30 reduced the total infarct area by almost 50%. FIG. 3B.

[0069] n-3 TriDHA but not n-3 TriEPA was neuroprotective after H/I. Total infarct size was reduced by a mean of 48% and 55% with treatment of 0.1 and 0.375 g TG/kg of 99% pure n-3 Tri-DHA, respectively, compared with saline control. FIG. 4A-4B. However, neuroprotection was not observed with 99% pure n-3 Tri-EPA compared with saline treatment. The timing of administration is also important. No protective effect was seen when n-3 TG90-DHA30 was administered after a 4-hour delay post H/I compared with control. FIG. 5. However when n-3 TG90-DHA30 was administered at 0 hour immediately post- H/I, and then again at 1- hr, and 2- hr post stroke, similar reduced brain infarct volumes (~ 50%) were observed compared to control. FIG. 5.

[0070] Coronal brain sections of adult mice were processed for Nissl staining (FIG. 6) to examine the effects of H/I and TG90-DHA30 treatment on brain and neuronal cell loss for long- term outcome at 8 weeks after H/I insult. As compared to the left control (contralateral hemisphere), the injured areas of the right hemisphere display gross neuronal cell loss. FIG. 6 shows brain tissue loss was markedly increased by 1.67 fold in the right hemisphere of saline- treated mice (n=5) as compared to TG90-DHA30 treated mice (n=6), 25.0+/- 2.4% vs. 15.0 +/- 2.5%, respectively (p=0.02). Thus, neuroprotection after injury and TG90-DHA30 Injection that are observed 24 hours after H/I can be demonstrated histologically almost 2 months after the initial stroke insult.

[0071] Additional experiments besides those in Examples 6 and 7 were done to study the effect of administering the n-3 TG [n-3 TG90-DHA30] emulsions on cerebral hypoxia-ischemia, the results of which are shown in FIGs. 9-15. Animals were handled and treated as described in Example 6. TTC staining showed that cerebral infarct size decreased after i.p. injections (FIG. 9) in three different rodent models including, the juvenile mouse, the adult mouse, and the neonatal mouse. Each injection provided about 15 mg DHA per mouse, or about 500 mg DHA/Kg.

[0072] FIG. 10 shows that n-3 TG90-DHA30 attenuated brain injury after hypoxia/ischemia. Using the protocol described in Example 7, it was shown that injection of n-3 TG90-DHA30 markedly protected different regions of the brain from stroke injury after hypoxia/ischemia. Each injection provided about 15 mg DHA per mouse, or about 500 mg DHA/Kg. Other unpublished data show that n-3 TG90-DHA30 also reduced 3-Nitrotyrosine protein oxidation and 4- Hydroxynonenal lipid peroxidation 24 hours after hypoxia/ischemia. [0073] FIG. 11 shows that i.p. injection n-3 TG90-DHA30 after hypoxia/ischemia reduced infarct volume which correlated with maintenance of the integrity of the mitochondrial membrane as is evidenced by a decrease in brain Ca2+-induced opening of mitochondrial permeability transition pores (mPTP) that typically occurs in untreated animals after hypoxia/ischemia. Each injection provided about 15 mg DHA per mouse, or about 500 mg DHA/Kg.

[0074] In the next experiment pure DHA or pure EPA omega-3 triglyceride emulsions were administered to test memory and neurologic function. Navigational memory was assessed 8 weeks after mice were subjected to hypoxia/ischemia to cause a stroke as an indicia of neurofunction following treatment with either n-3 Triglyceride having 100% DHA (pure DHA) or with n-3 Triglyceride having 100% EPA (pure EPA). Navigational memory was recorded 8 weeks after the initial injury on day 3 of the water-maze testing period, on which the platform was removed. FIG. 12A shows the time spent in each quadrant. FIG. 12B shows the distance that the mouse swam in each quadrant: a, p<0.05 as compared to naive; b p<0.05 as compared to saline. The results show that navigation memory and neurofunction was maintained and in fact was significantly enhanced in mice that had been treated twice (both immediately after the hypoxic-ischemic injury and one hour later) with n-3 TG having pure (99%) DHA. Unexpectedly, it was discovered that the neurologic performance results were significantly better with n-3 TG having pure DHA than with n-3 TG having EPA, although EPA seemed to have no deleterious effects. Mice were given 375 mg of n-3 TG 99% DHA or 99% EPA per Kg immediately after hypoxic-ischemic injury and 1 hour later, 8 weeks before testing was done

[0075] FIG. 13 shows that both n-3 TG and n-3 DG (diglyceride) emulsions reuce cerebral infarct size..

[0076] FIG. 14 illustrates DHA content in isolated brain mitochondria. These data show that at 4 hours after injection of n-3 TG90-DHA30 after hypoxia/ischemia-induced stroke that the DHA content in brain mitochondria is increased. It is speculated that this increase likely contributes to the beneficial effects of DHA. It is note that EPA content was not increased in brain mitochondria (Data not shown). Each injection provided about 15 mg DHA per mouse, or about 500 mg DHA/Kg. [0077] FIG. 15 shows that cerebral infarct volume at 24 hrs post hypoxia/ischemia was reduced in mice post- treated with neuroprotectin Dl (NPDl) (20ng) which is a catabolic product of DHA, as opposed to treatment with saline vehicle). In addition, this product of DHA also maintains close to normal brain mitochondrial permeability in isolated mitochondria exposed to high oxygen or after hypoxic-ischemic injury in mice. Note that while NPDl did have beneficial effects, it was not as effective as treatment with omega-3 triglyceride emulsions with pure (99%) DHA. Dosage of NPDl was 20 ng (nanogram) per mouse.

Cardiac Hypoxia/ischemia

[0078] The effect of administering n-3 TG90-DHA30 omega-3 triglyceride emulsions to an animal after cardiac hypoxia/ischemia were also studied. (Example 9.) To summarize, FIG. 7 shows that acute i.p. injection of n-3 TG emulsion (Table 1; n-3 TG90-DHA30) decreased infarct size in mouse heart after hypoxic-ischemic injury; decreased LDH release which is a marker for heart cell damage, and also maintained heart function as shown by the echocardiogram being more normal. Bcl-2 (B-cell lymphoma 2), encoded in humans by the BCL2 gene, is the founding member of the Bcl-2 family of regulator proteins that regulate cell death (apoptosis) and it is specifically considered as an important anti-apoptotic protein and is thus classified as an oncogene. FIG. 9 shows that Bcl-2 is increased after myocardial infarction in untreated animals, but is increased even more (by up to about 3.5 times) the level observed in normoxic conditions in animals treated with n-3 TG.

Embodiments of the invention comprising n-3 Tri-DHA Emulsions and Methods of Treating H/I

[0079] Based on these observations showing effective treatment of both cerebral and cardiac hypoxia-ischemia and reperfusion damage, certain embodiments are directed to n-3 Tri-DHA lipid-based oil-in-water emulsions wherein: (a) the emulsion comprises at least 7% to about 35% omega-3 oil and less than 10% omega-6 oil by weight in grams per 100 ml of emulsion; (b) the omega-3 oil comprises at least 20% to 100% triglyceride by weight per total weight of the omega-3 oil, and at least 20% wt.-% to 100% of the acyl-groups of the omega-3 triglycerides consist of DHA; (c) the omega-3 oil comprises less than 10% omega-6 fatty acids; and (d) the mean diameter of lipid droplets in the emulsion is less than about 5 microns. [0080] In certain embodiments the omega-3 oil component comprises 10%, 20%, 30% or 35% of the emulsion by weight in grams per 100 ml of emulsion; the triglyceride component in the described n-3 Tri-DHA oil of the present invention is 20-50%, 50-75%, 75-90%, 90-95%, 95%- 100% weight per total weight of the omega-3; and the DHA content of the omega-3 triglyceride is from 20-50%, 50-75%, 75-90%, 90-95%, 95%-100% wt.%. of the acyl groups of the TG. It is important to note that in these omega-3 emulsions it is not necessary to exclude EPA or the triglycerides in order to treat hypoxia/ischemia. In the case of TG with pure DHA, EPA would be excluded. For the n-3 TG-DHA emulsions the presence of at least 20% triglycerides in the omega-3 oil of which at least 20% are DHA are needed to treat hypoxia/ischemia and reperfusion damage.

[0081] Yet other embodiments include methods of treating H/I and reperfusion damage including any cerebral or cardiac H/I including stroke and myocardial infarction, respectively; and also methods of treating H/I in organs or tissue before it is harveste for transplantation, or after transplantation to minimize cell death and cell damage. In some embodiments the methods include (a) identifying a subject who has undergone hypoxia-ischemia, (b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount the described n-3 Tri-DHA oil of the present invention to reduce reperfusion damage caused by the hypoxia-ischemia. In some embodiments the hypoxia-ischemia causes cerebral hypoxia-ischemia including stroke and the described n-3 Tri-DHA emulsion is administered as soon as possible after the hypoxia-ischemia, such as within 20 minutes , or less than 1 hour, less than 2 hours, less than 3 hours and less than 4 hours after the H/I, and in some cases less than 6 hours after. In other embodiments the hypoxia-ischemia is in the heart and it causes myocardial infarction. Again, treatment is optimal as soon as possible after the diagnosis of the cardiac hypoxia- ischemia to reduce reperfusion damage. In some embodiments the described n-3 Tri-DHA emulsions are administered to reduce cell damage or cell death either before tissue or organs are harvested for transplantation following organ transplant or hypoxia-ischemia in organs including organ selected from the group consisting of brain, heart, kidney, spinal cord, large or small intestine, pancreas and lung.

