ES2365628T3 - COMPOSITIONS AND METHODS TO IMPROVE FUNCTIONAL VASCULAR INTEGRITY, CELL SURVIVAL AND REDUCTION OF APOPTOSIS AFTER AN ISCHEMICAL EPISODE IN THE BRAIN. - Google Patents

Abstract

Use of one or more long-chain polyunsaturated fatty acids (LCPUFA), and one or more nitric oxide-releasing compounds (NORC) in the preparation of a drug or co-administration medications for the enhancement of vascular integrity, reducing cell apoptosis in the place where an ischemia is located in an animal.

Description

FIELD OF THE INVENTION

The present invention relates to mammalian nutrition and its effects on the enhancement of vascular integrity and protection against cellular damage associated with ischemia or an episode of ischemia in the brain. In particular, the present invention uses combinations of compounds that release nitric oxide, long chain polyunsaturated fatty acids, and optionally other ingredients including antioxidants, anti-inflammatory agents, growth factors and B vitamins, administered as part of a regular diet, to improve vascular integrity, promote cell survival and thereby reduce ischemic injury in the brain and / or other organs, for example, in the eventuality of cerebral ischemia.

BACKGROUND OF THE INVENTION

Several publications, including patents, published applications, technical articles and scholarly articles, are cited throughout the specification.

Hypoxic ischemic brain injury occurs as a result of a decreased supply of oxygen to the brain due to a temporary or permanent reduction or blockage of blood flow to the brain. Ischemia of the brain results in an attack with subsequent apoptosis and necrosis of brain tissue, which leads to a heart attack. Similar to cardiovascular ischemia, cerebral ischemia can be caused by several factors such as blood clots, thrombosis, embolisms, atherosclerotic plaque blockage or other vascular obstructions. Hypercholesterolemia, hypertension, diabetes, obesity, among other factors, have been identified as risk factors for ischemic attacks. Ischemic attacks are a leading cause of death of human beings worldwide and also affect other animals, including companion animals.

Systemic lesions are also known to contribute significantly to brain aging and dementia. Dementia is an important disease that affects the older population. The incidence of dementia increases from 0.3 to 1% in people aged 60 to 64 and 50% in people over 95 with a double dominance every 5 years (Mc Dowell, I, Aging (Milano ) 13: (2001) 143-162). In western populations, Alzheimer's disease (AD) is considered the most important form of dementia, while vascular dementia (RV) seems to be the predominant form in eastern populations (Fratiglioni, L. et al., Drugs Aging (1999) 15: 365-375; Ott, A., et al., The Rotterdam Study, BMJ (1995) 310: 970-973). Increasing evidence indicates that vascular dementia and Alzheimer's disease often coexist in the population with dementia. For example, Kalaria (Neurobiol Aging (2000) 21: 321-330) reports that approximately 90% of patients with AD have a cerebrovascular pathology and approximately 30% of RV patients have an AD pathology. The coexistence of a cerebrovascular lesion and AD in the population with premature dementia was confirmed by Snowdon et al., (J. Am. Med. Soc. (1997) 277: 813-817). In addition, the hallmark of the pathological diagnosis of AD (amyloid position, and neurofibrillary tangles) was determined to exist in the elderly without dementia (Snowdon et al., 1997, supra). These data suggest that amyloid deposition and neurofibrillary tangles are not sufficient to cause dementia in AD patients, highlighting the important contribution of ischemic brain lesions to brain aging, and the development and progression of both RV and AD (Korczyn, AJ Neurol. Sci. (2005) 230: 3-6). Therefore, the prevention of ischemia-induced brain injury and apoptosis of neurons in humans and animals can delay the aging of the brain, reduce the risk of cognitive disability, and delay the progression of dementia.

Ischemia can occur in any tissue, but is more commonly associated with brain, heart or kidney lesions. As already described above, ischemia in the brain can cause serious injury, for example, stroke, or chronic disability such as dementia and related conditions. In the heart, ischemic episodes can result in heart failure; similarly it can result in kidney failure from ischemic episodes in the kidney.

During stroke or ischemic episodes of the brain or any other tissue, biochemical reactions occur in the vascular system that can lead to edema, hemorrhagic transformation, and a further danger in the affected tissue, for example neurological. As such, the treatment and protection of the vascular system has been identified as a potential way to explore acute ischemic attack in order to develop new therapies. In addition, vascular protection has implications for reducing tissue injury that occurs as a result of an ischemic episode.

The vascular endothelium has been shown to serve several regulatory functions, including modulation of vascular tone, inflammation, and homeostasis by maintaining a non-adhesive, antithrombocytic surface. (Boak, L., et al., Cur. Vasc, Pharmacol. (2004) 2: 45-52). Vascular homeostasis is partly a function of the contraction and relaxation of vascular smooth muscle, which is mediated by a variety of factors, including the free nitric oxide (NO) radical, which is a strong relaxant of vascular smooth muscle. .

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NO is produced in three different ways: through the enzymes NO synthase (NOS), through the neuronal NOS (nNOS), which are primarily located in the nervous tissue and generate NO for neurotransmission; inducible NOS (iNOS), which are primarily found in macrophages and can respond to pro-inflammatory mediators; and endothelial NOS (eNOS), which are produced by endothelial cells such as those found in the vascular endothelium (Michel, T., et al., J. Clin. Invest. (1927) 100: 2146-2152 , Moncada, S., et al., Pharmacol. Rev. (1991) 43: 109-142, and Nathan, C. (1992) FASEB J 6: 3051-3064). The NO released by the eNOS plays an important role in vasodilation, smooth muscle proliferation, blood pressure regulation, and has anticoagulant and anti-inflammatory effects through platelet inhibition and leukocyte adhesion and aggregation. (Gewaltig, MT, et al., Cardiovasc. Res. (2002) 55: 250-260, De Graaf, JC et al. Circulation (1992) 85: 2284-2290, Freedman, JE, et al. Circ. Res. (1999) 84: 14161421, Furchgott, RF, et al. FASEB J (1989) 3: 2007-2018, Gaboury, J., et al. Am J Physiol. Heart. Circ. Physiol. (1993) 265: H862- H867, and, Kubes, P., et al. Proc. Natl. Acad. Sci. USA (1991) 88: 4651-4655). A decrease in NO production during prolonged ischemia has been associated with an endothelial lesion (Laude, L., et al. Braz. J. Med. Biol. Res. (2001) 34: 1-7).

