JP2014505730A - Formulation containing ω3 fatty acid and anti-obesity agent for weight loss in patients with cardiovascular disease (CVD) and diabetics - Google Patents

Abstract

Approximately 90% by weight of one or more anti-obesity drugs combined with eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) (EPA: DHA weight ratio 5.7-6.3) In combination with a mixture of ω3 fatty acid formulations containing the above ω3 fatty acids, wherein the total amount of EPA, DHA, and DPA comprises about 82% by weight of the total formulation and about the total ω3 fatty acid content of the composition Teach 92% of the combinations. EPA + DHA is about 80% of the total formulation and about 89% of the total omega-3 fatty acid content of the composition. The formulation further comprises a specific amount of arachidonic acid (AA) and an ω3 fatty acid having 18 carbon atoms including one or more of stearidonic acid (SDA) and α-linolenic acid (ALA). Also good. The present application further teaches how to administer such a combination and relates to a unit dosage of such a combination to reduce the weight of patients with cardiovascular disease (CVD) and diabetics.

Description

This application claims the benefit of the priority date of US Provisional Application No. 61 / 457,269, filed February 16, 2011. This content is incorporated herein in its entirety.

FIELD OF THE INVENTION The present invention relates to a combination of one or more anti-obesity drugs and a mixture of ω3 fatty acid compositions designed to modulate ω3 deficiency in individuals who require ω3 fatty acids; and such It relates to a method of administering the combination and to a unit dose of such a combination for weight loss in patients with cardiovascular disease (CVD) and diabetics. In particular, the present invention is effective in raising ω3 levels to a point where ω3 formulations are designed to modulate ω3 deficiency in individuals who require ω3 fatty acids, particularly to reduce cardiovascular risk factors. Enables effective in providing a sustained vasodilator defined as a vasodilator that lasts at least 6 hours, especially a composition containing a specific ratio of highly purified long chain fatty acid composition It relates to a composition having an EPA: DHA ratio and an ω3 purity level.

Background of the Invention
According to US 2005 Dietary Guidelines Advisory Committee knowledge, 70% of Americans are deficient in omega-3 fatty acids. Further studies show that over 84% of CVD patients are deficient in omega-3 fatty acids, specifically eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA) Yes.

  Cardiovascular disease (CVD) is the leading cause of death for men and women in developed countries worldwide. These premature deaths are quite expensive for individuals and their families and are a heavy burden on the national health system. Risk factors for cardiovascular disease are well-recognized: higher than average serum cholesterol, high levels of LDL, low levels of HDL versus LDL levels, higher than average serum triglycerides, and plaques and lines that cause coronary artery occlusion Includes high levels of lipid oxidation products and inflammatory processes that create streak. An additional risk factor for cardiovascular disease and stroke is hypertension. Reduction of these risk factors is useful in reducing the prevalence of CVD and its many costs.

  In some cases, genetic predisposition contributes to the incidence of CVD, but poor quality diets and cedentary lifestyles are the main factors contributing to the increased risk of developing and progression of CVD. For this reason, clinical management of CVD often involves attempts to increase exercise and improve patient lifestyles to incorporate a balanced diet rich in omega-3 fatty acids. Because of non-compliance, patients often cannot protect lifestyle improvements, so these efforts alone do not provide optimal patient care and consider other treatment interventions or strategies. There must be.

  Treatment options include lipid modulators such as statins, or low-density lipoprotein (LDL), a metabolic component that contributes to increased atherosclerotic lesions and increases the risk of heart attack or stroke Fibrates that act to lower cholesterol and / or triglycerides (TG) can be included. However, many of these treatment options are accompanied by unwanted side effects that can add additional health risks or cause bodily discomfort.

  Obesity is a condition often present simultaneously with ω3 deficiency in patient populations that exhibit risk factors for CVD. Obesity is prevalent in many of the world's developed countries, and the majority of patient populations do not respond to monotherapy. Accordingly, there is a recognized need for combination therapy, particularly in obese patient populations suffering from obesity, type II diabetes, hypertension, metabolic syndrome, and risk factors for cardiovascular disease.

Omega 3 fatty acids are natural polyunsaturated fats found in marine products such as fish and are now also available as dietary supplements. Omega 3 fatty acids contain multiple double bonds in the aliphatic chain. ω3 fatty acids are named according to the number of double bonds (> 1), position, and configuration. The three main types of ω3 fatty acids are α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). These ω3 polyunsaturated fatty acids are found in several types of cardiovascular diseases such as myocardial infarction, arrhythmia, atherosclerosis, and hypertension (Abeywardena and Head, 2001; Kris-Etherton et al., 2003). Has been shown to prevent. (EPA) (C20: 5n-3) and (DHA) (C22: 6n-3) contribute to the prevention of various cardiovascular disorders by improving endothelium-dependent vasodilation and blocking platelet activation It is widely accepted that it is the main biologically active polyunsaturated fatty acid. Fish oil, EPA, and DHA have been shown to induce relaxation and inhibit contraction by an endothelium-dependent mechanism (Shimokawa et al., 1987; Yanagisawa and Lefer, 1987). Ω3 polyunsaturated fatty acids of high content, in particular, EPA is likely to inhibit platelet aggregation by generating reduction of thromboxane A _2, prolonged the bleeding time. Previous studies have also shown that supplementation with cod liver oil purified ω3 fatty acids enhances endothelium-dependent relaxation in isolated porcine coronary arteries (Shimokawa et al., 1988).

  A combination therapy that includes a combination of a novel ω3 formulation and one or more anti-obesity agents to relieve obesity symptoms while improving the patient's lipid profile and reducing various cardiovascular risk factors If it can be provided, a long-standing need will be realized.

  EPA: DHA weight ratio of 5.7 to 6.3 with a combination of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), containing about 90% by weight or more of ω3 fatty acid, total amount of EPA, DHA, and DPA A pharmaceutical formulation as shown in the invention disclosed herein, which is about 82% by weight and about 92% of the total ω3 fatty acid content of the composition is not disclosed in the prior art . EPA + DHA is about 80% of the total formulation and about 89% of the total omega-3 fatty acid content of the composition. Furthermore, the ω3 composition in combination with an anti-obesity agent is not disclosed in the prior art.

  By adjusting the ratio, content, and purity of omega fatty acid formulations, a meaningful and specific set of parameters can be provided to those skilled in the art, thereby obtaining formulations with desirable utility or pharmacological effects. It is worth noting that there is sex.

  We have discovered that the ability of omega-3 fatty acid preparations to cause endothelium-dependent relaxation depends on the content ratio of EPA and DHA and the purity of all formulations and the presence of additional important omega-3 fatty acids (especially DPA) did.

  Indeed, the formulations of the present invention with an EPA: DHA ratio of about 6: 1 induced significantly greater relaxation than the EPA: DHA 1: 1 preparation despite similar ω3 fatty acid content. These findings also suggest that EPA is likely to be a more potent endothelium-dependent vasorelaxant agonist than DHA. An excellent commercial omega-3 preparation (Lovaza®, GSK, Middlesex, UK) has an EPA: DHA ratio of 1.2: 1, so two major omega-3 fatty acids have similar organisms that cause endothelium-dependent relaxation. The fact that there is no pharmacological activity is important. Therefore, by optimizing the EPA: DHA ratio in the ω3 preparation, new products with improved vascular protection capabilities may be obtained.

  The present invention provides a novel composition that can be incorporated into an oral dosage formulation and a novel method of treatment to reduce risk factors associated with CVD. This book is for the treatment and / or prevention of risk factors for CVD and stroke, including the reduction of hypertension and the improvement of total lipid profiles such as low density lipoprotein (LDL), high density lipoprotein (HDL), and triglycerides. The composition of the inventive formulation can be used orally. Without being bound by theory, the inventors believe that the composition works by acting on different sites and aspects of cardiovascular disease. The compositions of the invention are preferably human and animal in unit dosage forms containing appropriate amounts of the active ingredients, such as tablets, capsules, pills, powders, granules, and oral solutions or suspensions. To be administered.

  The present invention also provides a method of treatment. For example, a patient who is deficient in ω3 fatty acids that may exhibit one or more risk factors for CVD is administered a therapeutically effective amount of a formulation of the invention to achieve a therapeutic level of ω3, thereby One or more risk factors for CVD are reduced. In embodiments, the present invention is also a method for providing a sustained vasodilatory effect to a patient by administering a therapeutically effective amount of a formulation of the present invention, whereby indomethacin-independent persistent blood vessels Expansion action is realized.

  By providing a therapeutic method for modulating ω3 deficiency, for improving heart health by delivering a composition of the present invention to an individual, and for reducing risk factors associated with cardiovascular disease The use of the present invention is realized. Delivery of the composition of the present invention, for example, delivery of the composition of the present invention by oral administration, can prevent oxidation of low density lipoprotein (LDL), increase high density lipoprotein (HDL), and lower total cholesterol. It has been shown to be useful. Delivery of the compositions of the present invention is also useful for lowering triglycerides and lowering homocysteine. Desirably, the compositions of the invention are formulated such that an effective amount is delivered by multiple tablets (or other suitable formulation) per day. Suitably, these doses may be taken with meals, mixed into food, or taken on an empty stomach. Generally, improvements are observed after daily use for 2-8 weeks. Optionally, the compositions of the invention may be delivered daily in a suitable form (eg, a chewable treat or bar). Other suitable dosage regimens can be readily developed by those skilled in the art. Such a dosing regime is not a limitation of the present invention. The compositions of the present invention may also be useful in the treatment of non-human animals, particularly mammals, in addition to their use in human CVD therapy. For example, these dietary supplements can be useful for companion animals such as dogs and cats, cows, horses, and pigs, among other animals.

