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EP2958559A1 - Utilisation d'une émulsion à base de lipides oméga-3 pour protéger les organes humains d'un accident ischémique - Google Patents

Utilisation d'une émulsion à base de lipides oméga-3 pour protéger les organes humains d'un accident ischémique

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Publication number
EP2958559A1
EP2958559A1 EP14754815.0A EP14754815A EP2958559A1 EP 2958559 A1 EP2958559 A1 EP 2958559A1 EP 14754815 A EP14754815 A EP 14754815A EP 2958559 A1 EP2958559 A1 EP 2958559A1
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EP
European Patent Office
Prior art keywords
omega
lipid
oil
ischemia
emulsion
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14754815.0A
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German (de)
English (en)
Other versions
EP2958559A4 (fr
Inventor
Richard J. Deckelbaum
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Columbia University in the City of New York
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Columbia University in the City of New York
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Publication of EP2958559A1 publication Critical patent/EP2958559A1/fr
Publication of EP2958559A4 publication Critical patent/EP2958559A4/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A61K31/232—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having three or more double bonds, e.g. etretinate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/60—Fish, e.g. seahorses; Fish eggs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles

Definitions

  • Cerebral hypoxia-ischemia 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.
  • 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.
  • 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.
  • 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.
  • the present invention fulfills this need. Stroke is the 3 ⁇ (1 -4 ⁇ 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.
  • 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.
  • 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.
  • Methods of the present invention also provide further comprise administering a conventional stroke treatment or preventative medication.
  • Omega-3 lipid-based emulsions of the present invention comprise at least 10%, preferably at least 20%, omega-3 oil, by weight.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 1A-1B are graphs that illustrate blood TG and glucose levels after TG emulsion injections.
  • FIG. 2 is a graph that illustrates n-3 TG injection and cerebral blood flow after H/I.
  • Cerebral blood flow was measured by laser Doppler flowmetry (LDF) in neonatal mice after carotid artery ligation.
  • 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.
  • TTC grey for living tissue and white for the infarcted tissue
  • 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. 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
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 11A-11B are graphs that illustrate acute n-3 TG injection decrease of brain Ca 2+ induced opening of mitrochondrial permeability transition pores (mPTP) after H/I. After H/I and after n-3 TG injection, mitochondrial function is maintained.
  • mPTP mitrochondrial permeability transition pores
  • 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.
  • FIG. 13A-13B are graphs that illustrate n-3 DG decreases brain injury and infarct volume in H/I neonatal mice.
  • 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).
  • 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.
  • FIG. 16 is a diagram that illustrates an in vivo left anterior descending coronary artery (LAD) occlusion model.
  • LAD left anterior descending coronary artery
  • FIG. 17 is a graph showing that n-3 TG decreases mitochondrial production of reactive oxidation species [ROS].
  • 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.
  • omega-3 oils means any omega-3 fatty acid, including free omega-3 fatty acids and omega-3 triglycerides, diglycerides and monoglycerides.
  • 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; A 3 ' 6 ' 9 ), eicosapentaenoic acid (20:5n-3; EPA; A 5 ' 8 ' 11 ' 14 ' 17 ), docosahexaenoic acid (22:6n-3; DHA) and docosapentaenoic acid (22:5n-3; DPA; A 7 ' 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.”
  • 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.
  • omega-3 tri/diglyceride means that omega-3 fatty acid comprises an omega-3 triglyceride and/or a diglyceride or any combination thereof.
  • 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.
  • 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.
  • the amount of fatty acid such as EPA or DHA in a glyceride is expressed as the wt. of the acyl groups of the respective glyceride.
  • hypoxia refers to a shortage of oxygen in the body or in a specific organ or tissue
  • 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.
  • stenosis chronic narrowing of a supply artery that is often caused by atherosclerotic disease
  • 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.
  • organ ischemia-hypoxia e.g., stroke, myocardial infarction, intestinal volvulus, etc.
