Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/56—Materials from animals other than mammals
  • A61K35/612—Crustaceans, e.g. crabs, lobsters, shrimps, krill or crayfish; Barnacles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• This invention relates generally to methods of using krill oil to treat risk factors for metabolic, cardiovascular, and inflammatory disorders, including, but not limited to, modulating endocannabinoid concentrations; reducing ectopic fat; reducing triacylglycerides in the liver and heart; reducing monoacylglyceride lipase activity in the visceral adipose tissue, liver, and heart; increasing levels of DHA in the liver; increasing the levels of EPA and DHA in the phospholipid fractions of tissues that exhibit changes in endocannabinoid concentration; reducing susceptibility to inflammation, modulating glucose and lipid homeostasis; reducing fatty liver disease (alcoholic and non-alcoholic); reducing MAGL activity in the heart; increasing levels of plasma ALA/LA; decreasing levels of ALA/LA in the heart; decreasing levels of ARA in the subcutaneous adipose tissue; and decreasing availability of substrates to decrease the activity of the endocannabinoid system.
• the present invention also relates to methods of using compositions comprising krill oil to modulate biological processes selected from the group consisting of glucose metabolism, lipid biosynthesis, fatty acid metabolism, cholesterol biosynthesis, and the mitochondrial respiratory chain.
• the present invention further includes pharmaceutical and/or nutraceutical formulations made from krill oil, methods of making such formulations, and methods of administering them to treat risk factors for metabolic, cardiovascular, and inflammatory disorders.
• Krill is a small crustacean which lives in all the major oceans worldwide. For example, it can be found in the Pacific Ocean (Euphausia pacifica), in the Northern Atlantic (Meganyctiphanes norvegica) and in the Southern Ocean off the coast of Antarctica (Euphausia superba).
• Kriil is a key species in the ocean as it is the food source for many animals such as fish, birds, sharks and whales.
• Krill can be found in large quantities in the ocean and the total biomass of Antarctic krill (Euphausia superba) is estimated to be in the range of 300-500 million metric tons.
• Antarctic krill feeds on phytoplankton during the short Antarctic summer.
• This autoproteolysis is highly efficient also post mortem, making it a challenge to catch and store the krill in a way that preserves the nutritional quality of the krill. Therefore, in order to prevent the degradation of krill the enzymatic activity is either reduced by storing the krill at low temperatures or the krill is made into a krill meal.
• krill meal process the krill is cooked so that all the active enzymes are denatured in order to eliminate all enzymatic activity.
• Krill is rich in phospholipids which act as emulsifiers. Thus, it is more difficult to separate water, fat, and proteins using mechanical separation methods than it is in a regular fish meal production line.
• krill becomes solid, gains weight and loses liquid more easily when mixed with hot water. Eventually this may lead to a gradual build up of coagulated krill proteins in the cooker and a non-continuous operation due to severe clogging problems. In order to alleviate this, hot steam must be added directly into the cooker.
• Omega-3 fatty acids have been shown to have potential effect of preventing cardiovascular disease, cognitive disorders, joint disease and inflammation-related diseases such as rheumatoid arthritis and osteoarthritis. Astaxanthin is a strong antioxidant and may also assist in promoting optimal health.
• Published PCT Application No. WO 00/23546 discloses isolation of krill oil from krill using solvent extraction methods.
• Krill lipids have been extracted by placing the material in a ketone solvent (e.g., acetone) in order to extract the lipid soluble fraction.
• a ketone solvent e.g., acetone
• This method involves separating the liquid and solid contents and recovering a lipid rich fraction from the liquid fraction by evaporation. Further processing steps include extracting and recovering by evaporation the remaining soluble lipid fraction from the solid contents by using a solvent such as ethanol.
• the compositions produced by these methods are characterized by containing at least 75 ⁇ g/g astaxanthin, preferably 90 ⁇ g/g astaxanthin.
• Another krill lipid extract disclosed contained at least 250 ⁇ g/g canastaxanthin, preferably 270 ⁇ g/g canastaxanthin.
• WO 02/102394 discloses methods of treating and/or preventing cardiovascular disease, rheumatoid arthritis, skin cancer, premenstrual syndrome, diabetes, and enhancing transdermal transport.
• the methods include administering a krill or marine oil to a patient.
• the application also describes a test that was carried out to evaluate the effects of krill and/or marine oils on arteriosclerotic coronary artery disease and hyperlipidemia, and resulted in a cholesterol decrease of about 15%, a triglyceride decrease of about 15%, an HDL increase of about 8%, an LDL decrease of about 13%, and a cholesterokHDL ratio decrease of about 14%
• Korean Published Application No. 2006008155 discloses an oral composition comprising a mixture of glucosamine and krill oil (provided in a ratio of 2:3) for use in methods of inhibiting osteoarthritis.
• U.S. Patent No. 7,666,447 discloses compositions including krill extracts and conjugated linoleic acid.
• the compositions are used in methods for treating an individual having a disease state selected from the group consisting of a joint ailment, PMS, Syndrome X, cardiovascular disease, bone disease and diabetes.
• the methods comprise administering to the individual a therapeutically effective amount of a composition including conjugated linoieic acid and a krill extract comprising krill oil.
• compositions comprising krill oil to treat risk factors for metabolic, cardiovascular, and inflammatory disorders.
• the present invention provides methods of using compositions comprising krill oil (KO) to treat risk factors for metabolic, cardiovascular, and inflammatory disorders, including, but not limited to, modulating endocannabinoid concentrations; reducing ectopic fat; reducing triacylglycerides in the liver and heart; reducing monoacylglyceride lipase activity in the visceral adipose tissue, liver, and heart; increasing levels of DHA in the liver; increasing the levels of EPA and DHA in the phospholipid fractions of tissues that exhibit changes in endocannabinoid concentration; reducing susceptibility to inflammation, modulating glucose and lipid homeostasis; reducing fatty liver disease (alcoholic and non-alcoholic); reducing MAGL activity in the heart; increasing levels of plasma ALA/LA; decreasing levels of ALA/LA in the heart; decreasing levels of ARA in the subcutaneous adipose tissue; and decreasing availability of substrates to decrease the activity of the endocannabinoid system.
• the present invention also provides methods of using compositions comprising krill oil to modulate biological processes selected from the group consisting of glucose metabolism, lipid biosynthesis, fatty acid metabolism, cholesterol biosynthesis, and the mitochondrial respiratory chain.
• the present invention further includes pharmaceutical and/or nutraceutical formulations made from the compositions, methods of making such formulations, and methods of administering them to treat risk factors for metabolic, cardiovascular, and inflammatory disorders.
• the present invention provides methods of administering compositions comprising krill oil to treat risk factors for metabolic, cardiovascular, and inflammatory disorders in a human subject, where the method includes the step of administering compositions containing krill oil.
• the risk factors that are treated are selected from the group consisting of modulating endocannabinoid concentrations; reducing ectopic fat; reducing triacylglycerides in the liver and heart; reducing monoacylglyceride lipase activity in the visceral adipose tissue, liver, and heart; increasing levels of DHA in the liver; increasing the levels of EPA and DHA in the phospholipid fractions of tissues that exhibit changes in endocannabinoid concentration; reducing susceptibility to inflammation, modulating glucose and lipid homeostasis; reducing fatty liver disease (alcoholic and nonalcoholic); reducing MAGL activity in the heart; increasing levels of plasma ALA/LA; decreasing levels of ALA/LA in the heart; decreasing levels of ARA in the subcutaneous
• the present invention provides methods of administering compositions comprising krill oil to modulate biological processes in a human subject, where the method includes the step of administering compositions containing krill oil.
