Patent Application
20160081962
Embodiments of the invention presented herein are directed to nutritional or dietary supplements, and other nutraceutical compositions that include an unsaturated fatty acid and nitrite and/or nitrate.
In some embodiments, the dietary supplements may include a non-animal based unsaturated fatty acid such as algae-derived docosahexaenoic acid (DHA) or some other plant based oil, vegetable based oil, nut based oil, or seed based oil. In addition, the dietary supplement may include a nitrite and/or nitrate component, such as beet root juice, either in liquid form or as a powder, or in some other embodiments a source of nitrite and/or nitrate.
In certain embodiments, the fatty acid component and the nitrite and/or nitrate component of the dietary supplement may be formulated as separate capsules, one for each component, wherein the capsules are packaged together and the consumer of the dietary supplement is instructed to consume one of each capsule simultaneously.
In other embodiments, the fatty acid component and the nitrite and/or nitrate component of the dietary supplement may be formed in a single capsule, where the two components are kept separate until ingestion of the dietary supplement by the consumer, wherein the capsule breaks down and the separate components come into contact with one another in the stomach or gut of the consumer.
In some embodiments, the fatty acid component and the nitrite and/or nitrate component may be combined in a single capsule where the components are allowed to mix with one another.
In some embodiments, the fatty acid component and the nitrite and/or nitrate component of the dietary supplement are combined in the stomach or gut of the consumer of the dietary supplement. The human stomach and gut represent suitable “bioreactors”, containing desirable temperature and pH levels that create suitable conditions for the reaction of the fatty acid component with the nitrite and/or nitrate component, wherein nitro fatty acids are formed in the stomach and gut and absorbed by the gut into the body where they are known to have beneficial effects in mammals.
In some embodiments, the nitrite and/or nitrate component will be present in a greater quantity than the fatty acid component of the dietary supplement.
In some embodiments, the dietary supplement may be packaged by known methods used in the dietary supplement industry such as gelatin capsules (gel caps), tablets, caplets, and the like.
In some embodiments, the dietary supplement may include a vegan formulation, wherein the non-animal-based unsaturated fatty acid component is combined with the nitrite and/or nitrate component in a gelatin capsule formed from materials taken from non-animal sources.
In some embodiments, the dietary supplement may include a non-vegan formulation, wherein the unsaturated fatty acid component is derived from animal products, such as fish oil, and combined with the nitrite and/or nitrate component in a conventional capsule formed from materials taken from animal based or other sources.
In some embodiments, the dietary or nutritional supplement may include an alimentary oil, such as olive oil, combined with a source of nitrite and/or nitrate, such as sodium nitrite and/or sodium nitrate, for example beet root juice or beet root powder.
In some embodiments, the dietary or nutritional supplement may include fish oil and a source of nitrite and/or nitrate. The fish oil may, in some embodiments, be a mixture of DHA and eicosapentaenoic acid (EPA) derived from fish oil or in some cases may include other fatty acids such as for example omega-3 fatty acids that are naturally found in fish oil. In some embodiments, the fish oil may be enriched with one or more fatty acids such as, for example, EPA or DHA or an omega-3 fatty acid. The nitrite and/or nitrate component may be beet root juice or beet root powder, sodium nitrite, sodium nitrate or another source of nitrite and/or nitrate.
In some embodiments, the dietary or nutritional supplement may include an activated oil that contains nitrated fatty acids that are produced by a process of reacting unsaturated fatty acids with a nitrite and/or nitrate source to produce nitro fatty acids, also referred to herein as activated fatty acids that are then encapsulated for later consumption. The source of the unsaturated fatty acid may be fish oil or alimentary oil. In some embodiments, the fatty acid may be enriched for one or more components such as for example, DHA or EPA.
In some embodiments, the dietary or nutritional supplement may include additional components. other than the fatty acid and nitrite and/or nitrate, such as rice bran oil, enzyme-treated stabilized rice bran, a solubilized fraction of rice bran oil, and derivatives thereof, glucosamine derivatives, methylsulfonylmethane, yucca concentrate, grape seed extract, beta-carotene, ephedra, gingko biloba, goldenseal, valerian, ginseng, and echinacea.
In some embodiments, the unsaturated fatty acids may be isolated from a natural source such as fish oil and may be derived from omega-3 fatty acids, conjugated linoleic acid, linoleic acid, α-linoleic acid, oleic acid, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid or a derivative or combination thereof.
In some embodiments, the nutritional supplement may be an additive for food.
Some embodiments of the invention are directed to the selection, formulation, and use of compounds which act with a protective response to prevent and attenuate inflammation to provide a therapeutic effect in their control of the pathological inflammation processes, and are also important in providing useful biochemical tools for mechanistic investigation of the enzymes involved.
Some embodiments are directed to a dietary supplement including a fatty acid component derived from an omega-3 fatty acid, an omega-6 fatty acid, an omega-9 fatty acid, and combinations thereof.
In some embodiments, the dietary supplement may further include one or more nutraceutical selected from vitamin A, vitamin B, vitamin B-1, vitamin B-2, vitamin B-6, vitamin B-12, vitamin C, vitamin D, vitamin D3, vitamin E, selenium, β-carotene, ginkgo biloba, goldenseal, valerian, ginseng, echinacea, grape seed extracts, ephedra, yucca concentrates, green tea extract, rice bran extract, wheat germ, wheat genii extract, beeswax, red yeast rice extract, stevia leaf extract, flaxseed oil, borage seed oil, coenzyme Q10, glucosamine derivatives, methylsulfonylmethane, pantothenic acid, biotin, thiamin, riboflavin, niacin, folic acid, palmitic acid, and derivatives thereof.
In other embodiments, the dietary supplement may include one or more secondary agent including but not limited to vitamin A, vitamin B, vitamin B-1, vitamin B-2, vitamin B-6, vitamin B-12, vitamin C, vitamin D, vitamin D3, vitamin E, selenium, β-carotene, ginkgo biloba, goldenseal, valerian, ginseng, echinacea, grape seed extracts, ephedra, yucca concentrates, green tea extract, rice bran extract, wheat germ, wheat germ extract, beeswax, red yeast rice extract, stevia leaf extract, flaxseed oil, borage seed oil, coenzyme Q10, glucosamine derivatives, methylsulfonylmethane, pantothenic acid, biotin, thiamin, riboflavin, niacin, folic acid, palmitic acid, and derivatives thereof. In some embodiments, the dietary supplement may include one or more secondary agent selected from policosanols, guggulipids, rice bran extract, wheat germ, wheat germ extract, beeswax, and red yeast rice extract, and such a dietary supplement may be formulated to promote a healthy heart and circulatory system. In other embodiments, the dietary supplement may include one or more secondary agent selected from vitamin B-1, vitamin B-2, vitamin B-6, vitamin B-12, vitamin C, vitamin D, vitamin D3, vitamin E, selenium, goldenseal, valerian, ginseng, and echinacea and such a dietary supplement may be formulated to promote healthy cell proliferation. In still other embodiments, the dietary supplement may include one or more secondary agent selected from vitamin A, vitamin C, vitamin E, and β-carotene, and such a dietary supplement may be formulated to promote healthy eyes. In yet other embodiments, the dietary supplement may include one or more secondary agent selected from vitamin A, vitamin C, vitamin E, selenium, ginkgo biloba, goldenseal, valerian, ginseng, echinacea, ephedra, green tea extract, and yucca concentrate, and such a dietary supplement may be formulated to promote general health.
Further embodiments are directed to methods for improving the health of an individual by administering to the individual a dietary supplement including a fatty acid component and a nitrite and/or a nitrate. In some embodiments, the dietary supplement may include a fatty acid component selected from linoleic acid, α-linoleic acid, γ-linoleic acid, oleic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), conjugated linoleic acid, or derivatives thereof, and in particular embodiments, the dietary supplement may further include vitamin E or a derivative thereof. In some embodiments, the dietary supplement may further include one or more secondary agent selected from vitamin A, vitamin B, vitamin B-1, vitamin B-2, vitamin B-6, vitamin B-12, vitamin C, vitamin D, vitamin D3, vitamin E, selenium, β-carotene, ginkgo biloba, goldenseal, valerian, ginseng, echinacea, grape seed extracts, ephedra, yucca concentrates, green tea extract, rice bran extract, wheat germ, wheat germ extract, beeswax, red yeast rice extract, stevia leaf extract, flaxseed oil, borage seed oil, coenzyme Q10, glucosamine derivatives, methylsulfonylmethane, pantothenic acid, biotin, thiamin, riboflavin, niacin, folic acid, palmitic acid, and derivatives thereof. In some embodiments, the dietary supplement may include one or more secondary agent selected from policosanols, guggulipids, rice bran extract, wheat germ, wheat germ extract, beeswax, and red yeast rice extract, and such a dietary supplement may be formulated to promote a healthy heart and circulatory system. In other embodiments, the dietary supplement may include one or more secondary agent selected from vitamin B-1, vitamin B-2, vitamin B-6, vitamin B-12, vitamin C, vitamin D, vitamin D3, vitamin E, selenium, goldenseal, valerian, ginseng, and echinacea and such a dietary supplement may be formulated to promote healthy cell proliferation. In still other embodiments, the dietary supplement may include one or more secondary agent selected from vitamin A, vitamin C, vitamin E, and β-carotene, and such a dietary supplement may be formulated to promote healthy eyes. In yet other embodiments, the dietary supplement may include one or more secondary agent selected from vitamin A, vitamin C, vitamin E, selenium, ginkgo biloba, goldenseal, valerian, ginseng, echinacea, ephedra, green tea extract, and yucca concentrate, and such a dietary supplement may be formulated to promote general health.
Embodiments of the invention also include methods for preparing a nitro-fatty acid by contacting an existing unsaturated fatty acid with a nitro containing compound; and reacting an existing unsaturated fatty acid with a nitro containing compound to form a nitro fatty acid. Still other methods for preparing activated fatty acids include the steps of contacting an unsaturated fatty acid with a mercuric salt and a selenium compound; contacting an intermediate resulting from step 1 with an electron withdrawing group donating reagent; reacting the intermediate resulting from step 2 with an oxidizing agent. Yet other methods for preparing nitro fatty acids include the steps of combining a first component at least comprising an aliphatic hydrocarbon having an electron withdrawing group at one end and a second component at least comprising aliphatic hydrocarbon chain having an aldehyde at one end in the presence of a base to form a first intermediate; generating an alkene from the first intermediate.
Nitric oxide (NO) is an endogenously generated, lipophilic signaling molecule that has been implicated in the maintenance of vascular homeostasis, modulation of oxygen radical reactions, inflammatory cell function, post-translational protein modification and regulation of gene expression. In addition, nitric oxide-derived species display separate and unique pharmacological properties, specifically can mediate oxidation and nitration of biomolecules such as, for example, unsaturated fatty acids.
Various reactions yield products capable of concerted oxidation, nitrosation and nitration of target molecules. For example, nitric oxide may react with superoxide (O2−) to yield peroxynitrite (ONOO−) and its conjugate acid, peroxynitritrous acid (ONOOH), the latter of which may undergo homolytic scission to form nitrogen dioxide (.NO2) and hydroxyl radical (.OH). In some instances, biological conditions may favor the reaction of ONOO− with CO2 which yields nitrosoperoxycarbonate (ONOOCO2−), which rapidly yields .NO2 and carbonate (.CO3−) radicals via homolysis or rearrangement to NO3− and CO2. During inflammation, neutrophil myeloperoxidase and heme proteins such as myoglobin and cytochrome c catalyze H2O2-dependent oxidation of nitrite (NO2−) to .NO2, resulting in biomolecule oxidation and nitration that is influenced by the spatial distribution of catalytic heme proteins. The reaction of .NO with O2 can also produce products that can be substrates or reactants for nitrosation and nitration. For example, the small molecular radius, uncharged nature and lipophilicity of .NO and O2 facilitate concentration of these species in biological membranes in a process referred to as the “molecular lens” effect. The increase in concentration induced by .NO and O2 solvation in hydrophobic cell compartments accelerates the normally slow reaction of .NO with O2 to yield N2O3 and N2O4. Finally, environmental sources also yield .NO2 as a product of photochemical air pollution and tobacco smoke.
Nitration of fatty acids by .NO2 can occur through several methods. For example, during both basal cell signaling and tissue inflammatory conditions, .NO2 can react with membrane and lipoprotein lipids. In both in vivo and in vitro systems, .NO2 has been shown to initiate radical chain auto-oxidation of polyunsaturated fatty acids via hydrogen abstraction from the bis-allylic carbon to form nitrous acid and a resonance-stabilized bis-allylic radical. Depending on the radical environment, the lipid radical species can react with molecular oxygen to form a peroxyl radical, which can react further to form lipid hydroperoxides then oxidized lipids. During inflammation or ischemia, when O2 levels are lower, lipid radicals can react to an even greater extent with .NO2 to generate multiple nitration products including singly nitrated, nitrohydroxy- and dinitro-fatty acid adducts. These products can be generated via hydrogen abstraction, direct addition of .NO2 across the double bond, or both, and in some cases, such reactions may be followed by further reactions of the intermediate products that are formed. Hydrogen abstraction causes a rearrangement of the double bonds to form a conjugated diene; however, the addition of .NO2 maintains a methylene-interrupted diene configuration to yield singly nitrated polyunsaturated fatty acids. This arrangement is similar to nitration products generated by the nitronium ion (NO2+), which can be produced by ONOO− reaction with heme proteins or via secondary products of CO2 reaction with ONOO−.