  • parenteral injections such as from lipid emulsions for immediate action.
  • hypoxia-ischemia refers to the occurrence of both hypoxia and ischemia in a tissue or organ.
  • 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.
  • 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.
  • 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.
  • this process takes place as quickly as possible.
  • the human body is capable of synthesizing certain types of fatty acids.
  • 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.
  • 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%.
  • C18:2n-6 Linoleic acid
  • Arachidonic acid C20: 4n-6
  • Most vegetable oils i.e., soybean and safflower
  • high omega-6 polyunsaturated fatty acids most in the form of 18:2 ( ⁇ 9 ' 12 ) -linoleic acid) content but low omega-3 (predominantly 18:3( ⁇ 9 ' 12 ' 15 )-a-linolenic acid) content.
  • Essential fatty acids may be obtained through diet or other enteral or parenteral administration.
  • 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.
  • 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.
  • 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.
  • DHA docosahexaenoic acid
  • AA membrane arachidonic acid
  • Cao investigated the effects of chronic ethyl docosahexaenoate (E-DHA) administration on mortality and cerebral edema induced by transient forebrain ischemia in gerbils.
  • GB 2388026 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • omega-3 triglyceride particles VLCT
  • LCT sterol regulatory element
  • 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.
  • 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.
  • 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.)
  • 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%).
  • Intralipid® a soy oil based emulsion containing 55% of its fatty acids as omega-6 linoleic acid
  • DHA DHA
  • 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.
  • 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 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.
  • n-3 Tri-DHA emulsions are administered after H/I had a neuroprotective effect against cerebral infarction and reperfusion damage.
  • 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.
  • 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.
  • 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.
  • n-6 TG emulsions having no DHA or EPA 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.
  • n-3 TG90-DHA30 administered 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.
  • FIG. IA shows 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.
  • 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 shows that
  • 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.
  • 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.
  • FIG. 10 shows that n-3 TG90-DHA30 attenuated brain injury after hypoxia/ischemia.
  • 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.
  • 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.
  • mPTP mitochondrial permeability transition pores
  • FIG. 13 shows that both n-3 TG and n-3 DG (diglyceride) emulsions reuce cerebral infarct size.
  • 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).
  • NPDl neuroprotectin Dl
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • TG-DHA triglycerides in the omega-3 oil of which at least 20% are DHA are needed to treat hypoxia/ischemia and reperfusion damage.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • 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%.
  • 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%.
  • the oil is typically synthesized.
  • 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.
  • enterally for example, orogastric or nasogastric
  • parenterally for example, subcutaneous, intravenous, intramuscular, intraperitoneal.
  • Most preferably the emulsion is administered intravenously.
  • Omega-3 lipid-based emulsions of the present invention are preferably provided at a dose capable of providing a protective benefit.
  • 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.
  • 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.
  • 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.
  • 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.
  • therapies such as surgery and angioplasty
  • medications given to prevent or treat hypoxia-ischemia.
  • 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.
  • Omega-3 lipid-based emulsions may be oil-in-water (o/w) emulsions in which the outer continuous phase consists of distilled water purified or sterilized for parenteral purposes.
  • 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.
  • 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.
  • 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.
  • 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.
  • median lipid droplet sizes may be less than about 1 ⁇ 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.
  • mean diameter of the lipid droplet can be larger, for example, from about 1 ⁇ and 5 ⁇ .
  • 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.
  • 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.
  • the omega-3 tri/diglyceride contains at least 40% (up to 100%) of their fatty acids as EPA and/or DHA.