• the biological processes are selected from the group consisting of glucose metabolism, lipid biosynthesis, fatty acid metabolism, cholesterol biosynthesis, and the mitochondrial respiratory chain.
• Ppargda peroxisome proliferator-activated receptor gamma coactivator 1a
• Hnf4a hepatocyte nuclear factor 4 alpha
• Pck1 phosphoenolpyruvate carboxykinase 1a
• G6pc glucose-6- phosphatase, catalytic
• Cptia carnitine palmitoyl transferase 1a
• Acads acyl- coenzyme A dehydrogenase, short chain
• Acadm acyl-coenzyme A dehydrogenase, medium chain
• Acadl acyl-coenzyme A dehydrogenase, long chain
• Hmgcr 3-hydroxy-3-methylglutaryl-coenzyme A reductase
• Pmvk phosphomevalonate kinase
• Sbref2 sterol regulatory element binding
• biological processes may also be affected by enhanced or increased expression of NADH (nicotinamide adenine dinucleotide) dehydrogenase and subunits thereof.
• NADH nicotinamide adenine dinucleotide
• the biological processes are also affected by factors including reduced hepatic glucose production, reduced hepatic gluconeogenesis, and reduced hepatic lipid synthesis.
• the present invention provides methods of decreasing lipid content in the liver of a human subject, comprising: administering to said subject an effective amount of a krill oil composition under conditions such that lipid content in the liver of the subject is decreased.
• the human subject is clinically obese.
• the present invention provides methods comprising providing a krill oil composition to a human subject under conditions such that the cardiovascular disease risk factors of the subject are improved.
• the cardiovascular risk factors are selected from the group consisting of elevated blood pressure, elevated serum total cholesterol and low-density lipoprotein cholesterol (LDL-C), low serum high-density lipoprotein cholesterol (HDL- C), diabetes mellitus, abdominal obesity, elevated serum triglycerides, small LDL particles, elevated serum homocysteine, elevated serum lipoprotein(a), prothrombotic factors, fatty liver and inflammatory markers.
• the human subject is clinically obese.
• the present invention provides methods comprising providing a krill oil composition to a human subject under conditions such that cannabinoid receptor signaling is reduced.
• inhibition of the endocannabinoid system of the subject comprises lowering the levels of arachidonylethanolamide (AEA) and/or 2-arachidonyl glycerol (2-AG).
• the human subject is clinically obese.
• the present invention provides methods comprising providing a krill oil composition to a human subject; and administering the krill oil composition to the human subject under conditions such that the appetite of the subject is reduced. In some embodiments, the human subject is clinically obese. [00019] In certain embodiments, the present invention provides methods comprising providing a krill oil composition to a human subject; and administering the krill oil composition to the human subject under conditions such that fat accumulation in the subject is reduced. In some embodiments, the human subject is clinically obese.
• the present invention provides uses of a krill oil composition in a human subject for improvement of cardiovascular disease risk factors, reduction of cannabinoid receptor signaling, reduction of appetite, reduction of fatty heart or reduction of fat accumulation.
• the present invention provides uses of krill oil for the preparation of a medicament for improvement of cardiovascular disease risk factors, reduction in cannabinoid receptor signaling, reduction of appetite, or reduction of fat accumulation.
• the present invention provides methods comprising providing a krill oil composition to a human subject; and administering the krill oil composition to the human subject under conditions such that the reproductive performance is increased.
• reproductive performance is improved chance of ovulation in females.
• reproductive performance is spermatogenesis, sperm motility and/or acreosome reaction.
• the present invention provides methods comprising providing a krill oil composition to a human subject; and administering the krill oil composition to the human subject under conditions such the liver and/or kidney functions are improved.
• FIG. 1 31 P NMR analysis of polar lipids in krill oil.
• FIG. 3 Plasma glucose concentration in Zucker rats fed different forms of omega-3 fatty acids.
• FIG. 4 Plasma insulin concentration in Zucker rats fed different forms of omega-3 fatty acids.
• FIG. 5 Estimated HOMA-IR values in Zucker rats fed different forms of omega-3 fatty acids.
• FIG. 6 The effect of dietary omega-3 fatty acids on TNF- ⁇ production by peritoneal macrophages.
• FIG. 7 The effect of dietary omega-3 fatty acids on lipid accumulation in the liver.
• FIG. 8 The effect of dietary omega-3 fatty acids on lipid accumulation in the muscle.
• FIG. 9 The effect of dietary omega-3 fatty acids on lipid accumulation in the heart.
• FIG. 10 Relative concentrations of DHA in the brain in Zucker rats supplemented with omega-3 fatty acids.
• FIG. 11 Mean group body weights (g) in the collagen-induced male
• B-PL2 is the krill oil group. * p ⁇ 0.05, significantly different from
• Group A (Positive Control-Fish Oil) and Group C (Control).
• FIG. 12 Body weight for the various treatment groups.
• FIG. 13 Muscle weight for the various treatment groups.
• FIG. 14 Muscle to body weight ratio for the various treatment groups.
• FIG. 15 Serum adiponectin levels (ng/ml) for the various treatment groups.
• FIG. 16 Serum insulin levels for the various treatment groups.
• FIG. 17 Blood glucose (mmol/l) levels in the various treatment groups.
• FIG. 18 HOMA-IR values for the various treatment groups.
• FIG. 19 Liver triglyceride levels ( ⁇ mol/g) for the various treatment groups.
• FIG. 20A-B Levels of anandamide (arachidonoyl ethanolamide) and 2- arachidonoyl glycerol in visceral adipose tissue in Zucker rats.
• FIG. 21 A-B Levels of anandamide (arachidonoyl ethanolamide) and 2- arachidonoyl glycerol in subcutaneous adipose tissue in Zucker rats.
• FIG. 22A-B Levels of anandamide (arachidonoyl ethanolamide) and
• FIG. 23A-B Levels of anandamide (arachidonoyl ethanolamide) and 2- arachidonoyl glycerol in heart tissue in Zucker rats.
• FIG. 24 Triacylglyceride content in liver.
• FIG. 25 Triacylglyceride content in heart.
• FIG. 26 Cholesterol profile in plasma.
• FIG. 27 Fatty acid analyses of monocytes.
• FIG. 28 TNF alpha release in peritoneal monocytes after ex vivo challenge with LPS.
• FIG. 30A-B Visceral AEA (A) and 2-AG (B) concentrations in obese
• FIG. 31 A-D. Liver (A and B) and heart (C and D) AEA (A and C) and
• 2-AG (B and D) concentrations in rats fed control, fish oil, or krill oil diets for four weeks. Values are expressed as mean +/- SD, n 6. Means that do not have a common number differ, P ⁇ 0.05.
• FIG. 33 Treatment-induced changes in the expression of the mitochondrial reactive oxygen species detoxification enzyme Sod2. Expression was significantly decreased by a KO diet.
• FIG. 34 Genes suggesting decreased giucose uptake and increased fructose metabolism. KO diet showed a trend for increased Aldob expression
• FIG. 35 Key genes regulating hepatic glucose production
• FIG. 36 Key genes involved in fatty acid metabolism.
• FIG. 37 Key genes regulating cholesterol biosynthesis in the liver 3- hydroxy-3-methylglutaryl-Coenzyme A
• FIG. 38 Transcriptional cofactors and gene targets proposed to mediate the effect of krill-supplements on hepatic glucose metabolism and lipid biosynthesis.