NOS enzymes oxidize L-arginine in citrulline, resulting in NO formation as a byproduct. In this way it has been suggested that supplementation with NO precursors such as L-arginine, can improve vascular health, and may facilitate the repair of vascular disease states. Indeed, arginine supplementation of the diet has been shown to induce the restoration of vasodilation and improves coronary circulation in animal models and human patients with hypercholesterolemia, as well as also improves endothelial function in general, in patients with artery disease. coronary artery (Boak, L., et al., 2004). In addition, supplementation with L-arginine has also been shown to increase NO levels and attenuate myocardial injury mediated by free radicals of O2 in human patients (Kiziltepe, U., et al., Int. J. Cardiol. ( 2004) 97: 93-100). More recent studies have shown that arginine supplementation facilitates endothelial repair in the intestine of rats followed by induced intestinal ischemic reperfusion (Sukhotnik, I., et al, Pediatr. Surg. Int. (2005) 3: 191-196). Thus, the administration of NO precursors such as L-arginine has implications for cell activation and protection, especially with respect to vascular endothelial cells.

Fatty acids have also been shown to modulate the activation of the endothelial cell. Administration of long chain polyunsaturated fatty acids (LCPUFA) was found to inhibit lymphocyte adhesion to vascular endothelial cells (Khalfoun, B., et al. Transplantation (1996) 62: 1649-1657). Subsequent investigations determined that the administration of LCPUFA reduces the expression of endothelial cells of adhesion molecules and cytokines in response to stimulation, suggesting that LCPUFAs have antiheterogenic and anti-inflammatory properties. (From Caterina R., et al., Am. J. Clin. Nutr. (2000) 71 (supl) 213S-223S). Increasing evidence indicates that regular consumption of LCPUFA, particularly n-3 fatty acids, protects the cardiovascular system, reduces the formation of atherosclerotic plaques and reduces the risk of mortality from cardiovascular disease, particularly following myocardial infarction. . (Calder, P.C. Clin. Sci. (Lond). 2004 107: 1-11). In contrast, there has been little investigation of the effect of diet LCPUFAs on the vascular system and brain tissue, particularly with respect to an ischemic episode in the brain.

It has long been known that oxidative stress and injury is involved in the aging process. Oxidative stress has also been shown to play a role in the pathogenesis of a number of neurodegenerative diseases, including Alzheimer's disease and certain forms of dementia (eg, Smith, MA, Dementia & Geriatric Cognitive Disorders ("Dementia and cognitive disorders geriatrics ") (1999), vol. 10, supplement 1). Consequently, antioxidant supplementation has been used to delay the aging process and protect against the degenerative effects of oxidative stress. Similarly, the folic acid of vitamins B, vitamin B-6, and vitamin B-12, are important for the well-being and normal functioning of the brain. The status of these vitamins is often inadequate in the elderly, and these inadequacies may result in hyperhomocysteinemia, a newly identified risk factor for atherosclerosis (Temple ME, et al., Ann. Pharmacother 2000; 34: 57-65; Hankey GJ, et al., Lancet 1999; 354: 407413) and Alzheimer's disease (Clarke R, et al., Arch. Neurol. 1998 55: 1449-1455). These inadequacies can result in ischemia of the brain or other tissues via occlusive vascular disease, stroke, or thrombosis.

DE 10214005 discloses a composition comprising 5-15 g of pectin and / or guar and 1-3 g of EPA / DHA in a preferred ratio of 2 to 3, whereby omega-3 fatty acids are stabilized against oxidation with vitamin C. and / or vitamin E, and before mixing with pectin / guar, they are flavored with caramel or vanilla. The described composition may also include 5 g of arginine. The document describes that omega-3 fatty acids have a positive effect on blood pressure, cardiac arrhythmia, heart attack, stroke risk, arteriosclerosis, and on inflammatory diseases such as rheumatism, psoriasis, arthritis. , ulcerative cellitis and Crohn's disease.

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US 2002/0182196 refers to a composition and method for the normalization of affected or impaired neurological function. The document describes that the affected or impaired neurological functions are manifested by a variety of conditions that encompass a spectrum of nervous system dysfunction states. Non-limiting examples of such conditions include memory impairment, certain behavioral problems, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), other attention deficits and / or hyperactivity syndromes, dementia or cognitive decline of multiple etiologies, various genetic disorders (for example, Down syndrome, fragility X syndrome, etc.), trauma of the central nervous system (CNS), poisoning (acute and chronic) or poisoning, autoimmune mechanisms, anoxic conditions -ischemic, neurodegenerative disorders, metabolic disorders of the nervous system, vascular disorders, hypertensive encephalopathy, rheological disorders, demyelination, cerebral edema, inflammatory neuronal conditions, learning problems, impulsive behavior, specific emotional problems or mood problems, low difficult functioning pressure condition It is several latrogenic, infections, eleptogenic foci and congenital malformations of the brain.

The document describes that the composition contains effective amounts of:

(TO)

at least one agent that promotes the synthesis of adenosine triphosphates (ATP) and / or creatine phosphate in the body;

(B)

at least one antioxidant for free radical scavenging in at least one body route;

(C)

at least one agent for the normalization or maintenance of the functioning of the membrane and the structure in the body;

(D)

at least one agent for normalization or maintenance of the normal functioning of the neurotransmitter in the body;

(AND)

at least one agent for the downward cortisone regulatory action; Y

(F)

at least one agent to suppress the activation of apoctotic pathways in the body, with possible inclusion of one or more of the following:

(G)

at least one agent to suppress inflammation in the body,

(H)

at least one agent to normalize or maintain the function of the vascular wall and the structure in the body;

(I)

at least one people to normalize or maintain the function of nerve growth factors and / or neurotropic factors in the body;

(J)

at least one agent to suppress the toxic effects of metal ions;

(K)

at least one agent to normalize or maintain the metabolism of methyl in the body;

(L)

at least one agent for the normalization or maintenance of the metabolism of insulin and glucose in the body; Y

(M)

at least one agent for the maximum regulatory activity of heat shock proteins in the body.