  Cardiovascular disease patients, impaired glucose tolerance, fasting hyperglycemia, insulin resistance syndrome, dyslipidemia, hypertension, hyperuricemia, gout, annular artery disease, myocardial infarction, metabolic syndrome, lipemia, coronary heart Compositions useful in the treatment of obesity in patients with disease, cardiac arrhythmia, cerebrovascular disease, stroke, peripheral defect disease, sleep apnea, and diabetic patients at risk for cardiovascular events, cardiac events, and vascular events and A method is disclosed. Anti-obesity agents useful in the treatment of obesity include energy expenditure, glycolysis, gluconeogenesis, glycogenolysis, lipolysis, fat absorption, fat storage, fat excretion, hunger and / or satiety and / or desire mechanisms, appetite / An agent that affects motivation, food intake, or gastrointestinal motility.

  The main object of the present invention is to provide a minimum of 90% by weight comprising a combination of one or more anti-obesity drugs and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) with an EPA: DHA weight ratio of 5.7-6.3 A mixture of ω3 fatty acid formulations containing ω3 fatty acids, wherein the total amount of EPA, DHA, and DPA comprises about 82% by weight of the total formulation and about 92% of the total ω3 fatty acid content of the composition Is to disclose the combination. EPA + DHA is about 80% of the total formulation and about 89% of the total omega-3 fatty acid content of the composition. The fatty acids of the present invention are in the form of biologically active glycerides (eg, triglycerides), biologically active esters (eg, ethyl esters), and biologically active phospholipids. , Derivatives, conjugates, precursors, and pharmaceutically acceptable salts and mixtures thereof. The combination of the ω3 formulation and the anti-obesity drug may be provided as a single unit dosage form, or may be provided as separate and separate unit dosage forms.

  It is a further object of the present invention to disclose such combinations and methods of administering unit doses of such combinations to reduce the weight of patients with cardiovascular disease (CVD) and diabetics.

  It is a further object of the present invention to provide a method for regulating omega 3 deficiency in a patient in need of omega 3 and at the same time assisting in the reduction of excess weight in such patients, and a system for implementing it.

  A still further object of the present invention is to provide a novel combination therapy to assist in the reduction of excess weight in such patients, while at the same time providing a sustained vasodilatory effect.

  These and other advantages of the invention will be apparent from the following description in conjunction with any accompanying drawings. In the following description, certain specific embodiments of the present invention are shown for purposes of illustration and example. Any drawing described herein forms a part of the specification and includes exemplary aspects of the invention, illustrating its various objects and features. In view of this disclosure, all examples are illustrative and not limiting.

2 shows the study design of a VASCAZEN ™ open-label study. Plot of improved whole blood EPA + DHA + DPA baseline levels up to 6 weeks. The normal distribution curve of the A group-C group during an open-label test is shown. Figure 5 shows the effect of different EPA: DHA ratios on the relaxation of coronary arteries with and without endothelium. Disclose the effects of eNOS inhibitors and EDHF inhibitors on the relaxation effects of the EPA: DHA 6: 1 control. Disclose how the presence of Src kinase and PI3-kinase affects the relaxation effects of EPA: DHA 6: 1 products. Figure 2 shows the changes in relaxation of EPA: DHA 6: 1 product by membrane permeable analogues. Figure 3 shows the effect of EPA: DHA 6: 1 on Akt phosphorylation and eNOS phosphorylation. Western blot data showing sustained eNOS activation and 40 μg protein by Vascazen at a concentration of 0.4% for 6 hours. The relationship between the purity, which is the ratio of the total of EPA + DHA to the ratio of total ω3, and relaxation of the coronary artery ring in the presence or absence of endothelium is shown. The relaxant effect of this EPA: DHA 6: 1 formulation is insensitive to the presence of indomethacin. FIGS. 11A and 11B show the indomethacin sensitivity of the relaxant action of the EPA: DHA 6: 1 formulation compared to several ω3 products that can be purchased without a doctor's prescription. The indomethacin sensitivity of the relaxant effect of the EPA: DHA 6: 1 formulation is shown compared to a similar ratio formulation containing certain additives. The comparative vasorelaxant effect of EPA: DHA 6: 1 of the present invention is shown compared to EPA: DHA 1: 1, EPA alone, and DHA alone. EPA: DHA 6: 1 shows a mechanism for stimulating NO endothelium formation through redox-sensitive activation of the phosphoinositide 3-kinase (PI3-kinase) / protein kinase (Akt) pathway.

Detailed Description of the Invention The present invention relates to patients with cardiovascular disease, as well as impaired glucose tolerance, fasting hyperglycemia, insulin resistance syndrome, dyslipidemia, hypertension, hyperuricemia, gout, annular artery disease, myocardial infarction, metabolic Syndrome, hyperlipidemia, coronary heart disease, cardiac arrhythmia, cerebrovascular disease, stroke, peripheral defect disease, patients suffering from sleep apnea, and the risk of cardiovascular events, cardiac events, and vascular events Combination therapy and methods for its use in the treatment of obesity in diabetic patients with gender are provided. Anti-obesity agents useful in the treatment of obesity include energy expenditure, glycolysis, gluconeogenesis, glycogenolysis, lipolysis, fat absorption, fat storage, fat excretion, hunger, satiety, desire mechanism, appetite, food intake, gastrointestinal motility, and Selected from anti-obesity agents that affect at least one mechanism of action selected from the group consisting of those combinations.

In certain embodiments, the method of treatment comprises an active compound (antagonist or inverse agonist) for the receptor product of the cannabinoid 1 (CB1) gene; cathepsin K inhibitor; PYY; PYY _3-36 ; PYY agonist; 5HT transporter inhibitor; NE transporter inhibitor; ghrelin antagonist; H3 antagonist / inverse agonist; MCH1R antagonist; MCH2R agonist / antagonist; MC3R agonist; NPY1 antagonist; NPY4 antagonist; NPY5 antagonist; leptin; leptin agonist / modulator; leptin derivative; opioid antagonist; orexin antagonist BRS3 agonist; 11βHSD-1 inhibitor; CCK-A agonist; CNTF; CNTF agonist / modulator; CNTF derivative; Cox-2 inhibitor; GHS agonist; 5HT2C agonist; CB1 antagonist; neuropeptide Y5, appetite suppressant; lipase inhibition Agents; 5HT6 antagonists; Nonamine reuptake inhibitors; UCP-1, 2, and 3 activators; β3 agonists; thyroid hormone β agonists; PDE inhibitors; FAS inhibitors; DGAT1 inhibitors; DGAT2 inhibitors; ACC2 inhibitors; glucocorticoid antagonists; acyl -Estrogen; Lipase inhibitor; Fatty acid transporter inhibitor; Dicarboxylic acid transporter inhibitor; Glucose transporter inhibitor; Serotonin reuptake inhibitor; Aminolex; Amphetoral; Amphetamine; Axokine; Benzphetamine; Chlorphene Theremin; clobenzolex; cloforex; chrominolex; chlortermine; cyclexamine; dextroamphetamine; difemethoxyzine, N-ethylamphetamine; fenbutrazate; phenisolex; fenpropo Lex; full drex (fludorex); full minorex; full Rilmethylamphetamine; levamfetamine; levofacetoperan; mefenolex; methanefepramon; methamphetamine; nalmefene; nor pseudoephedrine; pentrex; phendimetrazine; phenmetrazine; Picsilex; Topiramate; Zonisamide; IGF-IR antagonist; MetAP2 modulator; α-arrestin ARRDC3 modulator; Single Minded1 (SIM1) modulator; Methionine aminopeptidase 2 (MetAP2); Sirtuin 1 (SIRT1) modulator; or any combination thereof It relates to an anti-obesity agent that can be selected from the group comprising

Since endocannabinoid receptor blockers can attenuate weight loss, scientists have sought their usefulness as anti-obesity drug candidates. Pharmaceutical companies have worked together to develop drugs that target this receptor complex, resulting in multiple anti-obesity drug candidates. These anti-obesity drug candidates include Sanofi-Aventis' ACOMPLIA (RIMONIBANT) chemical formula 5- (4-chlorophenyl) -1- (2,4-dichloro-phenyl) -4-methyl-N- (piperidin-1-yl) ) -1H-pyrazole-3-carboxamide, Merck TARANABAN T, chemical formula N-[(1S, 2S) -3- (4-chlorophenyl) -2-, and Pfizer CP-945,598, chemical formula C ₂₅ H ₂₅ Cl ₂ N ₇ O.HCl is contained. All CB1R blockers reached Phase 3 clinical trials and failed. These trials failed in late development because of psychiatric adverse reactions including severe depression and anxiety.