• ether phospholipid as used herein preferably refers to a phospholipid having an ether bond at position 1 of the glycerol backbone.
• ether phospholipids include, but are not limited to, alkylacylphosphatidylcholine (AAPC), lyso-alkylacylphosphatidylcholine (LAAPC), and alkylacylphosphatidylethanolamine (AAPE).
• a "non-ether phospholipid” is a phospholipid that does not have an ether bond at position 1 of the glycerol backbone.
• omega-3 fatty acid refers to polyunsaturated fatty acids that have the final double bond in the hydrocarbon chain between the third and fourth carbon atoms from the methyl end of the molecule.
• Non-limiting examples of omega-3 fatty acids include, 5,8,11 ,14,17-eicosapentaenoic acid (EPA), 4,7,10, 13,16,19-docosahexaenoic acid (DHA) and 7, 10,13,16,19-docosapentaenoic acid (DPA).
• w/w weight/weight refers to the amount of a given substance in a composition on weight basis.
• a composition comprising 50% w/w phospholipids means that the mass of the phospholipids is 50% of the total mass of the composition (i.e., 50 grams of phospholipids in 100 grams of the composition, such as an oil).
• the present invention relates to methods of using krill oil and/or compositions comprising krill oil to treat risk factors for metabolic, cardiovascular, and inflammatory disorders, including, but not limited to, modulating endocannabinoid concentrations; reducing ectopic fat; reducing triacylglycerides in the liver and heart; reducing monoacylglyceride lipase activity in the visceral adipose tissue, liver, and heart; increasing levels of DHA in the liver; increasing the levels of EPA and DHA in the phospholipid fractions of tissues that exhibit changes in endocannabinoid concentration; reducing susceptibility to inflammation, modulating glucose and lipid homeostasis; reducing fatty liver disease (alcoholic and nonalcoholic); reducing MAGL activity in the heart; increasing levels of plasma ALA/LA; decreasing levels of ALA/LA in the heart; decreasing levels of ARA in the subcutaneous adipose tissue; and decreasing availability of substrates to decrease the activity of the endocannabinoid concentrations
• the present invention also relates to method of using krill oil and/or compositions comprising krill oil to modulate biological processes selected from the group consisting of glucose metabolism, lipid biosynthesis, fatty acid metabolism, cholesterol biosynthesis, and the mitochondrial respiratory chain.
• the present invention further includes pharmaceutical and/or nutraceutical formulations made from krill oil, methods of making such formulations, and methods of administering them to treat risk factors for metabolic, cardiovascular, and inflammatory disorders.
• the present invention relates to methods of using krill oil and compositions comprising krill oil to treat one or more risk factors for metabolic, cardiovascular, and inflammatory disorders.
• risk factors associated with metabolic, cardiovascular, inflammatory, and other disorders
• krill oil significantly modulates a substantial number of risk factors associated with metabolic, cardiovascular, inflammatory and other disorders.
• disorders may include, but are not limited to, obesity, type Il diabetes, type I diabetes, gestational diabetes, metabolic syndrome, dyslipidemia, hypercholesterolemia, hypertension, coronary artery disease, atherosclerosis, stroke, rheumatoid arthritis, and osteoarthritis.
• the level(s) of the risk factor(s) to be treated may be assessed in one or more body fluids of interest, including, but not limited to, blood, plasma, urine, sweat, tears, and cerebrospinal fluid.
• the Ievel(s) of the risk factor(s) may also be assessed in one or more organs of interest, including, but not limited to, the brain, heart, liver, blood vessels, visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), lungs, intestines, blood vessels, lymph nodes, kidneys, and pancreas.
• Specific risk factors that may be modulated by the krill oil-based compositions in the methods of the present invention include endocannabinoid concentrations (particularly AEA (N-arachidonoylethanolamine (anandamide)) and 2- AG (2-arachidonoylglycerol) in the liver, heart, and VAT, although the present invention is not limited to these endocannabinoids); ectopic fat; triacylglycerides in the liver and heart; monoacylglyceride lipase activity in the VAT, liver, and heart; susceptibility to inflammation, glucose and lipid homeostasis; fatty liver disease (alcoholic and non-alcoholic); MAGL (monoacylglycerol lipase) activity in the heart; levels of ALA/LA (alpha-linolenic acid/linoleic acid) in the heart; levels of ARA (arachidonic acid) in the SAT; and availability of substrates to decrease the activity of the endocannabinoid system
• the levels or concentrations of these risk factors are decreased in a subject suffering from or at risk for a metabolic, cardiovascular, or inflammatory disorder by administering a krill oil composition.
• the levels or concentrations of these risk factors are decreased in a patient population, by administering a krill oil composition to a patient population including individuals suffering from or at risk for a metabolic, cardiovascular, or inflammatory disorder.
• a krill oil composition may be administered to a subject or patient population in accordance with methods for reducing levels of one or more of these risk factors relative to the level of expression or activity seen in an individual or population not suffering from a metabolic, cardiovascular, inflammatory disorder.
• risk factors that modulated by the krill-based compositions in the methods of the present invention include levels of DHA (docosahexaenoic acid) in the liver; levels of EPA (eicosapentaenoic acid) and DHA in the phospholipid fractions of tissues that exhibit changes in endocannabinoid concentration; and levels of plasma ALA/LA.
• DHA docosahexaenoic acid
• EPA eicosapentaenoic acid
• DHA in the phospholipid fractions of tissues that exhibit changes in endocannabinoid concentration
• levels of plasma ALA/LA levels or concentrations of these risk factors are increased in a subject suffering from or at risk for a metabolic, cardiovascular, or inflammatory disorder by administering a krill oil composition.
• the levels or concentrations of these risk factors are increased in a patient population, by administering a krill oil composition to a patient population including individuals suffering from or at risk for a metabolic, cardiovascular, or inflammatory disorder.
• a krill oil composition may be administered to a subject or patient population in accordance with methods for increasing levels of one or more of these risk factors relative to the level of expression or activity seen in an individual or population not suffering from a metabolic, cardiovascular, inflammatory disorder.
• Biological processes that may be modulated by krill oil and compositions containing krill oil in the methods of the present invention include glucose metabolism, gluconeogenesis, lipid biosynthesis, fatty acid metabolism, cholesterol biosynthesis, and the mitochondrial respiratory chain.
• Ppargda peroxisome proliferator-activated receptor gamma coactivator 1 a
• Hnf4a hepatocyte nuclear factor 4 alpha
• Pck1 phosphoenolpyruvate carboxykinase 1 a
• G6pc glucose-6-phosphatase, catalytic
• Cptia Carnitine palmitoyl transferase 1 a
• Acads acyl-coenzyme A dehydrogenase, short chain
• Acadm acyl-coenzyme A dehydrogenase, medium chain
• Acadl acyl- coenzyme A dehydrogenase, long chain
• Hmgcr 3-hydroxy-3-methylglutaryl- coenzyme A reductase
• Pmvk phosphomevalonate kinase
• Sbref2 sterol regulatory element binding factor 2
• biological processes may also be affected by enhanced or increased expression of NADH (nicotinamide adenine dinucleotide) dehydrogenase and subunits thereof.
• NADH nicotinamide adenine dinucleotide
• the biological processes are also affected by factors including reduced hepatic glucose production, reduced hepatic gluconeogenesis, and reduced hepatic lipid synthesis.
• risk factors may be modulated by increasing or decreasing (as appropriate) the expression of a gene, activity of an enzyme, etc., relative to the level of expression or activity seen in an individual or population not suffering from a metabolic, cardiovascular, inflammatory, or other disorder.