[0082] In some embodiments the therapeutically effective amount is from about 0.2 g/kg/administration to about 4 g/kg/administration, but higher doses can be administered if a crisis warrants treatment as the emulsions are non-toxic. In some cases the omega-3 emulsions of the present invention including n-3 DG and n-3 TG emulsions can be administered continuously for a period of time after the H/I.

[0083] Other embodiments include a method comprising: (a) identifying a subject who is at risk of having a cerebral hypoxia-ischemia, and (b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the described n-3 Tri-DHA emulsion, thereby reducing the risk of the subject developing the reperfusion damage caused by the hypoxia-ischemia. Subjects at risk of developing cerebral H/I and cariac H/I can often be identified before they develop H/I; such subjects come within treatment with the present methods. Another method comprises (a) identifying a subject who has inflammation in an organ, and (b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the described n-3 Tri-DHA emulsion, thereby reducing the inflammation in the subject. Yet another method comprises administering to a subject an amount of a pharmaceutical composition comprising the described n-3 Tri-DHA emulsion, to reduce production of reactive oxygen species or inflammatory cytokines in the blood or an organ in the subject.

[0084] Sources of omega-3 fatty acids may be from any suitable source such as from fish oils, other oils or may be synthesized. Although EPA and DHA are preferred omega-3 fatty acids, other omega-3 fatty acids may be used. Suitable exemplary fish oils include oils from cold-water fish such as salmon, sardine, mackerel, herring, anchovy, smelt and swordfish. Fish oils generally contain glycerides of fatty acids with chain lengths of 12 to 22 carbons. Highly purified fish oil concentrates obtained, for example, from sardine, salmon, herring and/or mackerel oils may have an eicosapentaenoic acid (EPA) content of from about 20 to 40 wt.-%, preferably at least 25 wt.-%, and a docosahexaenoic acid (DHA) content of >10%, preferably at least 12%, based on the fatty acid methyl esters of the fish oil concentrate as determined by gas chromatography (percent by area). U.S. Pat. No. 6,159,523, incorporated herein by reference in its entirety, discloses a method for making fish oil concentrates. Generally, the amount of the polyunsaturated fatty acids of the omega-6 series (such as linoleic acid) in natural fish oils is low, i.e. less than 10%, preferably less than 5%. In the described n-3 Tri-DHA emulsions used in the present embodiments, the amount of omega-6 oil in the emulsions is less than 10%, preferably less than 5%. The amount of omega-6 oil in the fish oil is also less than 10%. To make n-3 TG with a high wt. % DHA of more than about 25 wt.%, the oil is typically synthesized.

[0085] Methods of the present invention preferably include administering the omega-3 emulsions including the described n-3 Tri-DHA emulsions of the present invention by any suitable route including enterally (for example, orogastric or nasogastric) or parenterally (for example, subcutaneous, intravenous, intramuscular, intraperitoneal). Most preferably the emulsion is administered intravenously.

[0086] Omega-3 lipid-based emulsions of the present invention are preferably provided at a dose capable of providing a protective benefit. Those skilled in the art would be able to determine the appropriate dose based on the experimental data presented herein. However, for example a suitable effective and tolerable dose for a human would be about 0.05 g/kg to about 4.0 g/kg. Higher doses may be given as necessary. Administration may be continuous or in the form of one or several doses per day. One skilled in the art would appreciate appropriate dosage and routes of administration based upon the particular subject and condition to be treated.

[0087] Omega-3 lipid-based emulsions of the present invention are preferably administered parenterally and/or enterally as soon after the ischemic insult as possible (or in some embodiments, before the insult when it can be predicted). The emulsion may be administered to prevent/reduce tissue damage and cell death from reperfusion injury after hypoxia-ischemia, including from organ transplant, in any organ including brain, heart, kidney, spinal cord, large or small intestine, pancreas and lung. For example, in a preferred embodiment an omega-3 lipid- based emulsion is administered from 0-20 minutes to two hours after the insult. In some embodiments the emulsion is administered within 1 hour, 2-3 hours, and 3-4 hours and in less than six hours after the insult. The present invention also provides for multiple administrations of the omega-3 lipid-based emulsion on the same day or even continuous infusion since these emulsions are not toxic. Routine experimentation will determine the optimal dose for the injury. For example, the emulsion may be first administered within 20 minutes of the insult, followed by a second administration 1-24 hours after the insult. For cerebral H/I administration of the described n-3 Tri-DHA emulsion is optimal within 4 hours. [0088] In another embodiment of the invention, methods of limiting or preventing cell death and cell/tissue damage resulting from hypoxic-ischemia further comprise administering an omega-3 lipid-based emulsion of the present invention including the described n-3 Tri-DHA emulsion, in conjunction with standard available therapies (such as surgery and angioplasty) and/or medications given to prevent or treat hypoxia-ischemia. For example, the following drugs are often administered to prevent or treat strokes: antiplatelet medications such as aspirin, clopidogrel, dipyridamole, ticlopidine; anticoagulants such as heparin and warfarin; and thrombolytic agents such as tissue plasminogen activator.

[0089] Preparation of lipid-based emulsions suitable for intravenous delivery are known in the art. Omega-3 lipid-based emulsions according to the invention may be oil-in-water (o/w) emulsions in which the outer continuous phase consists of distilled water purified or sterilized for parenteral purposes. Such oil-in-water emulsions may be obtained by standard methods, i.e. by mixing the oil components followed by emulsification and sterilization. The pH value of the lipid emulsion may be adjusted to a physiologically acceptable value, preferably to a pH of from about 6.0 to about 9.0, more preferably from about 6.5 to about 8.5. Auxiliary agents and additives may be added to the oil mixture prior to emulsification or prior to sterilization. The Omega-3 emulsions are preferably isotonic. Methods for making emulsions are well known in the art and are described for example in Kirk-Othmer, Encyclopedia of Chemical Technology, 3 rd Ed., v. 8, pp. 900- 933 (1979). See also U.S. Patent Nos. 2,977,283; 3,169,094; 4,101,673; 4,563,354; 4,784,845; 4,816,247, all of which are incorporated by reference in their entirety.

[0090] Omega-3 lipid-based emulsions according to the invention can be prepared by known standard procedures with inertization. Typically, first the lipids, emulsifier and other auxiliary agents and additives are mixed and then filled up with water with dispersing. The water may optionally contain additional water-soluble components (e.g. glycerol). The omega-3 emulsions of the present invention have lipid particles less than 5 microns in diameter. Preferably the omega-3 lipid-based emulsions of the present invention contain lipid particles having a diameter of about 100-400 nanometer, with an average size of 300 nanometers. For parenteral application, median lipid droplet sizes may be less than about 1 μιη and preferably in the range from about 100-500 nm, more preferably from about 100 nm to about 400 nm, most preferably from about 200 nm to about 350 nm. For other applications, such as transdermal applications, mean diameter of the lipid droplet can be larger, for example, from about 1 μιη and 5 μιη.

[0091] The present invention also provides omega-3 lipid-based emulsions suitable for enteral or parenteral administration to provide a protective benefit on cells against cell death following a hypoxic-ischemic insult. Omega-3 lipid-based emulsions of the present invention comprise at least 10%, preferably at least 20% (up to 100%) by weight of omega-3 oil, preferably 7% to about 35% omega-3 oil by weight in grams perlOO ml of emulsion. Preferably the omega-3 oil comprises at least 10%, preferably at least 20% (up to 100%) omega-3 tri/diglyceride. Fatty acids in the omega-3 tri/diglyceride preferably comprise at least 40% (up to 100%) EPA and/or DHA. The omega-3 lipid-based emulsion preferably comprises 10% to 100% omega-3 tri/diglyceride. In a preferred embodiment, the omega-3 tri/diglyceride contains at least 40% (up to 100%) of their fatty acids as EPA and/or DHA. Preferably, omega-3 emulsions of the present invention are sterile and have a particle size that is preferably between 100-400 nanometer mean diameter, with an average size of 300 nm. For the described n-3 Tri-DHA emulsions, the omega-3 oil comprises at least 20% and up to 100% of the triglyceride by weight per total weight of the omega-3 oil, and at least 20% wt.-% up to 100% of the acyl-groups of the omega-3 triglycerides consist of DHA.

Pharmaceutical Formulations

[0092] The term "the omega-3 lipid-base emulsions" generally includes the described n-3 Tri- DHA emulsions. Administration of the omega-3 lipid-based emulsions of the present invention, including the described n-3 Tri-DHA emulsions, may be either enteral, parenteral, or transdermal. The methods of administration of a pharmaceutical composition of the omega-3 lipid-base emulsions may further comprise any additional administrations of other conventional stroke treatment or preventative medication.

[0093] The omega-3 lipid-base emulsions of the present may contain from about 2 wt.-% to about 5 wt.-% of a stabilizing or isotonizing additive, such as a polyhydric alcohol, based on the emulsion. Preferred stabilizing or isotonizing additives include glycerol, sorbitol, xylitol or glucose. Glycerol is most preferred. [0094] In addition to distilled water, the omega-3 lipid-base emulsions of the present invention may contain conventional auxiliary agents and/or additives, such as emulsifiers, emulsifying aids (co- emulsifiers), stabilizers, antioxidants, and isotonizing additives.