  • 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.
  • 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.
  • 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.
  • 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.
  • a stabilizing or isotonizing additive such as a polyhydric alcohol
  • Preferred stabilizing or isotonizing additives include glycerol, sorbitol, xylitol or glucose. Glycerol is most preferred.
  • 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.
  • auxiliary agents and/or additives such as emulsifiers, emulsifying aids (co- emulsifiers), stabilizers, antioxidants, and isotonizing additives.
  • Emulsifiers may include physiologically acceptable emulsifiers (surfactants) such as phospholipids of animal or vegetable origin.
  • 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.
  • the emulsifier is 1.2 mg of egg yolk lecithin/100 ml emulsion.
  • Alkali metal salts preferably sodium salts, of long chain, C 16 to C 2 8 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.
  • 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.
  • 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 ⁇ - and ⁇ -tocopherol, and/or ascorbyl palmitate as antioxidants and thus for protection from peroxide formation.
  • an antioxidant such as vitamin E, in particular a-tocopherol (the most active isomer of vitamin E in humans) as well as ⁇ - and ⁇ -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.
  • 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.
  • 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.
  • 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.
  • compositions of the invention may further comprise various pharmaceutically active ingredients.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Absorption accelerators may include, but are not limited to, quaternary ammonium base, sodium laurylsulfate, and the like.
  • 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.
  • 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.
  • a liquid carrier such as water, vegetable oil, alcohol, polyethylene glycol, propylene glycol or glycerin.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a buffer such as guconic acid, lactic acid, citric acid or acetic acid, sodium guconate, sodium lactate, sodium citrate or sodium acetate.
  • injectable pharmaceutical compositions 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.
  • 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.
  • 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.
  • dissolving agents such as 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
  • compositions of the invention may further comprise various pharmaceutically active ingredients.
  • 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.
  • 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.
  • 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.
  • PUPA omega-3 long chain polyunsaturated fatty acids
  • Incorporation of EPA and DHA also increases membrane fluidity and flexibility.
  • Example 1 60 minutes of hypoxia-ischemia
  • omega-3 lipid-based emulsion 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)
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • 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 Triphenyl-2H-tetrazolium chloride
  • 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:
  • Example 3 Treatment of rats with omega-3 triglyceride lipid emulsion prior to 60 minutes of hypoxia
  • the rats were euthanized and their brains removed, cut into 2 mm sections and stained with 2,3,5 triphenyl-2H-tetrazolium chloride (TTC).
  • TTC 2,3,5 triphenyl-2H-tetrazolium chloride
  • 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.
  • 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.
  • SEM serum-derived microsomal protein
  • control 0.78 + 0.16 emulsion-treated
  • 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.
  • Example 5 Quantification of effects of omega-3 triglyceride treatment on cellular targets
  • ROS reactive oxygen species
  • 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 ⁇ £ ⁇ ). Sections are co-stained with antibodies recognizing neuronal specific proteins (Tau), astrocytes (GFAP) or microglia.
  • activated proteins known to participate in neuronal apoptosis caspase 3, Jun N- terminal kinases
  • neuronal survival activated Akt, phosphorylated BAD, FKHR
  • CAM KII protein kinase C isoforms, in particular PKCy and ⁇ £ ⁇
  • Sections are co-stained with antibodies recognizing neuronal specific
  • 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).
  • omega-3 n-3) triglyceride fish oil- based
  • omega-6 n-6) soy oil-based emulsions were commercially prepared intravenous phospholipid- stabilized emulsions.
  • the omega-3 triglyceride contained high concentrations of n-
  • 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.
  • 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.
  • Oleic acid (CI 8: ln-9) 6-13 19-30
  • Linoleic acid (C18:2n-6) 1-7 44-62
  • Tri-DHA 99% DHA
  • 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 N 2 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.
  • LPB lipoprotein-free buffer
  • sucrose 100 mg/1 mL LPB