  During a phase III clinical evaluation of these drugs, it was discovered that patients predisposed to depression, anxiety, or other mood disorders are most susceptible to adverse events with RIMONIBANT treatment. This suggests that blockade of CB1R complex and natural endocannabinoid regulation exacerbates existing conditions. Patients who did not exhibit psychological health problems were less sensitive to adverse events of this characteristic.

  During the analysis of the combination of the CB1 receptor blocker and the novel ω3 formulation of the present invention, we often found that the presence of anxiety and major depressive disorder was associated with the nutritional deficiencies of ω3 fatty acids I noticed the fact. The inventors have noted that these disorders are often reduced or alleviated when this deficiency is cured using high purity prescription ω3 fatty acids.

  Endocannabinoids produced by the body help regulate mood by binding to CB1R, a Gi / Go receptor complex. Drugs such as RIMONIBANT and other drugs that block the interaction between endocannabinoids and their receptors interfere with this regulatory mechanism, putting patients at risk of unstable mood and depressive disorder.

  The cannabinoid-1 receptor (CB1R), a G (i / o) receptor complex, is regulated by ω3, and chronic ω3 deficiency is mediated by regulation of the CB1R, a G (i / o) receptor complex Directly affects the function of presynaptic neurons. In the absence of ω3, the strong interactions normally observed in this receptor complex are weakened. Eventually, G (i / o) leaves the receptor complex, limiting the ability of endocannabinoid signaling through CB1R, resulting in an unstable mood. Although not bound by any particular theory or mechanism of action, the present inventors have stabilized the CB1R receptor complex by curing ω3 deficiency, resulting in improved endocannabinoid mood stabilization effects. Then I advocate. Thus, patients predisposed to depression episodes in clinical studies of RIMONIBANT or similar CB1R targeted therapies will benefit from ω3 pretreatment and ongoing treatment combined with RIMONIBANT or similar drugs as an adjunct therapeutic approach Will be able to. These patients would be excellent candidates for safe administration of RIMONIBANT or related drugs.

  Formulations containing a combination of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in an EPA: DHA weight ratio of 5.7 to 6.3 with a minimum of about 90% by weight of ω3 fatty acids with respect to the ω3 component of the combination therapy and method of use A long chain fatty acid composition comprising a total amount of EPA, DHA, and DPA of about 82% by weight of the total formulation and about 92% of the total ω3 fatty acid content of the composition I will provide a. EPA + DHA is about 80% of the total formulation and about 89% of the total omega-3 fatty acid content of the composition. The fatty acids of the present invention are in the form of biologically active glycerides (eg, triglycerides), biologically active esters (eg, ethyl esters), and biologically active phospholipids. , Derivatives, conjugates, precursors thereof, and pharmaceutically acceptable salts and mixtures thereof.

  The pharmaceutical formulations of the present invention are intended to be administered in an amount that provides a daily dosage of 1-4 gm of said formulation. Such dosage level pharmaceutical formulations are effective in treating or preventing risk factors for cardiovascular disease and are effective in preventing sudden death in CVD patients.

  Pharmaceutical formulations of the present invention may be provided in which the unit form is a gel or liquid capsule.

  Exemplary unit dosage forms include about 645 to about 715 mg / gm EPA (eg, about 680 mg / gm EPA), and about 105 to 115 mg / gm (eg, about 110 mg / gm) DHA. The unit dose may comprise about 22 to about 28 mg / gm DPA (eg, about 25 mg / gm DPA). The unit dose may further comprise up to about 0.5% (eg, about 0.15% to about 0.25%, or about 0.2%) of a stabilizer (eg, tocopherol). Effective unit dosages are generally 3gm to 4gm pharmaceutical formulations, which are given to CVD patients in one or more unit doses, for example, about 3-4 1 gram capsules per day. Offered daily. As shown below, one or more optional ingredients may be included in the formulation. Such components may be added separately or may be components of the source from which the ω3 fatty acid in the formulation is obtained.

  In certain embodiments, the formulation may further comprise about 30 mg / gm arachidonic acid (AA). In certain embodiments, the formulation may further contain up to about 5% (eg, about 3% or about 30 mg / gm) of arachidonic acid (AA). It has also been discovered that aspirin-acetylated COX-2 can convert ω-6 AA to lipoxin (LX), a potent anti-inflammatory mediator, via lipoxygenase (LOX) (Nature Chemical Biology, Vol. 6, June 2010). , Pp 401-402).

  Some embodiments of the formulation contain> 2% (eg,> 3%) 18 carbon omega 3 fatty acids individually or in total. Exemplary 18 carbon atom ω3 fatty acids include α-linolenic acid (ALA) and stearidonic acid (SDA) alone or in combination. Studies have shown that the presence of 18 carbon ω3 such as ALA induces anti-inflammatory effects (Zhao et al, Am J Clin Nutr 2007; 85: 385-91). The composition is formulated with a specific amount of DHA consisting of about 400 mg per day.

  The composition may contain additional fatty acids in even smaller amounts, and typically less than about 1% of each fatty acid is present. In exemplary embodiments, any additional fatty acid is contained from about 0.3-0.7%, or about 5%. These additional fatty acids are, for example, ω-6 fatty acids such as dihomo-γ-linolenic acid (DGLA; 20: 3, n6), docosapentaenoic acid (male bond acid; 22: 5, n6); ω-9 fatty acids Oleic acid (18: 1, n9), and other fatty acids such as 7,10,13,15-hexadecatetraenoic acid (16: 4, n1), 9,12,15,17-octa Decatetraenoic acid (18: 4, n1) may be included. Other fatty acids may be present in higher amounts. For example, eicosatetraenoic acid (ETA; 20: 4, n3) may be present in an amount up to about 2%, such as up to about 1.5%. Heneicosapentaenoic acid (HPA; 21: 5, n3) may be present in an amount up to about 3%, for example about 2.3%. These additional fatty acids may be added separately and may be present in formulations obtained from specific sources using specific methods. Other additional ingredients and fatty acids may also be present in small amounts, for example, 0-0.25% of the formulation.

  The composition is formulated with a DHA content that provides about 400 mg per day.

  Daily administration of the formulation can lower triglyceride (TG) levels and increase high density phospholipid (HDL) levels in CVD patients.

  The extremely potent ω3 formulation of the present invention is commercially available from Pivotal Therapeutics, Inc. under the trade name VASCAZEN ™ to mitigate cardiovascular risks associated with ω3 deficiency. VASCAZEN ™ has been prescribed to manage ω3 deficiency in patients with CVD by providing EPA and DHA to a level that cannot be reached with normal dietary improvement. More specifically, VASCAZEN (TM) products each comprise about 90% or more omega-3 fatty acids with an eicosapentaenoic acid (EPA): docosahexaenoic acid (DHA) ratio in the range of 5.7: 1 to 6.3: 1. This is an example of the present invention. This formulation contains about 680 mg / g EPA, about 110 mg / g DHA, and about 25 mg / g DPA per capsule. The total weight of each capsule is about 1000 mg. The daily dosage regimen of VASCAZEN ™ is generally intended to give 4 tablets / day in a single dose or in separate doses throughout the day. Based on the 1000 mg amount, the formulation contains at least about 90% or more omega-3 fatty acids, the total amount of EPA + DHA is about 80%, and the total amount of EPA + DPA + DHA is about 82%. In some embodiments, about 30 mg / g arachidonic acid, omega-6 fatty acids, and / or> 3% 18 carbon omega 3 fatty acids may be included.

  Low density lipid (LDL), HDL, and TG levels are monitored.

  According to research in the United States and the Dietary Guidelines Committee, 70% of Americans are deficient in omega-3 fatty acid intake. This is because the typical “Western food” lacks ω3 fatty acid intake, whereas “Western food” includes excessive intake of pro-inflammatory ω6 fatty acids. This dietary trend can be particularly dangerous in CVD patients. Together with other cardiovascular metabolic risk factors, ω3 deficiency further exacerbates the chronic progression of the disease. An increasing body of evidence has demonstrated cardiovascular health risks associated with chronic ω3 deficiency. In particular, a dietary deficiency of EPA acid and DHA allows a pro-inflammatory down pressure created by the metabolism of arachidonic acid (AA). Arachidonic acid (AA) is typically very abundant in most American diets. Overall, the lack of ω3 fatty acids contributes to the pro-inflammatory state, and the consequences of the pro-inflammatory state are dyslipidemia (high cholesterol, high triglycerides), increased atherosclerotic lesions, hypertension, And adverse effects on cardiovascular health, including increased risk of developing cardiac arrhythmias.

  Chronic ω3 deficiency can later lead to an increased risk of suffering a life-threatening heart attack. Maintenance of blood levels of EPA, DHA, and DPA above 6.1% of total blood fatty acids is associated with a risk of sudden cardiac death that is 80.0% lower compared to levels of 2.1% to 4.3%. To counteract the cardiovascular risks associated with excess AA and the pro-inflammatory effects on this metabolic pathway, intake of EPA and DHA may need to be increased to levels that cannot be reached by dietary changes alone. Therefore, further supplementation with extremely powerful EPA and DHA formulations is necessary to fill the “lack of ω3 nutrition”. Extremely powerful EPA and DHA formulations provide high levels of EPA and DHA for full clinical benefit, eliminating important risk factors for CVD patients.