• the various genes, enzymes, and other risk factors may be modulated by increasing or decreasing (as appropriate) the expression of a gene, activity of an enzyme, etc., relative to the level of expression or activity seen in an individual or population suffering from a metabolic, cardiovascular, inflammatory, or other disorder to be treated or prevented.
• the risk factors modulated in accordance with the methods of preventing or treating a metabolic, cardiovascular, inflammatory, or other disorder may be modulated to a degree that results in improvement in the symptoms of the disorder, elimination of the disorder, or reduction in risk for developing the disorder.
• it may be useful to establish a baseline level for the risk factor in a subject or patient population being treated by determining the amount of the risk factor present in a body fluid or organ of interest.
• Such a baseline could be determined by assessing the amount of one or more risk factors present in a body fluid or tissue sample taken from a subject or patient population, prior to any treatment with krill oil.
• the krill oil or krill oil composition is then administered in an amount that is sufficient to result in an increase/decrease (as appropriate) in a level of a risk factor observed in a subject or patient population being treated by the methods of the invention.
• the increase/decrease is at least 5% relative to the baseline level.
• the level of the risk factor is increased/decreased by at least 10%, at least 20%, at least 35%, at least 50%, at least 65%, at least 80%, at least 90%, or at least 95%, relative to the baseline level.
• the level of the risk factor may be increased/decreased by 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more as compared to the baseline level by the methods of the present invention.
• the present invention can be used in methods of decreasing cardiovascular disease risk factors of a subject.
• the cardiovascular risk factors are selected from the group consisting of elevated blood pressure, elevated serum total cholesterol and low-density lipoprotein cholesterol (LDL-C), low serum high-density lipoprotein cholesterol (HDL- C), diabetes mellitus, abdominal obesity, elevated serum triglycerides, small LDL particles, elevated serum homocysteine, elevated serum lipoprotein(a), prothrombotic factors, fatty liver and inflammatory markers.
• the subject is a human, and in other embodiments, the subject is clinically obese.
• the krill oil composition of the present invention find use in the treatment of fatty heart disease, alcoholic fatty liver disease, and nonalcoholic fatty liver disease.
• the krill oil compositions are useful for decreasing the lipid content of the heart and/or liver of a subject.
• the present invention provides methods of providing a krill oil composition to a subject; and administering the krill oil composition to the subject under conditions such the liver and/or kidney functions are improved.
• the subject is a human, and in other embodiments, the subject is clinically obese.
• a diet and exercise regime may fail to result in a consistent decrease in weight over the long term, the effect of omega-3 fatty acids alleviating the inflammatory condition in the adipose tissue may persist, generating a condition that can be described as "healthy adipose tissue".
• Reduction in adipose tissue inflammation may be achieved by increasing circulating levels of adiponectin.
• Adiponectin is an adipose tissue derived anti-inflammatory hormone.
• This aspect of the invention therefore relates to the discovery that krill oil is highly effective in alleviating negative health effects caused by obesity, such as reducing LDL cholesterol, reducing ectopic fat deposition and reducing susceptibility to inflammation. These negative health effects may lead to increased cardiovascular disease risk.
• another embodiment of the invention is to use krill oil in overweight and obese subjects for alleviating diet-induced adipose tissue dysfunction and diet induced changes in lipid metabolism.
• the present invention provides methods comprising providing a krill oil composition to a subject; and administering the krill oil composition to the subject under conditions such that the appetite of the subject is reduced.
• the subject is a human, and in other embodiments, the subject is clinically obese.
• the present invention provides methods comprising providing a krill oil composition to a subject; and administering the krill oil composition to the subject under conditions such that fat accumulation in the subject is reduced.
• the subject is a human, and in other embodiments, the subject is clinically obese.
• the krill oil compositions of the present invention find use in increasing or inducing diuresis. In some embodiments, the krill oil compositions of the present invention find use in decreasing protein catabolism and increasing the muscle mass of a subject.
• Type 2 diabetes is a metabolic disorder characterized by impaired glycemic control (high blood glucose levels).
• tissue-wide insulin resistance contributes to the development of the disease.
• Strategies for reducing insulin resistance or improving tissue sensitivity to insulin are recognized as beneficial in preventing type 2 diabetes.
• krill oil is effective in reducing risk factors of type 2 diabetes such as hyperinsulinemia and insulin resistance.
• the methods of the present invention may be used to treat and/or prevent type Il diabetes and metabolic syndrome in a subject, or reduce the incidence of type Il diabetes and/or metabolic syndrome in a patient population comprising individuals at risk for developing diabetes and/ metabolic syndrome.
• Another embodiment of the invention provides a krill oil composition effective for improving the blood lipid profile by increasing the HDL cholesterol levels, decreasing the LDL cholesterol and triglyceride levels.
• the novel krill oil composition is effective for treating metabolic syndrome, which is defined as the coexistence of 3 or more components selected from the group: abdominal obesity, high serum triglyceride levels, low HDL levels, elevated blood pressure and high fasting plasma glucose levels.
• krill oil compositions are provided that are effective and safe for the treatment of type Il diabetes and metabolic syndrome in humans.
• the present invention provides methods comprising providing a krill oil composition to a subject under conditions such that cannabinoid receptor signaling is reduced.
• inhibition of the endocannabinoid system of the subject comprises lowering the levels of arachidonylethanolamide (AEA) and/or 2-arachidonyl glycerol (2-AG).
• the subject is a human, and in other embodiments, the subject is clinically obese.
• the endocannabinoid system consists of cannabinoid (CB) receptors, endocannabinoids (EC) and enzymes involved in the synthesis and degradation of these molecules.
• Cannabionoid-1 (CB-1) receptors are located in the central nervous system such as the brain (basal ganglia, limbic system, cerebellum and hippocampus) and the reproductive system (both male and female), but also peripherally in liver, muscle, and different adipose tissues.
• Cannabionoid-2 (CB-2) receptors are located on immune cells and in the spleen.
• a dysregulated endocannabinoid system results in excessive eating and fat accumulation and is therefore likely to play an important role in the pathogenesis of obesity. This chronic activation may not only be caused by obesity, but also by high fat diets, which can predispose the body to enhanced endocannabinoid biosynthesis.
• the present invention discloses that krill oil can be used to effectively modulate the endocannabinoid system in Zucker fatty rats. It is shown that krill oil is more effective than fish oil and the control diet in reducing the level of endocannabinoids AEA and 2-AG in visceral adipose in this model. Visceral fat is the metabolically more active fat and accumulation of visceral fat has been associated with insulin resistance, glucose intolerance, dyslipidemia, hypertension and coronary heart disease. Accumulation of visceral fat is initiated where the capacity for storing subcutaneous fat is saturated.
• the Zucker rats are leptin receptor-deficient animals, and therefore they became obese due to the increased feed intake which gradually results in the development of metabolic syndrome (the rats develop hyperglycemia, ectopic fat deposition, and elevated LDL cholesterol levels).
• the data show that the reduction in 2-AG levels in subcutaneous adipose is the most pronounced while the level of AEA in liver and heart were also clearly reduced after intake of krill oil compared to all the other treatments.
• Subcutaneous fat is less metabolic active than visceral adipose tissue. Functional effects of a dysregulated endocannabinoid system were observed in Example 12, as the rats developed fatty heart, fatty liver, hyperglycemia and elevated LDL cholesterol levels.
• Krill oil is an effective agent for modulating the endocannabinoid system, and thereby alleviating the negative health effects of obesity.