Sections (B) and (H) may include arginine, and

Section (C) may include gamma linolenic acid and higher polyunsaturated LCPUFAS, such as DHA.

SUMMARY OF THE INVENTION

The invention provides uses and compositions for use as defined in the appendix claims. Compositions comprising one or more long chain polyunsaturated fatty acids (LCPUFA) and one or more nitric oxide releasing compounds (NORC) in an amount effective for the enhancement of vascular integrity, for example in the brain, and / are described herein. or the reduction of apoptosis in an animal. The enhanced vascular integrity, for example of the brain, and / or cell survival serves to reduce ischemic tissue damage, for example the brain, in the event of an ischemic episode.

In certain versions, the composition is a food composition for pets, a dietary supplement, or a food product formulated for human consumption. In several versions, LCPUFAs include at least one arachidonic acid, an eicosapentaenoic acid, a docosapentaenoic acid or an acid. docosahexaenoic acid, and the NORCs include at least one L-arginine or derivatives thereof. The compositions may comprise additional ingredients. For example, one or more antioxidants, B vitamins, cell growth factors, or anti-inflammatory agents may be included.

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In certain versions, the compositions are formulated for pets, such as a dog or a cat. In other versions, the compositions are formulated for human consumption.

Methods for the enhancement of vascular integrity and for the reduction of ischemia-induced brain injury in an animal are described herein, which comprise administration to the animal, on a regular basis, of a composition comprising one or more LCPUFA and one or more NORC, optionally supplemented with additional ingredients as described above, in an amount effective to enhance vascular integrity in the animal's tissue (s) and / or reduce ischemia-induced brain injury in the eventuality of an ischemic episode in the brain of the animal. In certain versions, the method is applied to a companion animal such as a dog or a cat. In other versions, the method is applied to humans.

In certain versions, regular administration of the composition to the animal results in a reduction of brain injury in the case of an ischemic episode in the animal's brain. In another version, regular administration of the composition to the animal results in a reduction in cellular apoptosis at the site of the animal where ischemia has occurred, which can be any tissue including the brain, heart and kidney.

Other features and advantages of the invention will be apparent with reference to the drawings, for the detailed description and for the following examples:

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Bar graph showing the effect of control and test diets on the volume of an ischemic lesion in all rats subject to transient cerebral ischemia. The compositions of the control and the test diets are described in example 1 (* p <0.01 versus the control). Figure 2. Bar graph showing the effect of control and test diets on the volume of ischemic lesion in rats that show lesions after transient cerebral ischemia. The compositions of the control and test diets are described in example 1 (* p <0.01 versus control). Figure 3. Bar graph showing the effect of control and test diets on cerebral apoptosis induced by ischemia in rats. The compositions of the control and test diets are described in example 1 (* p <0.001 versus control). Figure 4. Graph showing the correlation between the volume of the ischemic lesion (X axis) and the percentage of apoptotic cells as defined in TUNEL staining (Y axis) in rats subject to transient cerebral ischemia. The compositions of the control and test diets are described in example 1.

DETAILED DESCRIPTION OF ILLUSTRATIVE VERSIONS

Definitions

A variety of terms related to the methods and other aspects of the present invention are employed in the course of the specification and of the claims. These terms have the ordinary meaning given to them in the technique unless otherwise indicated. Other specifically defined terms must be interpreted in a manner consistent with the definition provided here.

The following abbreviations may be used in the specification and in the examples: AA, arachidonic acid; ALA, alfalinolenic acid; ANOVA, analysis of variance; DHA, docosahexaenoic acid; DM, dry matter; DPA, docosapentaenoic acid; EPA eicosapentaenoic acid; LA linoleic acid; LCPUFA, long chain polyunsaturated fatty acid; NORC, nitric oxide release compounds; L-Arg, L-arginine.

Within the context of this specification the term "approximately" is interpreted to mean optionally more or less 20%, more preferably optionally more or less 10%; still more preferably optionally more or less 5%, more preferably optionally more or less 2%.

"Effective amount" refers to an amount of a compound, material, or composition as described herein, which is effective in achieving a particular biological result. Such results include, but are not limited to, the enhancement of vascular integrity or the reduction of ischemic brain injury. This effective activity can be achieved, for example, by administering the compositions of the present invention to the animal.

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The term "vascular integrity" refers to the total health of blood vessels in an animal, including, without limitation, healthy vascular tone and structure, healthy vascular growth, healthy constriction and procrastination, low permeability, low proliferation, low inflammation, low adhesion of cells and / or platelets, maintenance of normal blood pressure, and blood flow, and the like. "Enhanced vascular integrity" refers to any improvement in the overall health of blood vessels in an animal, measured by any suitable means of the technique.

As used herein, "long chain polyunsaturated fatty acids" or "LCPUFA" refers to any monocarboxylic acid having at least 20 carbon atoms and at least two double bonds. Non-limiting examples of LCPUFA include fatty acids (n-6) such as arachidonic acid, and fatty acids (n-3) such as eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid.

As used herein, the term "compounds that release nitric oxide" or "NORC" refers to any compound that causes the release of free nitric oxide in an animal. Preferred examples of such compounds include L-arginine and analogues or derivatives of L-arginine such as, for example, arginine alpha-ketoglutarate, GEA 3175, sodium nitroprusside, glyceryl trinitrate, S-nitroso-N- acetyl-penicillamine, nitroglycerin, SNO-glutathione, non-steroidal anti-inflammatory drugs conjugated to NO, such as NOnaproxen, NO-aspirin, NO-ibuprofen, NO-diclofenac, NO-flurbiprofen and NO - ketoprofen, NO releasing compound 7, NO releasing compound 5, NO releasing compound 12, NO releasing compound 18, diazenium diolates and derivatives thereof, diethylamine NONOate or any organic or inorganic compound, biomolecules, or analogs, homologues, conjugates, or derivatives thereof, which cause the release of free nitric oxide in an animal.