  In an open-label study to analyze the safety and efficacy of VASCAZENTM, 143 patients were tested for whole blood omega-3 fatty acid levels and over about 2800 mg / day EPA and about 2 weeks or 6 weeks of follow-up. The formulation of the present invention was administered to patients to provide 480 mg / day of DHA. The primary endpoint was the change in the total amount of blood EPA + DHA + DPA. Levels (Omega-Score ™) were expressed as a percentage of total blood fatty acid levels over 2 or 6 weeks.

  The normalized baseline Omega-Score ™ was 3.4% (N = 143). In the 2-week and 6-week treatment groups, the formulations of the present invention have Omega-Score ™ levels of 52.8% (N = 63, p = 5, respectively) compared to the baseline levels measured in each group. <0.0001) and 120.6% (N = 31, p = <0.0001). Six weeks after the intervention, the maximum and stable level was maintained with an average score of 7.5%. The formulations of the present invention were generally well tolerated and reported only minor adverse events in a small percentage of study participants. (See Table 4).

Method:
A 6-week open-label study was conducted at a single facility in Canada. Subjects were eligible for the trial if they met all inclusion and exclusion criteria described in the clinical trial protocol. Prior to study registration, informed consent was obtained from all qualified subjects and enrolled in Group A (Figure 1). 63 subjects received 4 VASCAZEN ™ capsules (Group B) per day, an oral dose of 2720 mg EPA and 440 mg DHA per day. Two weeks after treatment, whole blood ω3 blood levels were assessed and 31 subjects were enrolled in Group C for continued treatment. Whole blood samples were obtained from Group C subjects at weeks 4 and 6 for follow-up Omega-Score ™ evaluation.

  The primary endpoint was the change in Omega-Score ™ values expressed as a percentage of total blood fatty acid levels over 2 weeks for Group B and 6 weeks for Group C. Baseline Omega-Score ™ values for Group A were calculated as the average percent at Week 0 before VASCAZEN ™ intervention. Group B and Group C Omega-Score ™ averages were evaluated as appropriate at designated time points.

  The study consisted of men and women over 15 years of age in stable medical condition. Exclusion criteria include ventricular arrhythmia, known bleeding or coagulopathy, liver or kidney disease, autoimmune disorder or immune system decline, seizure disorder or history of taking anticonvulsants, fish allergy, or implantable defibrillation A subject with motivation was included. Medical history and current medications were also recorded.

Laboratory analysis of total blood fatty acids in whole blood was performed by a central laboratory (University Health Network Laboratory, Toronto, Ontario) certified by the College of American Pathologists' Laboratory Accreditation Program. The analysis was performed by derivatizing the fatty acid to the methyl ester followed by gas chromatography-mass spectrometry (GC-MS analysis) (Agilent Technologies 6890N series gas chromatograph, 5975C detector, Mississauga, Ontario) . Fatty acids were extracted from 200 μL whole blood samples using a mixture of methanol and chloroform. The fatty acids were then methylated by incubating with 10% (w / v) BCl ₃ in methanol for 25 minutes at 90 ° C. to form fatty acid methyl esters (FAME). After cooling, FAME was extracted with a water / hexane mixture and 1 uL of n-hexane extract was injected for GC-MS analysis.

  The sample size was adjusted appropriately. Assuming an average baseline level of blood Omega-Score ™ levels of at least 3.0% and a standard deviation of changes in blood Omega-Score ™ levels of 1.8% in the study population, the minimum sample size is 63 The minimum to detect at a significant level of α = 0.05 that blood Omega-Score ™ levels increased by at least 25.0% relative to baseline after 2 weeks of study intervention The power will be 90.2%. For 30 subjects with a minimum sample size of VASCAZEN (TM) for 6 weeks, blood Omega-Score (TM) levels increased by at least 40.0% relative to baseline after 6 weeks of study intervention The minimum power to detect this with a significance level of α = 0.05 would be 94.2%. The safety population was defined as the group of patients taking VASCAZEN ™ at a dose of 4 capsules per day for a minimum of 2 weeks and a maximum of 6 weeks. A primary analysis of therapeutic efficacy was performed on some of the enrolled study subjects. Blood measurements were performed on enrolled study subjects at baseline and after 2 weeks of study treatment. For each test subject, the change in blood Omega-Score ™ level (expressed as a percent change from baseline) over a two week period was calculated. The Pearson-D'Agostino test was used to test the normality of the distribution of changes in blood Omega-Score ™ levels over two weeks. To test for changes in blood Omega-Score ™ levels over a two week period, a paired t-test was performed.

  A secondary analysis of therapeutic efficacy was performed on some of the enrolled study subjects. Blood Omega-Score ™ levels in enrolled study subjects were measured at baseline and at 2 weeks, 4 weeks, and 6 weeks after baseline. To test changes in blood Omega-Score ™ levels between any time point pairs over a 6 week period, analysis of variance (ANOVA) was performed using subjects as blocks. Multiple comparisons were made at the family-wide significance level of α = 0.05 to see which time pair (if any) were significantly different with respect to the mean of blood EPA + DHA + DPA levels. A linear contrast was performed to test the hypothesis that the mean blood EPA + DHA + DPA level increased linearly in some of the test subjects over a 6 week period.

result:
The baseline characteristics of each test group are outlined in Table 1. Age demographics were comparable across all groups and the majority of study participants were middle-aged. In group A consisting mainly of men (74.1%), the average age of all groups (N = 143) was 50.9 years, and a similar age distribution was observed for men (52.1) and women (46.9). The mean age of the 2-week treatment group B (N = 63, 74.2% were male) was 53.7 years, and the average age of males (55.8) and females (47.9) was similar. Finally, the mean age of the study subjects in group C (N = 31, 87% male) was 55.0 years (male, 54.0; female, 61.5). Baseline Omega-Score ™ values were measured and all three groups, including men and women, had an equivalent ω3 deficiency with a score of 3.3% to 3.8% (Omega-Score defined as less than 6.1%) (N Engl J Med, Vol. 346, No. 15, April 11, 2002, Pp. 1113-1118).

Omega-Score(商標)は、生データの正規分布から平均+/-SD(N=対象数)として計算した。C群(女性)には、正規分布曲線にフィットするのに十分な数が無かった。この群の生データの平均ベースラインスコアは2.98％であった。 (Table 1) Baseline characteristics ^* :

Omega-Score ™ was calculated as mean +/- SD (N = number of subjects) from the normal distribution of raw data. Group C (female) did not have enough numbers to fit a normal distribution curve. The average baseline score for the raw data in this group was 2.98%.

  The results of the main endpoints are shown in FIG. 2 and Table 2 and fitted to the normal distribution calculated in FIG. 3 and Table 3. From baseline levels of whole blood ω3 blood levels, ω3 deficiency (group A) in a large test group (N = 143) was revealed. In this group, the mean score for subjects is 4.4% (3.4% for normal distribution curve fit), and 84.5% of individuals have a cut-off score of 6.1% and are in the cardiovascular risk quartile Is shown. Scores of study participants (Group B) who received VASCAZEN (TM) intervention for 2 weeks improved significantly (P <0.0001) (Figure 2, Table 2), and the average value improved from 3.6% to 5.5% ( Figure 3, Table 3), score increased by 52.8%. Over 2 weeks of intervention, the number of study participants considered “hazardous” decreased from 80.6% to 46.8% (Table 3). Over the 6-week VASCAZEN ™ intervention, the mean score for Group C subjects improved (P <0.0001) (Figure 2, Table 2), and the mean improved from 3.4% to 7.5% from baseline to week 6. (Figure 3, Table 3). This corresponds to a 120.6% increase in whole blood concentration of EPA + DHA + DPA Omega-Score ™ values. After 6 weeks of VASCAZEN ™ intervention, 13.2% of participants remained at high risk (Omega-Score ™ <6.1%), Table 3.

表2.主要評価項目:2週間または6週間の介入にわたる血中総脂肪酸レベルのパーセントとして表された血中EPA+DHA+DPAレベルの総量の変化 (Table 2)

Table 2. Primary endpoint: change in total blood EPA + DHA + DPA levels expressed as a percentage of total blood fatty acid levels over 2 or 6 weeks of intervention

表3.主要評価項目:2週間または6週間の介入にわたる血中総脂肪酸レベルのパーセントとして表され、正規分布として示された血中EPA+DHA+DPAレベルの総量の変化 (Table 3)

Table 3. Primary endpoint: change in total blood EPA + DHA + DPA levels expressed as a percent distribution, expressed as a percentage of total blood fatty acid levels over 2 or 6 weeks of intervention

  Patients with a> 6.1% (ideal) score were 80% less likely to die from sudden cardiac arrest compared to individuals in the risk quartile (score) range of 2.1% to 4.3%. In this study, 84.5% of study participants, many of whom have cardiovascular health problems and are taking statins and / or blood pressure medications, have a score of less than 6.1% and are at high risk of adverse events In particular, patients with known dyslipidemia, type 2 diabetes, and / or hypertension are at increased risk of adverse events from the mean baseline value of the study population. Six weeks after VASCAZEN ™ intervention, 71.3% of Group C participants who had a previous baseline score of less than 6.1% were able to increase their scores to levels above this threshold.