• the invention also relates to the discovery that krill oil is effective in reducing the level of the endocannabinoid precursors, i.e., the arachidonic acid content in phospholipids in the heart, subcutaneous adipose tissue, and visceral adipose tissue.
• the TAG fraction of the visceral and subcutaneous adipose tissue was influenced by omega-3 supplementation, showing an increased incorporation of EPA (30 fold), DHA (10 fold) and DPA (10 fold).
• the large increase in TAG is less metabolically important than the small increase in the phospholipids.
• the AEA and 2-AG concentration in visceral and adipose tissue mirrors the fatty acid profiles.
• Liver TAG omega-3 were significantly increased in fish oil and krill oil groups whereas no changes were found in arachidonic acid or other omega-6 fatty acids.
• Heart TAGs fatty acids showed increased levels of EPA, DPA and DHA and decrease in ARA in the phospholipid fraction.
• krill oil is effective in changing endocannabinoid receptor signaling by modulating the level of the cannabinoid receptor ligands.
• the present invention also relates to modulation of the endocannabinoid system in tissues such as kidney, testis, different brain areas, intestines, pancreas, thyroids glands, etc
• a preferred embodiment of this invention is the use of k ⁇ ll oil for modulation of a dysfunctional endocannabinoid system in all tissues in order to obtain improved health
• Non-limiting examples of such health effects are treatment of obesity, reduction in feed intake, increased energy expenditure, reduction in cholesterol, improvement in male reproduction (spermatogenesis, sperm motility and acreosome reaction) and female reproduction (increased ovulation), increased sexual drive (libido), treatment of atherosclerosis, improvement in bone metabolism, improvement in lipid metabolism, treatment of ectopic fat deposition, treatment of liver disease such as fibrosis and cirrhosis, control of glucose homeostasis, improvement in insulin resistance, treatment of fatty heart and cardiomyopathy
• the various methods of the present invention may be carried out using k ⁇ ll oil, or compositions comprising krill oil
• the krill oil is characterized by containing high levels of astaxanthin, phospholipids, included an enriched quantities of ether phospholipids, and omega-3 fatty acids
• KnII oil is obtained from Antarctic krill (Euphausia Superba) by extracting the lipids with supercritical and/or liquid solvents
• Krill oil is different from fish oil at least in the respect that it contains astaxanthin, and the majority of the omega-3 fatty acids are attached to phospholipids
• compositions for use in accordance with the invention may include, but are not limited to, SuperbaTM krill oil (Aker Bioma ⁇ ne, Norway)
• compositions for use in accordance with the invention comprise krill oil that is obtained from krill meal by ethanol extraction and/or CO 2 extraction
• any suitable methods for extracting oil from krill may be used in accordance with the present invention
• the krill oil-containing compositions that are preferably used in order to carry out the methods of the present invention are distinguished from previously- described krill oil products, such as those described in U S Pat No 6,800,299 or WO 03/01 1873 and Neptune brand krill oil (NKO®, Neptune Technologies & Bioressources, Laval, Quebec, Canada), by having substantially higher levels of non-
• the krill compositions that may be used in accordance with the present invention are preferably derived from Euphausia superba. Regardless of the krill used, the compositions preferably comprise from about 40% to about 60% w/w phospholipids, preferably from about 45% to 55% w/w phospholipids and from about 300 mg/kg astaxanthin to about 2500 mg/kg astaxanthin, preferably from about 1000 to about 2200 mg/kg astaxanthin, more preferably from about 1500 to about 2200 mg/kg astaxanthin. In some preferred embodiments, the compositions comprise greater than about 1000, 1500, 1800, 1900, 2000, or 2100 mg/kg astaxanthin.
• the krill compositions of the present invention comprise from about 1%, 2%, 3% or 4% to about 8%, 10%, 12% or 15% w/w ether phospholipids or greater than about 4%, 5%, 6%, 7%, 8%, 9% or 10% ether phospholipids.
• the ether phospholipids are preferably alkylacylphosphatidylcholine, lyso-alkylacylphosphatidylcholine, alkylacylphosphatidyl-ethanolamine or combinations thereof.
• the krill compositions comprise from about 1%, 2%, 3% or 4% to about 8%, 10%, 12% or 15% w/w ether phospholipids and from about 30%, 33%, 40%, 42%, 45%, 48%, 50%, 52%, 54%, 55% 56%, 58% to about 60% non-ether phospholipids so that the total amount of phospholipids (both ether and non-ether phospholipids) ranges from about 40% to about 60%.
• the range of 40% to 60% total phospholipids, as well as the other ranges of ether and non-ether phospholipids can include other values not specifically listed within the range.
• the compositions comprise from about 20% to 45% w/w triglycerides; and from about 400 to about 2500 mg/kg astaxanthin.
• the compositions comprise from about 20% to 35%, preferably from about 25% to 35%, omega-3 fatty acids as a percentage of total fatty acids in the composition, wherein from about 70% to 95%, or preferably from about 80% to 90% of the omega-3 fatty acids are attached to the phospholipids.
• the krill oil extracted for use in the methods of the present invention contains few enzymatic breakdown products. Examples of the krill oil compositions of the present invention are provided in Tables 4-19.
• the present invention provides a polar krill oil comprising at least 65% (w/w) of phospholipids, wherein the phospholipids are characterized in containing at least 35% omega-3 fatty acid residues.
• the present invention is not limited to the presence of any particular omega-3 fatty acid residues in the krill oil composition.
• the krill oil comprises EPA and DHA residues.
• the krill oil compositions comprise less than about 5%, 4%, 3% or preferably 2% free fatty acids on a weight/weight (w/w) basis.
• the krill oil compositions comprise less than about 25%, 20%, 15%, 10% or 5% triglycerides (w/w).
• the krill oil compositions comprise greater than about 30%, 40%, 45%, 50%, 55%, 60%, or 65% phosphatidyl choline (w/w). In some embodiments, the krill oil compositions comprise greater than about 100, 200, 300, 400, or 500 mg/kg astaxanthin esters and up to about 700 mg/kg astaxanthin esters. In some embodiments, the present invention provides kriii oil compositions comprising at least 500, 1000, 1500, 2000, 2100, or 2200 mg/kg astaxanthin esters and at least 36% (w/w) omega-3 fatty acids.
• the krill oil compositions of the present invention comprise less than about 1.0 g/100 g, 0.5 g/100 g, 0.2 g/100 g or 0.1 g/100 g total cholesterol. In some embodiments, the krill oil compositions of the present invention comprise less than about 0.45.
• the present invention is carried out using a neutral krill oil extract comprising greater than about 70%, 75% 80%, 85% or 90% triglycerides.
• the krill oil compositions comprise from about 50 to about 2500 mg/kg astaxanthin esters.
• the krill oil compositions comprise from about 50, 100, 200, or 500 to about 750, 1000, 1500 or 2500 mg/kg astaxanthin esters.
• the compositions comprise from about 1% to about 30% omega-3 fatty acid residues, and preferably from about 5%-15% omega-3 fatty acid residues.
• the krill oil compositions comprise less than about 20%, 15%, 10% or 5% phospholipids.
• the present invention is carried out using krill oil containing less than about 70, 60, 50, 40, 30, 20, 10, 5 or 1 micrograms/kilogram (w/w) astaxanthin esters.
• the krill oil is clear or only has a pale red color.
• the low-astaxanthin krill oil is obtained by first extracting a krill material, such as krill oil, by supercritical fluid extraction with neat carbon dioxide. It is contemplated that this step removes astaxanthin from the krill material.