The reaction of polyunsaturated fatty acids with acidified nitrite (HNO2) can generate a complex mixture of products similar to those formed by direct reaction with .NO2, including the formation of singly nitrated products that maintain the bis-allylic bond arrangement. The acidification of NO2− can create a labile species, HNO2, which is in equilibrium with secondary products, including N2O3, .NO and .NO2, all of which can participate in nitration reactions. The relevance of this pathway as a mechanism of fatty acid nitration is exemplified by physiological and pathological conditions wherein NO2− is exposed to low pH (e.g., <pH 4.0). This may conceivably occur in the gastric compartment, following endosomal or phagolysosomal acidification or in tissues following-post ischemic reperfusion.
Nitrated linoleic acid (LNO2) and conjugated nitro-linoleic acid (CLNO2) have been shown to display robust cell signaling activities that are generally anti-inflammatory in nature. Synthetic LNO2 can inhibit human platelet function via cAMP-dependent mechanisms and inhibits neutrophil O2− generation, calcium influx, elastase release, CD11b expression and degranulation via non-cAMP, non-cGMP-dependent mechanisms. LNO2 may also induce vessel relaxation in part via cGMP-dependent mechanisms. In aggregate, these data, derived from a synthetic fatty acid infer that nitro derivatives of fatty acids (NO2—FA) represent a novel class of lipid-derived signaling mediators. To date, a gap in the clinical detection and structural characterization of nitrated fatty acids has limited defining NO2—FA derivatives as biologically-relevant lipid signaling mediators that converge .NO and oxygenated lipid signaling pathways.
Nitrite has the chemical formula NO2− and includes a symmetric anion with equal N—O bond lengths. In vivo nitrite has often been considered an inert end product of nitric oxide metabolism and, therefore, was looked on unfavorably as a dietary constituent. Recently, however, a new view of nitrite metabolism has led scientists to the understanding that metabolism of nitrite occurs in the blood and tissues of the body to form nitric oxide (NO) and other bioactive nitrogen oxides. Thus nitrite can be viewed as a storage pool for supporting NO signaling during metabolic stress.
Moreover both nitrate (NO3−) and nitrite are found readily in the everyday diet and their levels can be especially high in certain vegetables. For example, a single serving of spinach, lettuce or beet root is known to contain relatively high levels of nitrate. Moreover, diets such as the Mediterranean diet or the Japanese diets, that are rich in vegetables, have shown beneficial results with respect to reducing blood pressure and protecting against cardiovascular disease.
The metabolism of arachidonic acid is a key element of inflammation. In acute inflammation, there is typically a respiratory burst of neutrophil activity that initiates cascades involving a change in the oxidation state of the cell. Alteration in the redox state of the cell activates transcription factors such as NFκB as well as AP1, which then causes production of proinflammatory mediators. These mediators, such as Tumor necrosis factorA (TFα) and various interleukins, cause a burst of other cytokines. Arachadonic acid is released, which is oxidized to biologically active mediators. When arachadonic acid is oxidized via the cyclooxygenase or lipoxygenase pathways, eicosanoids e.g. prostaglandins, leukotrines, and hyroxyeicosatetraenoic acid (HETE) are produced, which cause erythma, edema, and free radical production.
Acute inflammation is often characterized by the generation of excited oxygen species, e.g. superoxide anion, which damages the lipid-rich membranes and activate the chemical mediators of the proinflammation and inflammation cascades. These oxygenated species tend to concentrate in hydrophobic regions. Both in or near these hydrophobic compartments, .NO and NOx undergo a rich spectrum of reactions with oxygen species, transition metals, thiols, lipids, and a variety of organic radicals. These multifaceted reactions yield reactive species that transduce .NO signaling and modulate tissue inflammatory responses.
During inflammation, adaptive and protective responses are elicited by vascular and other tissues to protect the host from its own mechanisms directed at destroying invading pathogens. Heme oxygenase 1 (HO-1) plays a central role in vascular inflammatory signaling and mediates a protective response to inflammatory stresses such as atherosclerosis, acute renal failure, vascular restenosis, transplant rejection, and sepsis. Heme oxygenase 1 catalyzes the degradation of heme to billverdin, iron, and CO, the last of which has been shown to display diverse, adaptive biological properties, including anti-inflammatory, anti-apoptotic, and vasodilatory actions. During inflammation, HO-1 gene expression is up-regulated, with induction typically occurring transcriptionally. Neutrophil myeloperoxidase and heme proteins such as myoglobin and cytochrome c catalyze H2O2-dependent oxidation of nitrite (NO2) to NO2, resulting in biomolecule oxidation and nitration that is influenced by the spatial distribution of catalytic heme proteins. These and other products are capable of concerted oxidation, nitrosation and nitration of target molecules.
The body contains an endogenous antioxidant defense system made up of antioxidants such as vitamins C and E, glutathione, and enzymes, e.g., superoxide dismutase. When metabolism increases or the body is subjected to other stress such as infection, extreme exercise, radiation (ionizing and non-ionizing), or chemicals, the endogenous antioxidant systems are overwhelmed, and free radical damage takes place. Over the years, the cell membrane continually receives damage from reactive oxygen species and other free radicals, resulting in cross-linkage or cleavage or proteins and lipoproteins, and oxidation of membrane lipids and lipoproteins. Damage to the cell membrane can result in myriad changes including loss of cell permeability, increased intercellular ionic concentration, and decreased cellular capacity to excrete or detoxify waste products. As the intercellular ionic concentration of potassium increases, colloid density increases and m-RNA and protein synthesis are hampered, resulting in decreased cellular repair. Some cells become so dehydrated they cannot function at all.
Natural products such as Royal Jelly comprises water, crude protein, including small amounts of many different amino acids, simple sugars (monosaccharides), and fatty acids. It also contains many trace minerals, some enzymes, antibacterial and antibiotic components and vitamins. The major type of fatty acids contained in Royal Jelly are hydroxyl fatty acids such as 10-hydroxy-2-decenoic acid. Royal Jelly is harvested from bees and has been reported as a possible immunomodulatory agent in Graves' disease. It has also been reported to stimulate the growth of glial cells and neural stem cells in the brain. To date, there is preliminary evidence that it may have some cholesterol-lowering, anti-inflammatory, wound-healing, and antibiotic effects. Research also suggests that the 10-Hydroxy-2-decenoic acid may inhibit the vascularization of tumors and has also been hypothesized to correct cholesterol level imbalances due to nicotine consumption. Holistically, Royal Jelly is believed to have anti-aging properties making it a component in skin care and natural beauty products.
Diabetes is a metabolic disease in which the body's inability to produce any or enough insulin causes elevated levels of glucose in the blood. Increased levels of glucose in the blood can potentially lead to serious health problems. Chronic diabetes conditions include type 1 diabetes and type 2 diabetes. Pre-diabetes is a condition where an individuals' blood sugar levels are higher than normal, but not high enough to be classified as diabetes. Individuals with pre-diabetes have an increased risk of developing type 2 diabetes, heart disease and stroke. Studies have shown that individuals with pre-diabetes who lose weight and increase their physical activity can reverse the pre-diabetes categorization.
Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
It must also be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “cell” is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.
As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%.
“Administering” when used in conjunction with a therapeutic means to administer a therapeutic directly into or onto a target tissue or to administer a therapeutic to a patient, whereby the therapeutic positively impacts the tissue to which it is targeted. Thus, as used herein, the term “administering”, when used in conjunction with a nitrated lipid can include, but is not limited to, providing a nitrated lipid to a subject systemically by, for example, intravenous injection, whereby the therapeutic reaches the target tissue. “Administering” a composition may be accomplished by, for example, injection, oral administration, topical administration, or by these methods in combination with other known techniques. Such combination techniques include heating, radiation, ultrasound and the use of delivery agents.
The term “animal” as used herein includes, but is not limited to, humans and non-human vertebrates such as wild, domestic and farm animals.
The term “improves” is used to convey that the present invention changes either the characteristics and/or the physical attributes of the tissue to which it is being provided, applied or administered. The term “improves” may also be used in conjunction with a diseased state such that when a diseased state is “improved” the symptoms or physical characteristics associated with the diseased state are diminished, reduced or eliminated.
The term “inhibiting” includes the administration of a compound of the present invention to prevent the onset of the symptoms, alleviating the symptoms, or eliminating the disease, condition or disorder.
By “pharmaceutically acceptable”, it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
“Nutraceutical” as used herein generally refer to natural, bioactive chemical compounds that provide physiological benefits, including, disease prevention and health promotion which may be used to supplement the diet. Nutraceuticals can be either purified or concentrated by using bioengineering methods and can be enhanced through genetic methods, which contain elevated levels of natural substances. Examples of nutraceuticals include isolated nutrients and herbal products and generally contain at least one of the following ingredients: a vitamin, a mineral, an herb or other botanical, an amino acid, a metabolite, constituent, extract, or combination of these ingredients. Common examples of nutraceuticals include beta-carotene, ephedra, ginkgo biloba, goldenseal, valerian, ginseng, and echinacea. The nutraceuticals described herein may be useful for maintenance and support of, for example, healthy joints, skin, and eye and brain function.
As used herein, the term “therapeutic” means an agent utilized to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a patient. In part, embodiments of the present invention are directed to the treatment of inflammation, obesity-related diseases, metabolic diseases, cardiovascular diseases, cerebrovascular and neurodegenerative diseases, cancer or the aberrant proliferation of cells.
A “therapeutically effective amount” or “effective amount” of a composition is a predetermined amount calculated to achieve the desired effect, i.e., to inhibit, block, or reverse the activation, migration, or proliferation of cells. The activity contemplated by the present methods includes both medical therapeutic and/or prophylactic treatment, as appropriate. The specific dose of a compound administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, and the condition being treated. However, it will be understood that the effective amount administered will be determined by the physician in the light of the relevant circumstances including the condition to be treated, the choice of compound to be administered, and the chosen route of administration, and therefore, the above dosage ranges are not intended to limit the scope of the invention in any way. A therapeutically effective amount of compound of this invention is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in the tissue.
The terms “treat,” “treated,” or “treating” as used herein refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
As used herein and in the attached claims, the term “enriched” shall mean that the composition or portion of the composition includes a concentration of the identified component that is greater than the amount of the component naturally occurring in the composition. For example, with reference to fatty acids a composition enriched for fatty acids may include greater than at least 50 nM fatty acids. Therefore, a composition that is enriched for fatty acids may be at least 0.05% by weight fatty acid, at least 0.1% by weight fatty acid, at least 0.15% by weight fatty acid, at least 0.25% by weight fatty acid, at least 0.5% by weight fatty acid, at least 1.0% by weight fatty acid, at least 2% by weight fatty acid, and so on.
Generally speaking, the term “tissue” refers to any aggregation of similarly specialized cells which are united in the performance of a particular function.
The fatty acids of various embodiments may be any unsaturated and polyunsaturated fatty acid known in the art. The term “fatty acid” describes aliphatic monocarboxylic acids. Various embodiments include fatty acid having an aliphatic hydrocarbon chain identical or similar to identified, naturally occurring fatty acids. For example, aliphatic hydrocarbon chains of known naturally occurring fatty acids are generally unbranched and contain an even number of from about 4 to about 24 carbons. Embodiments of the invention encompass such naturally occurring fatty acids as well as non-naturally occurring fatty acids which may contain an odd number of carbons and/or a non-naturally occurring linker. Some embodiments of the invention include fatty acids having from 8 to 23 carbons, and others include fatty acids having from 12 to 18 carbons in the aliphatic hydrocarbon chain. In still other embodiments, fatty acids may have greater than 24 carbons in the aliphatic hydrocarbon chain. The fatty acids of the invention may also be branched at one or more location along the hydrocarbon chain, and in various embodiments, each branch may include an aliphatic hydrocarbon chain of from 1 to 24 carbons, 2 to 20 carbons or 4 to 18 carbons.