[0095] Emulsifiers may include physiologically acceptable emulsifiers (surfactants) such as phospholipids of animal or vegetable origin. Examples of phospholipids are egg yolk lecithin, a biologic phospholipid, a phosphatidylcholine with fixed fatty acyl chain composition, a glycophospholipid or a phosphatidylethanolamine. Particularly preferred are purified lecithins, especially soybean lecithin, egg lecithin, or fractions thereof, or the corresponding phosphatides. The emulsifier content may vary from about 0.02.wt.- to about 2.5 wt.- , preferably from about 0.6 wt.-% to about 1.5 wt.-% and most preferably about 1.2 wt.- , based on the total emulsion. In one embodiment the emulsifier is 1.2 mg of egg yolk lecithin/100 ml emulsion.

[0096] Alkali metal salts, preferably sodium salts, of long chain, C₁₆ to C₂8 fatty acids may also be used as emulsifying aids (co-emulsifiers). The co-emulsifiers are employed in concentrations of from about 0.005 wt-% to about 0.1 wt.-%, preferably about 0.02 wt-% to about 0.04 wt.-%, based on the total emulsion. Further, cholesterol or a cholesterol ester alone or in combination with other co-emulsifiers may be employed as an emulsifying aid in a concentration of from about 0.005 wt-% to about 0.1 wt-%, preferably from about 0.02 wt-% to about 0.04 wt-%, based on the emulsion.

[0097] The omega-3 lipid-base emulsions of the present invention may further comprise an effective amount of an antioxidant, such as vitamin E, in particular a-tocopherol (the most active isomer of vitamin E in humans) as well as β- and γ-tocopherol, and/or ascorbyl palmitate as antioxidants and thus for protection from peroxide formation. The total amount of alpha tocopherol may be up to 5000 mg per liter. In a preferred embodiment the total amount of said antioxidant is from about 10 mg to about 2000 mg, more preferably from about 25 mg to about 1000 mg, most preferably from about 100 mg to 500 mg, based on 100 g of lipid.

[0098] The omega-3 lipid-base emulsions of the invention may be administered orally, enterally, parenterally, transdermally, intravascular, intravenously, intramuscularly, intraperitonealy or transmucosally, and are preferably administered by intravenous injection. Thus the present invention also relates to a pharmaceutical composition comprising omega-3 diglyceride emulsions as described herein, preferably for injection into the human or animal body.

[0099] Pharmaceutical compositions of the invention may further comprise various pharmaceutically active ingredients. In particular, the pharmaceutically active ingredient may be delivered to a particular tissue of the body (drug targeting) in combination with emulsions of the present invention. The omega-3 lipid-base emulsions may include carriers for such targeted tissue treatment. Suitable carriers may be, for example, macromolecules linked to the emulsion droplet, lipid microspheres comprising soybean oil or lecithin or fish oil U.S. Pub. No. 2002/0155161, incorporated herein by reference in its entirety, discloses tissue-targeted delivery of emulsions.

[0100] The pharmaceutical composition may be formulated into a solid or a liquid dosage form. Solid dosage forms include, but are not limited to, tablets, pills, powders, granules, capsules, suppositories, and the like. Liquid dosage forms include, but are not limited to liquids, suspensions, emulsions, injection preparations (solutions and suspensions), and the like. The choice of dosage form may depend, for example, on the age, sex, and symptoms of the patient.

[0101] The pharmaceutical composition may optionally contain other forms of omega-3 Tri- DHA or diglyceride emulsions and/or additional active ingredients. The amount of omega-3 Tri- DHA/diglyceride emulsions or other active ingredient present in the pharmaceutical composition should be sufficient to treat, ameliorate, or reduce the target condition.

[0102] The pharmaceutically acceptable excipient may be any excipient commonly known to one of skill in the art to be suitable for use in pharmaceutical compositions. Pharmaceutically acceptable excipients include, but are not limited to, diluents, carriers, fillers, bulking agents, binders, disintegrants, disintegration inhibitors, absorption accelerators, wetting agents, lubricants, glidants, surface active agents, flavoring agents, and the like.

[0103] Carriers for use in the pharmaceutical compositions may include, but are not limited to, lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, or silicic acid. [0104] Absorption accelerators may include, but are not limited to, quaternary ammonium base, sodium laurylsulfate, and the like.

[0105] Wetting agents may include, but are not limited to, glycerin, starch, and the like. Adsorbing agents used include, but are not limited to, starch, lactose, kaolin, bentonite, colloidal silicic acid, and the like.

[0106] In liquid pharmaceutical compositions of the present invention, the omega-3 emulsions of the present invention and any other solid ingredients are dissolved or suspended in a liquid carrier, such as water, vegetable oil, alcohol, polyethylene glycol, propylene glycol or glycerin.

[0107] Liquid pharmaceutical compositions can contain emulsifying agents to disperse uniformly throughout the composition an active ingredient or other excipient that is not soluble in the liquid carrier. Emulsifying agents that can be useful in liquid compositions of the present invention include, for example, gelatin, egg yolk, casein, cholesterol, acacia, tragacanth, chondrus, pectin, methyl cellulose, carbomer, cetostearyl alcohol, and cetyl alcohol.

[0108] Liquid pharmaceutical compositions of the present invention can also contain viscosity enhancing agents to improve the mouth-feel of the product and/or coat the lining of the gastrointestinal tract. Such agents include for example acacia, alginic acid bentonite, carbomer, carboxymethylcellulose calcium or sodium, cetostearyl alcohol, methyl cellulose, ethylcellulose, gelatin guar gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, polyvinyl alcohol, povidone, propylene carbonate, propylene glycol alginate, sodium alginate, sodium starch glycolate, starch tragacanth and xanthan gum.

[0109] Sweetening agents such as sorbitol, saccharin, sodium saccharin, sucrose, aspartame, fructose, mannitol and invert sugar can be added to improve the taste. Preservatives and chelating agents such as alcohol, sodium benzoate, butylated hydroxy toluene, butylated hydroxyanisole and ethylenediamine tetraacetic acid can be added at safe levels to improve storage stability.

[0110] A liquid composition according to the present invention can also contain a buffer such as guconic acid, lactic acid, citric acid or acetic acid, sodium guconate, sodium lactate, sodium citrate or sodium acetate. [0111] Selection of excipients and the amounts to use can be readily determined by an experienced formulation scientist in view of standard procedures and reference works known in the art.

[0112] When preparing injectable pharmaceutical compositions, solutions and suspensions are sterilized and are preferably made isotonic to blood. Injection preparations may use carriers commonly known in the art. For example, carriers for injectable preparations include, but are not limited to, water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and fatty acid esters of polyoxyethylene sorbitan. One of ordinary skill in the art can easily determine with little or no experimentation the amount of sodium chloride, glucose, or glycerin necessary to make the injectable preparation isotonic. Additional ingredients, such as dissolving agents, buffer agents, and analgesic agents may be added. If necessary, coloring agents, preservatives, perfumes, seasoning agents, sweetening agents, and other medicines may also be added to the desired preparations.

[0113] Pharmaceutical compositions of the invention may further comprise various pharmaceutically active ingredients. In particular, the pharmaceutically active ingredient may be delivered to a particular tissue of the body (drug targeting) in combination with micro emulsions of the present invention. Ornega-3 emulsions may include carriers for such targeted tissue treatment. Suitable carriers may be, for example, macromolecules linked to the emulsion droplet, lipid microspheres comprising soybean oil or lecithin or fish oil. U.S. Pub. No. 2002/0155161, incorporated herein by reference in its entirety, discloses tissue-targeted delivery of emulsions.

[0114] Omega-3 compositions of the invention allow for rapid and efficient uptake of omega-3 fatty acids, including EPA and DHA, into cell membranes of organs and tissues. Accordingly, there is provided a method for delivering an emulsion of omega-3 Tri-DHA or diglycerides enriched with EPA or DHA to cells and organs by administering omega-3 emulsions of the present invention.

[0115] Lipolysis of emulsions of the invention facilitates the release of free omega-3 fatty acids and monoglycerides into the bloodstream or in cells. Free fatty acids may be transported into mitochondria for use as an energy source, or may be incorporated into cell membranes. Enriching cell membranes and phospholipids with omega-3 long chain polyunsaturated fatty acids (PUPA) may help promote or restore an adequate balance between omega-3 and omega-6 fatty acids. Incorporation of EPA and DHA also increases membrane fluidity and flexibility.

EXAMPLES

Example 1 : 60 minutes of hypoxia-ischemia

[0116] Postnatal day 19-21 Wistar rats of both genders were subjected to unilateral (right) carotid artery ligation. See Rice, J. E., 3rd, R. C. Vannucci, et al. (1981), "The influence of immaturity on hypoxic-ischemic brain damage in the rat," Ann Neurol 9(2): 131-41 and Vannucci, S. J., L. B. Seaman, et al. (1996), "Effects of hypoxia-ischemia on GLUT1 and GLUT3 glucose transporters in immature rat brain," Journal of Cerebral Blood Flow & Metabolism 16(1): 77-81.