The term "ischemia" refers to any decrease or interruption in the blood supply to any body organ, tissue, or part of the body, caused by any constriction or obstruction of the vascular system. "Ischemic episode" refers to any transient or permanent period of ischemia.

"Vascular system" refers to any network of blood vessels in an animal's body, including unrestricted blood vessels, arteries, veins, and capillaries.

"Vascular endothelial cells" or "vascular endothelium" refers to cells that comprise the thin flat cell layer that line the inner surface of the blood vessels, forming an interface between the blood circulating in the lumen and the rest of the The wall of the glass.

The present invention relates to any animal, preferably a mammal, more preferably companion animals and more preferably humans. A "companion animal" is any domesticated animal, and includes without limitation, cats, dogs, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, horses, cows, goats, lambs, donkeys, pigs, and the like.

As used herein the term "pet feed" or "pet feed composition" means a composition that is intended to be ingested by an animal and preferably by pets. A "feed for nutritionally balanced and complete pet" is a feed that contains all the required nutrients known in appropriate amounts and proportions based on the recommendations of recognized authorities in the field of pet nutrition, and as a consequence is able to serve as the sole source of dietary intake to maintain life or promote production, without the addition of supplementary nutritional sources. Feed compositions for nutritionally balanced domestic animals are widely known and widely used in the art.

As used herein, a "dietary supplement" is a product that is intended to be ingested additionally to an animal's normal diet.

As used herein, "a food product formulated for human consumption" is any composition that is intended to be ingested by a human.

Description

The inventors observed that arginine as a supplement for dogs increased the expression of multiple growth factors and anti-inflammatory proteins in a dose-dependent manner. Many of these growth factors, including NGF, nIGF, and BDNF have potent neuronal protective effects. (Mattson, MP et al. Neurobiol. Aging (2002) 23: 695-705, and Kruttgen, A. et al. Proc. Natl. Acad. Sci. USA (1998) 95: 9614-9619). In subsequent investigations of dogs using natural sources of arginine including herring food (containing fish oil rich in LCPUFA), the inventors discovered a response equal to or better than that observed using pure L-arginine. As described in detail in the examples herein, functional studies of transient cerebral ischemia in an ovarioectomized rat model, showed that a diet containing a combination of L-arginine, fish oil, antioxidants, and B vitamins markedly reduced brain injuries and apoptosis more than diets containing arginine or fish oil alone. Consequently, several aspects of the present invention utilize these findings by providing dietary compositions and methods to improve the vascular integrity of an animal and reduce ischemic injury in the episode of ischemia in the animal, for example a brain injury in the case of cerebral ischemia. in the animal

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Dietary compositions are expected to be effective in a variety of ischemic situations, including but not limited to, brain aging (strokes and mini strokes) and related disorders, ischemic cardiac episodes, induced ischemia and reperfusion surgery (pre and post operative) peripheral ischemia induced by diabetes, and as an alternative to estrogen treatment of post menopause, to reduce the risk of stroke and cardiovascular disease in post menopausal women.

In several versions, the compositions and methods of the present invention apply a multi-component method for the improvement of functional vascular integrity, the reduction of apoptosis and the enhancement of cell survival after an ischemic episode. These components include (1) enhancement of integrity and vascular function, (2) enhancement of cell repair, (3) enhancement of anti-inflammatory effects

(4) optimization of cellular metabolism, and (5) reduction of oxidative stress.

Compositions

Compositions comprising one or more long chain polyunsaturated fatty acids (LCPUFA) and one or more nitric oxide releasing compounds (NORC) in an amount effective for the enhancement of vascular integrity in animals are described herein. The LCPUFA and NORC may be present in the composition as an ingredient or additive. In preferred versions of the composition, the LCPUFAs comprise at least one of the fatty acids (n-3) such as ALA, EPA, DPA and DHA, and the NORCs comprise at least one of the L-Arg and derivatives thereof. The compositions enrich the blood plasma with LCPUFA and NORC in those animals to which the composition is administered.

The compositions of the invention may include a number of additional components for the purpose of providing a multi-component method for promoting vascular integrity and enhancing cell survival in ischemia as indicated above. These include antioxidants, B vitamins, growth factors and anti-inflammatory agents.

In a preferred version, the compositions of the invention are feed compositions for domestic animals. These will advantageously include the feeds that are intended for the supply of the necessary dietary requirements, as well as treats (for example, cookies) or other dietary supplements. Optionally, the compositions of a pet feed can be a dry composition (eg croquettes), a semi-wet composition, a wet composition or any mixture thereof.

In another preferred version, the compositions of the invention are food products formulated for human consumption. These will advantageously include foods and nutrients that are intended to provide the necessary dietary requirements of a human being as well as other human dietary supplements. In a detailed version, food products formulated for human consumption are completely and nutritionally balanced.

In another preferred version, the composition is a dietary supplement, such as a sauce, drinking water, a beverage, a liquid concentrate, a yogurt, a powder, a granulate, a paste, a suspension, a chiclet, a snack, a candy, a snack, a pill, a pill, a capsule, a tablet or any other form of supply. Dietary supplements can be specially formulated for the consumption of a particular animal such as a companion animal, or a human being. In a detailed version the dietary supplement may comprise a high concentration of LCPUFA and NORC so that the supplement can be administered to the animal in small amounts, or alternatively it can be diluted before administration to an animal. The dietary supplement may require mixing with water before being administered to the animal.

The composition may be in frozen form. The LCPUFA and NORC may be premixed with other components of the composition to provide the necessary beneficial amounts, may be coated on a composition of an animal feed, a dietary supplement or a food product formulated for human consumption, or may be added to the composition before being offered to the animal, such as using a powder or a mixture.