The aliphatic hydrocarbon chain of fatty acids of various embodiments may be unsaturated or polyunsaturated. The term “unsaturated” refers to a fatty acid having a aliphatic hydrocarbon chain that includes at least one double bond and/or substituent. In contrast, a “saturated” hydrocarbon chain does not include any double bonds or substituents. Thus, each carbon of the hydrocarbon chain is ‘saturated’ and has the maximum number of hydrogens. “Polyunsaturated,” generally, refers to fatty acids having hydrocarbon chains with more than one double bond. The double bonds of the unsaturated or polyunsaturated fatty acids of various embodiments may be at any location along the aliphatic hydrocarbon chain and may be in either cis or trans configuration. The term “cis,” refers to a double bond in which carbons adjacent to the double bond are on the same side and the term “trans” refers to a double bond in which carbons adjacent to the double bond are on opposite sides. Typically “cis” is the same as Z, and “trans” is the same as E but sometimes the IUPAC rules for naming compounds will give the opposite of this, which is the typical case in nitroalkenes. For example, a nitroalkene can have the two carbon groups “cis” but the two groups that take priority for the naming of compounds (a nitro group on one carbon of the alkene and a carbon group on the other carbon of the alkene) are on opposite sides and thus are E. Therefore the nitroalkene analog of a “cis” double bond is actually an E nitroalkene. Similarly, the nitroalkene analog of a “trans” double bond is actually a Z nitroalkene. Without wishing to be bound by theory, double bonds in cis configuration along the carbon chain (cis carbon chain but E nitroalkene) may induce a bend in the hydrocarbon chain. Double bonds in “trans,” configuration along the carbon chain (trans carbon chain but Z nitroalkene) may not cause the hydrocarbon chain to bend.
Many unsaturated and polyunsaturated fatty acids have been identified and are known to be naturally occurring. Such unsaturated or polyunsaturated naturally occurring fatty acids, generally, include an even number of carbons in their aliphatic hydrocarbon chain. For example, a naturally occurring unsaturated or polyunsaturated fatty acid may have, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and so on carbons and may include omega(ω-3, ω-5, ω-6, ω-7, ω-9 fatty acids and the like. Any such fatty acid may be useful in embodiments of the invention. The symbol ‘ω’ is used to refer to the terminal methyl carbon of the aliphatic hydrocarbon chain. The placement of the double bond of the ω-X fatty acid is the carbon-carbon bond X number of carbons from the ω carbon. For example, an ω-6 fatty acid has a double bond between the 6th and 7th carbons counting backward from the ω carbon and an ω-3 fatty acid has a double bond between the 3rd and 4th carbons counting backward from the co carbon. Various embodiments of the invention include ω-3 fatty acids, including, but not limited to, linoleic acid, alpha-linoleic acid, eicosapentanoic acid, docosapentaenoic acid, docosahexanoic acid and stearidonic acid; ω-5 fatty acids including, but not limited to, myristoleic acid; ω-6 fatty acids including, but not limited to, linoleic acid, gamma-linoleic acid, dihomo-gamma-linoleic acid and arachidonic acid; ω-7 fatty acids including, but not limited to, conjugated linoleic acid, palmitoleic acid; and ω-9 fatty acids including, but not limited to, oleic acid and erucic acid. Of course, the fatty acids of the invention may also be referred to using IUPAC nomenclature in which the placement of the double bond is determined by counting from the carbon of the carboxylic acid, and ‘C-X’ denotes the carbon in aliphatic hydrocarbons using IUPAC nomenclature wherein X is the number of the carbon counting from the carboxylic acid. Embodiments of the invention also include synthetic equivalents to naturally occurring fatty acids and derivatives thereof.
In particular embodiments, the fatty acids utilized in embodiments of the invention may be omega-3 fatty acids. As used herein, the term “omega-3 fatty acids” or “ω-3 fatty acids” may include natural or synthetic omega-3 fatty acids, or pharmaceutically acceptable esters, derivatives, conjugates (see, e.g., U.S. Publication No. 2004/0254357 to Zaloga et al. and U.S. Pat. No. 6,245,811 to Horrobin et al., each of which is hereby incorporated by reference in its entirety), precursors or salts thereof and mixtures thereof. Examples of ω-3 fatty acid oils include but are not limited to ω-3 polyunsaturated, long-chain fatty acids such as a eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and α-linolenic acid; esters of ω-3 fatty acids with glycerol such as mono-, di- and triglycerides; and esters of the ω-3 fatty acids and a primary, secondary or tertiary alcohol such as fatty acid methyl esters and fatty acid ethyl esters. In certain embodiments, the ω-3 fatty acid oils may be long-chain fatty acids such as EPA or DHA, triglycerides thereof, ethyl esters thereof and mixtures thereof. The ω-3 fatty acids or their esters, derivatives, conjugates, precursors, salts and mixtures thereof can be used either in their pure form or as a component of an oil, such as fish oil, preferably purified fish oil concentrates.
Various fish oils are known and useful as sources for ω-3, ω-6, and ω-9 fatty acids, and any such oil may be used in embodiments of the invention. For example, oils derived from herring, sardines, mackerel, lake trout, flounder, albacore tuna, krill, and salmon are useful sources of ω-3, ω-6, and ω-9 fatty acids.
Commercially available ω-3 fatty acids suitable for use in the invention may include, but are not limited to, Life's DHA (Martek Biosciences Corporation, Columbia, Md.) Incromega F2250, F2628, E2251, F2573, TG2162, TG2779, TG2928, TG3525 and E5015 (Croda International PLC, Yorkshire, England), and EPAX6000FA, EPAX5000TG, EPAX4510TG, EPAX2050TG, K85TG, K85EE, K80EE and EP AX7010EE (Pronova Biocare a.s., 1327 Lysaker, Norway). In certain embodiments, the ω-3 fatty acids may be a mixture of several ω-3 fatty acids such as OMACOR™ omega-3 fatty acids which are combinations of EPA and DHA ω-3 fatty acids, and are described in U.S. Pat. Nos. 5,502,077, 5,656,667 and 5,698,594, which are hereby incorporated by reference in their entireties.
Similarly various plant oils are known and useful as sources for ω-3, ω-6, and ω-9 fatty acids, and any such oil may be used in embodiments of the invention. For example, vegetable oils, such as safflower oil, sunflower oil, olive oil, nut oils, such as peanut oil, walnut oil, pecan oil, hazelnut oil, or seed oil, such as kiwifruit seed oil, perilla seed oil, chia seed oil, flax seed oil, lingonberry seed oil, camelina seed oil, purslane seed oil, black raspberry seed oil, hemp seed oil, canola oil, grape seed oil, sesame seed oil, and poppy seed oil are useful sources of ω-3, ω-6, and ω-9 fatty acids, and in particular ω-9 fatty acids, such as, oleic acid http://en.wikipedia.org/wiki/Oleic_acid-cite_note-pmid17093176-2#cite_note-pmid17093176-2.
In particular embodiments of the invention the fatty acids utilized in embodiments of the invention may be conjugated fatty acids. As used herein, the term “conjugated fatty acid” may include natural or synthetic conjugated fatty acids, or pharmaceutically acceptable esters, derivatives, conjugates, precursors or salts thereof and mixtures thereof. As the nomenclature implies, the double bonds of CLAs are conjugated, with only one single bond between them. Examples of conjugated fatty acids include (9Z,11E)-octadeca-9,11-dienoic acid and 10E, 12Z-octadeca-10,12-dienoic acid, both of which are isomers of conjugated linoleic acid an ω)-7 polyunsaturated fatty acid.
Conjugated fatty acids such as conjugated linoleic acid can be readily found in nature. In particular, in meat and dairy products derived from ruminant animals. Conjugated linoleic acid is also produced by humans from certain trans isoforms of oleic acid.
Other embodiments of the invention include unsaturated or polyunsaturated non naturally occurring fatty acids which may have an odd number of carbons such as, for example, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21 and so on. As in naturally occurring fatty acids, the one or more double bonds associated with non-naturally occurring fatty acids may be at any position along the aliphatic hydrocarbon chain, and the double bonds may be in either cis or trans configuration. In yet other embodiments, the non-naturally occurring fatty acids may include one or more linker groups which interrupt the aliphatic hydrocarbon chain. For example, in some embodiments, activated fatty acids may have one or more non-carbon-carbon linkage such as, for example, ester, ether, vinyl ether, amino, imine and the like at any position within the aliphatic hydrocarbon chain.
As set forth above, nitrite has the chemical formula NO2− and includes a symmetric anion with equal N—O bond lengths. In vivo nitrite has often been considered as an inert end product of nitric oxide metabolism and, therefore, was looked on unfavorably as a dietary constituent. Recently, however, a new view of nitrite metabolism has led scientists to the understanding that metabolism of nitrite occurs in the blood and tissues of the body to form nitric oxide (NO) and other bioactive nitrogen oxides. Thus nitrite can be viewed as a storage pool for supporting NO signaling during metabolic stress.
Moreover both nitrate (NO3−) and nitrite are found readily in the everyday diet and their levels can be especially high in certain vegetables. For example, a single serving of spinach, lettuce or beet root is known to contain relatively high levels of nitrate. Moreover, diets such as the Mediterranean diet or the Japanese diets, that are rich in vegetables, have shown beneficial results with respect to reducing blood pressure and protecting against cardiovascular disease.
In particular embodiments of the invention, the dietary supplement includes an unsaturated fatty acid component and a nitrite component. The unsaturated fatty acid component may be non-animal based, such as algae-derived DHA or it could also be a plant based oil, a vegetable based oil, such as safflower oil, sunflower oil or olive oil, and the like, a nut based oil, such as peanut oil, walnut oil, pecan oil or hazel nut oil, and the like, or a seed based oil, such as kiwifruit seed oil, perilla seed oil, chia seed oil, flax seed oil, lingonberry seed oil, camelina seed oil, purslane seed oil, black raspberry seed oil, hemp seed oil, or canola oil, and the like.
The dietary supplement also preferably includes a nitrite and/or a nitrate component. In some embodiments, the nitrite and/or nitrate component may be beet root juice, either in liquid form or in powder form. In other embodiments the nitrite and/or nitrate component may be sodium nitrate, sodium nitrite or other source of nitrite or nitrate. The source of nitrite and/or nitrate could be any source that would be capable of providing nitrite and/or nitrate for combination with the unsaturated fatty acid component as described in detail below.
In some embodiments of the invention, the unsaturated fatty acid component and the nitrite and/or nitrate component are formulated as separate components that are each formed into separate gel caps, tablets, caplets, or the like. The separate tablets are preferably packaged together, in a single foil wrapper, or similar packaging vehicle, and the consumer of the dietary supplement is instructed to consume one of each separate tablet simultaneously.
In another embodiment of the invention, the unsaturated fatty acid component and the nitrite and/or nitrate component are combined in a single capsule where the components are allowed to mix with one another prior to ingestion by the consumer of the dietary supplement.
Once the dietary supplement has been consumed or ingested by the individual, the unsaturated fatty acid component and the nitrate and/or nitrite component of the dietary supplement are allowed to combine with one another and with other components, such as stomach acid and digestive enzymes, present in the stomach and gut of the consumer of the dietary supplement. The gut and stomach provides a suitable environment or “bioreactor” for reaction of the fatty acid component with the nitrite component of the dietary supplement in order to form a nitro fatty acid. More specifically, the temperature and pH of the stomach and gut provide a suitable environment for conversion of the fatty acid component to a nitro fatty acid by the nitrite and/or nitrate component. Once the nitro fatty acid has been produced in the stomach and gut of the consumer of the dietary supplement as set forth above, the nitro fatty acid, or activated fatty acid, is free to be absorbed by the gut into the body of the consumer, where they are known to have beneficial effects in mammals, including humans as are set forth in more detail below.
In certain embodiments of the invention, the unsaturated fatty acid component and the nitrite and/or nitrate component are formulated as a single capsule where the components are kept separate until the time of ingestion, wherein the capsule is broken down by acid and digestive enzymes present in the stomach and gut of the consumer, and the separate components are combined with one another in the stomach and gut of the consumer.
In some embodiments, it is expected that activated fatty acids will be formed from the combination of the fatty acid component and the nitrite and/or nitrate component. In some embodiments, the activated fatty acids formed include nitro fatty acids. Table 1 below, illustrates the formation of activated fatty acids from the combination of various fatty acid components and nitrite components in several exemplary dietary supplement formulations. It is expected that the combination of the fatty acid component and nitrite and/or nitrate component will result in the conversion of about 5% to about 40% of the fatty acid component in the dietary supplement. In some embodiments, about 10%, about 20%, or about 30% of the fatty acid component will be converted to an activated fatty acid.
In some embodiments of the invention, the quantity of the nitrite and/or nitrate component will be greater than the quantity of the fatty acid component. In some embodiments, of the invention, the ratio of fatty acid component to nitrite and/or nitrate component may be from about 1:2 to about 1:1,000. In yet other embodiments, the quantity of the nitrite and/or nitrate component can be less than or equal to fatty acid component. In some embodiments, the ratio of fatty acid component to nitrite and/or nitrate component may be about 2:1 to about 1:1.
As set forth above, in certain embodiments of the invention, the dietary supplement is formulated from only non-animal based components and is known as a vegan formulation. In this vegan formulation, the fatty acid component is derived from plant based oil as set forth above. Moreover, the capsule in the vegan formulation is also preferably made from non-animal sources rendering a finished product that is vegan and completely free of any animal based material.