^*治療して2週間後に軽微な挫傷が現れ、3日以内に消失した。対象は、有害事象なく、さらに4週間、VASCAZEN(商標)の服用を続けた。 (Table 4)

^* Minor contusion appeared 2 weeks after treatment and disappeared within 3 days. Subjects continued taking VASCAZEN ™ for 4 additional weeks without adverse events.

  The study participant score in group B increased significantly by 52.8% from 3.6% to 5.5% (P <0.0001). Long-term VASCAZEN (TM) intervention significantly improved the scores of group C individuals over 6 weeks (P <0.0001, ANOVA), with similar improvements within group B within 2 weeks. After 4 weeks, VASCAZEN ™ significantly increased the average score from 3.4% to 7.9% (P <0.0001). This corresponds to an improvement of 132.4%. This brought the average overall score well within the low-risk quartile of> 6.1%. In fact, after 4 weeks, only 15% of study participants remained below this benchmark level, and this level is maintained in the 6-week VASCAZEN ™ intervention study group. VASCAZEN ™ is generally well tolerated and has a low incidence of minor adverse events typical of ω3 polyunsaturated fatty acid ethyl esters. This trial highlighted the prevalence of chronic ω3 deficiency in the majority (84%) of both men and women.

  There is a good record of the consequences of ω3 deficiency in CVD patients. A great deal of research has linked EPA and DHA deficiencies. Many studies and current therapeutic approaches have classified ω3 as a therapeutic agent for treating symptoms associated with CVD. Unfortunately, the general thought path for ω3 fatty acid therapy does not lead to optimal results. EPA and DHA should not be considered therapeutic agents, and ideally should be considered essential nutrients that must be taken regularly as part of a healthy balanced diet. Ω3 deficiency in CVD patients adds unnecessary risk. This risk can be avoided by proper ω3 supplementation. The present invention provides balanced essential levels of EPA and DHA that many CVD patients, as exemplified by VASCAZEN ™ intervention, are difficult to incorporate into their daily diet with food alone. In a typical Western diet, the average American takes only 1/15 of the omega-3 fatty acids needed to reach and maintain clinically beneficial levels of EPA and DHA. In order to provide a sufficient amount of this essential nutrient so as to provide a daily dose that the present invention can provide, fish would have to be eaten daily on multiple meals per day. This is unrealistic for most people.

  From this study, EPA + DHA + DPA can be maintained at a level of> 6.1% within 4 weeks of intervention using the present invention, 4 per day supplying approximately 2720 mg EPA and 440 mg DHA. Over 85% of patients at doses of capsules proved to be able to achieve these levels. These findings are measured routinely in CVD patients (via Omega-Score ™ assessment) and the ω3 fatty acid supplement of the present invention to maintain clinically beneficial EPA + DHA + DPA blood levels Support the use of.

Sustained vasodilatory effect:
In addition to the benefits outlined above with respect to ω3 supplementation to ω3 deficient patient populations, the formulations of the present invention have been shown to provide sustainable eNOS vasodilatory activity, defined as vasodilatory effects lasting more than 6 hours. Has been. This sustainable eNOS vasodilator action could not be achieved in the past using either prescription ω3 supplements or OTC grade ω3 supplements.

  In order to understand this vasodilatory effect in connection with the treatment and prevention of cardiovascular disease, it is first necessary to understand the mechanism of vasodilation through the endothelial lining of blood vessels.

Rely on the following list of abbreviations for the following discussion.
Abbreviation list <br/> Abbreviation Meaning
[Ca ²⁺ ] i intracellular free calcium concentration
APA Apamine
CaM calmodulin
CaMK-2 calmodulin kinase-2
cAMP cyclic adenosine 3 ': 5' monophosphate
cGMP cyclic guanosine 3 ': 5' monophosphate
EDHF Endothelial hyperpolarizing factor
eNOS Endothelial NO synthase
ET-1 Endothelin-1
H ₂ O ₂ hydrogen peroxide
IKCa calcium-dependent intermediate conductance potassium channel
Indo indomethacin
L-NA N-ω-Nitro-L-arginine
MnTMPyP Mn (III) tetrakis (1-methyl-4-pyridyl) porphyrin
NO Nitric oxide
O ₂ °-Superoxide anion
PEG-catalase Polyethylene glycol-catalase
PGI ₂ Prostacyclin I2
PI3-K Phosphoinositide-3 kinase
PKC protein kinase C
PP2 4-Amino-5- (4-chlorophenyl) -7- (t-butyl) pyrazolo [3,4]
Pyrimidine
ROS reactive oxygen species
sGC soluble guanylyl cyclase
SKCa Ca2 ⁺ -dependent small conductance potassium channel
SOD conductance superoxide dismutase
TRAM34 1-[(2-Chlorophenyl) diphenylmethyl] -1H-pyrazole
TX _A2 Thromboxane A2
U46619 9,11-dideoxy-9-prostaglandin F2 methanoepoxy

The endothelium consists of a monolayer of endothelial cells that lines the luminal surface of all blood vessels. Endothelial cells play an important function in the regulation of vascular homeostasis. Endothelial cells regulate the contact between blood and the underlying thrombogenic artery wall. Endothelial cells release a large number of physiological stimuli such as nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) by releasing several short-lived powerful endothelium-derived vasoactive factors such as nitric oxide (NO). Responds to circulating hormones and shear stress. These two factors play a major role in the control of vascular tone (Busse et al., 2002; Michel and Feron, 1997). In addition, endothelial cells can also produce prostacyclin (PGI ₂ ), a prostanoid that causes relaxation of some blood vessels.

Endothelial nitric oxide (NO):
NO is produced from L-arginine by endothelial nitric oxide synthase (eNOS). NO plays an important role in normal vascular biology and pathophysiology. NO induces vascular smooth muscle relaxation by activating soluble guanylyl cyclase to form cyclic guanosine 3′-5 ′ monophosphate (cGMP). In addition to regulating vascular tone and inhibiting platelet aggregation, NO is associated with vascular smooth muscle cell proliferation, monocyte adhesion (Dimmeler et al., 1997; Hermann et al., 1997; Tsao et al., 1996), and It also inhibits many important steps involved in atherogenesis, including cell death. Receptor-dependent and receptor-independent agonists increase free Ca intracellular concentration ([Ca ²⁺ ] i), Ca ²⁺ / calmodulin (CaM) complex and eNOS bind, and eNOS becomes active As a result, eNOS can be activated (Fleming et al., 2001). Indeed, by removing Ca ²⁺ from the extracellular space, and by CaM antagonists, both agonist-induced NO formation and subsequent vascular relaxation disappear. eNOS is also regulated at the post-translational level in endothelial cells, mainly by protein / protein interactions and multi-site phosphorylation at Ser116, Thr497, Ser635, and Ser1179 (both sequence residue numbers are human Corresponding to the sequences Ser114, Thr495, Ser633, and Ser1177) (Bauer et al., 2003; Boo et al., 2002; Dimmeler et al., 1997). Indeed, eNOS has been shown to be regulated by interaction with positive and negative protein modulators such as caveolin (Cav-1) and heat shock protein 90 (Garcia-Cardena et al., 1998; Ju et al., 1997; Pritchard et al., 2001). In the basal state, most of the eNOS appears to be bound to caveolin-1, and its enzymatic activity is suppressed in vesicles (Michel et al., 1997). This tonic inhibition of eNOS can be released by replacing caveolin-1 with Ca ²⁺ / CaM in response to a Ca ²⁺ mobilization agonist (Ju et al., 1997). In addition to these modulators, phosphorylation of key regulatory sites in eNOS plays an important role in the regulation of enzyme activity in response to several physiological stimuli (Ju et al., 1997). Phosphorylation of eNOS Ser1179 has been shown to be associated with increased enzyme activity (Gallis et al., 1999; McCabe et al., 2000). The phosphorylation of eNOS-Ser1179 is regulated by a PI3-kinase-dependent mechanism (Gallis et al., 1999). Akt, one of the key regulatory targets of PI3-kinase, directly phosphorylates eNOS Ser1179 and activates enzymes in response to vascular endothelial growth factor (VEGF), sphingosine-1-phosphate, and estrogen (Dimmeler et al., 1997; Fulton et al., 1999). However, eNOS-Ser1179 can also be phosphorylated by AMP-activated protein kinase (Busse et al., 2002), protein kinase A (PKA), and protein kinase G (PKG). It appears that exactly which protein kinase phosphorylates eNOS-Ser1179 in intact cells depends on the type of endothelial cell and the stimulus. For example, shear stress phosphorylates eNOS Ser1179 in a PI3-kinase-dependent and PKA-dependent manner without Akt being involved, whereas EGF phosphorylates eNOS Ser1179 in a PI3-kinase-dependent and Akt-dependent manner. Oxidizes (Boo et al., 2002). Furthermore, ischemia reperfusion injury activates the PKA pathway and phosphorylates eNOS Ser1179 and Ser635 (Li et al., 2010). In addition, shear stress (Butt et al., 2000), hypoxia, low density lipoprotein (LDL) (Chen et al., 2008; Chen et al., 1999), and oxidized fatty acids (Corson et al., 1996) Several factors can regulate eNOS expression levels.