• the krill material is then subjected to supercritical fluid extraction with carbon dioxide and a polar entrainer such as ethanol, preferably about 20% ethanol.
• the oil extracted during this step is characterized in containing low amounts of astaxanthin.
• krill oil comprising astaxanthin is extracted by countercurrent supercritical fluid extraction with neat carbon dioxide to provide a low-astaxanthin krill oil.
• the present invention is carried out using krill oil that is substantially odorless.
• substantially odorless it is meant that the krill oil lacks an appreciable odor as determined by a test panel.
• the substantially odorless krill oil comprises less than about 10, 5 or 1 milligrams/kilogram trimethylamine.
• the odorless krill oil is produced by first subjecting krill material to supercritical fluid extraction with neat carbon dioxide to remove odor causing compounds such as trimethylamine, followed by extraction with carbon dioxide with a polar entrainer such as ethanol.
• the present invention provides encapsulated Euphausia superba krill oil compositions.
• the present invention provides a method of making a Euphausia superba krill oil composition comprising contacting Euphausia superba with a polar solvent to provide an polar extract comprising phospholipids, contacting Euphausia superba with a neutral solvent to provide a neutral extract comprising triglycerides and astaxanthin, and combining said polar extract and said neutral extract to provide the Euphausia superba krill oils described above.
• fractions from polar and non-polar extractions are combined to provide a final product comprising the desired ether phospholipids, non-ether phospholipids, omega-3 moieties and astaxanthin.
• the present invention provides methods of making a Euphausia superba (or other krill species) krill oil comprising contacting a Euphausia superba preparation such as Euphausia superba krill meal under supercritical conditions with CO 2 containing a low amount of a polar solvent such as ethanol to extract neutral lipids and astaxanthin; contacting meal remaining from the first extraction step under supercritical conditions with CO 2 containing a high amount of a polar solvent such as ethanol to extract a polar lipid fraction containing ether and non-ether phospholipids; and then blending the neutral and polar lipid extracts to provide the compositions described above.
• the compositions of this invention are contained in acceptable excipients and/or carriers for oral consumption.
• the actual form of the carrier, and thus, the composition itself, is not critical.
• the carrier may be a liquid, gel, gelcap, capsule, powder, solid tablet (coated or non-coated), tea, or the like.
• the composition is preferably in the form of a tablet or capsule and most preferably in the form of a soft gel capsule.
• Suitable excipient and/or carriers include maltodextrin, calcium carbonate, dicalcium phosphate, tricalcium phosphate, microcrystalline cellulose, dextrose, rice flour, magnesium stearate, stearic acid, croscarmellose sodium, sodium starch glycolate, crospovidone, sucrose, vegetable gums, lactose, methylcellulose, povidone, carboxymethylcellulose, corn starch, and the like (including mixtures thereof).
• Preferred carriers include calcium carbonate, magnesium stearate, maltodextrin, and mixtures thereof.
• the various ingredients and the excipient and/or carrier are mixed and formed into the desired form using conventional techniques.
• the tablet or capsule of the present invention may be coated with an enteric coating that dissolves at a pH of about 6.0 to 7.0.
• a suitable enteric coating that dissolves in the small intestine but not in the stomach is cellulose acetate phthalate. Further details on techniques for formulation for and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
• the composition may comprise one or more inert ingredients, especially if it is desirable to limit the number of calories added to the diet by the composition.
• the dietary supplement of the present invention may also contain optional ingredients including, for example, herbs, vitamins, minerals, enhancers, colorants, sweeteners, flavorants, inert ingredients, and the like.
• composition of the present invention may contain one or more of the following: ascorbates (ascorbic acid, mineral ascorbate salts, rose hips, acerola, and the like), dehydroepiandosterone (DHEA), Fo-Ti or Ho Shu Wu (herb common to traditional Asian treatments), Cat's Claw ( ancient herbal ingredient), green tea (polyphenols), inositol, kelp, dulse, bioflavinoids, maltodextrin, nettles, niacin, niacinamide, rosemary, selenium, silica (silicon dioxide, silica gel, horsetail, shavegrass, and the like), spirulina, zinc, and the like.
• ascorbates ascorbic acid, mineral ascorbate salts, rose hips, acerola, and the like
• DHEA dehydroepiandosterone
• Fo-Ti or Ho Shu Wu hereb common to traditional Asian treatments
• Cat's Claw
• the composition may further comprise vitamins and minerals including, but not limited to, calcium phosphate or acetate, tribasic; potassium phosphate, dibasic; magnesium sulfate or oxide; salt (sodium chloride); potassium chloride or acetate; ascorbic acid; ferric orthophosphate; niacinamide; zinc sulfate or oxide; calcium pantothenate; copper gluconate; riboflavin; beta- carotene; pyridoxine; folic acid; thiamine; biotin; chromium chloride or picolonate; potassium iodide; sodium selenate; sodium molybdate; phylloquinone; retinoic acid; cyanocobalamin; sodium selenite; copper sulfate; vitamin A; vitamin C; inositol; potassium iodide; vitamin E, vitamin K; niacin; and pantothenic acid.
• vitamins and minerals including, but not limited to, calcium phosphate
• the particles comprise an amino acid supplement formula in which at least one amino acid is included (e.g., 1- camitine or tryptophan).
• the composition comprises at least one food flavoring such as acetaldehyde (ethanal), acetoin (acetyl methylcarbinol), anethole (parapropenyl anisole), benzaldehyde (benzoic aldehyde), N butyric acid (butanoic acid), d or I carvone (carvol), cinnamaldehyde (cinnamic aldehyde), citral (3,7- dimethyl-2,6 ⁇ octadienal, geranial, neral), decanal (N-decylaldehyde, capraldehyde, capric aldehyde, caprinaldehyde, aldehyde C 10), ethyl acetate, ethyl butyrate, 3- methyl-3-phenyl glycidic acid ethyl ester (ethyl methyl phenyl glycidate, strawberry
• compositions comprise at least one synthetic or natural food coloring (e.g., annatto extract, astaxanthin, beet powder, ultramarine blue, canthaxanthin, caramel, carotenal, beta carotene, carmine, toasted cottonseed flour, ferrous gluconate, ferrous lactate, grape color extract, grape skin extract, iron oxide, fruit juice, vegetable juice, dried algae meal, tagetes meal, carrot oil, corn endosperm oil, paprika, paprika oleoresin, riboflavin, saffron, tumeric, and oleoresin).
• synthetic or natural food coloring e.g., annatto extract, astaxanthin, beet powder, ultramarine blue, canthaxanthin, caramel, carotenal, beta carotene, carmine, toasted cottonseed flour, ferrous gluconate, ferrous lactate, grape color extract, grape skin extract, iron oxide, fruit juice, vegetable juice, dried algae meal, tage
• compositions comprise at least one phytonutrient (e.g., soy isoflavonoids, oligomeric proanthcyanidins, indol-3-carbinol, sulforaphone, fibrous ligands, plant phytosterols, ferulic acid, anthocyanocides, triterpenes, omega 3/6 fatty acids, conjugated fatty acids such as conjugated linoleic acid and conjugated linolenic acid, polyacetylene, quinones, terpenes, cathechins, gallates, and quercitin).