In other embodiments of the invention, the dietary supplement may be formulated from animal based components and is known as a non-vegan formulation. In this non-vegan formulation, the fatty acid component is derived from animal based oil, such as fish oil. The capsule for the non-vegan formulation is preferably made from conventional encapsulation materials.
In some embodiments of the invention, the dietary or nutritional supplement may include alimentary oil, such as olive oil, combined with a source of nitrite and/or nitrate, such as sodium nitrite, sodium nitrate, beet root juice, beet root powder or the like. Once consumed or ingested by the consumer of the dietary or nutritional supplement, the unsaturated fatty acids present in the olive oil are converted by the nitrite and/or nitrate into nitro fatty acids, which can then be absorbed by the gut into the body, where they are known to have beneficial effects in mammals, including humans as are set forth in greater detail below.
In some embodiments of the invention, the dietary or nutritional supplement may include fish oil and a source of nitrite and/or nitrate. The fish oil may, in some embodiments, be a mixture of DHA and EPA derived from fish oil or in some cases may include other fatty acids such as for example, omega-3 fatty acids that are naturally found in fish oil. In some embodiments, the fish oil can be enriched with one or more fatty acid such as, for example, EPA or DHA or an omega-3 fatty acid. The dietary or nutritional supplement further includes a source of nitrite and/or nitrate such as, for example, beet root juice, beet root powder or, in some embodiments, sodium nitrite, sodium nitrate or another source of nitrite and/or nitrate. Once ingested by the consumer of the dietary or nutritional supplement, the unsaturated fatty acids present in the fish oil are converted by the nitrite and/or nitrate into nitro fatty acids, which can then be absorbed by the gut of the individual into the body, where they are known to have beneficial effects in mammals, including humans as are set forth in greater detail below.
In some embodiments of the invention, the dietary or nutritional supplement may include nitrated fatty acids that are produced by a process of reacting fatty acids with a nitrite and/or nitrate source to produce nitro fatty acids that are then encapsulated for later consumption. The source of the unsaturated fatty acid may be fish oil or alimentary oil. In some embodiments, the fatty acid can be enriched for one or more components such as, for example, DHA or EPA. Enriching as used herein means adding an additional amount of a fatty acid to an existing fatty acid mixture to form a fatty acid blend, which has amounts of one or more fatty acid that are not found naturally or not otherwise found in the fatty acid mixture to be enriched.
Other sources of nitrite and/or nitrate may be suitable for the nitrite and/or nitrate component of the dietary supplement, and include vegetables, such as celery, arugula, butter lettuce, lettuce, spinach, carrots and other food sources that are high in nitrites and/or nitrates. Because nitrates are reduced to nitrites in the gut by commensal bacteria, in the above embodiments any form of these vegetables, for example, powder, paste or oil that is suitable for encapsulation in a dietary supplement would be suitable.
It is contemplated that the dietary and nutritional supplements described above may be packaged by any known methods used in the dietary and nutritional supplement industry, such as gel caps, tablets, caplets and the like.
As used herein an “activated fatty acid” refers to a fatty acid having at least one electron withdrawing group covalently bound to a carbon of the saturated or unsaturated aliphatic chain of a fatty acid. Such activated fatty acids may be substituted by any number of electron withdrawing groups at any number of positions on the hydrocarbon chain, and an electron withdrawing group may be positioned in either cis or trans configuration at a double bond or in either R or S absolute stereochemistry at an sp3 chiral/stereogenic center. For example, an activated fatty acid may have one electron withdrawing group, and in another, an activated fatty acid may be substituted with multiple electron withdrawing groups at multiple positions along the hydrocarbon chain. While the activated fatty acids may have an electron withdrawing group positioned at any carbon along the aliphatic hydrocarbon chain between the carboxy terminal carbon to the terminal methyl (ω), in some cases, the electron withdrawing group may be positioned within about 1 carbon from the carboxy terminal carbon and within about 1 carbon from the terminal methyl. The electron withdrawing group may be positioned within about 3 carbons of either the carboxy terminal carbon and/or the methyl terminal carbon, and in still others cases, the electron withdrawing group may be positioned within 5 carbons of either of the carboxy terminal carbon and/or the methyl terminal carbon.
The electron withdrawing group may be positioned on a carbon directly attached to a double bond of the activated fatty acid forming an “electron withdrawing vinyl” group. The electron withdrawing group of such vinyl groups may be on either side of the double bond.
A mono or polyunsaturated fatty acid may have two electron-withdrawing groups, and there are several ways that an unsaturated fatty acid can have two electron-withdrawing groups.
The activated fatty acids may include compounds of general formulae I and II:
wherein R1 and R2 are independently selected from —H and any electron withdrawing groups including, but not limited to —NO2− wherein at least one of R1 and R2 is an electron withdrawing group and m and n are, independently, 1-20.
The activated fatty acids may also include compounds of general formula III:
wherein R1, R2, m and n are as described above, R3 and R4 are, independently, selected from —H, —OH, —COH, —COR, —CO, —COOH, —COOR, —Cl, —F, —Br, —I, —CF3, —CN, —SO3−, —SO2R, —SO3H, —NH3+, —NH2R+, —NHR2+, —NR3− and —NO2−, k and p are, independently, 0 to 5 and x and y are independently, 0 to 3, and wherein each double bond is in either cis or trans configuration. In still other embodiments, any carbon associated with m, n, k or p may be substituted.
The activated fatty acids may also include compounds of general formula IV:
herein R1, R2, m and n are as described above, R3 and R4 are, independently, selected from —H, —OH, —COH, —COR, —CO, —COOH, —COOR, —Cl, —F, —Br, —I, —CF3, —CN, —SO2−, —SO2R, —SO3H, —NH3+, —NH2R+, —NHR2+, —NR3+ and —NO2−, k and p are, independently, 0 to 5 and x and y are independently, 0 to 3, and wherein each double bond is in either cis or trans configuration. In still other embodiments, any carbon associated with m, n, or p may be substituted.
The dietary supplement as described in various embodiments of the invention above, may be administered to individuals to treat, ameliorate, modulate and/or prevent a number both acute and chronic inflammatory and metabolic conditions. In particular embodiments, the dietary supplement may be used to treat acute conditions including general inflammation, arterial stenosis, organ transplant rejection and burns, and chronic conditions such as, chronic lung injury and respiratory distress, diabetes, hypertension, obesity, rheumatoid arthritis, neurodegenerative disorders and various skin disorders. However, in other embodiments, the dietary supplement may be used to treat any condition having symptoms including chronic or acute inflammation, such as, for example, arthritis, lupus, Lyme's disease, gout, sepsis, hyperthermia, ulcers, enterocolitis, osteoporosis, viral or bacterial infections, cytomegalovirus, periodontal disease, glomerulonephritis, sarcoidosis, lung disease, lung inflammation, fibrosis of the lung, asthma, acquired respiratory distress syndrome, tobacco induced lung disease, granuloma formation, fibrosis of the liver, graft vs. host disease, postsurgical inflammation, coronary artery bypass graft (CABG), acute and chronic leukemia, B lymphocyte leukemia, neoplastic diseases, arteriosclerosis, atherosclerosis, myocardial inflammation, psoriasis, immunodeficiency, disseminated intravascular coagulation, systemic sclerosis, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, encephalomyelitis, edema, inflammatory bowel disease, hyper IgE syndrome, cancer metastasis or growth, adoptive immune therapy, reperfusion syndrome, radiation burns, alopecia and the like.
For example, in one embodiment, the dietary supplement may be administered to treat hypertension by lowering blood pressure to normal levels without reducing the blood pressure of the individual below normal levels even if the dietary supplement is over-administered. Thus, without wishing to be bound by theory, the dietary supplement of the invention may provide treatment of an individual without the negative effects associated with over-administration or over-treatment using traditional medications.
In another embodiment, the dietary supplement as described in various embodiments of the invention above may be administered to treat pre-diabetes of an individual by controlling and/or lowering the blood sugar of the individual. More specifically, a method of controlling and/or lowering blood sugar levels, HBA1C levels or a combination thereof, of an individual with pre-diabetes includes administering the dietary supplement to the individual and monitoring the blood sugar levels, HBA1C levels or a combination thereof, of the individual.
In a still further embodiment, the dietary supplement may be useful for ischemic preconditioning or protecting the heart from ischemic injury due to vessel spasm or blockage. For example, nitrated fatty acids produced by mitochondria in cells under ischemic conditions cause a number of physiological changes within the cell that increases cell survival under ischemic conditions. By providing activated fatty acids to an individual, similar ischemic preconditioning or protection may be achieved allowing for improved survival of, for example, cardiac tissue under ischemic conditions or organs being preserved for optimizing viability and function upon transplantation. In particular embodiments, the dietary supplement may be provided to individuals at risk of heart disease, heart attack, heart failure, vascular blockage, arrhythmia, atrial fibrillation, heart valve diseases, cardiomyopathy, and the like to both reduce or alleviate the symptoms of such maladies and to increase the likelihood of survival in the event of, for example, a heart attack, arrhythmia, or arterial fibrillation or to more generally improve heart or circulatory system function.
In addition, administration of the dietary supplement may be useful for activating a number of other factors important for cell signaling. For example, in one embodiment, activated fatty acids may induce gene expression and tissue activity of heme oxygenase-1 (HO-1) which has been shown to mediate adaptive and protective responses during inflammation, and activation of an adaptive or protective inflammatory response mediated by HO may be useful in treating inflammatory diseases such as, but not limited to, atherosclerosis, acute renal failure, vascular restenosis, transplant rejection, and sepsis. Thus, activated fatty acids may be useful for treating general inflammation resulting from surgery, injury or infection.
The dietary and nutritional supplements of the invention can be administered in any conventional manner by any route where they are active. Administration can be systemic or local. For example, administration can be, but is not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, buccal, ocular, intravaginally, or inhalation. In certain embodiments, the administration may be parenteral. In some embodiments, the supplement may be prepared in the presence or absence of stabilizing additives that favors extended systemic uptake, tissue half-life and intracellular delivery. Thus, modes of administration for the compounds of the present invention (either alone or in combination with other pharmaceuticals) can be injectable (including short-acting, depot, implant and pellet forms injected subcutaneously or intramuscularly). In some embodiments, an injectable formulation including the supplement may be deposited to a site of injury or inflammation, such as, for example, the site of a surgical incision or a site of inflammation due to arthroscopy, angioplasty, stent placement, by-pass surgery and so on.
When administered, activated fatty acids produced by the mixing of the saturated fatty acid component and the nitrite and/or nitrate component, may interact with a number of cellular receptors and/or proteins that mediate inflammation, either by inhibiting or stimulating their activity thereby inhibiting or reducing inflammation. Without wishing to be bound by theory, activated fatty acids may modulate important signaling activities including, for example, neurotransmission, gene expression, vascular function and inflammatory responses, and chemical properties of activated fatty acids that may facilitate these activities include, but are not limited to, the strong, reversible electrophilic nature of the (3 carbon adjacent to the electron withdrawing vinyl group, an ability to undergo Nef-like acid base reactions to release NO, an ability to partition into both hydrophobic and hydrophilic compartments, and a strong affinity for G-protein coupled receptors and nuclear receptors.
For example, in one embodiment, the dietary supplement may be administered to mediate cell signaling via multiple G-protein coupled receptors and nuclear receptors such as, but not limited to, peroxisome proliferator-activated receptors (PPAR) including PPARα, PPARγ, and PPARδ. PPAR is a nuclear receptor that is expressed throughout an organism, including in monocytes/macrophages, neutrophils, endothelial cells, adipocytes, epithelial cells, hepatocytes, mesangial cells, vascular smooth muscle cells, neuronal cells and when “activated” induces transcription of a number of target genes. Activation of PPAR has been shown to play various roles in regulating tissue homeostasis including, for example, increasing insulin sensitivity, suppress chronic inflammatory processes, reduce circulating free fatty acid levels, correct endothelial dysfunction, reduce fatty streak formation, delay plaque formation, limit blood vessel wall thickening and enhance plaque stabilization and regression. The activated fatty acids produced as a result of ingesting the dietary supplement embodied herein may perform each of these functions associated with PPAR activation.
Moreover, activated fatty acids may perform these functions without significantly altering normal cellular process. For example, in one embodiment, the dietary supplement may be administered to treat hypertension by lowering blood pressure to normal levels without reducing the blood pressure of the individual below normal levels even if the activated fatty acid is over-administered. Thus, without wishing to be bound by theory, the dietary supplement of the invention may provide treatment of an individual without the negative effects associated with over-administration or over-treatment using traditional medications.