[0117] Immediately after ligation, six rats were given 50mg 20% omega-3 lipid-based emulsion (0.25cc)(a 20% long chain omega-3 triglyceride-based formula having >45% of total omega-3 fatty acid as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) via orogastric feeding tube, and six control rats were given 0.25cc water, both enterally. The 20% omega-3 lipid-based emulsion was made placing 20 gm of omega-3 triglyceride in 100 ml of water, and emulsifying with 1.2 gm of egg yolk lecithin. Rats were allowed to recover for 2 hours and then they underwent hypoxia-ischemia for 60 minutes of 8% oxygen at a constant temperature. The six pre-treated rats were given another dose of 50mg omega-3 lipid-based emulsion immediately after the hypoxia-ischemia and control rats were given 0.25cc water. All rats were euthanized at 72 hours of reperfusion. The brains were removed and cut into 2mm sections and stained with 2, 3, 5, Triphenyl-2H-tetrazolium chloride (TTC). TTC is a vital die that stains cells red that have respiring mitochondria. Dead tissue (infarct) appears white. The sections were scored as follows:

• 0 - no evidence of edema or cell death

• 1 - edema without cell death

• 2 - edema with minimal cell death

• 3 - edema with significant cell death

[0118] All rats survived 60 minutes of hypoxia-ischemia. Six of the six control rats had edema and/or cell death with a mean score of 2 +/- 0.83 (standard deviation), while two of the six treated rats had damage with a mean score of 0.42 +/- 0.62 (p<0.005). Example 2: 65 minutes of hypoxia-ischemia

[0119] Postnatal day 19-21 Wistar rats of both genders were subjected to unilateral (right) carotid artery ligation. Immediately after ligation, six rats were given 50mg 20% omega-3 lipid- based emulsion (0.25cc)(20% omega-3 fatty acid based formula having >40% of total omega-3 fatty acid as EPA and DHA)) via orogastric feeding tube and six control rats were given 0.25cc water, both enterally. The emulsion was made as described in Example 1. The rats recovered for two hours, and then underwent hypoxia-ischemia for 65 minutes of 8% oxygen at a constant temperature. The six pre-treated rats were given another dose of 50mg omega-3 lipid-based emulsion immediately after the hypoxia-ischemia and control rats were given 0.25 cc water. All rats were euthanized at 72 hours of reperfusion. The brains were removed and cut into 2mm sections and stained with 2, 3, 5, Triphenyl-2H-tetrazolium chloride (TTC). The sections were scored as follows:

• 0 - no evidence of edema or cell death

• 1 - edema without cell death

• 2 - edema with minimal cell death

• 3 - edema with significant cell death

[0120] The 65 minutes of hypoxia-ischemia produced damage in all rats. Four of the six control rats survived with a mean score of 2.75 +/- 0.50, while five of the six treated rats survived with a mean score of 1.70 +/= 0.76 (p<0.05).

Example 3: Treatment of rats with omega-3 triglyceride lipid emulsion prior to 60 minutes of hypoxia

[0121] Postnatal day 19-21 Wistar rats were subjected to unilateral (right) carotid artery. Immediately after ligation, six rats were given 50mg of a 20% omega-3 lipid-based emulsion (0.25cc), and six control rats were given 0.25cc water, both enterally. The emulsion was as described above in Example 1. Rats were allowed to recover for two hours, and then underwent hypoxia-ischemia for 60 minutes of 8% oxygen at a constant temperature. The six pre-treated rats were given another dose of 50mg omega-3 triglyceride lipid emulsion immediately after the hypoxia/ischemia and control rats were given 0.25cc water. At 72 hours of reperfusion, the rats were euthanized and their brains removed, cut into 2 mm sections and stained with 2,3,5 triphenyl-2H-tetrazolium chloride (TTC). The damage in each animal was then given a score from 0 (no damage) to 4 (> 60% ipsilateral hemisphere infarcted). All of the vehicle-treated animals suffered brain damage, with a mean damage score of 2.00+0.89; the omega-3 triglyceride lipid emulsion-treated rats were significantly less damaged, having a mean damage score 0.33+0.52, p < 0.05. The size of brain infarcts was determined by TTC staining.

[0122] These results show that when omega -3 triglycerides were administered either immediately before and/or after hypoxia-ischemia they confer a significant neuroprotection. Very similar results were obtained when the omega-3 triglycerides were injected parenterally.

Example 4: Treatment following hypoxic ischemia

[0123] Post-natal day 19-21 rat pups were subjected to unilateral carotid artery ligation and 60 minutes of hypoxic ischemia, according to the previously described protocol. On four separate occasions, rats were treated by parenteral injection of omega-3 lipid-based emulsion (100 mg) immediately after the insult, and again at four hours after the insult. The emulsion was as described above in Example 1. Brain damage was evaluated by TTC staining at 72 hours of reperfusion. In each instance, administration of the omega-3 lipid-based oil emulsion provided greater than 50% protection, i.e. reduction of tissue damage.

[0124] Figure 4 shows the results of these experiments. Figure 4 represents at total of 14 control subjects (saline-treated) and 21 treated subjects (omega-3 lipid-based emulsion treated). Mean damage scores were: 1.93 + 0.22 (SEM), control, 0.78 + 0.16 emulsion-treated; p < 0.0001 by two-tailed test. Thus, in addition to the significance of the overall protection, it can be seen that 40% of the treated animals were 100% protected (no damage at all, compared to 1/14 untreated; 40 % suffered only mild damage, compared to 1/14 mildly damaged untreated animals. These results indicate that treatment following hypoxic-ischemia provides a neuroprotective benefit as indicated by a reduction of tissue damage.

[0125] Preliminary experiments conducted in the adult mouse show a comparable level of neuroprotection from hypoxic-ischemic damage.

[0126] Fatty acyl composition analyses of brain lipids (by gas liquid chromatography) after hypoxia/ischemia show no relative differences between infarcted brain versus non infarcted brain indicating that effects of acute administration of omega-3 emulsions are not dependent on fatty acid compositional changes in brain membranes. In the infarcted areas, however, absolute concentrations of all fatty acids fell to similar degrees by about 15% ^g fatty acid per gram wet brain) indicating brain edema. This decrease did not occur with administration of omega-3 emulsions indicating that these omega-3 fatty acids prevented the brain edema as well as infarction.

Example 5: Quantification of effects of omega-3 triglyceride treatment on cellular targets

[0127] Studies on the effects of omega-3 triglyceride treatment on the generation of reactive oxygen species (ROS) and markers of oxidative damage, as well as indices of inflammation at 2, 4, 8 and 24h after the hypoxic-ischemic insult are performed. Lasting protection is confirmed by brain histopathology at eight weeks following the original hypoxia-ischemic. Sections of the brain are stained (including both involved and non-involved hemispheres) with antibodies recognizing activated proteins known to participate in neuronal apoptosis (caspase 3, Jun N- terminal kinases), neuronal survival (activated Akt, phosphorylated BAD, FKHR) or to mediate the effects of NMDA-R signaling (CAM KII, and protein kinase C isoforms, in particular PKCy and ΡΚ£ζ). Sections are co-stained with antibodies recognizing neuronal specific proteins (Tau), astrocytes (GFAP) or microglia. These analyses allow quantification of the effect of omega-3 triglycerides on hypoxia-ischemia induced changes in apoptotic versus anti-apoptotic signaling in neurons, as well as gain indices of astrocytic or microglia involvement. Further, whole brain extracts from involved and non-involved hemispheres are prepared to quantify the extent of caspase, JNK and Akt activation by immunoblotting.

Example 6: Materials and Methods for Studying Cerebral Hypoxia-ischemia Ethics Statement

[0128]. All research studies were carried out according to protocols approved by the Columbia University Institutional Animal Care and Use Committee (IACUC) and in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines.

Materials [0129] Tri-DHA and Tri-EPA were purchased from Nu-Chek Prep, Inc. (Elysian, MN). Egg yolk phosphatidylcholine was obtained from Avanti Polar-Lipids, Inc. (Alabaster, AL).

Lipid Emulsions

[0130] Four different types of lipid emulsions were used, omega-3 (n-3) triglyceride fish oil- based and omega-6 (n-6) soy oil-based emulsions were commercially prepared intravenous phospholipid- stabilized emulsions. The omega-3 triglyceride contained high concentrations of n-

3 fatty acids (FA) as described. 1 ' 2 See Table 1. These omega-3 triglyceride emulsions are referred to as "n-3 TG" in FIGS. 7-16, they comprisel0% omega-3 fish oil (n-3) having less than 10% omega-6 oil by weight in grams per 100 ml of emulsion, wherein the omega-3 oil is >90 % triglyceride (TG) by weight per total weight of the omega-3 oil, and in which up to about 30% wt.-% of the acyl groups are DHA and up to about 28% wt.-% are EPA. The n-3 TG emulsions are also called n-3 TG90-DHA30. Other emulsions having pure (99%) DHA or pure (99%) EPA was also tested as described. For doses of injected n-3 TG emulsions, an amount was calculated to achieve an administration containing 50% of the TG-FA as DHA and EPA (Table 1). Thus, 1 gm of TG emulsions is expressed as 0.5 gm n-3 TG.

[0131] The n-6 TG emulsions described in Table 1 and shown in the Figures, comprise 20% omega-6 oil (n-6) by weight in grams per 100 ml of emulsion, 0% DHA, 0% EPA and 55% TG from linoleic acid (Table 1). The n-6 TG emulsions were produced from soy bean oil rich in n-6 FA: linoleic acid constituting about 55% of total FA.