• phytonutrient e.g., soy isoflavonoids, oligomeric proanthcyanidins, indol-3-carbinol, sulforaphone, fibrous ligands, plant phytosterols, ferulic acid, anthocyanocides, triterpenes, omega 3/6 fatty acids, conjugated fatty acids such as conjugated linole
• Sources of plant phytonutrients include, but are not limited to, soy lecithin, soy isoflavones, brown rice germ, royal jelly, bee propolis, acerola berry juice powder, Japanese green tea, grape seed extract, grape skin extract, carrot juice, bilberry, flaxseed meal, bee pollen, ginkgo biloba, primrose (evening primrose oil), red clover, burdock root, dandelion, parsley, rose hips, milk thistle, ginger, Siberian ginseng, rosemary, curcumin, garlic, lycopene, grapefruit seed extract, spinach, and broccoli.
• soy lecithin soy isoflavones
• brown rice germ royal jelly
• bee propolis acerola berry juice powder
• Japanese green tea grape seed extract
• grape skin extract grape juice
• carrot juice bilberry
• flaxseed meal bee pollen
• bee pollen ginkgo biloba
• primrose evening primrose oil
• Example 1 The krill meal obtained in Example 1 was then ethanol extracted according to the method disclosed in JP 02-215351 , the contents of which are incorporated herein by reference. The results showed that around 22% fat from the meal could be extracted.
• Table 1 shows the fatty acid composition of the krill meal and the krill oil extracted from the meal using ethanol.
• Table 2 shows the composition and properties of the krill meal and products before and after extraction, whereas Table 3 shows the lipid composition.
• the krill meal obtained in Example 1 was then subjected to a supercritical fluid extraction method in two stages. During stage 1 , 12.1% fat (neutral krill oil) was removed using neat CO 2 only at 300 bars, 60° C and for 30 minutes. In stage 2, the pressure was increased to 400 bar and 20% ethanol was added (v/v) for 90 minutes. This resulted in further extraction of 9% polar fat which hereafter is called polar krill oil.
• the total fatty acid composition of the polar krill oil, the neutral krill oil and a commercial product obtained from Neptune Biotech (Laval, Quebec, Canada) are listed in Table 4.
• fatty acid composition for the phospholipids (Table 5), the neutral lipids (Table 6), the free fatty acids, diglycerides (Table 7), triglycerides, lyso-phosphatidylcholine (LPC) (Table 8), phosphatidylcholine (PC), phosphatidylethanolamine (PE) (Table 9), phosphatidylinositol (Pl) and phosphatidylserine (Table 10) are shown.
• Table 11 shows the level of astaxanthin and cholesterol for the different fractions.
• Neutral lipids were extracted from krill meal (138 kg) using SFE with neat CO 2 (solvent ratio 25 kg/kg) at 500 bar and 75°C.
• the neutral lipids were fractionated at 200 bar (75°C) and at 60 bar (35°C) at two separators.
• the extract obtained at the first separator (S 1 - 19.6 kg) was characterized, and the results can be found in Table 12.
• the extract obtained at the second separator (S2 - 0.4 kg) was rich in water, and was not further used.
• the polar lipids were extracted using CO 2 at 500 bar, 20% ethanol and at a temperature of 75°C.
• Example 17 The asta oil obtained in Example 1 was blended with the polar lipids obtained in Example 4 in a ratio of 46:54 (v/v). Next the ethanol was removed by evaporation and a dark red and transparent product was obtained. The product was analyzed and the results can be found in Table 15. Furthermore, the product was encapsulated into soft gels successfully. During the encapsulation it was observed that any further increase in phospholipids, and thereby viscosity, will make it very difficult to encapsulate the final product.
• Krill lipids were extracted from krill meal (a food grade powder) using supercritical fluid extraction with co-solvent. Initially, 300 bar pressure, 333 0 K and 5% ethanol (ethanol: CO 2 , w/w) were utilized for 60 minutes in order to remove neutral lipids and astaxanthin from the krill meal. Next, the ethanol content was increased to 23% and the extraction was maintained for 3 hours and 40 minutes. The extract was then evaporated using a falling film evaporator and the resulting krill oil was finally filtered. The product obtained was then analyzed and the results can be found in Table 16.
• Krill oil was prepared according to the method described in Example 6 by extracting from the same krill meal. The oil was subjected to 31 P NMR analysis for the identification and quantification of the various forms of phospholipids. The analysis was performed according to the following methods: Samples (20-40 mg) were weighed into 1.5 ml centrifuge tubes. Next, NMR detergent (750 ⁇ l-10% Na cholate, 1% EDTA, pH 7.0 in H 2 O+D 2 O, 0.3 g L- 1 PMG internal standard) was added. Next, the tube was placed in an oven at 6O°C and periodically shaken/sonicated until completely dispersed. The solution was then transferred to a 5 ml NMR tube for analysis.
• NMR detergent 750 ⁇ l-10% Na cholate, 1% EDTA, pH 7.0 in H 2 O+D 2 O, 0.3 g L- 1 PMG internal standard
• Phosphorus NMR spectra were recorded on the two- channel Bruker Avance300 with the following instrument settings: spectrometer frequency 121.498 MHz, sweep width 24,271 Hz, 64,000 data points, 30 degree excitation pulse, 576 transients were normally taken, each with an 8 second delay time and f.i.d. acquisition time of 1.35 sec. Spectra were processed with a standard exponential weighting function with 0.2 Hz line broadening before Fourier transformation.
• Peaks were identified using known chemical shifts. Deacylation of samples with monomethylamine was also used on two samples for confirmation of peak identity and to achieve better peak resolution.
• the main polar ether lipids of the krill meal are alkylacylphosphatidylcholine (AAPC) at 7-9% of total polar lipids, lyso-alkylacylphosphatidylcholine (LAAPC) at 1% of total polar lipids (TPL) and alkylacylphosphatidyl-ethanolamine (AAPE) at ⁇ 1% of TPL.
• AAPC alkylacylphosphatidylcholine
• LAAPC lyso-alkylacylphosphatidylcholine
• TPL total polar lipids
• AAPE alkylacylphosphatidyl-ethanolamine
• the purpose of this experiment was to investigate the effect of different omega-3 fatty acid sources on metabolic parameters in the Zucker rat.
• the Zucker rat is a widely used model of obesity and insulin resistance. Obesity is due to a mutation in the leptin receptor which impairs the regulation of intake.
• Omega-3 sources compared in this study were fish oil (FO) and two types of krill oil.
• the krill oil was either from a commercial supplier (Neptune® krill oil (NKO)) or prepared according to Example 6 (SuperbaTM).
• the effect induced by the novel krill oil is often more pronounced than the effect of FO an in several cases greater than the effect induced by NKO.
• the effects of the two types of krill oil differentiated with respect to the reduction of blood LDL cholesterol levels as well as lipid accumulation in the liver and muscle (FIGS. 2-9).
• the efficacy of transfer of DHA from the diet to the brain tissue was greatest with the kriil oil prepared as in Example 6 (FIG. 10).
• Treatment 4 also contained 0.36% omega-3 fatty acids obtained from regular 18-12 fish oil.
• the diets were fed to the mice for 7 weeks with free access to drinking water.
• Data represented in this example means +/- SE. Columns not sharing a common letter are significantly different (P ⁇ 0.05) by ANOVA followed by Tukey's multiple comparison test.
• the data are presented in FIGS. 12-19.
• Omega-3 fatty acid sources were fish oil (FO) and krill oil (KO).
• the KO was prepared by extracting the triacylglycerides and the phospholipids from the krill meal using supercritical CO 2 with ethanol so that the final oil consisted of at 50% phospholipids, 30% omega-3 fatty acids and around 1300 ppm astaxanthin.