Endothelial hyperpolarizing factor (EDHF):
Endothelium-dependent vasorelaxation has also been observed after inhibition of NO and PGI2 synthesis in some blood vessels and has been attributed to endothelium-derived hyperpolarizing factor (EDHF). EDHF relaxes blood vessels by hyperpolarization of vascular smooth muscle. This action closes the voltage-gated Ca ²⁺ channel, lowering intracellular free Ca ²⁺ levels, and then relaxing vascular smooth muscle. Potassium (K ⁺ ) channels underlie the hyperpolarization induced by EDHF, with intermediate conductance Ca ²⁺ activated K ⁺ (IKCa) channels and small conductance Ca ²⁺ activated K ⁺ (SKCa channels). In some disease states, including the presence of cardiovascular risk factors, the endothelium undergoes functional and structural changes, losing its protective role and becoming proatherosclerotic (Vanhoutte, 1989). Loss of normal endothelial function is called endothelial dysfunction and is characterized by reduced NO production by eNOS and / or NO bioavailability failure after increased NO destruction by reactive oxygen species (ROS), particularly superoxide anions. (Vanhoutte, 1989).

  From previous studies by the inventors, natural products such as Concord grape juice (Anselm et al., 2007) and red wine polyphenols (Ndiaye et al., 2005) have been found to be redox-sensitive Ser / PI3-kinase / eNOS Ser1177. It has been shown to activate NO endothelialization by causing Akt pathway-dependent phosphorylation.

  Fish oil ω3 is a rich source of EPA and DHA. Omega 3 fatty acids have been shown to cause endothelium-dependent vasorelaxation in rat aortic and coronary arterial rings in vitro by stimulating the formation of NO in the endothelium (Engler et al., 2000; Omura et al. , 2001). However, signal transduction pathways leading to eNOS activation have not been fully studied. Furthermore, little information is currently available regarding the optimal ratio of EPA: DHA for eNOS activation. Therefore, the following experiments were performed to characterize fish oil-induced eNOS activation in isolated blood vessels and cultured endothelial cells.

  Initial experiments confirmed the ability of ω3 fatty acids (EPA, DHA, and different ratios of EPA: DHA) to cause endothelium-dependent relaxation in the porcine coronary annulus, thereby playing a role of NO and EDHF in endothelium-dependent relaxation. Designed to characterize and identify the signaling pathways involved.

  Additional experiments were designed to verify the ability of ω3 fatty acids (EPA, DHA, and different ratios of EPA: DHA) to cause eNOS activation in endothelial cultured cells, as well as to verify the underlying signaling.

To perform the above confirmation, we designed an experiment that classifies vascular reactivity. First, fat and adhering tissue were removed from the left circumflex coronary artery taken from a fresh pig heart and the rings were cut to a length of 2-3 mm. An endothelium-free ring was obtained mechanically by rubbing with a single pliers inserted into the vessel lumen. Endothelial or non-endothelial rings were infused with Krebs bicarbonate solution (composition, mM: NaCl 118.0, KCl 4.7, CaCl ₂ 2.5, MgSO ₄ 1.2, infused with a mixture of 95% O ₂ and 5% CO _2. , NaHCO ₃ 23.0; KH ₂ PO ₄ 1.2, and glucose 11.0, pH 7.4, 37 ° C.). The rings were kept in equilibrium for 5 minutes at 5 g basic tension and then contracted with KCl (80 mM) to verify vascular smooth muscle reactivity. Endothelial integrity was verified after a 30 minute wash period. Rings were contracted to 80% of maximal contraction using U46619 (1-60 nM, thromboxane A2 analog) and bradykinin (0.3 μM) was added at the contraction plateau to check for the presence of functioning endothelium. After repeated washing and return to baseline, the rings were recontracted with U46619 and then tested in increasing ranges of ω3 fatty acids (0.001% -0.4% v to test the ability to induce relaxation of the coronary artery ring. / v) applied. During the stabilization phase (30 minutes before contraction with U46619), the following different pharmacological tools were added to the Krebs bicarbonate solution to characterize signaling pathways that lead to endothelium-dependent relaxation:
indomethacin (10 μM): a cyclooxygenase (COX) inhibitor that blocks the formation of vasoactive prostanoids (particularly prostacyclin),
b. Nω-nitro-L-arginine (L-NA, 300 μM): NO synthase (NOS) competitive inhibitor that overcomes the NO component, and
c. TRAM34 (100 nM) and apamin (100 nM): inhibitors of Ca ²⁺ activated potassium channels (IKCa and SKCa), respectively, that overcome the EDHF component.

Porcine coronary artery endothelial cells were collected and washed with a phosphate buffered saline solution (PBS) containing no calcium to remove residual blood. Endothelial cells were isolated using collagenase (Type I, Worthington, 1 mg / ml, 14 minutes at 37 ° C.), 15% v / v fetal calf serum, 2 mM glutamine, 100 U / mL penicillin, 100 U / mL streptomycin, and 250 mg Culture medium MCDB131 (Invitrogen) supplemented with / ml fungizone (Sigma, St Louis, MO) in 5% CO ₂ at 37 ° C. All experiments were performed using confluent endothelial cells used for the first passage. Endothelial cells were exposed to MCDB131 containing 0.1% v / v fetal calf serum 5 hours prior to treatment with different substances.

After treatment, the endothelial cells were rinsed twice with PBS and extracted buffer (composition, mM: Tris / HCl 20, pH 7.5 (QBiogene), NaCl 150, Na ₃ VO ₄ 1, Na ₄ P ₂ O ₇ 10, Elution was performed using NaF 20, okadaic acid 0.01 (Sigma), protease inhibitors (Complete, Roche), and 1% Triton X-100). 25 μg of total protein was separated with SDS-polyacrylamide (Sigma, 8%) at 100V for 2 hours. The separated protein was transferred to a polyvinylidene fluoride membrane (Amersham) by electrophoresis at 100 V for 2 hours. Membranes were blocked for 1 hour with blocking buffer containing 3% bovine serum albumin dissolved in TBS-T (Tris buffered saline solution (Biorad) containing 0.1% Tween20 (Sigma)). To detect the protein, the membrane was separated from each primary antibody (p-eNOS Ser1177, p-eNOS Thr495, and p-Akt Ser473 (1: 1000 dilution), β-tubulin (1: 5000 dilution, Cell Signaling Technology)) Incubated overnight at 4 ° C. in TBS-T containing After the wash period, the membrane was washed with a secondary antibody conjugated to horseradish peroxidase (p-eNOS, anti-rabbit for p-Akt, anti-mouse for β-tubulin (Cell Signaling Technology, 1: 5000 dilution)) and room temperature. Incubated for 1 hour. A stained protein marker (Invitrogen) was used to confirm the molecular weight of the separated protein. Immunoreactive bands were detected using chemiluminescence (Amersham).

  All results were expressed as mean ± standard error of the mean (SEM). n indicates the number of different coronary arteries tested. Statistical analysis was performed using Student's t test or analysis of variance (ANOVA) test followed by Bonferroni post-hoc test. A P value of <0.05 was considered statistically significant.

result:
The ω3 fatty acid preparation EPA: DHA 1: 1 induced concentration-dependent relaxation of the coronary artery ring containing endothelium, whereas the coronary artery ring without endothelium contracted with U46619 There was only a slight relaxation (Figure 4). Relaxation to EPA: DHA 1: 1 was observed in amounts exceeding 0.01% v / v, reaching approximately 75% at 0.4% v / v (FIG. 4). In addition, ω: 3 fatty acid preparation EPA: DHA 6: 1 also induced endothelium-dependent relaxation. This was more potent than endothelium-dependent relaxation induced by EPA: DHA 1: 1 (FIG. 4). Relaxation to EPA: DHA 6: 1 started at 0.01% v / v and reached approximately 98% at 0.4% v / v (FIG. 4). These findings indicate that the ω3 fatty acid preparation EPA: DHA 6: 1 is more effective than the EPA: DHA 1: 1 preparation in inducing endothelium-dependent relaxation of the coronary artery rings. This was followed by all subsequent experiments using the ω3 fatty acid preparation EPA: DHA 6: 1.

  ω: 3 fatty acid preparation EPA: DHA 6: 1 was confirmed to induce endothelium-dependent relaxation involving both NO and EDHF.

Previous studies have shown that EPA and DHA induce relaxation of the coronary annulus primarily through mechanisms that are endothelium-dependent and sensitive to inhibitors of NO and EDHF formation (Omura et al. al., 2001). Thus, whether NO and EDHF are involved in endothelium-dependent relaxation induced by omega-3 fatty acid formulations of the present invention (referred to herein as EPA: DHA 6: 1) with an EPA: DHA ratio of about 6: 1 A test was conducted to confirm the above. Endothelium-dependent relaxations against EPA: DHA 6: 1 are EDHF component inhibitors TRAM34 and apamin (inhibitors of intermediate conductance Ca ²⁺ -dependent potassium channel IKCa and small conductance Ca ²⁺ -dependent potassium channel SKCa, respectively) It was not significantly affected by Fig. 5). In contrast, relaxation was partially inhibited by L-NA (a competitive inhibitor of eNOS) but was inhibited in a statistically significant amount. This shows that NO is involved (Figure 5). Furthermore, the combination of L-NA + TRAM34 and apamin eliminated endothelium-dependent relaxation towards EPA: DHA 6: 1 (FIG. 5). Taken together, these findings indicate that EPA: DHA 6: 1 induces endothelium-dependent relaxation mediated primarily by NO and to a lesser extent also by EDHF.