Table 1. Fatty Acid Composition of Lipid Emulsions¹

Stearic acid (C18:0) 0.5-2 1.4-5.5

Oleic acid (CI 8: ln-9) 6-13 19-30

Linoleic acid (C18:2n-6) 1-7 44-62

Arachidonic acid (C20: 4n-6) 1-4 <0.5

a-linolenic acid (C18:3n-3) 2 4-11

Eicosapentaenoic acid 12.5-28.2 - (C22:6n-3)

Docosahexaenoic acid (C22: 14.4-30.9 - 6n-3)

Data provided by Fresenius Kabi AG; FA, Fatty acids

[0132] The pure Tri-DHA (99% DHA) and Tri-EPA (99% EPA) emulsions were VLDL-sized and laboratory-made with TG oil and egg yolk phospholipid using sonication and centrifugation procedures that are known in the art.³'⁴ Briefly, 200 mg Tri-DHA (Tri-DHA oil >99%) or Tri- EPA (Tri-EPA oil > 99%) was mixed with a 5: 1 weight ratio of egg yolk phosphatidylcholine (40 mg). The mixture was fully evaporated under N₂ gas, and was further desiccated under vacuum overnight at 4°C. The dried lipids were resuspended in 1 mL of lipoprotein-free buffer (LPB) (150 mmol/L NaCl, 0.5 ml of 0.1% glycerol and 0.24 mmol/L EDTA, pH 8.4, density 1.006 g/mL) at 60°C with added sucrose (100 mg/1 mL LPB) to remove excess phospholipid liposomes. The lipid emulsions were then sonicated for 1 hr at 50° C, 140 W under a stream of N₂ using a Branson Sonifier model 450 (Branson Scientific, Melville, NY). After sonication, the solution was dialyzed in LPB for 24 hr at 4°C to remove sucrose. The final emulsions comprising VLDL-sized particles were analyzed for the amount of TG and PL by enzymatic procedure using GPO-HMMPS, glycerol blanking method (Wako Chemicals USA, Inc., Richmond, VA) and choline oxidase-DAOS method (Wako Chemicals USA, Inc., Richmond, VA). The TG: phospholipid mass ratio was 5.0+1.0: 1 similar to that of VLDL-sized particles. The emulsions were then stored under argon at 4°C and were used within 2 weeks of preparation.

Induction of Unilateral Cerebral H/I

[0133] Three-day-old C57BL/6J neonatal mice of both genders were purchased from Jackson Laboratories (Bar Harbor, ME) with their birth mother. The Rice-Vannucci model of H/I was used and modified to plO neonatal mice. Briefly, on postnatal day 10 H/I was induced by the ligation of the right common carotid artery, which was further cauterized and cut under isoflurane anesthesia. The investigator was blinded to the lipid emulsion treatment during the surgery and after the surgery. The entire surgical procedure was completed within 5 min for each mouse. Pups were then allowed to recover with their dams for 1.5 hr. Surrounding temperature during experiments was kept at 28° C. Mice were then exposed to systemic hypoxia for 15 min in a hypoxic chamber in a neonatal isolette (humidified 8% oxygen/nitrogen, Tech Air Inc., White Plains, NY).⁵ The ambient temperature inside the chamber during hypoxia was stabilized at 37+ 0.3°C. To minimize a temperature-related variability in the extent of the brain damage, during the initial 15 hr of reperfusion mice were kept in an isolette at the ambient temperature of 32°C.

Quantification of Brain Infarction

[0134] After 24 hr of reperfusion, the animals were sacrificed by decapitation and brains were immediately harvested. 1-mm coronal slices were cut by using a brain slicer matrix. Slices were then immersed in a PBS solution containing 2% triphenyl-tetrazolium chloride (TTC) at 37°C for 25 min. TTC is taken up into living mitochondria, which converts it to a red color.⁶ Thus, viable tissue stains brick-red, and nonviable (infarcted) tissue can be identified by the absence of staining (white). Using Adobe Photoshop and NIH Image J imaging applications, planar areas of infarction on serial sections were summed to obtain the volume (mm ) of infarcted tissue, which was divided by the total (infarcted + non-infarcted) volume of the hemisphere ipsilateral to carotid artery ligation, and expressed as a percentage of total volume.

Experimental Groups

[0135] H/I brain injury was induced in different groups of animals, which received specific treatments before and after H/I injury. Animals followed different treatment protocols.

[0136] Protocol 1: Pre-H/I treatment of n-3 TG (containing both DHA and EPA) or n-6 TG emulsions.

[0137] Two doses of n-3 TG or n-6 TG emulsions or vehicle (saline, equal volumes/kg) were administered to non-fasting rodents at a fixed dose of 3 mg of n-3 or n-6 TG-FA per mouse for each injection (equivalent to a maximum of 1.5 g of total TG/kg; plO mice weighed 4 - 6 gm for these experiments). The first dose was i.p. administered immediately after surgery, and the second immediately at the end of the 15 min hypoxic period. Volumes injected for TG emulsions and saline were always equal.

[0138] Since n-3 emulsions contain low concentrations of alpha-tocopherol as an anti-oxidant agent, in separate experiments an equivalent dose of pure alpha-tocopherol to match the content of n-3 emulsion content (0.8 g/L) was given to neonatal mice by i.p. injection of alpha- tocopherol (Vital E®, Intervet, Schering Plough) at a dose of 5 mg alpha-tocopherol/kg body weight, the amount contained in each i.p. injection of the n-3 TG emulsions.

Protocol 2: Post-H/I treatment of n-3 TG ( containing both DHA and EPA ).

[0139] Two doses of the commercially available n-3 TG emulsion or saline were i.p. injected into non-fasting rodents at 0.75 g of n-3 TG/kg body weight for each dose (equivalent to 1.5 g of total TG/kg). The first dose was administered immediately after 15-min hypoxia, and the second at 1 hr after start of the reperfusion period.

Protocol 3: Dose response, timing and specificity of n-3 TG.

[0140] Two types of n-3 containing lipid emulsions either Tri-DHA or Tri-EPA (0.1 g n-3 TG/kg or 0.375 g n-3 TG/kg body weight for each dose) were administered twice to non-fasting rodents according to the amount of DHA and EPA in the mixed n-3 TG emulsions. See Table 1. The first dose was initially administered immediately after 15-min hypoxia, and the second after 1 hr of reperfusion. Then in different sets of experiments, the efficacy of Tri-DHA emulsions was determined, with the initial injection administered at four-time points (0 hr, or at 1-hr, 2-hr or 4- hr after H/I), 0.375 g n-3 TG/kg body weight for each dose. For the immediate treatment of 0 hr, the first dose was injected immediately after 15-min hypoxia, with a second injection after 1 hr of reperfusion, whereas in the "delayed" treatments, the first dose was given after the 1^st or 2^nd or 4^th hr of reperfusion and a second dose was administered 1 hr after the 1^st dose.

Measurement of Blood TG and Glucose Levels

[0141] Blood samples for blood TG were directly taken from left ventricle of hearts under isoflurane inhalation from a separate cohort of non-fasting, 10-day-old mice. Samples were taken over a 5 hr period after a single i.p. injection of either 0.75 g n-3 TG/kg commercially available n-3 rich TG (DHA and EPA) emulsions or saline. Total plasma TG was enzymatically measured by GPO-HMMPS, glycerol blanking method (Wako Chemicals USA, Inc., Richmond, VA). For glucose levels, blood samples were taken from mouse tails from a separate cohort of non-fasting 10-day-old mice. Samples were taken at two time points from each mouse. The first sample was taken at time zero before surgery and TG injection, and the second at about 10 min after H/I and TG injection (approximately 100 min after surgery as described under the Unilateral Cerebral H/I protocol above). Blood glucose levels were electrochemically measured in mg/dL by a glucose meter (OneTouch Ultra, LifeScan, Inc., Milpitas, CA).

Measurement of Cerebral Blood Flow ( CBF) by Laser Doppler Flowmetry (LDF)

[0142] In a cohort of neonatal C57BL/6J mice pups subjected to carotid artery ligation and recovery as described above, relative CBF was measured during hypoxia in ipsilateral (right) hemispheres using a laser Doppler flow meter (Periflux 5000). In these mice, in preparation for CBF measurement the scalp was dissected under isoflurane anesthesia and Doppler probes were attached to the skull (2 mm posterior and 2 mm lateral to the bregma) using fiber optic extensions. Only local anesthesia (1% lidocaine) was used postoperatively. Mice were then placed into a hypoxia chamber (8%0₂/92%N₂). Changes in CBF in response to hypoxia were recorded for 20 min and expressed as percentage of the pre -hypoxia level for n-3 treated and saline treated neonatal mice.

Measurement of Bleeding Time after n-3 TG injection

[0143] Bleeding times were measured in mice after severing a 3-mm segment of the tail.⁷ Two doses of saline were administered vs. n-3 TG in a similar time frame as the original protocol: an initial injection followed by a second injection at 2 hr later. Bleeding times were measured at 45 min after the second dose. The amputated tail was immersed in 0.9% isotonic saline at 37° C, and the time required for the stream of blood to stop was defined as the bleeding time. If no cessation of bleeding occurred after 10 min, the tail was cauterized and 600 s was recorded as the bleeding time.

Long-Term Assessment of Brain Tissue Death [0144] A long-term assessment of cerebral injury was performed at 8 wk after neonatal H/I insult. This cohort of mice at plO underwent unilateral H/I followed by post H/I injections with either 0.375 g Tri-DHA/kg (n = 6) or saline (n = 5) as described above. At 8 wk after H/I, mice were sacrificed by decapitation. Brains were removed, and embedded in Tissue Tek-OTC- compound (Sakura Fineteck, Torrance, CA) with subsequent snap freezing in dry ice-chilled isopentane (-30°C), and stored at -80°C. For analysis, coronal sections (10 μιη every 500 μιη) were cut serially in a Leica cryostat and mounted on Superfrost slides (Thermo Scientific, Illinois). Sections were processed for Nissl staining by using Cresyl Violet Acetate (Sigma- Aldrich, St. Louis, MO). Using Adobe Photoshop and NIH Image J imaging applications, 9 sections from each brain containing both the right and left hemispheres were traced for brain tissue area. As previously described the area of left control or contralateral hemisphere which had not had injury was given a value in 100% for each animal. The brain area remaining in the right injured ipsilateral hemisphere was then compared to the left hemisphere, and the difference was taken as the percent right brain tissue loss, for each animal.