The compositions of the invention comprise LCPUFA and NORC in an amount effective to enhance vascular integrity in an animal to which the composition is to be administered. For animal feed and food products formulated for human consumption, the amount of LCPUFA as a percentage of the composition is in the range of about 0.1% to about 13% of the composition based on dry matter, although a higher percentage can be supplied. In several versions the amounts approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0, 9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1, 9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2, 9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3, 9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4, 9%, 5.0%, or more, for example, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% or more, of the composition based on dry matter, and the amount of NORC as a percentage of the composition is in the range of about 0.1% to about 12% of the composition on the basis of dry matter, although it can be supplied somewhat higher. In several versions, the amount is approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, or more, for example, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or more, of the composition based on dry matter. In a specific version, 2-2.5% of LCPUFA and 2-2.5% of pure L-arginine are used. Dietary supplements may be formulated to contain several times higher concentrations of LCPUFA and NORC, easy to administer to the animal in the form of a tablet, capsule, liquid concentrate, or other similar dosage form, or to be diluted before administrations, such as for example by dissolution in water, by dilution with water, spraying or spraying on a feed for domestic animals, and other similar modes of administration.

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In an alternative version, the amount of LCPUFA and NORC in the composition is a function of the amount required to establish a specified concentration of LCPUFA and NORC in the animal's blood serum. The specified concentration of LCPUFA in the blood serum can be calculated by determining the blood serum levels of animals fed with the recommended amounts of LCPUFA and NORC specified above, as can be appreciated by one skilled in the art.

The sources of each of the LCPUFAs can be any suitable, synthetic or natural source. Preferred sources of LCPUFA include, without limitation, primroses, dark green vegetables such as spinach, algae, and blue-green algae such as spirulina, vegetable seeds and oils such as flax, canol, soy beans, walnut, squash, safflower, sesame, wheat germ, sunflower, corn, and hemp, and fish, especially cold-water fish such as Salmon, tuna, mackerel, herring, sea bass, striped sea bass, shark, grouper, catfish, sardines, shrimp and clams, and their extracted oils, or LCPUFA can be synthesized from de novo according to any suitable means of the technique.

The sources of each NORC can be any suitable, synthetic or natural source. Preferred sources of arginine include, without limitation, animal and vegetable proteins. Non-limiting examples of vegetables particularly rich in arginine content include vegetables such as soybeans, lupins and carob beans, grains such as wheat and rice, and fruits such as grapes. Seeds and nuts such as cocoa and peanuts are also rich in arginine content. Non-limiting examples of animal proteins particularly rich in arginine content include the products of poultry and fish. The NORCs can be synthesized de novo, according to any suitable means of the art, or provided by a commercial source.

The compositions of the invention may optionally comprise supplementary substances such as minerals, vitamins, salts, condiments, dyes, and preservatives. Non-limiting examples of supplementary minerals include calcium, phosphorus, potassium, sodium, iron, chloride, boron, copper, zinc, magnesium, manganese, iodine, selenium and the like. Non-limiting examples of supplemental vitamin include vitamin A, various B vitamins, vitamin C, vitamin D, vitamin E and vitamin K. Additional dietary supplements may also be included, for example, niacin, pantothenic acid, inulin, folic acid, biotin, amino acids, and the like.

The compositions of the invention may optionally comprise one or more supplementary substances that promote or maintain vascular health, or further enhance vascular integrity or cell survival. Such substances include, without limitation, one or more of vinpocetine, coenzyme Q10, phosphatidylserine, acetyl-Lcarnitine, alpha-lipoic acids, extracts such as Bilberry (Vaccinium myrtilis) or antioxidants, including tocopherols, tocotrienols , carotinoids such as alpha-and beta-carotene, lycopene, lutein, astaxanthin, zeaxanthin and flavonoids such as flavanols, flavanones, flavones, flavan-3-oles. (catechins), anthocyanins, isoflavones (isoflavonoids), and the like, as well as anti-inflammatory agents and growth factors (particularly applicable to promote cell survival) as would be understood by one skilled in the art.

In various versions, animal feed or dietary supplements of compositions of the invention may comprise on the basis of dry matter, from about 15% to about 50% crude protein, by weight of the composition. The crude protein material may comprise vegetable proteins such as soybeans, cottonseed, and peanuts, or animal proteins such as casein, albumin and meat protein. Non-limiting examples of meat protein for use herein include pork, lamb, equines, poultry, fish and mixtures thereof.

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The compositions may further comprise, on the basis of dry matter, from about 5% to about 40% fat, by weight of the composition. The compositions may further comprise a source of carbohydrates. The compositions may comprise, on a dry matter basis, from about 15% to about 60% carbohydrates, by weight of the composition. Non-limiting examples of these carbohydrates include grains or cereals such as rice, corn, sorghum, alfalfa, barley, soy beans, canola, oats, wheat, and mixtures thereof. . The compositions may also optionally comprise other materials such as dried cow's milk whey and other dairy by-products.

The compositions may also comprise at least one source of fibers. A variety of soluble fibers

or insoluble can be used as is known to those skilled in the art. The source of fibers can be beet pulp (sugar beet), gum arabic, talha gum, psyllium, rice bran, locust bean gum, citrus pulp, pectin, fructooligosaccharide additional to short-chain oligofructose, mannoligofructose, soy fiber, lupine fiber, arabinogalactan, galactooligosaccharide, arabinoxylan or mixtures thereof. Alternatively, the source of fibers may be a fermentable fiber. A fermentable fiber has been previously described to provide a benefit to the immune system of a companion animal. Fermentable fiber or other compositions known to those skilled in the art provide a prebiotic composition to enhance the growth of probiotic microorganisms within the intestine, and can also be incorporated into the composition to aid in enhancing the benefit provided by the present invention to the system. Immune of an animal. Additionally, probiotic microorganisms such as Lactobacillus or Bifidobacterium species, for example, can be added to the composition. In a detailed version, the composition is a complete and nutritionally balanced animal feed. In this context, the feed for domestic animals may be wet feed, a dry feed or an intermediate moisture content, as would be recognized by those skilled in the art of formulating and manufacturing feed for domestic animals. "Wet feed" refers to a feed for domestic animals, which is typically sold in cans or paper bags, and has a moisture content typically in the range of about 70% to about 90%. "Dry feed" refers to a feed for domestic animals that is of a composition similar to wet feed, but contains a limited moisture content, typically in the range from about 5% to about 15%, and therefore presents for example as small croquettes similar to cookies. Compositions and dietary supplements can be specially formulated for adult animals, or for older animals

or young people, for example, a "puppy think" "kitty think" or a "senior" formulation. In general, specialized formulations will include energy and nutritional requirements appropriate for animals in different stages of development or age.