Shopfer et al. have found that conjugated linoleic is an endogenous ligand for PPARγ. Activation of PPAR has been shown to be induced by a locking reaction in which a critical thiol in a highly conserved cysteine (Cys 285 of human PPARγ) which is located in a ligand binding domain of PPAR. Partial activation of PPAR has been shown to occur when relatively high concentrations of known thiol reactive compounds, such as 15-deoxy-Δ12,14-prostaglandin J2 (15-d PGJ2), are administered. Without wishing to be bound by theory, activated fatty acids may bind to PPAR covalently at the reactive thiol in the ligand binding domain of PPAR. Moreover, activated fatty acids may induce a conformational change in PPAR. More specifically, activated fatty acid binding may result in the C-terminus of the ligand binding domain (α-helix 12) to adopt an active conformation that may promote a beneficial pattern of co-repressor release and co-activator recruitment. Thus, activated fatty acids may enhance PPAR activation and transcription of PPAR regulated genes beyond that of known PPAR activating compounds.
Activation of PPAR has been shown to be induced by a locking reaction in which a critical thiol in a highly conserved cysteine (Cys 285 of human PPARγ) which is located in a ligand binding domain of PPAR. Partial activation of PPAR has been shown to occur when relatively high concentrations of known thiol reactive compounds, such as 15-deoxy-Δ12,14-prostaglandin J2 (15-d PGJ2), are administered. Without wishing to be bound by theory, activated fatty acids may bind to PPAR covalently at the reactive thiol in the ligand binding domain of PPAR. Moreover, activated fatty acids may induce a conformational change in PPAR. More specifically, activated fatty acid binding may result in the C-terminus of the ligand binding domain (α-helix 12) to adopt an active conformation that may promote a beneficial pattern of co-repressor release and co-activator recruitment. Thus, activated fatty acids may enhance PPAR activation and transcription of PPAR regulated genes beyond that of known PPAR activating compounds.
In addition to activation of PPAR, activated fatty acid administration may be useful for activating a number of other factors important for cell signaling. For example, in one embodiment, the dietary supplement may be administered to induce gene expression and tissue activity of heme oxygenase-1 (HO-1) which has been shown to mediate adaptive and protective responses during inflammation, and activation of an adaptive or protective inflammatory response mediated by HO may be useful in treating inflammatory diseases such as, but not limited to, atherosclerosis, acute renal failure, vascular restenosis, transplant rejection, and sepsis. In addition, the dietary supplement may be useful for activating a number of other factors important for cell signaling. For example, in one embodiment, the dietary supplement may be administered to induce gene expression and tissue activity of heme oxygenase-1 (HO-1) which has been shown to mediate adaptive and protective responses during inflammation, and activation of an adaptive or protective inflammatory response mediated by HO may be useful in treating inflammatory diseases such as, but not limited to, atherosclerosis, acute renal failure, vascular restenosis, transplant rejection, and sepsis. Thus, the dietary supplement may be useful for treating general inflammation resulting from surgery, injury or infection. In another embodiment, activated fatty acids produced by the combination of the fatty acid component and the nitrite may induce a reversible post-translational modification of proteins, such as, for example, glutathione (GSH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by covalently binding to catalytic cysteines on such proteins. Without wishing to be bound by theory, the covelent modification of these proteins by activated fatty acids may increase the hydrophobicity of these proteins inducing translocation to membranes and suggests a role for redox regulation of enzyme function, cell signaling and protein trafficking. In yet another embodiment, activated fatty acids may be administered to repress NF-κB dependent gene expression and endothelial tumor necrosis factor-α induced expression of vascular cell adhesion molecules in monocytes and macrophages which results in inhibition of rolling and adhesion during inflammation. Thus, activated fatty acids may be useful for treating general inflammation resulting from surgery, injury or infection. In a further embodiment, activated fatty acids may be administered to limit tissue inflammatory injury and inhibit the proliferation of vascular smooth muscle cells by increasing cellular levels of nuclear factor erythroid 2-related factor-2 (Nrf-2) which may be useful in the treatment of a number of vascular diseases. In a still further embodiment, activated fatty acids may be useful for ischemic preconditioning. For example, nitrated fatty acids produced by mitochondria in cells under ischemic conditions cause a number of physiological changes within the cell that increases cell survival under ischemic conditions. By providing activated fatty acids to an individual, similar ischemic preconditioning may be achieved allowing for improved survival of, for example, cardiac tissue under ischemic conditions or organs being preserved for optimizing viability and function upon transplantation.
In some embodiments, the dosage regimen as described above may be combined with a secondary form of treatment or a secondary agent. For example, the dietary supplement such as those described above may be combined with antioxidants, statins, squalene synthesis inhibitors, azetidinone-based compounds, LDL catabolism activators, PPAR antagonists or agonsits, antiarrhythmic agent, NSAIDs and the like, and combinations thereof.
In particular embodiments, the dietary supplement of the invention may be mixed with one or more nutraceutical equivalents to any of the agents described above. For example, in some embodiments, the dietary supplement of the invention may be mixed with a nutraceutical statin equivalent such as, for example, from rice bran oil, enzyme-treated stabilized rice bran, a solubilized fraction of rice bran oil, and derivatives thereof and the like. In other embodiments, one or more nutraceutical including, but not limited to, glucosamine derivatives, methylsulfonylmethane, yucca concentrates, grape seed extracts, beta-carotene, ephedra, ginkgo biloba, goldenseal, valerian, ginseng, echinacea, and the like may be combined with the dietary supplement.
Embodiments further include nutraceuticals including the nutraceutical equivalents to any of the agents described above. Thus, in certain embodiments, the nutraceuticals may include one or more other nutraceutical compound or one or more other secondary agent. Nutraceuticals containing various combinations of ingredients are well known in the art, and any known nutraceutical may be combined to produce a combination nutraceutical. For example, in various embodiments, the dietary supplement may be combined with vitamins including vitamins A, B, including vitamin B-1, B-2, B-6, B-12, C, D including vitamin D3, and E, and the like and derivatives thereof, minerals such as selenium and the like, plant extracts such as β-carotene, ginkgo biloba, goldenseal, valerian, ginseng, echinacea, grape seed extracts, ephedra, yucca concentrates, green tea extract, rice bran extract, wheat germ, wheat germ extract, beeswax, red yeast rice extract, stevia leaf extract, and the like, nutraceutical oils such as flaxseed oil, borage seed oil, and other know nutraceutical components such as coenzyme Q10, glucosamine derivatives, methylsulfonylmethane, pantothenic acid, biotin, thiamin, riboflavin, niacin, folic acid, palmitic acid, and the like. Thus, without wishing to be bound by theory, nearly any nutraceutical can be incorporated into the dietary supplement described herein.
In particular embodiments, one or more additional ingredients may be provided to produce a nutraceutical for treating or preventing specific diseases or indication. For example, in some embodiments, the dietary supplement may be combined with other nutraceutically active components that can act as antioxidants such as vitamin C, vitamin E, vitamin D, selenium and the like to create a nutraceutical for treating aging and cancer. In other embodiments, a nutraceutical for treating or preventing diseases of the eye may be prepared by combining the dietary supplement with, for example, vitamin A and/or β-carotene, and in still other embodiments, a nutraceutical with neuroprotective activities or that enhances cognitive abilities may be prepared by combining the dietary supplement with, for example, ginkgo biloba. In yet other embodiments, nutraceuticals for treating or preventing heart or circulatory diseases may be prepared by combining the dietary supplement with policosanol, guggulipids, rice bran extract, enzyme-treated stabilized rice bran, a solubilized fraction of rice bran oil, wheat germ, wheat germ extract, beeswax, red yeast rice extract, and or other nutraceuticals known to exhibit statin-like activity. In further embodiments, components with various activities may be combined. For example, a nutraceutical with neuroprotective activities may include one or more antioxidants such as vitamin C, vitamin E, or selenium along with ginkgo biloba, since it is well known that antioxidants are also effective neuroprotectants. In yet other embodiments, vitamin E may be provided to any nutraceutical described herein to stabilize the activated fatty acids and increase the shelf life of the nutraceutical.
Nutraceuticals having fatty acids and one or more additional nutraceutically active components may be combined in a single dose formulation by known methods. For example, in some embodiments, lipophilic additional nutraceutically active components may be combined with the activated fatty acids directly. In other embodiments, the activated fatty acid may be separated from a non-lipophilic additional nutraceutically active component by, for example, preparing separate cores that are combined into a single capsule or incorporating the non-lipophilic additional nutraceutically active component into one or more coating layers.
The dietary supplement of various embodiments may be prepared by any method known in the art. For example, in particular embodiments, the dietary supplement may be derived from natural sources such as, for example, fish oils which may contain activated fatty acids, and in particular, nitro-fatty acids that can be isolated, purified or concentrated form the fish oil. In other embodiments, an activated fatty acid may be prepared by contacting a naturally occurring unsaturated fatty acids with one or more nitro containing compounds or nitrogenating agents. Such naturally occurring activated fatty acids may be useful in the production of nutraceuticals.
Other embodiments of the invention include a dietary supplement prepared as described above which are formulated as a solid dosage form for oral administration including capsules, tablets, pills, powders, and granules. In such embodiments, the active compound may be admixed with one or more inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents and can additionally be prepared with enteric coatings.
Preparation of the dietary supplement in solid dosage form may vary. For example, in one embodiment, a liquid or gelatin formulation of the dietary supplement may be prepared by combining the fatty acid and the nitrite with one or more fatty acid diluent, such as those described above, and adding a thickening agent to the liquid mixture to form a gelatin. The gelatin may then be encapsulated in unit dosage form to form a capsule. In another exemplary embodiment, an oily preparation of a fatty acid and nitrite prepared as described above may be lyophilized to form a solid that may be mixed with one or more pharmaceutically acceptable excipient, carrier or diluent to form a tablet, and in yet another embodiment, the fatty acid of an oily preparation may be crystallized to from a solid which may be combined with a pharmaceutically acceptable excipient, carrier or diluent to form a tablet.
Further embodiments which may be useful for oral administration of the dietary supplement include liquid dosage forms. In such embodiments, a liquid dosage may include a pharmaceutically acceptable emulsion, solution, suspension, syrup, and elixir containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
Other suitable diluents for formulations include, but are not limited to those described below:
Vegetable oil: As used herein, the term “vegetable oil” refers to a compound, or mixture of compounds, formed from ethoxylation of vegetable oil, wherein at least one chain of polyethylene glycol is covalently bound to the vegetable oil. In some embodiments, the fatty acids have between about twelve carbons to about eighteen carbons. In some embodiments, the amount of ethoxylation can vary from about 2 to about 200, about 5 to 100, about 10 to about 80, about 20 to about 60, or about 12 to about 18 of ethylene glycol repeat units. The vegetable oil may be hydrogenated or unhydrogenated. Suitable vegetable oils include, but are not limited to castor oil, hydrogenated castor oil, sesame oil, corn oil, peanut oil, olive oil, sunflower oil, safflower oil, soybean oil, benzyl benzoate, sesame oil, cottonseed oil, and palm oil. Other suitable vegetable oils include commercially available synthetic oils such as, but not limited to, Miglyol™ 810 and 812 (available from Dynamit Nobel Chemicals, Sweden) Neobee™ M5 (available from Drew Chemical Corp.), Alofine™ (available from Jarchem Industries), the Lubritab™ series (available from JRS Pharma), the Sterotex™ (available from Abitec Corp.), Softisan™ 154 (available from Sasol), Croduret™ (available from Croda), Fancol™ (available from the Fanning Corp.), Cutina™ HR (available from Cognis), Simulsol™ (available from CJ Petrow), EmCon™ CO (available from Amisol Co.), Lipvol™ CO, SES, and HS-K (available from Lipo), and Sterotex™ HM (available from Abitec Corp.). Other suitable vegetable oils, including sesame, castor, corn, and cottonseed oils, include those listed in R. C. Rowe and P. J. Shesky, Handbook of Pharmaceutical Excipients, (2006), 5th ed., which is incorporated herein by reference in its entirety. Suitable polyethoxylated vegetable oils, include but are not limited to, Cremaphor™ EL or RH series (available from BASF), Emulphor™ EL-719 (available from Stepan products), and Emulphor™ EL-620P (available from GAF).
Mineral oils: As used herein, the term “mineral oil” refers to both unrefined and refined (light) mineral oil. Suitable mineral oils include, but are not limited to, the Avatech™ grades (available from Avatar Corp.), Drakeol™ grades (available from Penreco), Sirius™ grades (available from Shell), and the Citation™ grades (available from Avater Corp.).