Statistical Analysis

[0145] Data are presented as mean + SEM. Plasma TG levels were compared at each time point after i.p. injection of n-3 TG emulsion. Student t tests were used for 2-group comparisons. 1-way ANOVA, followed by Bonferroni procedure for post hoc analysis to correct for multiple comparisons, was used to compare the differences among the emulsions on the infarct areas across coronal sections. Statistical significance, which was analyzed by using SPSS software 16.0 (SPSS Inc., Chicago, IL), was determined at p < 0.05.

Example 7: n3 Fatty Acid Rich TG Emulsions are Neuroprotective after Cerebral Hypoxic- Ischemic Injury in Neonatal Mice

Effects of n-3 TG on Blood Triglyceride and Glucose Levels, and Bleeding Time

[0146] To determine if TG from the n-3 TG emulsions were systemically absorbed, the blood TG levels were examined up to 5 hr after i.p. injection. After n-3 TG injection, there was a substantial increase of TG levels up to three fold higher at 1.5 hr (p < 0.05) compared to the baseline, followed by a decrease of levels to baseline at 3 and 5 hr. See FIG. 1A. This indicates that n-3 TG entered into the blood stream and was being catabolized. In comparison, TG levels of saline-injected mice remained constant over the 5 hr time period reflecting normal blood TG levels in neonates.

[0147] After H/I, blood glucose levels might affect infarct size.⁹ Therefore, blood glucose levels were measured in each group (n-3 TG vs. n-6 TG vs. saline control) prior to surgery and after 15- min H/I after TG or saline injection. See FIG. IB. No difference in blood glucose levels among groups was observed when comparing at the same time point. Still, after H/I insult, blood glucose levels decreased similarly, about 30% or more, in all groups (p < 0.05).

[0148] There was no difference in capillary bleeding times in n-3 treated mice (437+82 sec) as compared to saline controls (418+90 sec). n-3 TG does not Change Cerebral Blood Flow after H/I

[0149] There was no effect on CBF in the ipsilateral hemisphere of n-3 treated neonatal mice as compared to saline treated animals. Immediately after right common carotid artery ligation, and initiation of hypoxia at 8% oxygen, CBF was approximately 25% of initial (pre-H/I) level in the ipsilateral hemisphere in both control and n-3 treated groups, and this was maintained for the duration of hypoxia. In the contralateral (unligated) hemisphere blood flow was unchanged in both groups. Very similar blood flow levels were maintained in neonatal H/I mice whether they were saline treated or n-3 treated in this model. See FIG. 2. n-3 TG but not n-6 TG Protects Brain against H/I Injury

[0150] Coronal sections of brains were stained with TTC to quantify the extent of post H/I brain injury and the effect of n-3 TG injection. See FIG. 3. FIG. 3 A shows representative images of neonatal mouse brain from saline treated, n-6 TG emulsion treated and n-3 TG emulsion treated mice with pre-and-post injection after H/I, respectively. In all H/I animals, tissue death was localized to the right hemisphere (ipsilateral to ligation) as illustrated by the white areas in the upper panels of FIG. 3A. The image in the lower panels, FIG 3A, demonstrated tracings of the infarcted areas for quantifying infarct volume using NIH Image J. The brains from saline treated animals exhibited a consistent pannecrotic lesion involving both cortical and subcortical regions ipsilateral to the ligation. In the majority of the animals the neuroprotection after n-3 TG injection was most marked in the subcortical area, whereas saline treated mice had large cortical and subcortical infarcts. See FIG. 3 A.

[0151] Infarct volume was substantially decreased in n-3 TG treated mice (n = 28) compared to saline treated littermates (control) (n = 27), 19.9 + 4.4% vs. 35.1 + 5.1%, respectively (p = 0.02). See FIG. 3B. There was a significant increase in infarct volume with n-6 TG emulsion injection compared to saline control (p = 0.03) and the n-3 TG groups (p < 0.01).

[0152] Because alpha-tocopherol is a component of the TG emulsions (present in low concentrations to prevent FA oxidation) TTC staining was used to compare the extent of cerebral H/I injury in alpha-tocopherol treated and saline treated neonatal mice. There was no significant difference in infarct volume between brains in alpha-tocopherol injected mice compared to saline treated mice (data not shown).

[0153] It was then determined if n-3 TG are effective if injected only after H/I (without injection prior to H/I. See FIG. 3C. Similarly, the smaller n-3 TG associated lesions were mainly subcortical (data not shown). Compared to saline controls in the immediate post-H/I treatment the total infarct area was significantly reduced almost 50% in the n-3 TG post H/I treated group.

DHA but not EPA is Neuroprotective after H/I

[0154] To determine possible differences in neuroprotection of EPA vs. DHA, the extent of brain injury was studied using the post-H/I treatment protocol with Tri-DHA vs. Tri-EPA in two dosages (O. lg TG/kg vs. 0.375 g TG/kg). No statistical differences in brain infarct volume between 0.1 g TG/kg and 0.375 g TG/kg Tri-DHA treated groups were observed. However, compared to saline control, total infarct size was reduced by a mean of 48% and 55% by treatment with 0.1 and 0.375 g TG/kg Tri-DHA, respectively. See FIG. 4. Neuroprotection was not observed with Tri-EPA injection at either of the two doses compared with saline treatment.

[0155] To better approximate realistic timelines for neuroprotection after stroke for humans delayed treatment protocols were performed in an effort to study the therapeutic window of Tri- DHA emulsions. No protective effect from Tri-DHA after a 4-hr delay in treatment was noted compared with saline group. However, Tri-DHA administered at 0 hr immediately post H/I, and then delayed 1-hr and 2-hr post stroke showed similar reduced (-50 %) brain infarct volumes compared to saline treated animals. See FIG. 5. This substantial protection occurred mainly in subcortical areas similar to the findings described above.

Long-Term Neuroprotection

[0156] Coronal brain sections of adult mice were processed for Nissl staining (FIG. 6) to examine the effects of H/I and Tri-DHA treatment on brain and neuronal cell loss for long-term outcome at 8 wk after H/I insult. As compared to the left control (contralateral hemisphere), the injured areas of the right hemisphere display gross neuronal cell loss. As shown in FIG. 6, brain tissue loss was markedly increased by 1.67 fold in the right hemisphere of saline-treated mice (n = 5) as compared to Tri-DHA treated mice (n = 6), 25.0+2.4% vs. 15.0+2.5%, respectively (p =

0.02.. Thus, neuroprotection after injury and Tri-DHA injection that are observed 24 hr after H/I can be demonstrated histologically almost 2 months after the initial stroke insult.

References Cited in Example 6 and Example 7

1. Oliveira FL, Rumsey SC, Schlotzer E, Hansen I, Carpentier YA, et al. (1997) Triglyceride hydrolysis of soy oil vs fish oil emulsions. JPEN J Parenter Enteral Nutr 21: 224-229.

2. Qi K, Seo T, Al-Haideri M, Worgall TS, Vogel T, et al. (2002) Omega-3 triglycerides modify blood clearance and tissue targeting pathways of lipid emulsions. Biochemistry 41: 3119- 3127.

3. Qi K, Al-Haideri M, Seo T, Carpentier YA, Deckelbaum RJ (2003) Effects of particle size on blood clearance and tissue uptake of lipid emulsions with different triglyceride compositions. JPEN J Parenter Enteral Nutr 27: 58-64.

4. Schwiegelshohn B, Presley JF, Gorecki M, Vogel T, Carpentier YA, et al. (1995) Effects of apoprotein E on intracellular metabolism of model triglyceride -rich particles are distinct from effects on cell particle uptake. J Biol Chem 270: 1761-1769.

5. Ten VS, Bradley-Moore M, Gingrich JA, Stark RI, Pinsky DJ (2003) Brain injury and neurofunctional deficit in neonatal mice with hypoxic-ischemic encephalopathy. Behav Brain Res 145: 209-219.

6. Liszczak TM, Hedley-Whyte ET, Adams JF, Han DH, KoUuri VS, et al. (1984) Limitations of tetrazolium salts in delineating infarcted brain. Acta Neuropathol 65: 150-157.

7. Denis C, Methia N, Frenette PS, Rayburn H, Ullman-Cullere M, et al. (1998) A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis. Proc Natl Acad Sci U S A 95: 9524-9529. 8. Seo T, Blaner WS, Deckelbaum RJ (2005) Omega-3 fatty acids: molecular approaches to optimal biological outcomes. Curr Opin Lipidol 16: 11-18.

9. Bruno A, Biller J, Adams HP, Jr., Clarke WR, Woolson RF, et al. (1999) Acute blood glucose level and outcome from ischemic stroke. Trial of ORG 10172 in Acute Stroke Treatment (TOAST) Investigators. Neurology 52: 280-284.

Example 8: Omega-3 Triglyceride DHA emulsions in Cardiac Hypoxia-Ischemia Material and Methods Animal Care

[0157] All studies were performed with the approval of the Institutional Animal Care and Use Committee at Columbia University, New York University School of Medicine, and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Pub. No. 85-23, 1996). C57BL6 mice (weight between 25-30 g and 12-14 weeks old) were obtained from Jackson Laboratories for our studies. Mice were kept in an animal care facility for a week prior to the studies. All mice were fed a normal chow diet (Teklad Global diets, Harlan Laboratories).