Certain aspects of the invention are preferably used in combination with a complete and balanced food (for example, as described in the National Research Council, 1985, Nutritional Requirements for Dogs). "), National Academy Press, Washington

D.C. or Association of American Feed Control Officials (Official Publication 1996). That is, compositions comprising LCPUFA or DHA according to certain aspects of this invention are preferably used with a high quality commercial feed. As used herein, "high quality commercial feed" refers to a diet manufactured to produce nutrient digestibility of 80% or more, as mentioned for example in the recommendations of the National Research Council above. for dogs Similar high nutrient standards would be used for other animals.

Experts will understand how the appropriate amount of LCPUFA and NORC to be added to a given composition is determined. Such factors that can be taken into account include the type of composition (for example, a composition for domestic animal feed, a dietary supplement, or a food product formulated for human consumption), the average consumption of specific types of compositions by different animals and the manufacturing conditions, with which the composition is prepared. Preferably, the concentrations of LCPUFA and NORC to be added to the composition are calculated based on the energy and nutrition needs of the animal. In accordance with certain aspects of the invention, the LCPUFA and NORC may be added at any time during the manufacture and / or processing of the composition. This includes, without limitation, as part of the formulation of the composition of the feed for domestic animals, a dietary supplement, or a food product formulated for human consumption or as a coating applied to the composition of the feed for domestic animals, a dietary supplement, or a food product formulated for human consumption.

The compositions can be made according to any suitable method in the art such as that described in the Waltham Book of Dog and Cat Nutrition, "ATB Edney editorial, chapter by A. Rainbird, entitled "A balanced diet" on pages 57 to 74, Pergamon Press Oxford.

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Methods

Methods for enhancing vascular integrity and / or promoting cell survival in an animal are described herein, which comprise administration to the animal of a composition comprising one or more LCPUFA and one or more NORC, in an effective amount for the enhancement of vascular integrity and / or promote cell survival in the animal. Yet another aspect of the invention characterizes prophylactic methods for the reduction of ischemia-induced injury to the brain or other tissues of an animal, which comprise administration to the animal, on a regular basis, of a composition comprising one

or more LCPUFA and one or more NORC in an amount effective to reduce ischemia-induced injury, for example to the brain, in the eventuality of an ischemic episode in the animal, for example, in the animal's brain.

In detailed versions of any of the two aforementioned aspects of the invention, the composition is a feed composition for domestic animals, a dietary supplement, or a food product formulated for human consumption as exemplified herein. In another detailed version the LCPUFA are one or more of an LCPUFA (n-3), including but not limited to, the EPA, DPA and DHA, and the NORC is one or more of L-Arg and derivatives thereof. Animals may include any domesticated or companion animal as described above or may include humans. In certain versions, the animal is a companion animal such as a dog or cat. In another version the animal is a human.

The compositions can be administered to the animal by any of several alternative routes of administration. Such routes include, without limitation, the oral, intranasal, intravenous, intramuscular, intragastric, transpyloric, subcutaneous, rectal, and similar routes. Preferably the compositions are administered orally. As used herein, the term "oral administration" or "oral administration" means that the animal ingests or a human receives the indication to feed or feed the animal with one or more of the compositions of the invention described herein. .

When the human receives the indication to feed on the composition, said indication may instruct and / or inform the human that the use of the composition may and / or wants to provide the referenced benefit, for example the enhancement of vascular integrity in the animal or cell survival in the animal, the reduction of ischemia-induced injury, for example to the brain, heart and other tissues, protection from ischemia-related injury in the event of an ischemic episode, for example in the reperfusion surgery, peripheral ischemia induced by diabetes and others, as mentioned above. This indication may be an oral indication (for example through an oral instruction, for example by a doctor, veterinarian, or other health professional or through radio or television (ie advertisements) or it may be a written indication (for example, through a written indication for example by a doctor, by a veterinarian or by another health professional (for example, prescriptions), by a sales or organizational professional (for example through marketing brochures, pamphlets, or other instructional paraphernalia), written media (eg internet, email, or other computer-related media), and / or in the packaging associated with the composition (for example, a label present on the container containing the composition).

The administration can be carried out on a basis according to the needs or according to the wishes, for example once monthly, once weekly, once daily, or more than once daily. Similarly, administration may be every two days, every two weeks or every two months, every three days, every three weeks or every three months, every four days, every four weeks or every four months, etc. Administration can be several times per day. When used as a supplement for ordinary dietary needs, the composition can be administered directly to the animal or otherwise brought into contact or mixed with feed or daily food. When feed or daily food is used, the administration will be well known to ordinary experts.

Administration can also be carried out on a regular basis as for example as part of a dietary regimen in the animal. A dietary regimen may comprise the cause of regular ingestion by the animal of a composition comprising one or more LCPUFA and one or more NORC in an amount effective to enhance vascular integrity or to reduce ischemia-induced brain injury, in the eventuality of an ischemic episode in the animal. Regular ingestion may be once a day, or two, three, four, or more times per day, on a daily or weekly basis. Similarly, regular administration may be every two days or every two weeks, every three days or every three weeks, every four days or every four weeks, every five days or every five weeks or every six days or every six weeks and in This type of dietary regimen administration can be several times per day. The objective of a regular administration is to provide the animal with the preferred daily dose of LCPUFA and NORC, as exemplified herein.