• the Zucker rats were 4 wk old at the start of the study with average initial weight of 250 g. At this stage the Zucker rats can be non-insulin resistant. Rats were fed the test diets for 4 wk after which they were sacrificed and blood and tissue samples were collected. Table 20 shows the fatty acid composition of the triacylglycerides and the phospholipids for visceral adipose tissue, subcutaneous adipose tissue, liver and heart.
• FIGS 24 and 25 show the TAG deposition in the liver and heart, respectively.
• krill oil is the most effective in reducing ectopic fat deposition.
• FIG. 26 shows the cholesterol profile in rat plasma, and again krill oil is the most effective treatment.
• FIG 27 shows the fatty acid profile of the monocytes.
• krill oil is most effective in reducing the level of arachidonic acid and thereby reducing the inflammatory potential of the monocytes.
• FIG. 28 shows the level of TNF-alpha after lipopolysaccharide (LPS) challenge, and both krill and fish oils show a reduced level of TNF-alpha release compared to the control.
• LPS lipopolysaccharide
• the 3 diets were prepared by Altromin GmbH & Co. KG and stored in vacuum bags to reduce (n-3) LCPUFA oxidation.
• Rats were food-deprived overnight and macrophages were isolated from their peritoneal cavity.
• the rats were deeply anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally, Sigma-Aldrich) before being killed.
• Cells were obtained by peritoneal lavage with 60 ml_ of cold PBS containing 5 mmol/L EDTA
• the rats were subjected to a vigorous massage of the peritoneal area prior to collection of cells Immediately after death, blood was drawn from aorta, and liver, brain, heart, subcutaneous adipose tissues (SAT), and visceral adipose tissues (VAT) were removed and stored at 280°C.
• SAT subcutaneous adipose tissues
• VAT visceral adipose tissues
• Cells were centrifuged at 300 x g; 10 mm and the cell pellet was washed twice with cold sterile PBS and suspended in DMEM, 10% heat-inactivated fetal calf serum, penicillin (100 kU/L), and streptomycin (100 mg/L). The cell number was determined with a Coulter Counter corrected for viability determined by tryptan blue dye exclusion The cells were then seeded at the density of 4.0 x 10 5 cells cm 2 and incubated for 2 h at 37°C and 5% CO 2 atm.
• macrophages were cultured in DMEM with 10% fetal calf serum in the presence of lipopolysaccharide (LPS) from Escherichia coli 026. B6 (Sigma Aldrich) (100 mg/L) for 24 h. The incubation time was chosen based on preliminary experiments that showed no substantial difference in cytokine secretion between 24 and 48 h. At the time indicated, supernatants and cells were separated and stored at 28O°C until ELISA and fatty acid analysis were performed. Sandwich ELISA tests were carried out all at the same time to avoid variations during the assay conditions and performed as described by the manufacturer.
• LPS lipopolysaccharide
• Serum C-reactive protein (Chemicon International), tumor necrosis factor-a (TNFa), interleukin (IL)-IO, and tumor growth factor-b (TGFb) (Biosource) were determined by a sandwich ELISA.
• TNFa tumor necrosis factor-a
• IL interleukin
• TGFb tumor growth factor-b
• Biosource tumor growth factor-b
• SFA are transparent to UV, they were measured, after methylation, by means of a gas chromatograph (Agilent, Model 6890) equipped with split ratio of 20:1 injection port, a flame ionization detector, an autosampler (Agilent, Model 7673), a 100-m HP-88 fused capillary column (Agilent), and an Agilent ChemStation software system.
• the injector and detector temperatures were set at 250°C and 280°C, respectively.
• H 2 served as carrier gas (1 mL/min) and the flame ionization detector gases were H 2 (30 mL/min), N 2 (30 mL/min), and purified air (300 mL/min).
• the temperature program was as follows: initial temperature was 120°C, programmed at 10°C/min to 210°C and 5°C/min to 230°C, then programmed at 25°C/min to 250°C and held for 2 min.
• AEA and 2-AG were measured.
• MAGL and FAAH activities were determined in the heart, liver, VAT 1 and SAT from C and FO- and KO-supplemented rats.
• 2-AG hydrolysis (mostly by MAGL) was measured by incubating the 10,000 X g cytosolic fraction of tissues (100 mg per sample) in Tris-HCI 50 mmol/L, at pH 7.0 at 37°C for 20 min, with synthetic 2-arachidonoyl-[ 3 H]-glycerol (40 Ci/mmol, ARC) properly diluted with 2-AG (Cayman Chemicals). After incubation, the amount of [ 3 H]-glycerol produced was measured by scintillation counting of the aqueous phase after extraction of the incubation mixture with 2 volumes of
• Plasma proinflammatory TNFa, IL-6, IL-I b
• anti-inflammatory cytokines IL-10 and TGFb
• C-reactive protein did not differ among the experimental groups (see Table 22).
• liver TAG EPA, DPA, and DHA levels were significantly elevated in the FO and KO groups compared with C, whereas ARA levels did not differ among the groups.
• EPA and DPA concentrations were greater in both (n-3) LCPUFA supplemented groups than in C, whereas DHA was significantly higher than C only in the KO group.
• LA was significantly greater in the (n-3) LCPUFA- supplemented groups than in C, whereas the ARA level did not differ (see Table 28).
• the heart TAG fatty acid profile had higher levels of EPA, DPA, and DHA and lower levels of ARA in the FO and KO groups compared with C.
• LA and ALA concentrations were lower than in C only in the KO-supplemented rats.
• mice Male CBA/J mice were purchased from Jackson Laboratory at six weeks of age and were individually housed and fed 84 kcal/week of a control AIN93M diet. At two months of age, mice were transferred to one of six test diets (10 mice per diet): Control, a diet supplemented with fish oil (FO), and a diet supplemented with Superba krill oil (KO). All mice received 84 kcal/week. The supplemented diets were based on modifications of the Control diet as described in Table 29. Amounts of each component are shown as grams of that component per kilogram of diet.
• mice were euthanized by cervical dislocation, blood was collected from the body cavity, and tissues were rapidly dissected, flash frozen in liquid nitrogen, and stored at -80°C. Gene expression profiling was performed. Total RNA was extracted from liver tissue of seven mice per group and was processed according to standard protocols described by Affymetrix. Samples were hybridized on the Affymetrix Mouse Genome 430 2.0 array, which allows from the detection of approximately 20,000 known genes. To determine the effect of a test diet on the expression of a gene, the average signal intensity for the treated group was compared to the average signal intensity for that gene in the Control group. Comparisons between groups were made using two-tailed t-tests (experimental vs. Control); a gene was considered to be significantly changed by treatment at p ⁇ 0.01.
• PAGE Gene set Enrichment
• hepatic lipid accumulation hepatic steatosis
• fish oil did not significantly modulate lipid biosynthesis in this study.
• krill oil would be a novel dietary intervention to modulate key pathways of energy metabolism in the liver in a manner which would oppose the effect seen in type 2 diabetes.
• KO significantly suppressed the pathway "cholesterol biosynthesis.”
• Other studies have shown that krill oil has the ability to improve circulating triglycerides as well as cholesterol levels in rats and humans, and the current data provide molecular evidence to support those findings.
• KO resulted in a significant decrease in the expression of two key genes in the pathway of cholesterol metabolism including the gene encoding the rate- limiting enzyme for cholesterol synthesis (Hmgcr) and the Pmvk gene which encodes a protein that catalyzes the fifth condensation reaction in cholesterol synthesis (FIG. 37).
• liver pyruvate kinase PkIr
• AcIy ATP citrate lyase
• Acaca acetyl CoA carboxylase

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