  Several studies have shown that NO-mediated relaxation in response to grape-derived polyphenols involves the redox-sensitive Src / PI3-kinase / Akt pathway (Anselm et al., 2007; Ndiaye et al., 2005). Therefore, it was determined to see if this pathway is involved in NO-mediated relaxation to EPA: DHA 6: 1. In order to selectively test the NO component, all experiments were performed in the presence of an EDHF component inhibitor (apamin + TRAM34) and in the presence of a vasoactive prostanoid formation inhibitor (indomethacin). Relaxation induced by EPA: DHA 6: 1 was significantly reduced by PP2 (Src kinase inhibitor, FIG. 6) and wortmannin (PI3-kinase inhibitor, FIG. 6). Furthermore, relaxation to EPA: DHA 6: 1 was shifted to the right in a statistically significant amount by membrane-permeable SOD analogs MnTMPyP and catalase (PEG-catalase) and by native SOD and catalase. (Figure 7). Taken together, these findings suggest that Src kinase and PI3-kinase mediate EPA: DHA 6: 1 stimulation signals to eNOS via a redox-sensitive mechanism.

  To obtain direct evidence that EPA: DHA 6: 1 can activate the PI3-kinase / Akt pathway and activate eNOS, coronary cultured endothelial cells were exposed to EPA: DHA 6: 1 for up to 6 hours. Western blots were used to confirm the levels of phosphorylated Akt and eNOS. The data show that EPA: DHA 6: 1 increases Akt and eNOS phosphorylation levels from 15 minutes and this effect lasts up to 6 hours (FIGS. 8A and 8B). The level of total eNOS expression remained unaffected by EPA: DHA 6: 1 treatment (FIG. 8A). Furthermore, the stimulatory effect of EPA: DHA 6: 1 on Akt and eNOS phosphorylation was inhibited by MnTMPyP, PEG-catalase, and by native SOD and catalase (FIG. 8A). Thus, these data provide direct evidence that EPA: DHA 6: 1 activates eNOS via a redox-sensitive mechanism.

％で表したω3は、EE(エチルエステル)としての総脂肪酸に対する％で表した総ω3を示す。 (Table 5) Comparison of capsule contents VASCAZEN (TM) vs. Germany ω3 OTC brand

The ω3 expressed in% indicates the total ω3 expressed in% relative to the total fatty acid as EE (ethyl ester).

  Turning now to FIGS. 9-12, these figures serve to illustrate the importance of both the purity and presence of the additive in the formulation, respectively, in providing a maximum relaxation response. For this discussion, ω3 purity was defined as the percentage of total EPA + DHA per capsule. The use of indomethacin as a relaxing action determinant is based on the following explanation. In some blood vessels, vasorelaxant prostanoids such as prostacyclin have been identified as endothelium-derived vasorelaxing factors. These vasorelaxing prostanoids are produced from arachidonic acid metabolism by cyclooxygenase-1 (COX-1). Indomethacin is a COX-1 inhibitor and therefore prevents the formation of vasorelaxant prostanoids. The magnitude of endothelium-dependent relaxation depends on the purity of the formulation (Figure 9) and the EPA: DHA ratio (Figure 4). In addition, the EPA: DHA 6: 1 formulation is similar to the OTC ω3 product TETESEPT (TM), which has an ω3 purity of 22.2% compared to the EPA: DHA 6: 1 formulation ω3 purity (defined above) of 75.1%. Was more effective than the other OTCω3 tested (ABTEILACHSOL ™ 1300, DOPPELHERZ®, SCHAEBENS ™, and OPTISANA ™) (Figure 11A). Endothelium-dependent relaxation induced by VASCAZEN ™ (an example of EPA: DHA 6: 1) was not affected by 10 μM indomethacin. In contrast, relaxation induced by TETESEPT ™, similar to relaxation of EPA: DHA 6: 1, was significantly reduced by indomethacin (FIGS. 11A and B). Endothelium-dependent relaxation induced by SCHAEBENS ™ and OPTISANA ™ was significantly reduced, and relaxation to ABTEI® and DOPPELHERZ® was slightly reduced (FIGS. 11A and B). Furthermore, the data suggests that OTCω3's indomethacin-sensitive relaxation is most likely due to the presence of additives such as vitamin E (α-tocopherol), not due to EPA and DHA or their relative concentration ratios. I understand that. See Table 5. In fact, EPA: DHA 6: 1 has a vitamin E content of 0.2%, whereas the OTC ω3 formulation has a vitamin E content of 0.85-1.1% (Table 5). Furthermore, the importance of the vitamin E additive effect is suggested by the fact that the TETESEPT ™ vitamin E content is more than five times that of the EPA: DHA 6: 1 formulation. Thus, the selective inhibitory effect of indomethacin that is induced against TETESEPT ™ but not against EPA: DHA 6: 1 is explained by more than 5-fold vitamin E content per capsule. Most likely. Vitamin E causes endothelium-dependent relaxation, which has been shown to be inhibited by indomethacin (Wu et al., J Nutr. 135: 1847-1853, 2005). Both ω3 purity and additives contribute to the endothelium-dependent relaxation observed with ω3 products. This is further illustrated by comparing the relaxation induced by the EPA: DHA 6: 1 formulation with that of the METAGENICS ™ EPA-DHA 6: 1 formulation. In fact, the latter is significantly inhibited by indomethacin compared to the former (FIG. 12). Thus, in the presence of indomethacin, the relaxation observed in the presence of the ω3 product is clearly dependent on the ω3 purity. These experiments highlight the sustained (greater than 6 hours) vasodilator effect achieved by the unique ratio and ω3 purity of the novel EPA: DHA 6: 1 product of the present invention. When compared to EPA only or DHA only, EPA: DHA 1: 1, or 6: 1 product containing exogenous impurities, the combination of 6: 1 ratio and absence of exogenous impurities in the present invention is independent of indomethacin Leads to sex vasodilation (see FIGS. 4, 9, 11, 12, and 13).

  These findings indicate that the ω3 fatty acid preparation is a potent endothelium-dependent vasodilator, and this action depends on the ratio of EPA and DHA in the capsule and ω3 purity. Furthermore, these suggest that ω3 fatty acids activate eNOS via the redox-sensitive PI3-kinase / Akt pathway leading to changes in eNOS phosphorylation levels, as illustrated in FIG.

  All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which this invention pertains. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication was incorporated by reference in detail and individually.

  While a particular form of the invention has been illustrated, it should be understood that the invention is not limited to this particular form or arrangement described and shown herein. Various changes may be made without departing from the scope of the invention, and the invention is limited to what has been shown and described herein, and any drawings / drawings described herein. It will be apparent to those skilled in the art that this is not considered.

  Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objectives and obtain the stated goals and advantages as well as the inherent goals and benefits. The aspects, methods, procedures, and techniques described herein are representative of preferred embodiments of the invention, are intended to be illustrative, and are not intended to limit the scope. Changes therein and other uses will occur to those skilled in the art and are encompassed within the spirit of the invention and are defined by the scope of the appended claims. Although the invention has been described with reference to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims (30)