Reagents

[0158] The primary antibodies used were Bcl-2, Beclin-1, PPAR-γ, p-AKT, total- AKT, p-GSK- 3β, total- GSK-3P (Cell Signaling, USA); and β-actin (BD Biosciences Pharmingen, USA). The secondary antibodies used were anti-rabbit IRdye800, anti-mouse IRdye700 (1:50,000 dilution). SB216763 (3μΜ), Rosiglitazone (6mg/kg body weight) were purchased from Sigma- Aldrich, USA. Phosphatidylinositol 3-kinase (PI3K)/AKT inhibitor LY-294002 (10 μΜ) was purchased from Calbiochem. The doses of the inhibitors and agonist used in this study were based on publications in the literature.¹ n-3 fish oil-based emulsion (10 g of TG/100 mL) was commercially prepared intravenous phospholipid-stabilized emulsions, and contained high concentrations of n-3 FA as previously described 2 ' 3 n-3 TG emulsion was rich in EPA (up to 28%) and DHA (up to 30%).

In vivo Left Anterior Descending Coronary Artery (LAD) occlusion [0159] In vivo murine model of ischemia-reperfusion injury: Prior to surgery, mice were anesthetized with isoflurane inhalation (4% induction followed by 1-2.5% maintenance). Subsequent to anaesthesia, mice were orally intubated with polyethylene-60 (PE-60) tubing, connected to a mouse ventilator (MiniVent Type 845, Hugo-Sachs Elektronik) set at a tidal volume of 240 μΐ_^ and a rate of 110 breaths per minute, and supplemented with oxygen. Body temperature was maintained at 37°C. A median sternotomy was performed, and the proximal left coronary artery (LAD) was visualized and ligated with 7-0 silk suture mounted on a tapered needle (BV-1, Ethicon). After 30 min of ischemia, the prolene suture was cut and the LAD blood flow was restored. Immediately after, intraperitoneal (IP) injection of n-3 TG emulsion (1.5g/kg body weight) was performed and the second injection was done after 60 min of reperfusion. Control animals received IP injection of saline solution following the same time course. The chest wall was closed, and mice were treated with buprenorphine and allowed to recover in a temperature-controlled area⁴'⁵.

Echocardiogram

[0160] In vivo transthoracic echocardiography was performed using a Visual Sonics Vevo 2100 ultrasound biomicroscopy system. This high-frequency (40 MHz) ultrasound system has an axial resolution of -30-40 microns and a temporal resolution of >100 Hz. Baseline echocardiography images was obtained prior to myocardial ischemia and post-ischemic images were obtained after 48 hours of reperfusion. The mice were lightly anesthetized with isoflurane (1.5-2.0 L/min) in 100% C"2 and in vivo transthoracic echocardiography of the left ventricle (LV) using a MS-400 38-MHz microscan transducer was used to obtain high resolution two dimensional mode images. Images were used to measure LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), LV ejection fraction (EF) and LV fractional shortening (FS) as published earlier.⁴'⁵

Infarct size measurement

[0161] Myocardial infarct size determination: At 48h of reperfusion mice were re-anesthetized, intubated, and ventilated using a mouse ventilator. A catheter (PE-10 tubing) was placed in the common carotid artery to allow for Evans blue dye injection. A median sternotomy was performed and the LAD was re-ligated in the same location as before. Evans blue dye (1.25 ml of a 7.0% solution) was injected via the carotid artery catheter into the heart to delineate the non- ischemic zone from the ischemic zone. The heart was then rapidly excised and fixed in 1.5% agarose. After the gel solidified, the heart was sectioned perpendicular to the long axis in 1-mm sections using a tissue chopper. The 1-mm sections was placed in individual wells of a six- well cell culture plate and counterstained with 1% TTC for 4 min at 37 °C to demarcate the nonviable myocardium. Each of the 1 mm thick myocardial slices was imaged and weighed. Images were captured using a Q-Capture digital camera connected to a computer. Images were analysed using computer-assisted planimetry with NIH Image 1.63 software to measure the areas of infarction, and total risk area.⁴'⁵

Ex vivo Ischemia and Reperfusion (I/R)

[0162] Experiments were carried out and modified for use in mice hearts.⁴'⁵ C57BL6 mice weighting between 25-30 g and 12-14 weeks old were anesthetized by injecting ketamine/xylazine cocktail [80 mg/kg and 10 mg/kg respectively]. The hearts, rapidly excised, through the aorta were retrograde perfused in a non-recirculating mode, using an isovolumic perfusion system through Langendorff technique (LT), with Krebs-Henseleit buffer, containing (in mM) the following: 118 NaCl, 4.7 KC1, 2.5 CaCl₂, 1.2 MgCl₂, 25 NaHC0₃, 5 Glucose, 0.4 Palmitate, 0.4 BSA, and 70 mU/1 insulin. Perfusion p0₂ > 600 mmHg was maintained in the oxygenation chamber.

[0163] Left ventricular developed pressure (LVDP) was continuously monitored, using a latex balloon placed on the left ventricle and connected to a pressure transducer (Gould Laboratories; Pasadena, CA). Cardiac function measurements were recorded on a 2-channel ADI recorder. The experimental plan included an equilibration baseline period of 30 min normoxic perfusion followed by 30 min global zero-flow ischemia and 60 min of reperfusion. The flow rate was 2.5 ml/min. The perfusion apparatus was tightly temperature controlled for maintaining heart temperature at 37 + 0.1°C under all conditions. The control heart received Krebs-Henseleit buffer; in treated harts, we added to the standard buffer an emulsion with n-3 fatty acids packaged in triglyceride (TG); we used 300mg/100ml as final amount.

Assay of lactate dehydrogenase (LDH)

[0164] Myocardial injury was assessed by measuring the release of lactate dehydrogenase (LDH) from the effluent in the ex vivo VR system and from blood samples in the in vivo LAD system, using the commercially available enzymatic kits (Pointe Scientific, INC, MI USA) as published earlier.⁴'⁵

Western blot analysis

[0165] The tissue and cell protein concentration was determined using a DC Protein Assay kit (Bio-Rad). Equal amounts of protein were separated by SDS-PAGE (4-12% gradient gels), and proteins were loaded to a nitrocellulose membrane (Invitrogen), After blocking nonspecific binding with the Odyssey blocking buffer (Li-Cor Biosciences), membranes were incubated overnight at 4^CC with target primary antibodies (1 : 1,000 dilution), according to the manufacturers instructions. Successively, membranes were incubated with infrared labeled secondary antibodies for Ih at room temperature, The bound complex was visualized using the Odyssey infrared Imaging System (Li-Cor; Lincoln, NE), The images were analyzed using the Odyssey Application Software, version 1.2 (Li-Cor) to obtain the integrated intensities.

Statistical analysis

[0166] Data were expressed as the mean ± SD. For assessing the difference between values, the Student's t test was used, A value of p<0.05 was considered statistically significant. n-3 TG administration reduces infarct size and improved cardiac function in LAD model

[0167] To test the effect of acute n-3 TG administration in myocardial ischemic injury, mice were subjected to 30 min of ischemia induced by LAD occlusion; coronary flow was then restored and myocardial functional recovery during reperfusion was assessed. IP injection of n-3 TG emulsion was administered immediately after ischemia at the onset of reperfusion and at 60 min into reperfusion. At the end of 48h of reperfusion, sections of heart were stained with TTC to quantify the extent of VR damage in both groups. FIG, 1A shows quantification of the infarct area in mice hearts from saline treated compared to n-3 TG treated group. Myocardial infarct size was significantly reduced (p<().05) in n-3 TG emulsion treated mice (vs saline treated mice). The total area at risk was similar for both groups. Plasma LDPi release, a key marker of myocardial injury, was significantly reduced in n-3 TG treated mice (FIG. IB). These data indicate that acute treatment of n-3 TG during reperfusion markedly reduces injury due to myocardial infarction in mice. [0168] Echocardiography assessment showed substantial differences in fractional shortening ( FS) between control and n-3 TG treated mice. A significant recovery of FS was observed in n-3 TG treated group vs saline treated controls (p<0.0i) (FIG. 1C). These data along with infarct size changes and LDH levels reduction reveal that acute n-3 TG treatment protects mice from myocardial ischeniia-reperfusion injury and improves heart function, in ex-vivo model n-3 TG protects myocardium from Ϊ/R injury

[0169] To investigate further the effect of acute intervention with n-3 TG emulsion after I/R, we also utilized the ex vivo perfused heart (I/R model). Our experiments showed that administration of n-3 TG emulsion during reperfusion in the ex vivo model significantly improved LVDP recovery after I/R (FIG. 2A), compare to control hearts. Reperfusion of the heart with KREB'S buffer + n-3 TG maintained normal rhythm and LVDP was nearly restored to 100% similar to pre-ischemia time.

[0170] During reperfusion period, heart perfusates were collected to detect LDH release, as markers of ischemic injury. LDH release appeared significant different between n-3 TG treated and control hearts, showing that acute n-3 TG treatment exhibits a protective role (FIG. 2B). n-3 TG modulates key signalling pathways linked to I/R injury.