Castor oils: As used herein, the term “castor oil”, refers to a compound formed from the ethoxylation of castor oil, wherein at least one chain of polyethylene glycol is covalently bound to the castor oil. The castor oil may be hydrogenated or unhydrogenated. Synonyms for polyethoxylated castor oil include, but are not limited to polyoxyl castor oil, hydrogenated polyoxyl castor oil, microgolglyceroli ricinoleas, macrogolglyceroli hydroxystearas, polyoxyl 35 castor oil, and polyoxyl 40 hydrogenated castor oil. Suitable polyethoxylated castor oils include, but are not limited to, the Nikkol™ HCO series (available from Nikko Chemicals Co. Ltd.), such as Nikkol HCO-30, HC-40, HC-50, and HC-60 (polyethylene glycol-30 hydrogenated castor oil, polyethylene glycol-40 hydrogenated castor oil, polyethylene glycol-50 hydrogenated castor oil, and polyethylene glycol-60 hydrogenated castor oil, Emulphor™ EL-719 (castor oil 40 mole-ethoxylate, available from Stepan Products), the Cremophore™ series (available from BASF), which includes Cremophore RH40, RH60, and EL35 (polyethylene glycol-40 hydrogenated castor oil, polyethylene glycol-60 hydrogenated castor oil, and polyethylene glycol-35 hydrogenated castor oil, respectively), and the Emulgin® RO and EIRE series (available from Cognis PharmaLine). Other suitable polyoxyethylene castor oil derivatives include those listed in R. C. Rowe and P. J. Shesky, Handbook of Pharmaceutical Excipients, (2006), 5th ed., which is incorporated herein by reference in its entirety.
Sterol: As used herein, the term “sterol” refers to a compound, or mixture of compounds, derived from the ethoxylation of sterol molecule. Suitable polyethoxylated sterols include, but are not limited to, PEG-24 cholesterol ether, Solulan™ C-24 (available from Amerchol); PEG-30 cholestanol, Nikkol™ DHC (available from Nikko); Phytosterol, GENEROL™ series (available from Henkel); PEG-25 phyto sterol, Nikkol™ BPSH-25 (available from Nikko); PEG-5 soya sterol, Nikkol™ BPS-5 (available from Nikko); PEG-10 soya sterol, Nikkol™ BPS-10 (available from Nikko); PEG-20 soya sterol, Nikkol™ BPS-20 (available from Nikko); and PEG-30 soya sterol, Nikkol™ BPS-30 (available from Nikko). As used herein, the term “PEG” refers to polyethylene glycol.
Polyethylene glycol: As used herein, the term “polyethylene glycol” or “PEG” refers to a polymer containing ethylene glycol monomer units of formula —O—CH2-CH2-. Suitable polyethylene glycols may have a free hydroxyl group at each end of the polymer molecule, or may have one or more hydroxyl groups etherified with a lower alkyl, e.g., a methyl group. Also suitable are derivatives of polyethylene glycols having esterifiable carboxy groups. Polyethylene glycols useful in the present invention can be polymers of any chain length or molecular weight, and can include branching. In some embodiments, the average molecular weight of the polyethylene glycol is from about 200 to about 9000. In some embodiments, the average molecular weight of the polyethylene glycol is from about 200 to about 5000. In some embodiments, the average molecular weight of the polyethylene glycol is from about 200 to about 900. In some embodiments, the average molecular weight of the polyethylene glycol is about 400. Suitable polyethylene glycols include, but are not limited to polyethylene glycol-200, polyethylene glycol-300, polyethylene glycol-400, polyethylene glycol-600, and polyethylene glycol-900. The number following the dash in the name refers to the average molecular weight of the polymer. In some embodiments, the polyethylene glycol is polyethylene glycol-400. Suitable polyethylene glycols include, but are not limited to the Carbowax™ and Carbowax™ Sentry series (available from Dow), the Lipoxol™ series (available from Brenntag), the Lutrol™ series (available from BASF), and the Pluriol™ series (available from BASF).
Propylene glycol fatty acid ester: As used herein, the term “propylene glycol fatty acid ester” refers to a monoether or diester, or mixtures thereof, formed between propylene glycol or polypropylene glycol and a fatty acid. Fatty acids that are useful for deriving propylene glycol fatty alcohol ethers include, but are not limited to, those defined herein. In some embodiments, the monoester or diester is derived from propylene glycol. In some embodiments, the monoester or diester has about 1 to about 200 oxypropylene units. In some embodiments, the polypropylene glycol portion of the molecule has about 2 to about 100 oxypropylene units. In some embodiments, the monoester or diester has about 4 to about 50 oxypropylene units. In some embodiments, the monoester or diester has about 4 to about 30 oxypropylene units. Suitable propylene glycol fatty acid esters include, but are not limited to, propylene glycol laurates: Lauroglycol™ FCC and 90 (available from Gattefosse); propylene glycol caprylates: Capryol™ PGMC and 90 (available from Gatefosse); and propylene glycol dicaprylocaprates: Labrafac™ PG (available from Gatefosse).
Stearoyl macrogol glyceride: Stearoyl macrogol glyceride refers to a polyglycolized glyceride synthesized predominately from stearic acid or from compounds derived predominately from stearic acid, although other fatty acids or compounds derived from other fatty acids may be used in the synthesis as well. Suitable stearoyl macrogol glycerides include, but are not limited to, Gelucire® 50/13 (available from Gattefosse).
In some embodiments, the diluent component comprises one or more of mannitol, lactose, sucrose, maltodextrin, sorbitol, xylitol, powdered cellulose, microcrystalline cellulose, carboxymethylcellulose, carboxyethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, starch, sodium starch glycolate, pregelatinized starch, a calcium phosphate, a metal carbonate, a metal oxide, or a metal aluminosilicate.
Exemplary excipients or carriers for use in solid and/or liquid dosage forms include, but are not limited to:
Sorbitol: Suitable sorbitols include, but are not limited to, PharmSorbidex E420 (available from Cargill), Liponic 70-NC and 76-NC (available from Lipo Chemical), Neosorb (available from Roquette), Partech SI (available from Merck), and Sorbogem (available from SPI Polyols).
Starch, sodium starch glycolate, and pregelatinized starch include, but are not limited to, those described in R. C. Rowe and P. J. Shesky, Handbook of Pharmaceutical Excipients, (2006), 5th ed., which is incorporated herein by reference in its entirety.
Disintegrant: The disintegrant may include one or more of croscarmellose sodium, carmellose calcium, crospovidone, alginic acid, sodium alginate, potassium alginate, calcium alginate, an ion exchange resin, an effervescent system based on food acids and an alkaline carbonate component, clay, talc, starch, pregelatinized starch, sodium starch glycolate, cellulose floc, carboxymethylcellulose, hydroxypropylcellulose, calcium silicate, a metal carbonate, sodium bicarbonate, calcium citrate, or calcium phosphate.
Still further embodiments of the invention include activated fatty acids administered in combination with other active ingredients such as, for example, adjuvants, protease inhibitors, or other compatible drugs or compounds where such combination is seen to be desirable or advantageous in achieving the desired effects of the methods described herein.
In certain embodiments, the dietary supplement may be a gel capsule, and in some embodiments, the one or more activated fatty acids may be about 5% by weight to about 95% by weight of the total gel capsule.
In further embodiments, at least one of the one or more secondary agent may include one or more agents selected from solubilizers, stabilizers, colorants, plasticizers diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, antioxidants, or preservatives or a combination thereof.
The compositions of various embodiments may further include one or more film forming materials and/or binders and/or other conventional additives such as lubricants, fillers, antiadherents, antioxidants, buffers, solubilizers, dyes, chelating agents, disintegrants, and/or absorption enhancers. Surfactants may act as both solubilizers and absorption enhancers. Additionally, coatings may be formulated for immediate release, delayed or enteric release, or sustained release in accordance with methods well known in the art. Conventional coating techniques are described, e.g., in Remington's Pharmaceutical Sciences, 18th Ed. (1990), hereby incorporated by reference. Additional coatings to be employed in accordance with the invention may include, but are not limited to, for example, one or more immediate release coatings, protective coatings, enteric or delayed release coatings, sustained release coatings, barrier coatings, and combinations thereof. In some embodiments, an immediate release coating may be used to improve product elegance as well as for a moisture barrier, and taste and odor masking. Rapid breakdown of the film in gastric media is important, leading to effective disintegration and dissolution.
In some embodiments, the compositions may include at least one or more secondary agent. For example, in some embodiments, at least one polymer, such as, but not limited to cellulose derivatives such as hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, polyvinylpyrrolidone/vinyl acetate copolymer, ethyl cellulose aqueous dispersions and combinations thereof, preferably hydroxpropyl cellulose, ethyl cellulose, and mixtures thereof, may be added to the composition at a ratio of polymer to secondary agent of from about 1:20 to about 20:1 by weight or about 1:5 to about 10:1 by weight. In particular, where the amount of secondary agent is less than about 15, mg, the amount of polymer may be from about 1:2 to about 5:1 or from about 1:1 to about 4:1, and in embodiments where the amount of secondary agent is about 15 mg or more, the amount of polymer may be from about 1:4 to about 4:1 or about 1:3 to about 2:1.
In embodiments in which one or more secondary agents are included in the composition, the secondary agent may be provided as a homogenous solution or a heterologous suspension in a pharmaceutically acceptable solvent. Such pharmaceutically acceptable solvents may be an aqueous or organic solvent such as, for example, methanol, ethanol, isopropranol, ethylene glycol, acetone, or mixtures thereof. In other embodiments, pharmaceutically acceptable solvents may include, but are not limited to, polypropylene glycol; polypropylene glycol; polyethylene glycol, for example, polyethylene glycol 600, polyethylene glycol 900, polyethylene glycol 540, polyethylene glycol 1450, polyethylene glycol 6000, polyethylene glycol 8000, and the like; pharmaceutically acceptable alcohols that are liquids at about room temperature, for example, propylene glycol, ethanol, 2-(2-ethoxyethoxy)ethanol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 and the like; polyoxyethylene castor oil derivatives, for example, polyoxyethyleneglycerol triricinoleate or polyoxyl 35 castor oil, polyoxyethyleneglycerol oxystearate, RH 40 (polyethyleneglycol 40 hydrogenated castor oil) or RH 60 (polyethyleneglycol 60 hydrogenated castor oil), and the like; saturated polyglycolized glycerides; polyoxyethylene alkyl ethers, for example, cetomacrogol 1000 and the like; polyoxyethylene stearates, for example, PEG-6 stearate, PEG-8 stearate, polyoxyl 40 stearate NF, polyoxyethyl 50 stearate NF, PEG-12 stearate, PEG-20 stearate, PEG-100 stearate, PEG-12 distearate, PEG-32 distearate, PEG-150 distearate and the like; ethyl oleate, isopropyl palmitate, isopropyl myristate and the like; dimethyl isosorbide; N-methylpyrrolidinone; parafin; cholesterol; lecithin; suppository bases; pharmaceutically acceptable waxes, for example, carnauba wax, yellow wax, white wax, microcrystalline wax, emulsifying wax and the like; pharmaceutically acceptable silicon fluids; soribitan fatty acid esters such as sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan stearate and the like; pharmaceutically acceptable saturated fats or pharmaceutically acceptable saturated oils, for example, hydrogenated castor oil (glyceryl-tris-12-hydroxystearate), cetyl esters wax (a mixture of primarily C14-C18 saturated esters of C14-C18 saturated fatty acids having a melting range of about 43-47° C.), glyceryl monostearate and the like.
Other embodiments are directed to a gel capsule including a core having a fatty acid component and a nitrite and/or nitrate component and one or more coating layers encapsulating the core. In some embodiments, the gel capsule may be flavored, and in particular embodiments, the flavoring agent may be a flavor selected from berry, strawberry, chocolate, cocoa, lemon, butter, almond, cashew, macadamia nut, coconut, blueberry, blackberry, raspberry, peach, lemon, lime, mint, orange, banana, chili pepper, pepper, cinnamon, and pineapple. In some embodiments, at least one of the one or more coating layers may include at least one flavoring agent, and in other embodiments, the core may include at least one flavoring agent. In further embodiments, at least one of the one or more coating layers may be an enteric coating, and in still further embodiments, the core may further include one or more agents selected from solubilizers, stabilizers, colorants, plasticizers diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, antioxidants, or preservatives. In some embodiments, the core, at least one of the one or more coating layers, or a combination thereof further comprises one or more secondary agents.
In certain embodiments, such gel capsules may be formulated to include a core having from about 10 mg to about 500 mg of one or more fatty acid and from about 10 mg to about 1000 mg of nitrite and/or nitrate and one or more coating layers encapsulating the core, and the core, at least one of the one or more coating layers, or combinations thereof may include from about 0.25% by weight to about 3.0% by weight of one or more flavoring agents. In other embodiments, such gel capsules may be formulated to include a core having from about 10 mg to about 500 mg of one or more activated fatty acid and from about 2 mg to about 50 mg of vitamin E and one or more coating layers encapsulating the core, and the core, at least one of the one or more coating layers, or combinations thereof may include from about 0.25% by weight to about 3.0% by weight of one or more flavoring agents.