The administration of the compositions comprising one or more LCPUFA and one or more NORC, including administration as part of a dietary regimen, may cover a period of time ranging from gestation to the entire life of the animal.

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The following examples are provided to describe the invention in more detail. They are intended to illustrate the invention.

Example 1

Effect of β-estradiol 17 or dietary supplement with arginine, fish oil or a combination thereof on brain injury from transient cerebral ischemia in an ovarioectomized rat model

Chronic treatment with β-estradiol 17 (E2) was compared with three test diets containing arginine, LCPUFA in the form of fish oil or a combination thereof, respectively, for the study of its effects on brain injury caused by a transient cerebral ischemia in an ovarioectomized rat model.

Methods:

Animals. Charles Rivers Sprague-Dawley female rats (250 g, Wilmington, MA) were acclimatized to the animal facilities, three days before surgery with a 12-hour light and dark cycle. A bilateral ovarioectomy was performed 2 weeks before starting diet feeding. Four weeks after starting the diet diet, an occlusion of the transient central cerebral artery (tMCA) was performed with anesthesia after an intraperitoneal injection (i.p.) of ketamine (60 mg / kg) and xylazine (10 mg / kg)

Diet and administration of hormones. Fourteen to 15 rats per group were randomized from one to five treatment groups. These groups were as follows:

Group 1, control diet (white diet)

Group 2, control diet + SILASTIC® estradiol implant for 1 week (white + E2)

Group 3, diet I (pink diet)

Group 4, diet II (purple diet)

Group 5, diet III (gray diet)

The control diet was a standard rat diet containing 140 g / kg of casein, 100 g / kg of sucrose, 50 g / kg of fiber, 155 g / kg of dextrin, 466 g / kg of cornstarch, 35 g / kg of standard salt mixture, 40 g / kg of soybean oil, 10 g / kg of standard vitamin mixture, 1.8 g / kg of L-cystine and 2.5 g / kg of choline chloride. All three test diets (diet I, II and III) were supplemented with (1) additional B vitamins, including 45 mg / kg of nicotinic acid, 35 mg / kg of pantothenate, 24 mg / kg of pyridoxine, 15 mg / kg of thiamine, 9 mg / kg of riboflavin, 3 mg / kg of folic acid, 0.8 mg / kg of biotin and 0.255 mg / kg of vitamin B 12, and (2) an antioxidant coktail comprising 500 mg / kg of vitamin E, 150 mg / kg of vitamin C, 100 mg / kg of astaxanthin and 0.4 mg / kg of selenium. Diet I also included 2% arginine (20 g / kg), diet II also included 2% arginine and 2% lacha fish oil (20 g / kg). Diet III also included 2% of lacha fish oil.

All diets were administered ad libitum to the rats for four weeks. Changes in food took place every week. Also once a week, body weights and food intake were determined. One week before the induction of a tMCA occlusion, group 2 of rats were treated with E2 at a dose of 4 mg / ml in a SILASTIC® pill that was implanted subcutaneously. This procedure has been used to protect the brain from a variety of attacks and produces a physiologically important serum E2 level (Simpkins et al., 1997, Jung et al. 2003). This was a positive control since it had been previously shown that this dose and time of treatment with E2 resulted in the protection of the effects of tMCA occlusion (Sympkins et al., 1997, Yang et al., 2004a, Yang et al. ,, 2004b).

Occlusion of the middle cerebral artery and sample preparation. The animals were anesthetized by an intraperitoneal injection of ketamine (60 mg / kg) and xylazine (10 mg / kg). The tMCA occlusion was performed as previously described (Sympkins et al., 1997) with a slight modification. In short, the common left carotid artery, the external carotid artery, and the internal carotid artery were shown by an incision in the midline of the cervix. A monofilament suture of 3.0 was inserted into the volume of the internal carotid artery and gently advanced until resistance ceased to occur. The surgical procedure was performed within 20 minutes with minimal bleeding. The suture was held in place for 60 minutes and then removed to allow reperfusion. The rectal temperature was monitored and maintained between 36.5 and 37 ° C with a heating pad during the process. At 24 hours after the start of reperfusion, the animals were sacrificed and the brains were removed. The brains were then dissected coronally into 2 mm sections using a brain metal matrix (ASI Instruments Inc .; Warren, MI) and stained by incubation in a 2% solution of 2,3,5-triphenyltetrazolium chloride ( TTC) in physiological saline at 37 ° C, and then fixed in 10% formalin.

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DNA fragmentation analysis with the MCA occlusion marked with dUTP at the indented end, induced with TdT (TUNEL assay), is a widely used focal ischemic attack model (Bederson et al., 1986). This in vivo model for neuron death can rapidly induce a synchronized apoptotic process in a large number of neurons and other cells (Li et al., 1997). The effects of transient ischemia on apoptosis were therefore examined by DNA fragmentation analysis with the TUNEL assay. TUNEL tinting was carried out in accordance with the manufacturer's modified instructions (Gavrieli et al., 1992). The formalin-fixed sections, embedded in paraffin, were dewaxed with xylol, rehydrated with a graduated treatment with ethanol, and a final wash with PBS was given. Sections were subsequently fixed with 4% paraformaldehyde for 20 minutes. The sections were then washed and treated for 15 minutes with 100 µg / milliliter of Proteinase K in PBS, equilibrated with equilibration buffer for 10 minutes, and then incubated with buffer containing the TdT enzyme and FITC-labeled dUTP (Promega, Madison, WI) at 37 ° C in a humidification chamber. The reaction was terminated by incubation in 2xSSC buffer for 15 minutes at room temperature. The sections were then mounted with anti-bleaching reagents, containing DAPI (molecular tests, Eugene, OR). The positive control sections were immersed in a DNase I solution for 10 minutes at room temperature before equilibration in TdT buffer. Sections were observed by a fluorescence microscope with appropriate excitation / emission filter pairs.

Some animals were eliminated from the study during the protocol. The number of animals quantified by lesion volume ranged from 12 to 15 per group.