[0171] To determine if n-3 TG protects hearts by modulating changes in key signalling pathways linked to I/R injury, p-AKT, p~GSK-3(J, and Bcl-2 were probed in myocardial tissue by western blotting, n-3 TG emulsion significantly increased phosphorylation of A T and GS 3p (FIG. 3), and Bcl-2 protein expression (FIG. 4), indicating that n-3 TG likely reduces apoptosis by activating the PBK-A KT-G8 3p signalling pathway and anti-apoptotic protein Bcl-2, Since Bci-2 interacts with Beclin-1^6, and influences autophagy, as shown in FIG. 4, the expression of Beclin-1 increased after ischemia reperfusion condition; n-3 TG treated hearts showed a significant reduction in Beclin-1 protein expression, with concomitant increase of Bcl-2 protein expression as we mentioned above, To establish the link between n-3 TG and PI3K/AKT and GS 3p pathways in I/R injury, hearts were treated with GS -3p inhibitor SB216763 (3μΜ) or Phosphatidylinositol 3-kinase (PI3K)/AKT inhibitor LY-294002 (ΙΟμΜ); each of them was added at the beginning of the baseline period and continued throughout ischemia and reperfusion. The doses of the inhibitors used in this study were based on publications in the literature . LDH release was significantly reduced by η-·3 TG, and protection afforded by n-3 TG was abrogated PDK/AKT inhibitor, LY-294002 (FIG-, 6). Treatment with SB-2.16763 plus n-3

TG emulsion significantly inhibited LDH release compared to I R control hearts (FIG, 6).

[0172] Next, hypoxia- inducible factor 1 (HIF-l) was investigated, which is a key mediator of adaptive responses to decreased oxygen availability in ischemia, HIF-la protein expression (FIG. 5) increased rapidly after ischemia. Administration of n-3 TG during reperfusion significantly inhibited the protein expression of HIF-l . In our lab, previous studies showed that n-3 fatty acids, in contrast to saturated fatty acids, are able to lower macrophages and arterial endothelial lipase and inflammatory markers and these effects are linked to PPAR-y^b. Accordingly, the potential association of PPAR-γ and n-3 TG acute treatment in I/R condition was examined. Western blot analysis showed that in n-3 TG treated hearts protein expression of PPAR-γ was significantly lower compared to the control hearts (FIG. 5).

[0173] In order to establish the link between PPAR-γ and n-3 TG effect, mice were treated with Rosiglitazone (6mg/kg body weight, IP injection), a common agonist of PPAR-y, 30 min before I/R injury in the isolated perfused hearts. These hearts were perfused with Krebs-Henseleit buffer without or with n-3 TG emulsion during reperfusion time. LDH release was significantly higher in Rosiglitazone plus n-3 TG treated hearts vs Rosiglitazone treated hearts (FIG. 6). These data indicate that PPAR-y reduction is linked to cardioprotection afforded by n-3 TG during I/R.

[0174] Taken together, these results suggest that PDK/AKT, GSK-3p and PPAR-y are key pathways modulating n-3 TG cardioprotection.

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Claims

CLAIMS WHAT IS CLAIMED IS:

1. A triglyceride omega-3 lipid-based oil-in- water emulsion suitable for administration to a patient,

(a) the emulsion comprises at least 7% to 35% omega-3 oil and less than 10% omega-6 oil by weight in grams per 100 ml of emulsion,

(b) the omega-3 oil comprises at least 20% triglyceride by weight per total weight of the omega-3 oil, and at least 20% wt.-% of the acyl-groups of the omega-3 triglycerides consist of DHA,

(c) the omega-3 oil comprises less than 10% omega-6 fatty acids, and

(d) the mean diameter of lipid droplets in the emulsion is less than about 5 microns.

2. The triglyceride omega-3 lipid-based oil-in- water emulsion of claim 1, wherein the omega-3 oil is fish oil, synthetic omega-3 oil or a combination thereof.

3. The triglyceride omega-3 lipid-based oil-in- water emulsion of claim 1, wherein from 20 wt- % to 50 wt.-% of the acyl-groups of the omega-3 triglycerides consist of DHA.

4. The triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1, wherein from 50 wt- % to 75 wt.-% of the acyl-groups of the omega-3 triglycerides consist of DHA.

5. The triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1, wherein from 75 to 90 wt.-% of the acyl-groups of the omega-3 triglycerides consist of DHA.

6. The triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1, wherein from 90 to 95 wt.-% of the acyl-groups of the omega-3 triglycerides consist of DHA.

7. The triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1, wherein from 95 to 100 wt.-% of the acyl-groups of the omega-3 triglycerides consist of DHA.

8. The triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1, wherein the lipid droplets are less than about 1 micron diameter.

9. The triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1, wherein the lipid droplets are from about 100 to about 500 nm diameter.

10. A method comprising

(a) identifying a subject who has undergone hypoxia-ischemia, and

(b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount the triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1 to reduce reperfusion damage caused by the hypoxia-ischemia.

11. The method of claim 10, wherein the hypoxia-ischemia causes cerebral hypoxia-ischemia, and the therapeutically effective amount of the triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1 is administered within 20 minutes to 2 hours after the cerebral hypoxia- ischemia to reperfusion damage caused by the hypoxia-ischemia.

12. The method of claim 10, wherein the hypoxia-ischemia causes cerebral hypoxia-ischemia, and the therapeutically effective amount of the triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1 is administered within 2 to 4 hours or 4 to 6 hours after the cerebral hypoxia-ischemia to reperfusion damage caused by the hypoxia-ischemia.

13. The method of claim 10, wherein the cerebral hypoxia-ischemia causes a stroke.

14. The method of claim 10, wherein the therapeutically effective amount is from about 0.2 g/kg/administration to about 4 g/kg/administration.

15. The method of claim 10, wherein the hypoxia-ischemia and the reperfusion damage caused by the hypoxia-ischemia occurs in an organ selected from the group consisting of brain, heart, kidney, spinal cord, large or small intestine, pancreas and lung.

16. The method of claim 10, wherein hypoxia-ischemia and the reperfusion damage caused by the hypoxia-ischemia causes a myocardial infarction or a cerebral infarction.

17. A method for reducing cell death or damage in an organ or tissue caused by hypoxia- ischemia or reperfusion damage caused by the hypoxia-ischemia comprising administering to a patient in need thereof a pharmaceutical composition comprising a therapeutically effective amount the triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1.

18. The method of claim 17, wherein the hypoxia-ischemia or reperfusion damage caused by the hypoxia-ischemia is caused by organ transplantation.

19. The method of claim 17, wherein the cell death or cell damage occurs in an organ selected from the group consisting of brain, heart, kidney, spinal cord, large or small intestine, lung, liver, and pancreas.

20. The method of claim 17, wherein the hypoxia-ischemia causes a stroke.

21. The method of claim 17, wherein the hypoxia-ischemia causes a myocardial infarction.

22. The method of claim 17, wherein the therapeutically effective amount is from about 0.2 g/kg/administration to about 4 g/kg/administration.

23. A method comprising:

(a) identifying a subject who is at risk of having a cerebral hypoxia-ischemia, and

(b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1, thereby reducing the risk of the subject developing reperfusion damage caused by the hypoxia-ischemia.

24. A method comprising:

(a) identifying a subject who has inflammation in an organ, and

(b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1, thereby reducing the inflammation in the subject.

25. A method comprising administering to a subject an amount of a pharmaceutical composition comprising the triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1, to reduce production of reactive oxygen species in the blood or in an organ in the subject.

26. The method of claim 25, wherein the organ is selected from the group consisting of brain, heart, kidney, spinal cord, large or small intestine, lung, liver, and pancreas.

27. A method for reducing adverse cytokine production in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the triglyceride omega-3 lipid-based oil-in- water emulsion of claim 1.

28. The emulsion of claim 1, wherein the emulsion comprises at least 20% to 30% omega-3 oil by weight in grams perl 00 ml of emulsion.

29. The emulsion of claim 1, wherein the emulsion comprises at least 30% to 35% omega-3 oil by weight in grams per 100 ml of emulsion.

30. The emulsion of claim 1, wherein the omega-3 oil comprises at least 30% to 40% triglyceride by weight per total weight of the omega-3 oil.

31. The emulsion of claim 1, wherein the omega-3 oil comprises at least 40% to 50% triglyceride by weight per total weight of the omega-3 oil.

32. The emulsion of claim 1, wherein the omega-3 oil comprises at least 50% to 75% triglyceride by weight per total weight of the omega-3 oil.

33. The emulsion of claim 1, wherein the omega 3 oil comprises at least 75% to 100% triglyceride by weight per total weight of the omega-3 oil.

34. The triglyceride omega-3 lipid-based oil-in- water emulsion of claim 1, wherein the emulsion comprises less than 5% omega-6 fatty acid.

35. The triglyceride omega-3 lipid-based oil-in- water emulsion of claim 1, wherein the emulsion comprises less than 1% omega-6 fatty acid.

36. The triglyceride omega-3 lipid-based oil-in- water emulsion of claim 1, wherein the emulsion comprises at least 35% omega-3 oil by weight in grams per 100 ml of emulsion and less than 3% omega-6 oil.

37. A method comprising (a) identifying a subject who has inflammation in an organ or a tissue, and (b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount the triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1 thereby reducing the inflammation in the subject.

38. A method for reducing cell death or damage or hypoxia/ischemia in an organ or tissue comprising administering to a an organ donor a pharmaceutical composition comprising a therapeutically effective amount the triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1 prior to harvesting the organ or tissue from the organ donor.

39. A method for reducing cell death or damage or hypoxia/ischemia in an organ or tissue to be transplanted into an organ or tissue recipient, comprising administering to the recipient a pharmaceutical composition comprising a therapeutically effective amount the triglyceride omega-3 lipid-based oil-in-water emulsion of claim 1 prior to surgically implanting the organ or tissue in the recipient.