The fatty acid and nitrite and/or nitrate containing core may be coated with one or more coating layer. For example, in some embodiments, the gel capsule may include a water-soluble gel layer between the coating layer and the activated fatty acid core. In other embodiments, the gel capsules may include a number of additional coatings on the capsules such as, for example, immediate release coatings, protective coatings, enteric or delayed release coatings, sustained release coatings, barrier coatings, and combinations thereof. In some embodiments, one or more secondary agent or non-activated fatty acid may be mixed with the fatty acid component and nitrite and/or nitrate component, or be present in either a coating layer, a water-soluble gel layer, or an additional coating layer. Additionally, in various embodiments, the fatty acid component and nitrite and/or nitrate component of the invention may be formulated with one or more additional non-pharmaceutically active ingredients including, but not limited to, solubilizers, antioxidants, chelating agents, buffers, emulsifiers, thickening agents, dispersants, and preservatives. In some embodiments, the fatty acid component and nitrite and/or nitrate component may be encapsulated in a coating prepared from gelatin as described in U.S. Pat. No. 6,531,150, which is hereby incorporated by reference in its entirety. The gelatin layer may further include one or more other non-gelatin protein and/or one or more polysaccharide such as, for example, albumin, pectin, guaran gum, carrageenan, agar and the like, and/or one or more additive such as, for example, enteric materials, plasticizers, preservatives, and the like. Enteric materials used in embodiments of the invention include any material that does not dissolve in the stomach when the gel capsule is administered orally and include, but are not limited to, pectin, alginic acid, cellulose such as carboxyl methylcellulose, celluloseacetate phthalate, and the like, Eudragit™, an acrylic copolymer. Without wishing to be bound by theory, the addition of an enteric coating may provide a means for masking the flavor of the fatty acid component and/or nitrite component by limiting the release of the fatty acids and/or nitrites to the stomach. Plasticizers may include polyhydric alcohols, such as sorbitol, glycerin, polyethylene glycol and the like. In the embodiments described above, each coating layer may be from about 0.001 to about 5.00 mm or 0.01 to 1.00 mm thick.
The coatings of various embodiments may further include one or more film forming materials and/or binders and/or other conventional additives such as lubricants, fillers, antiadherents, antioxidants, buffers, solubilizers, dyes, chelating agents, disintegrants, and/or absorption enhancers. Surfactants may act as both solubilizers and absorption enhancers. Additionally, coatings may be formulated for immediate release, delayed or enteric release, or sustained release in accordance with methods well known in the art. Conventional coating techniques are described, e.g., in Remington's Pharmaceutical Sciences, 18th Ed. (1990), hereby incorporated by reference. Additional coatings to be employed in accordance with the invention may include, but are not limited to, for example, one or more immediate release coatings, protective coatings, enteric or delayed release coatings, sustained release coatings, barrier coatings, and combinations thereof. In some embodiments, an immediate release coating may be used to improve product elegance as well as for a moisture barrier, and taste and odor masking. Rapid breakdown of the film in gastric media is important, leading to effective disintegration and dissolution.
Capsular materials (i.e., the activated fatty acid containing core and/or one or more coating layers) may further include one or more preservatives, coloring and opacifying agents, flavorings and sweeteners, sugars, gastroresistant substances, or combinations thereof. Suitable preservative and colorant are known in the art and include, for example, benzoic acid, para-oxybenzoate, caramel colorant, gardenia colorant, carotene colorant, tar colorant and the like. In particular embodiments, one or more flavoring agents may be included the contents of the core of the gelatin capsule or in one or more coating layers of the capsule, or a combination thereof. For example, providing a palatable flavoring to the fatty acid component and/or nitrate component gel capsule may be achieved by providing a flavored coating layer having a water soluble flavor. In such embodiments, from about 0.25% and about 1.50% by weight of said coating layer may be the water soluble flavoring. Any suitable flavor known in the art may be provided to the coating layer, such as, berry, strawberry, chocolate, cocoa, vanilla, lemon, nut, almond, cashew, macadamia nut, coconut, blueberry, blackberry, raspberry, peach, lemon, lime, mint, peppermint, orange, banana, chili pepper, pepper, cinnamon, and pineapple. In some embodiments, an oil soluble flavoring may be mixed with a activated fatty acid core that is encapsulated within the capsule. In such embodiments, from about 0.25% and about 1.50% by weight of said core may be the oil soluble flavoring. Such oil soluble flavoring may be similar to the taste of the flavor of the capsule, e.g., strawberry and strawberry, or the taste of the oil flavoring may be complementary to the capsule flavoring, e.g., banana and strawberry. Such flavoring agents and methods for providing flavoring to fatty acid containing capsules may be found in U.S. Pat. Nos. 6,346,231 and 6,652,879 which are hereby incorporated by reference in their entireties.
In some embodiments, the gel capsules of embodiments may include at least one coating layer including one or more secondary agent. In such embodiments, a layer including one or more secondary agent may be of sufficient thickness to prevent oxidative degradation of the one or more secondary agent. For example, in some embodiments, the thickness of this layer may be from about 5 to about 400 microns, about 10 to about 200 microns, about 20 to about 100 microns, or in certain embodiments, from about 40 to about 80 microns. In other embodiments, the thickness of such layers may be expressed in terms of percentage weight gain based on the total weight of the capsule. For example, a layer including one or more secondary agents may create a weight gain of about 0.05 to about 20%, about 0.1 to about 10%, about 0.1 to about 5%, and in particular embodiments about 0.25 to about 1%. In certain embodiments, a coating layer containing one or more secondary agent may further include at least one compound to prevent oxidative degradation. For example, in some embodiments, at least one polymer, such as, but not limited to cellulose derivatives such as hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, polyvinylpyrrolidone/vinyl acetate copolymer, ethyl cellulose aqueous dispersions and combinations thereof, preferably hydroxpropyl cellulose, ethyl cellulose, and mixtures thereof, may be added to the coating layer at a ratio of polymer to secondary agent of from about 1:20 to about 20:1 by weight or about 1:5 to about 10:1 by weight. In particular, where the amount of secondary agent is less than about 15 mg, the amount of polymer may be from about 1:2 to about 5:1 or from about 1:1 to about 4:1, and in embodiments where the amount of secondary agent is about 15 mg or more, the amount of polymer may be from about 1:4 to about 4:1 or about 1:3 to about 2:1.
In embodiments in which one or more secondary agents are applied in a coating layer, the secondary agent may be provided as a homogenous coating solution or a heterologous suspension in a pharmaceutically acceptable solvent. Such pharmaceutically acceptable solvents may be an aqueous or organic solvent such as, for example, methanol, ethanol, isopropranol, ethylene glycol, acetone, or mixtures thereof. In other embodiments, pharmaceutically acceptable solvents may include, but are not limited to, polypropylene glycol; polypropylene glycol; polyethylene glycol, for example, polyethylene glycol 600, polyethylene glycol 900, polyethylene glycol 540, polyethylene glycol 1450, polyethylene glycol 6000, polyethylene glycol 8000, and the like; pharmaceutically acceptable alcohols that are liquids at about room temperature, for example, propylene glycol, ethanol, 2-(2-ethoxyethoxy)ethanol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 and the like; polyoxyethylene castor oil derivatives, for example, polyoxyethyleneglycerol triricinoleate or polyoxyl 35 castor oil, polyoxyethyleneglycerol oxystearate, RH 40 (polyethyleneglycol 40 hydrogenated castor oil) or RH 60 (polyethyleneglycol 60 hydrogenated castor oil), and the like; saturated polyglycolized glycerides; polyoxyethylene alkyl ethers, for example, cetomacrogol 1000 and the like; polyoxyethylene stearates, for example, PEG-6 stearate, PEG-8 stearate, polyoxyl 40 stearate NF, polyoxyethyl 50 stearate NF, PEG-12 stearate, PEG-20 stearate, PEG-100 stearate, PEG-12 distearate, PEG-32 distearate, PEG-150 distearate and the like; ethyl oleate, isopropyl palmitate, isopropyl myristate and the like; dimethyl isosorbide; N-methylpyrrolidinone; parafin; cholesterol; lecithin; suppository bases; pharmaceutically acceptable waxes, for example, carnauba wax, yellow wax, white wax, microcrystalline wax, emulsifying wax and the like; pharmaceutically acceptable silicon fluids; soribitan fatty acid esters such as sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan stearate and the like; pharmaceutically acceptable saturated fats or pharmaceutically acceptable saturated oils, for example, hydrogenated castor oil (glyceryl-tris-12-hydroxystearate), cetyl esters wax (a mixture of primarily C14-C18 saturated esters of C14-C18 saturated fatty acids having a melting range of about 43-47° C.), glyceryl monostearate and the like.
Still other embodiments are directed to a method for preparing a gel capsule including the steps of combining gelswatch ingredients, melting the gelswatch ingredients to form a liquefied gelswatch, combining the liquefied gelswatch with a fatty acid component and/or nitrite component, and encapsulating the fatty acid component and/or nitrite component to form a gel capsule. In some embodiments, the method may further include drying the gel capsule, washing the gel capsule, and packaging the gel capsules. In certain embodiments, the gelswatch ingredients may include, for example, gelatin or a gelatin substitute, modified starch or other suitable gelatin substitute, a softener, glycerol, sorbitol or other suitable polyol, a flavoring agent, a coloring agent, keratin and combinations thereof.
Any method for preparing gel capsules known in the art may be used in various embodiments of the invention. For example, in one embodiment, capsules may be produced by a method including the steps of preparing a sheet of an outer coating layer and one or more sheets of other layers, laminating the sheets, drying the laminated sheets to obtain a dried sheet, and encapsulating a fatty acid component and/or nitrite component and one or more secondary agents within the dried sheet on a rotary filler to form a seamed capsule. In another embodiment, seamless capsules may be produced using an instrument equipped with two or more nozzles arranged concentrically. In other embodiments, gelatin capsules may be manufactured as, for example, a two-piece, sealed or unsealed hard gelatin capsule.
In another embodiment, a gelatin capsule including a fatty acid component and a nitrite and/or nitrate component may be formed by the encapsulation of a dose of the fatty acid component and nitrite and/or nitrate component in a gelatin capsule. In such embodiments, the gelatin capsule may be made of, for example, gelatin, glycerol, water, a flavoring, a coloring agent and combinations thereof, and the nitro fatty acid dose may be, for example, 180 mg of nitrated DHA and 60 mg of nitrated EPA. The manufacturing process of such embodiments may include the steps of combining gelswatch ingredients, melting and forming a liquefied gelswatch, delivering the liquefied gelswatch and the fatty acid component and/or nitrite component to an encapsulation machine, encapsulating a dose of the fatty acid component and/or nitrite component, drying the encapsulated dose, washing the encapsulated dose and packaging the nitro fatty acid capsules for shipment. The gelswatch ingredients may include any ingredients described herein that are useful in the production of gelatin capsules such as, for example, gelatin or a gelatin substitute such as modified starch or other suitable gelatin substitute known in the art, a softener such as glycerol or sorbitol or other suitable polyol or other gelatin softener known in the art, a flavoring agent such as strawberry flavor Firmenich #52311A or other suitable gelatin capsule flavoring known in the art and optionally a coloring agent such as keratin or other suitable gelatin capsule coloring agent known in the art.
In particular embodiments, the gel capsule may be formed from a gelswatch mixture of about 45 parts by weight of gelatin, about 20 parts by weight of glycerol, about 35 parts by weight of water and about 0.5 or more parts by weight of flavoring. The gelswatch ingredients may be heated to about 60° C. to 70° C. and mixed together to form liquefied gelswatch. The liquefied gelswatch and the fatty acid component and/or nitrite component may then be poured into an encapsulation machine. The encapsulation machine then forms the fatty acid component and nitrite and/or nitrate component capsule by encapsulating the fatty acid component and/or nitrite component dose into a gelatin capsule.
The capsule can then be dried at a temperature of, for example, about 20° C. The water content of the capsule may be reduced by evaporation during the drying step. The capsule can then be washed and ready for packaging, selling, or shipping. In some embodiments, a sweetener or flavoring agent can be added to the capsule through a dipping process. In the dipping process, the gelatin capsule is dipped in a sweetener/flavoring solution and then dried, allowing for the sweetener to form a coating around the outside of the capsule. In some embodiments, a sweetener or flavoring agent may be added to the capsule through an enteric coating process, and in other embodiments, a liquefied sweetener or flavoring agent can be sprayed on to the outside of the gelatin capsule and dried. Other methods of making gelatin capsules are known in the art and contemplated.
In various embodiments, the one or more coatings on the capsule may be applied by any technique known in the art including, but not limited to, pan coating, fluid bed coating or spray coating, and the one or more coatings may be applied, for example, as a solution, suspension, spray, dust or powder. For example, in some embodiments, a polymeric coating may be applied as aqueous-based solutions, organic-based solutions or dispersions containing and, in some embodiments, one or more secondary agent. In such embodiments, polymer-containing droplets may atomized with air or an inert gas and sprayed onto the core containing the activated fatty acids, and in some embodiments, heated air or inert gas may be added to facilitate evaporation of the solvent and film formation. In the case of soft gelatin capsules, the processing parameters of spray rate and bed temperature must be controlled to limit solubilization and capsule agglomeration. Additionally, a high bed temperature may result in evaporation of residual water from the capsule shell, causing the capsule to become brittle. In addition, coating uniformity which includes mass variance of the coated capsules and variance of the content of the coated activated fatty acid and accuracy of deposition must be evaluated.