Statistical methods. The results were analyzed by analyzing a variance pathway (ANOVA) using the Prism program (Graphpad Inc; San Diego, CA). The significance of the differences between the groups was determined by the one-way ANOVA followed by Tukey multiple comparison tests. All values were expressed as the mean ± SEM.

Results:

Attack volume All four experimental conditions (estradiol and diets I, II and III reduced the size of the infarction (Figure 1). Treatment with estradiol (group 2) reduced the volume of the infarction by 68% to a typical value of estrogen protection of the lesions of the attack (Simpkins, et al., 1997; Fan et al., 2003; Yang et al., 2004ª; Yang et al., 2004b). Similarly, diet II reduced the volume of the infarction significantly in 67% The other two diets tested also reduced mean infarct volumes, but the data were too variable for the values to be statistically significant.

To the extent that each group had rats with non-observable lesions (value 0 in our lesion volume calculations), the data were evaluated after these values were omitted, to determine the extent to which the 0 values contributed to differences in the group As shown in Figure 2, the same two groups were significantly smaller than the control diet group with this modification in the data. Thus, the differences between the groups were driven by the animal in which the lesions were observed as well as by the number of animals without observable lesions. The protective effects of both estrogen treatment and diets took place mainly in the cortex, an area of the brain called the penumbra, since it is believed to be "salvageable" in an experimental attack. That is, in contrast to the basal ganglia called the nucleus of the infarction, which is not easily saved by any treatment tested to date in an experimental attack.

Apoptosis The apoptosis opinion using the TUNEL staining method was carried out to determine the consequences of estradiol treatment and the three test diets about the apoptotic response to experimental ischemia. Three regions of the brain for TUNEL staining were assessed in all animals that completed the study. The TUNEL method was assessed in the cerebral cortex nucleus, the cerebral cortex penumbra and the subcortex nucleus, based on our assessment of damaged brain regions, using TTC staining (Wen et al., 2004). The number of cells positive to the TUNEL assay was normalized to the total number of cells in the field using DAPI nuclear counterstain. Sections of all three brain regions were counted and the average cell counts of each animal were used to calculate the group mean. Cell counts were performed in randomly selected microscopic fields (320 µm square sections) in slices of the maximum extent of cerebral infarction, determined by staining with TTC. TUNEL staining was extensive in all three brain regions in control animals. When a comparison of the five treatment groups was made, TUNEL staining in each brain correlated with the results found using TTC staining to quantify the volume of the lesion (Figure 3). That is, there was a substantial reduction in TUNEL staining both in animals treated with E2 and also in animals fed with diet II. Animals fed diets I and III were intermediate between controls and animals in diet II. This correlation between TTC staining and TUNEL staining is shown in Figure 4. The characterization of the effects of hormones and diets on TUNEL staining indicates that a substantial portion of the protection by E2 and diet number II a From cell death, it is mediated by an inhibition of apoptosis.

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Conclusions: The three test diets reduced the volume of the mean infarction, as well as the TUNEL-positive cell counts, and diet II reduced these parameters to the level found with a known neuroprotector, estradiol.

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Claims (21)

one.

Use of one or more long-chain polyunsaturated fatty acids (LCPUFA), and one or more nitric oxide-releasing compounds (NORC) in the preparation of a drug or co-administration medications for the enhancement of vascular integrity, reducing cell apoptosis in the place where an ischemia is located in an animal.

2.

The use of claim 1, wherein the ischemia occurs in a selected tissue of the brain, heart and kidney.

3.

Use of one or more LCPUFA and one or more NORC in the preparation of a drug or medications for co-administration for the reduction of brain lesions induced by an ischemia in an animal.

Four.

Use according to any one of claims 1 to 3, wherein the medicament is a composition for pet feed, a dietary supplement, or a food product formulated for human consumption.

5.

Use according to any one of claims 1 to 3, wherein the LCPUFAs include at least one acid from the group consisting of arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid.

6.

Use according to any one of claims 1 to 3, wherein the NORC includes at least one L-arginine or one of the derivatives thereof.

7.

Use according to claim 6, wherein the LCPUFAs are in an amount from at least about 0.1% to about 13% by weight of the formulation and L-arginine in an amount from at least about one 0.1% to about 12% by weight of the formulation.

8.

Use according to any one of claims 1 to 3, wherein the animal is a companion animal.

9.

Use according to claim 8, wherein the companion animal is a cat or a dog.

10.

Use according to any one of claims 1 to 3, wherein the animal is a human.

eleven.

A composition comprising one or more LCPUFA and one or more NORCs for use in the reduction of ischemia-induced brain injury, in an animal where the composition is administered to the animal on a regular basis.

12.

The composition of claim 11, wherein the composition is a feed composition for a pet, a dietary supplement, or a food product formulated for human consumption.

13.

The composition of claim 11, wherein the LCPUFAs include at least one arachidonic acid, an eicosapentaenoic acid, a docosapentaenoic acid, or a docosahexaenoic acid.

14.

The composition of claim 11, wherein the NORCs include at least one L-arginine or derivatives thereof.

fifteen.

The composition of claim 14, wherein the LCPUFAs are in an amount from at least about 0.1% to about 13% by weight of the formulation, and the L-arginine in an amount from at least about one 0.1% to about 12% by weight of the formulation.

16.

The composition of claim 11, wherein the animal is a companion animal.

17.

The composition of claim 16, wherein the companion animal is a cat or a dog.

18.

The composition of claim 11, wherein the animal is a human.

19.

A composition comprising one or more LCPUFA and one or more NORC, for use in the enhancement of vascular integrity and the reduction of brain injury in the eventuality of an ischemic episode in the brain.

twenty.

A composition comprising one or more LCPUFA and one or more NORC, for use in the enhancement of vascular integrity and the reduction of cellular apoptosis at the location of an ischemia in an animal.

image 1 The composition of claim 20, wherein the ischemia takes place in a selected tissue of the brain, heart, and kidney. 22. A composition comprising one or more LCPUFA and one or more NORC for use in reducing the brain injury induced by an ischemia in an animal. 10