A pharmaceutical formulation for treating or preventing risk factors for cardiovascular disease (CVD), preventing sudden death in patients with cardiovascular disease, treating obesity-related disorders, preventing weight gain, or maintaining weight,
A mixture containing omega-3 fatty acids including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA), wherein the weight ratio of EPA: DHA is in the range of 5.7: 1 to 6.3: 1 Wherein the mixture contains about 90% by weight or more of ω3 fatty acids and EPA, DHA, and DPA constitute about 82% by weight of the contents of the mixture; and energy consumption, glycolysis, sugar Affects at least one mechanism of action selected from the group consisting of neoplasia, glycogenolysis, lipolysis, fat absorption, fat storage, fat excretion, hunger, satiety, craving mechanism, appetite, food intake, gastrointestinal motility, and combinations thereof A pharmaceutical formulation comprising at least one anti-obesity agent that affects   The formulation of claim 1, comprising about 25 mg / g DPA.   The formulation of claim 1, further comprising about 30 mg / g arachidonic acid (AA).   2. The formulation of claim 1, further comprising about 30 mg / g of one or more omega-3 fatty acids having 18 carbon atoms.   6. The formulation according to claim 5, wherein the one or more ω3 fatty acids having 18 carbon atoms are selected from the group consisting of α-linolenic acid (ALA), stearidonic acid (SDA), and combinations thereof. .   2. The formulation of claim 1, wherein the ω3 fatty acid is in the form of an ethyl ester and a pharmaceutically acceptable salt thereof.   The formulation according to claim 1, wherein the ω3 fatty acid is in the form of a triglyceride and a pharmaceutically acceptable salt thereof.   The formulation according to claim 1, wherein the ω3 fatty acid is in the form of a phospholipid and a pharmaceutically acceptable salt thereof.   The formulation of claim 1, wherein the dosage form comprises about 645 to about 715 mg / gm EPA, about 105 to about 115 mg / gm DHA, and about 22 to about 28 mg / gm DPA.   2. The formulation of claim 1, which is a unit dosage form comprising at least 680 mg EPA, at least 110 mg DHA, and at least 25 mg DPA.   10. The formulation of claim 9, wherein the unit dosage form further comprises about 30 mg AA.   10. The formulation of claim 9, wherein the unit dosage form further comprises ω3 fatty acids having 18 carbon atoms, about 30 mg / g.   13. The formulation of claim 12, wherein the one or more ω3 fatty acids having 18 carbon atoms are selected from the group consisting of α-linolenic acid (ALA), stearidonic acid (SDA), and combinations thereof. .   10. The formulation of claim 9, further comprising a stabilizer.   15. The formulation of claim 14, wherein the stabilizer is tocopherol in an amount of about 0.2%.   10. The formulation of claim 9, wherein the unit dosage form may comprise tablets, capsules, pills, powders, granules, and oral solutions or suspensions.   17. The formulation according to claim 16, wherein the unit dosage form is a gel or a liquid capsule. Anti-obesity agent is an antagonist or inverse agonist for the receptor product of the cannabinoid 1 (CB1) gene; cathepsin K inhibitor; PYY; PYY _3-36 ; PYY agonist; 5HT transporter inhibitor; NE transporter inhibitor; ghrelin antagonist H3 antagonist / inverse agonist; MCH1R antagonist; MCH2R agonist / antagonist; MC3R agonist; NPY1 antagonist; NPY4 agonist; NPY5 antagonist; leptin; leptin agonist / modulator; leptin derivative; opioid antagonist; orexin antagonist; BRS3 agonist; 11βHSD-1 inhibition Agents; CCK-A agonists; CNTF; CNTF agonists / modulators; CNTF derivatives; Cox-2 inhibitors; GHS agonists; 5HT2C agonists; CB-1 antagonists; neuropeptide Y5, appetite suppressors; lipase inhibitors; 5HT6 antagonists; monoamines Reuptake inhibitor; UCP-1, 2, and 3 activators; β3 agonists; thyroid hormone β agonists; PDE inhibitors; FAS inhibitors; DGAT1 inhibitors; DGAT2 inhibitors; ACC2 inhibitors; glucocorticoid antagonists; acyl-estrogens; lipase inhibitors; fatty acid transporters Inhibitors; Dicarboxylic acid transporter inhibitors; Glucose transporter inhibitors; Serotonin reuptake inhibitors; Aminolex; Amphetoral; Amphetamine; Axoquine; Benzphetamine; Chlorphentermine; Clobenzolex; Croforex; Chrominolex; Chlortermine; cyclexamine; dextroamphetamine; difemethoxyzine, N-ethylamphetamine; fenbutrazete; phenisolex; fenproporex; fluproprex; fluminorex; furfurylmethylamphetamine; Mefenolex; Methanefepramon; Methamphetamine; Nalmefene; Norseudoephedrine; Pentrex; Fendimetrazine; Fenmetrazine; Phytofarm Compound 57; Pisirolex; Topiramate; Zonisamide; IGF-IR antagonist; MetAP2 modulator; 2. The formulation of claim 1, selected from the group consisting of an α-arrestin ARRDC3 modulator; a Single Minded 1 (SIM1) modulator; a methionine aminopeptidase 2 (MetAP2); a sirtuin 1 (SIRT1) modulator; and combinations thereof. A pharmaceutical formulation for treating or preventing risk factors for cardiovascular disease (CVD), preventing sudden death in patients with cardiovascular disease, treating obesity-related disorders, preventing weight gain, or maintaining weight,
A mixture containing omega-3 fatty acids including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA), wherein the weight ratio of EPA: DHA is in the range of 5.7: 1 to 6.3: 1 The mixture contains about 90% by weight or more of ω3 fatty acids, EPA, DHA, and DPA constitute about 82% by weight of the contents of the mixture, and the mixture is about 25 mg / g DPA, about 30 mg / g arachidonic acid (AA) and about 30 mg / g of one or more ω3 fatty acids having 18 carbon atoms, the ω3 fatty acids having 18 carbon atoms are α- A mixture selected from the group consisting of linolenic acid (ALA), stearidonic acid (SDA), and combinations thereof; and energy consumption, glycolysis, gluconeogenesis, glycogenolysis, lipolysis, fat absorption, fat storage, fat excretion Hunger, satiety, desire mechanism, appetite, food intake, gastrointestinal motility, and combinations thereof Is selected from the group consisting of combined comprise at least one anti-obesity agent affects at least one mechanism of action, a pharmaceutical formulation. Anti-obesity agent is an antagonist or inverse agonist for the receptor product of the cannabinoid 1 (CB1) gene; cathepsin K inhibitor; PYY; PYY _3-36 ; PYY agonist; 5HT transporter inhibitor; NE transporter inhibitor; ghrelin antagonist H3 antagonist / inverse agonist; MCH1R antagonist; MCH2R agonist / antagonist; MC3R agonist; NPY1 antagonist; NPY4 agonist; NPY5 antagonist; leptin; leptin agonist / modulator; leptin derivative; opioid antagonist; orexin antagonist; BRS3 agonist; 11βHSD-1 inhibition Agents; CCK-A agonists; CNTF; CNTF agonists / modulators; CNTF derivatives; Cox-2 inhibitors; GHS agonists; 5HT2C agonists; CB-1 antagonists; neuropeptide Y5, appetite suppressors; lipase inhibitors; 5HT6 antagonists; monoamines Reuptake inhibitor; UCP-1, 2, and 3 activators; β3 agonists; thyroid hormone β agonists; PDE inhibitors; FAS inhibitors; DGAT1 inhibitors; DGAT2 inhibitors; ACC2 inhibitors; glucocorticoid antagonists; acyl-estrogens; lipase inhibitors; fatty acid transporters Inhibitors; Dicarboxylic acid transporter inhibitors; Glucose transporter inhibitors; Serotonin reuptake inhibitors; Aminolex; Amphetoral; Amphetamine; Axoquine; Benzphetamine; Chlorphentermine; Clobenzolex; Croforex; Chrominolex; Chlortermine; cyclexamine; dextroamphetamine; difemethoxyzine, N-ethylamphetamine; fenbutrazete; phenisolex; fenproporex; fluproprex; fluminorex; furfurylmethylamphetamine; Mefenolex; Methanefepramon; Methamphetamine; Nalmefene; Norseudoephedrine; Pentrex; Fendimetrazine; Fenmetrazine; Phytofarm Compound 57; Pisirolex; Topiramate; Zonisamide; IGF-IR antagonist; MetAP2 modulator; 20. The formulation of claim 19, selected from the group consisting of an α-arrestin ARRDC3 modulator; a Single Minded 1 (SIM1) modulator; a methionine aminopeptidase 2 (MetAP2); a sirtuin 1 (SIRT1) modulator; and combinations thereof.   A condition comprising one or more risk factors for CVD with a risk of sudden death due to CVD, and a therapeutically effective amount to achieve a therapeutic effect for treating an obesity-related disorder Use of the formulation according to 1.   A condition comprising one or more risk factors for CVD with a risk of sudden death due to CVD, and a therapeutically effective amount to achieve a therapeutic effect for treating an obesity-related disorder Use of the formulation according to 19.   Use of a therapeutically effective amount of the formulation according to claim 1 to achieve indomethacin-independent sustained vasodilatory action.   20. Use of a therapeutically effective amount of the formulation of claim 19 to achieve an indomethacin-independent sustained vasodilatory effect.   2. The formulation of claim 1, wherein the at least one anti-obesity agent is an endocannabinoid receptor blocker. The endocannabinoid receptor blocker is 5- (4-chlorophenyl) -1- (2,4-dichloro-phenyl) -4-methyl-N- (piperidin-1-yl) -1H-pyrazole-3-carboxamide; 26. The formulation of claim 25, selected from the group consisting of N-[(1S, 2S) -3- (4-chlorophenyl) -2-, C ₂₅ H ₂₅ Cl ₂ N ₇ O.HCl, and combinations thereof. .   20. The formulation of claim 19, wherein the at least one antiobesity agent is an endocannabinoid receptor blocker. The endocannabinoid receptor blocker is 5- (4-chlorophenyl) -1- (2,4-dichloro-phenyl) -4-methyl-N- (piperidin-1-yl) -1H-pyrazole-3-carboxamide; 28. The formulation of claim 27, selected from the group consisting of N-[(1S, 2S) -3- (4-chlorophenyl) -2-, C ₂₅ H ₂₅ Cl ₂ N ₇ O.HCl, and combinations thereof. .   A condition comprising one or more risk factors for CVD with a risk of sudden death due to CVD, and a therapeutically effective amount to achieve a therapeutic effect for treating an obesity-related disorder Use of the formulation according to 25.   A condition comprising one or more risk factors for CVD with a risk of sudden death due to CVD, and a therapeutically effective amount to achieve a therapeutic effect for treating an obesity-related disorder Use of the formulation according to 27.

Images