Gel capsules of various embodiments of the invention may be of any shape such as, but not limited to, round, oval, tubular, oblong, twist off, or a non-standard shape (e.g., animal, tree, star, heart, etc.), and the size of the capsule may vary in accordance to the volume of the fill composition intended to be contained therein. For example, in some embodiments, hard or soft gelatin capsules may be manufactured using conventional methods as a single body unit comprising the standard capsule shape. A single-body soft gelatin capsule typically may be provided, for example, in sizes from 3 to 22 minims (1 minim=0.0616 ml) and in shapes of oval, oblong or others. Similarly, hard gel capsules may be manufactured using conventional methods in standard shapes and various standard sizes, such as those designated (000), (00), (0), (1), (2), (3), (4), and (5) where the largest number corresponds to the smallest size. Non-standard shapes may be used as well.
Other pharmaceutical formulations containing the compounds of the invention and a suitable carrier can be in various forms including, but not limited to, solids, solutions, powders, fluid emulsions, fluid suspensions, semi-solids, and dry powders including an effective amount of a fatty acid and a nitrite and/or nitrate of the invention. It is also known in the art that the active ingredients can be contained in such formulations with pharmaceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers, antioxidants, preservatives and the like. The means and methods for administration are known in the art and an artisan can refer to various pharmacologic references for guidance. For example, Modern Pharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Gilman's, The Pharmaceutical Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., New York (1980) both of which are hereby incorporated by reference in their entireties can be consulted.
Other embodiments of the invention include a fatty acid component and/nitrite and/or nitrate component prepared as described above which are formulated as a solid dosage form for oral administration including capsules, tablets, pills, powders, and granules. In such embodiments, the active compound may be admixed with one or more inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents and can additionally be prepared with enteric coatings.
Further embodiments are directed to methods for improving the health of an individual by administering to the individual a dietary supplement including a fatty acid component and a nitrite and/or nitrate component, and a nutraceutically acceptable excipient. In some embodiments, the dietary supplement may further include one or more secondary agent selected from vitamin A, vitamin B, vitamin B-1, vitamin B-2, vitamin B-6, vitamin B-12, vitamin C, vitamin D, vitamin D3, vitamin E, selenium, β-carotene, ginkgo biloba, goldenseal, valerian, ginseng, echinacea, grape seed extracts, ephedra, yucca concentrates, green tea extract, rice bran extract, wheat germ, wheat germ extract, beeswax, red yeast rice extract, stevia leaf extract, flaxseed oil, borage seed oil, coenzyme Q10, glucosamine derivatives, methylsulfonylmethane, pantothenic acid, biotin, thiamin, riboflavin, niacin, folic acid, palmitic acid, and derivatives thereof. In some embodiments, the dietary supplement may include one or more secondary agent selected from policosanols, guggulipids, rice bran extract, wheat germ, wheat germ extract, beeswax, and red yeast rice extract, and such a dietary supplement may be formulated to promote a healthy heart and circulatory system. In other embodiments, the dietary supplement may include one or more secondary agent selected from vitamin B-1, vitamin B-2, vitamin B-6, vitamin B-12, vitamin C, vitamin D, vitamin D3, vitamin E, selenium, goldenseal, valerian, ginseng, and echinacea and such a dietary supplement may be formulated to promote healthy cell proliferation. In still other embodiments, the dietary supplement may include one or more secondary agent selected from vitamin A, vitamin C, vitamin E, and β-carotene, and such a dietary supplement may be formulated to promote healthy eyes. In yet other embodiments, the dietary supplement may include one or more secondary agent selected from vitamin A, vitamin C, vitamin E, selenium, ginkgo biloba, goldenseal, valerian, ginseng, echinacea, ephedra, green tea extract, and yucca concentrate, and such a dietary supplement may be formulated to promote cognitive health or formulated as a neuroprotectant.
Various embodiments of the invention are also directed to compositions including one or more fatty acid components, one or more nitrite and/or nitrate components and one or more coating layers encapsulating the core. As above, the compositions may include one or more additional secondary components such as, for example, rice bran oil, enzyme-treated stabilized rice bran, a solubilized fraction of rice bran oil, and derivatives thereof, glucosamine derivatives, methylsulfonylmethane, yucca concentrate, grape seed extract, beta-carotene, ephedra, ginkgo biloba, goldenseal, valerian, ginseng, green tea extract, and echinacea. The fatty acid may be derived from an omega-3 fatty acids, omega-6 fatty acids, omega-9 fatty acids, linoleic acid, conjugated linoleic acid, α-linoleic acid, oleic acid, eicosapentaenoic acid, docosahexaenoic acid or a derivative or combination thereof.
This invention and embodiments illustrating the method and materials used may be further understood by reference to the following non-limiting examples.
Although the present invention has been described in considerable detail with reference to certain preferred embodiments thereof, other versions are possible. Therefore the spirit and scope of the appended claims should not be limited to the description and the preferred versions contained within this specification. Various aspects of the present invention will be illustrated with reference to the following non-limiting examples.
Exemplary compositions may be prepared as described above including the ingredients listed in Table 2. In the present examples, Capsule 1 and capsule 2 are intended to be consumed simultaneously. The compositions can be prepared by any known methods and may comprise additional components. Furthermore, the capsules in the following examples can also be gel capsules.
Exemplary compositions may be prepared as described above including the ingredients listed in Table 3. In the present examples, the compositions can be prepared by any known methods and may comprise additional components. Furthermore, the capsules in the following examples can also be gel capsules.
A number of alimentary fats including plant oils such, olive oil (virgin, and refined), sunflower oil, vegetable oil flax seed oil, sesame oil, palm oil, soybean oil, canola oil, pumpkin seed oil, corn oil, safflower oil, peanut oil, grape seed oil, argan oil, avocado oil, mustard oil, Almond oil, cottonseed oil, diacylglycerol (DAG) oil, ghee, Walnut oil, rice bran oil as well as other vegetable oils contain abundant amount of unsaturated fatty acids and are suitable for human consumption. Unsaturated fatty acids can also be found in relative abundance in animal fats such as clarified butter, lard and fish oils such as cod liver oil and herring oil.
These unsaturated fatty acids are expected to be readily converted to nitro fatty acid include by contacting existing unsaturated fatty acid with a nitro containing compound; and reacting an existing unsaturated fatty acid with a nitro containing compound to form a nitro fatty acid. Foodstuffs enriched for nitro fatty acids are expected to improve the health of an individual as part of a balanced diet.
Activated fatty acids may be prepared by a method including the steps of reacting the unsaturated fatty acid with a mercuric salt (such as, for example, HgCl2, Hg(NO3)2, Hg(OAc)2) and a selenium compound (including but not limited to PhSeBr, PhSeCl, PhSeO2CCF3, PhSeO2H, PhSeCN), contacting the intermediate resulting from step a) with a reagent or reactant that can introduce an electron withdrawing group; and reacting the intermediate resulting from step b) with an oxidizing agent (including but not limited to oxygen (O2), ozone (O3), hydrogen peroxide (H2O2) and other inorganic peroxides, Fluorine (F2), chlorine (Cl2), and other halogens, nitric acid (HNO3) and nitrate compounds, sulfuric acid (H2SO4), persulfuric acids (H2SO5 and H2SO8), chlorite, chlorate, perchlorate, and other analogous halogen compounds, hypochlorite and other hypohalite compounds, including bleach (NaClO), hexavalent chromium compounds such as chromic and dichromic acids and chromium trioxide, pyridinium chlorochromate (PCC), and chromate/dichromate compounds, permanganate compounds sodium perborate, nitrous oxide (N2O) silver oxide (Ag2O), osmium tetroxide (OsO4), Tollens' reagent, 2,2′-dipyridyldisulfide (DPS) sodium periodate, meta-Chloroperoxybenzoic acid and t-butyl hydroperoxide.
The source of the electron withdrawing group may be any compound known in the art that is capable of generating an electron withdrawing group that can be incorporated into the activated fatty acid, such as, for example, NaNO2, AgNO2, HSO2OH. In certain embodiments, the process of forming nitrated fatty acids is performed in the absence of oxygen.
It is envisioned that a variety of alimentary fats as vegetable oils and animal fat can be treated with nitrite and/or nitrate to produce activated fatty acids from polyunsaturated fatty acids such as linoleic acid, conjugated-linoleic acid, α-linoleic acid, γ-linoleic acid, oleic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or derivatives thereof.
80% of the basal plasma nitrite (NO2−) level derives from oxidation of NO, reduction of peroxynitrate (NO3−) may also contribute to elevation of NO2−. It has been reported that exogenous NO3− intake (10 mg/kg in humans) may increase plasma NO2− concentration up to four- to five-fold in 30 min.
The largest dietary sources of NO3− for the human body include green vegetables, such as spinach, lettuce, and collard greens, and also radishes, beets and, and meat. Furthermore, NO2− itself can be found in cured meats.
Nitrated lipids or activated fatty acids can be formed by several different mechanisms, such as through a reaction of NO2− with unsaturated fatty acid derivatives at low pH. For example, The “Mediterranean diet”, which is particularly rich in NO2− and PUFA, and supplemented with acidic vinegar, may favor intragastric generation of nitrated lipids. Indeed, it has been shown that nitration of unsaturated fatty acids from extra virgin olive oil is possible under exposure to NO2− in mild acidic conditions.
Recent research by d'Ischia and colleagues has demonstrated that oxides of nitric oxide and its oxidation products including NO3−, NO2, HNO2 and NO2+, interact with unsaturated fatty acid such as linoleic acid and lipid peroxides to produce complex mixtures of products, including nitroepoxides and other nitrogen containing derivatives of oxidized lipids. At high concentrations and in the presence of oxygen, NO reacts with polyunsaturated fatty acids and esters to afford mixtures of nitration products, including isomeric nitroalkene and nitronitrate derivatives. For example, ethyl linoleate reacts smoothly with NO2− in acidic media, conditions that favor formation of HNO2, to afford complex, yet relatively well-defined patterns of nitration products, some of which were amenable to chromatographic isolation.
The presence of oxygen can have a dramatic effect on the nitration of unsaturated fatty acids. Species such as nitric oxide are highly lipophilic and will more readily react molecular oxygen that also preferentially partions into a hydrophobic milieu. In some embodiments, it is desirable to conduct the nitration of unsaturated fatty acids in the absence of molecular oxygen, such that the latter does not compete for free radicals with the fatty acid substrates. It is further expected that some of the initial unsaturated fatty acids will not become nitrated and will remain in their native state.
In a particular embodiment, Olive oil, which contains abundant amounts of unsaturated fatty acids (85% oleic acid and 5% linoleic acid) represents suitable substrate for nitration with NO2−. It is expected that the nitration of olive will yield a complex mixture of fatty acids including nitro-oleic acid, nitro-linoleic acid, oleic acid and linoleic acid. Based on the relative ratio of oleic acid to linoleic acid of about 18:1 it is expected that the ratio of nitrated oleic acid to linoleic acid will be similar. The higher the free fatty acid content of an oil the greater the acidity and therefore the more suitable such oils are to being nitrated. It is further expected that some of the initial unsaturated fatty acids will not become nitrated and will remain in their native state.
In another embodiment, fish oil is a suitable substrate for nitration. The typical composition of unsaturated fatty acids in fish oil is docosahexaenoic acid, eicosapentaenoic acid, linoleic acid, oleic acid. The ratios of oleic acid to linoleic acid to docosahexaenoic acid and eicosapentaenoic acid combined are about 1:10:26. The ratio of docosahexaenoic acid to eicosapentaenoic acid in turn is typically about 5:1.
In yet other embodiments, corn oil, palm oil, peanut oil and safflower oil also make suitable substrates for nitration due to high levels of oleic and, linoleic acid and conjugated linoleic acid.
In addition to fish oil and olive oil, safflower oil comprises upwards of 80% conjugated linoleic acid and also contains about 20%. It is therefore expected that nitration of safflower oil will result in the formation of conjugated nitro-linoleic acid and nitro-oleic acid at a ratio of about 4:1 under anaerobic conditions. It is further expected that some of the initial polyunsaturated fatty acids will not become nitrated and will remain in their native state.
It is envisioned that a alimentary oil such as olive oil, that is already known to impart overall health benefits to people that consume it regularly as part of their diet, will provide additional overall health benefits due to the presence of nitrated fatty acids as many of the health benefits observed may result from the nitration of unsaturated fatty acids in the gut upon consumption.
This application claims the benefit of U.S. Provisional Application No. 61/821,817, filed on May 10, 2013.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/37622 | 5/12/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61821817 | May 2013 | US |