  • the lipid emulsions were then sonicated for 1 hr at 50° C, 140 W under a stream of N 2 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.
  • 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.
  • TTC triphenyl-tetrazolium chloride
  • 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.
  • Protocol 1 Pre-H/I treatment of n-3 TG (containing both DHA and EPA) or n-6 TG emulsions.
  • n-3 TG or n-6 TG emulsions or vehicle 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.
  • n-3 emulsions contain low concentrations of alpha-tocopherol as an anti-oxidant agent
  • an equivalent dose of pure alpha-tocopherol to match the content of n-3 emulsion content 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.
  • alpha- tocopherol Vital E®, Intervet, Schering Plough
  • Protocol 2 Post-H/I treatment of n-3 TG ( containing both DHA and EPA ).
  • n-3 TG emulsion or saline 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • coronal sections (10 ⁇ every 500 ⁇ ) 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.
  • Example 7 n3 Fatty Acid Rich TG Emulsions are Neuroprotective after Cerebral Hypoxic- Ischemic Injury in Neonatal Mice
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • Example 8 Omega-3 Triglyceride DHA emulsions in Cardiac Hypoxia-Ischemia Material and Methods Animal Care
  • mice 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).
  • the primary antibodies used were Bcl-2, Beclin-1, PPAR- ⁇ , p-AKT, total- AKT, p-GSK- 3 ⁇ , total- GSK-3P (Cell Signaling, USA); and ⁇ -actin (BD Biosciences Pharmingen, USA).
  • the secondary antibodies used were anti-rabbit IRdye800, anti-mouse IRdye700 (1:50,000 dilution).
  • SB216763 (3 ⁇ ), Rosiglitazone (6mg/kg body weight) were purchased from Sigma- Aldrich, USA.
  • Phosphatidylinositol 3-kinase (PI3K)/AKT inhibitor LY-294002 (10 ⁇ ) was purchased from Calbiochem.
  • 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%).
  • mice 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 ⁇ ⁇ and a rate of 110 breaths per minute, and supplemented with oxygen. Body temperature was maintained at 37°C.
  • PE-60 polyethylene-60
  • 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 area 4 ' 5 .
  • IP intraperitoneal
  • 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.
  • isoflurane 1.5-2.0 L/min
  • LVEDD LV end-diastolic diameter
  • LVESD LV end-systolic diameter
  • EF LV ejection fraction
  • FS LV fractional shortening
  • 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.
  • 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
  • mice 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 CaCl 2 , 1.2 MgCl 2 , 25 NaHC0 3 , 5 Glucose, 0.4 Palmitate, 0.4 BSA, and 70 mU/1 insulin.
  • Perfusion p0 2 > 600 mmHg was maintained in the oxygenation chamber.
  • LVDP Left ventricular developed pressure
  • 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 hearts, we added to the standard buffer an emulsion with n-3 fatty acids packaged in triglyceride (TG); we used 300mg/100ml as final amount.
  • TG triglyceride
  • 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 C C 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.
  • 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.
  • 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.
  • n-3 TG modulates key signalling pathways linked to I/R injury.
  • 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.
  • 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.
  • 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 .
  • the potential association of PPAR- ⁇ 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- ⁇ was significantly lower compared to the control hearts (FIG. 5).
  • 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.
  • N-3 fatty acid rich triglyceride emulsions are neuroprotective after cerebral hypoxic-ischemic injury in neonatal mice. PLoS One; 8(2):e56233.
  • n-3 fatty acids are neuroprotective after cerebral hypoxia-ischemia in rodent models. FASEB J 23: 334.335 (Abstract).
  • Triglycerides in fish oil affect the blood clearance of lipid emulsions containing long- and medium-chain triglycerides in mice. J Nutr 136: 2766-2772.

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Abstract

La présente invention concerne des procédés visant à limiter la mort ou la dégradation cellulaire ou les lésions de reperfusion résultant d'une hypoxie-ischémie, lesdits procédés consistant à administrer une émulsion à base de lipides oméga-3 après un accident hypoxique-ischémique. L'émulsion à base de lipides oméga-3 comprend, de préférence, environ 10 à 35 % en poids en grammes d'huile d'oméga-6 par 100 ml d'émulsion; l'huile d'oméga-3 contient environ 20 à 100 % de triglycérides en poids par rapport à son poids total et environ 20 à 100 % en poids des groupes acyle des triglycérides oméga-3 correspondent à du DHA; l'huile d'oméga-3 contient moins de 10 % d'acides gras oméga-6; et le diamètre moyen des gouttelettes lipidiques de l'émulsion est inférieur à environ 5 microns.
EP14754815.0A 2013-02-20 2014-02-20 Utilisation d'une émulsion à base de lipides oméga-3 pour protéger les organes humains d'un accident ischémique Withdrawn EP2958559A4 (fr)

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